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COVID-19 impact will also be included and considered for forecast.

Global Cell Harvesting System Market research report provides detail information about Market Introduction, Market Summary, Global market Revenue (Revenue USD), Market Drivers, Market Restraints, Market Opportunities, Competitive Analysis, Regional and Country Level.

Cell Harvesting System Market Size Covers Global Industry Analysis, Size, Share, CAGR, Trends, Forecast And Business Opportunity.

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Cell Harvesting System Market: Increase in healthcare facilities and increase in bone marrow transplantation are key drivers for the Global Cell Harvesting System Market.

The global cell harvesting systems market size was valued USD 3533.27 Million in 2017 and is expected to grow at a CAGR of 14.01% over the forecast period.

Cell harvesting is a system which is used to cultivate, regenerate, transplant and repair the damages organs with the healthy one. Cell harvesting is one of the important parts of biopharmaceutical industry which directly relates with the quality of product. Stem cell harvesting also helps in the treatment of various diseases such as cancer, autoimmune disease, anemia and others. So, during the study of Global Cell Harvesting System market, we have considered Cell Harvesting System to analyze the market.

Global Cell Harvesting System Market report is segmented on the technique type, application type, end user type and by regional & country level. Based upon technique type, global Cell Harvesting System Market is classified as Altered Nuclear Transfer and Blastomere Extraction. Based upon Application type, global Cell Harvesting System Market is classified as Bone Marrow, Peripheral Blood, Umbilical Cord Blood, and Adipose Tissue. Based upon end users, global Cell Harvesting System Market is classified as Research Centers, Academics Institutes, Diagnostic Labs, and Hospitals.

The regions covered in this Cell Harvesting System Market report are North America, Europe, Asia-Pacific and Rest of the World. On the basis of country level, market of Cell Harvesting System is sub divided into U.S., Mexico, Canada, U.K., France, Germany, Italy, China, Japan, India, South East Asia, GCC, Africa, etc.

Key Players for Global Cell Harvesting System Market Reports

Global Cell Harvesting System market report covers prominent players like Tomtec, Bertin Technologies, PerkinElmer Inc., TERUMO BCT, INC., SP Scienceware, hynoDent AG, Avita Medical, BRAND GMBH Teleflex Incorporated., Argos Technologies, Inc., Thomas Scientific, Arthrex, Inc. and others.

Global Cell Harvesting System Market Dynamics

The commercialization and growth of global Cell Harvesting System market over the past 25 years has been highly impactful. Bone marrow transplantation is one of the major factors driving the growth of cell harvesting system over the forecast period. Due to the increase in blood cancer it has raised the demand for bone marrow transplantation which in turn increased the demand for cell harvesting system. As per The Leukemia & Lymphoma Society report 2018, an estimated combined total of 174,250 people in the US are expected to be diagnosed with leukemia, lymphoma or myeloma in 2018. There is also an increase in awareness about stem cells and its advantages which are helpful in the treatment of various disorders. Furthermore, various technological advancement have also increase the new and better technologies with better results are expected to promote the growth of cell harvesting system market over the forecast period. However, High cost, lack of reimbursement policies, immune rejection and others are the various factors which are expected to hamper the growth of cell harvesting system market over the forecast period.

Global Cell Harvesting System Market Regional Analysis

North America dominates the market with highest market share which is closely followed by the Europe over the forecast period. Due to the increased prevalence of leukemia, lymphoma and others coupled with increased healthcare facilities. As per The Leukemia & Lymphoma Society 2018 report, new cases of leukemia, lymphoma and myeloma are expected to account for 10 percent of the estimated 1,735,350 new cancer cases diagnosed in the US in 2018. Asia Pacific is expected to be the third largest and fastest growing region over the forecast period. Due to various technological advancements, increase in awareness among people and others are expected to support the growth of cell harvesting system market over the forecast period. Furthermore, Increase in healthcare facilities in the developing economies such as India, China and others are expected to fuel the growth of cell harvesting system market. Latin America, Middle East and Africa and expected to develop at a considerable rate over the forecast period.

Key Benefits for Global Cell Harvesting System Market Reports

Global Cell Harvesting System market report covers in depth historical and forecast analysis.Global Cell Harvesting System Market research report provides detail information about Market Introduction, Market Summary, Global market Revenue (Revenue USD), Market Drivers, Market Restraints, Market opportunities, Competitive Analysis, Regional and Country Level.Global Cell Harvesting System Market report helps to identify opportunities in market place.Global Cell Harvesting System Market report covers extensive analysis of emerging trends and competitive landscape.

By Techniques Type:

Altered Nuclear TransferBlastomere Extraction

By Application:

Bone MarrowPeripheral BloodUmbilical Cord BloodAdipose Tissue

By End User:

Research CentersAcademics InstitutesDiagnostic LabsHospitals

By Region

North AmericaU.S.CanadaEuropeUKFranceGermanyItalyAsia PacificChinaJapanIndiaSoutheast AsiaLatin AmericaBrazilMexicoThe Middle East and AfricaGCCAfricaRest of Middle East and Africa

Cell Harvesting System Market Key PlayersTomtecBertin TechnologiesPerkinElmer Inc.TERUMO BCT, INC.SP SciencewarehynoDent AGAvita MedicalBRAND GMBHTeleflex Incorporated.Argos Technologies, Inc.Thomas ScientificArthrex, Inc.

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Table of Content:

Market Overview: The report begins with this section where product overview and highlights of product and application segments of the Global Cell Harvesting System Market are provided. Highlights of the segmentation study include price, revenue, sales, sales growth rate, and market share by product.

Competition by Company: Here, the competition in the Worldwide Global Cell Harvesting System Market is analyzed, By price, revenue, sales, and market share by company, market rate, competitive situations Landscape, and latest trends, merger, expansion, acquisition, and market shares of top companies.

Company Profiles and Sales Data: As the name suggests, this section gives the sales data of key players of the Global Cell Harvesting System Market as well as some useful information on their business. It talks about the gross margin, price, revenue, products, and their specifications, type, applications, competitors, manufacturing base, and the main business of key players operating in the Global Cell Harvesting System Market.

Market Status and Outlook by Region: In this section, the report discusses about gross margin, sales, revenue, production, market share, CAGR, and market size by region. Here, the Global Cell Harvesting System Market is deeply analyzed on the basis of regions and countries such as North America, Europe, China, India, Japan, and the MEA.

Application or End User: This section of the research study shows how different end-user/application segments contribute to the Global Cell Harvesting System Market.

Market Forecast: Here, the report offers a complete forecast of the Global Cell Harvesting System Market by product, application, and region. It also offers global sales and revenue forecast for all years of the forecast period.

Research Findings and Conclusion: This is one of the last sections of the report where the findings of the analysts and the conclusion of the research study are provided.

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Covid 19 Outbreak Cell Harvesting System Market 2020 Product Type, Applications/end user, Key Players and Geographical Regions 2026 - Jewish Life...

People usually volunteer to donate stem cells for an allogeneic transplant either because they have a loved one or friend who needs a match or because they want to help people. Some people give their stem cells so they can get them back later if they need an autologous transplant.

People who want to donate stem cells or join a volunteer registry can speak with a health care provider or contact the National Marrow Donor Program to find the nearest donor center. Potential donors are asked questions to make sure they are healthy enough to donate and dont pose a risk of infection to the recipient. For more information about donor eligibility guidelines, contact Be the Match or the donor center in your area.

Be the Match (formerly the National Marrow Donor Program)Toll-free number: 1-800-MARROW-2 (1-800-627-7692)Website: http://www.bethematch.org

A simple blood test is done to learn the potential donors HLA type. There may be a one-time, tax-deductible fee of about $75 to $100 for this test. People who join a volunteer donor registry will most likely have their tissue type kept on file until they reach age 60.

Pregnant women who want to donate their babys cord blood should make arrangements for it early in the pregnancy, at least before the third trimester. Donation is safe, free, and does not affect the birth process.

If a possible stem cell donor is found to be a good match for a recipient, steps are taken to teach the donor about the transplant process and make sure he or she is making an informed decision. If a person decides to donate, a consent form must be signed after the risks of donating are fully discussed. The donor is not pressured to take part. Its always a choice.

If a person decides to donate, a medical exam and blood tests will be done to make sure the donor is in good health.

Stem cells may be collected from these 3 different sources:

Each method of collection is explained here.

This process is often called bone marrow harvest. Its done in an operating room, while the donor is under general anesthesia (given medicine to put them into a deep sleep so they dont feel pain). The marrow cells are taken from the back of the pelvic (hip) bone. The donor lies face down, and a large needle is put through the skin and into the back of the hip bone. Its pushed through the bone to the center and the thick, liquid marrow is pulled out through the needle. This is repeated several times until enough marrow has been taken out (harvested). The amount taken depends on the donors weight. Often, about 10% of the donors marrow, or about 2 pints, are collected. This takes about 1 to 2 hours. The body will replace these cells within 4 to 6 weeks. If blood was taken from the donor before the marrow donation, its often given back to the donor at this time.

After the bone marrow is harvested, the donor is taken to the recovery room while the anesthesia wears off. The donor may then be taken to a hospital room and watched until fully alert and able to eat and drink. In most cases, the donor is able to leave the hospital within a few hours or by the next morning.

The donor may have soreness, bruising, and aching at the back of the hips and lower back for a few days. Over-the-counter pain medications or nonsteroidal anti-inflammatory drugs are helpful. Some people may feel tired or weak, and have trouble walking for a few days. The donor might be told to take iron supplements until the number of red blood cells returns to normal. Most donors get back to their usual activities in 2 to 3 days. But it could take 2 or 3 weeks before they feel completely back to normal.

There arent many risks for donors and serious complications are rare. But bone marrow donation is a surgical procedure. Rare complications could include anesthesia reactions, infection, nerve or muscle damage, transfusion reactions (if a blood transfusion of someone elses blood is needed this doesnt happen if you get your own blood), or injury at the needle insertion sites. Problems such as sore throat or nausea may be caused by anesthesia.

Allogeneic stem cell donors do not have to pay for the harvesting because the recipients insurance company usually covers the cost. Even so, be sure to ask about insurance coverage before you decide to have the bone marrow harvest done.

Once the cells are collected, they are filtered through fine mesh screens. This prevents bone or fat particles from being given to the recipient. For an allogeneic or syngeneic transplant, the cells may be given to the recipient through a vein soon after they are harvested. Sometimes theyre frozen, for example, if the donor lives far away from the recipient.

For several days before starting the donation process, the donor is given a daily injection (shot) of a drug that causes the bone marrow to make and release a lot of stem cells into the blood. Filgrastim can cause some side effects, the most common being bone pain and headaches. These may be helped by over-the-counter pain medications or nonsteroidal anti-inflammatory drugs. Nausea, sleeping problems, low-grade (mild) fevers, and tiredness are other possible effects. These go away once the injections are finished and collection is completed.

After the shots, blood is removed through a catheter (a thin, flexible plastic tube) thats put in a large vein in the arm. Its then cycled through a machine that separates the stem cells from the other blood cells. The stem cells are kept while the rest of the blood is returned to the donor, often through the same catheter. (In some cases, a catheter may be put in each arm one takes out blood and the other puts it back.) This process is called apheresis. It takes about 2 to 4 hours and is done as an outpatient procedure. Often the process needs to be repeated daily for a few days, until enough stem cells have been collected.

Possible side effects of the catheter can include trouble placing the catheter in the vein, blockage of the catheter, or infection of the catheter or at the area where it enters the vein. Blood clots are another possible side effect. During the apheresis procedure, donors may have problems caused by low calcium levels from the anti-coagulant drug used to keep the blood from clotting in the machine. These can include feeling lightheaded or tingly, and having chills or muscle cramps. These go away after donation is complete, but may be treated by giving the donor calcium supplements.

The process of donating cells for yourself (autologous stem cell donation) is pretty much the same as when someone donates them for someone else (allogeneic donation). Its just that in autologous stem cell donation the donor is also the recipient, giving stem cells for his or her own use later on. For some people, there are a few differences. For instance, sometimes chemotherapy (chemo) is given before the growth factor drug is used to tell the body to make stem cells. Also, sometimes it can be hard to get enough stem cells from a person with cancer. Even after several days of apheresis, there may not be enough for the transplant. This is more likely to be a problem if the patient has had certain kinds of chemo in the past, or if they have an illness that affects their bone marrow.

Cord blood is the blood thats left in the placenta and umbilical cord after a baby is born. Collecting it does not pose any health risk to the infant or the mother. Cord blood transplants use blood that would otherwise be thrown away. After the umbilical cord is clamped and cut, the placenta and umbilical cord are cleaned. The cord blood is put into a sterile container, mixed with a preservative, and frozen until needed.

Some parents choose to donate their infants cord blood to a public blood bank, so that it may be used by anyone who needs it. Many hospitals collect cord blood for donation, which makes it easier for parents to donate. Parents can donate their newborns cord blood to volunteer or public cord blood banks at no cost. For more about donating your newborns cord blood, call 1-800-MARROW2 (1-800-627-7692) or visit Be the Match.

Other parents store their newborns cord blood in private cord blood banks just in case the child or a close relative needs it someday. If you want to donate or bank (save) your childs cord blood, youll need to arrange it before the baby is born. Some banks require you to set it up before the 28th week of pregnancy, although others accept later setups. Among other things, youll be asked to answer health questions and sign a consent form.

Parents may want to bank their childs cord blood if the family has a history of diseases that may benefit from stem cell transplant. There are several private companies offer this service. But here are some things to think about:

More information on private family cord blood banking can be found at the Parents Guide to Cord Blood Foundation. You can visit their website at http://www.parentsguidecordblood.org.

Continued here:
Donating Stem Cells and Bone Marrow

Bone marrowaspiration and trephine biopsy are usually performed on the back of the hipbone, or posterior iliac crest. An aspirate can also be obtained from the sternum (breastbone). For the sternal aspirate, the patient lies on their back, with a pillow under the shoulder to raise the chest. A trephine biopsy should never be performed on the sternum, due to the risk of injury to blood vessels, lungs or the heart.

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The need to selectively isolate and concentrate selective cells, such as mononuclear cells, allogeneic cancer cells, T cells and others, is driving the market. Over 30,000 bone marrow transplants occur every year. The explosive growth of stem cells therapies represents the largest growth opportunity for bone marrow processing systems.Europe and North America spearheaded the market as of 2016, by contributing over 74.0% to the overall revenue. Majority of stem cell transplants are conducted in Europe, and it is one of the major factors contributing to the lucrative share in the cell harvesting system market.

In 2016, North America dominated the research landscape as more than 54.0% of stem cell clinical trials were conducted in this region. The region also accounts for the second largest number of stem cell transplantation, which is further driving the demand for harvesting in the region.Asia Pacific is anticipated to witness lucrative growth over the forecast period, owing to rising incidence of chronic diseases and increasing demand for stem cell transplantation along with stem cell-based therapy.

Japan and China are the biggest markets for harvesting systems in Asia Pacific. Emerging countries such as Mexico, South Korea, and South Africa are also expected to report lucrative growth over the forecast period. Growing investment by government bodies on stem cell-based research and increase in aging population can be attributed to the increasing demand for these therapies in these countries.

Major players operating in the global bone marrow processing systems market are ThermoGenesis (Cesca Therapeutics inc.), RegenMed Systems Inc., MK Alliance Inc., Fresenius Kabi AG, Harvest Technologies (Terumo BCT), Arthrex, Inc. and others

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Bone Marrow Processing System Market Incisive Insights Regarding Major Regions, Key Players And Opportunities Up To 2025 - Kentucky Journal 24

Global Stem Cell Therapy Market: Overview

Also called regenerative medicine, stem cell therapy encourages the reparative response of damaged, diseased, or dysfunctional tissue via the use of stem cells and their derivatives. Replacing the practice of organ transplantations, stem cell therapies have eliminated the dependence on availability of donors. Bone marrow transplant is perhaps the most commonly employed stem cell therapy.

Osteoarthritis, cerebral palsy, heart failure, multiple sclerosis and even hearing loss could be treated using stem cell therapies. Doctors have successfully performed stem cell transplants that significantly aid patients fight cancers such as leukemia and other blood-related diseases.

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Global Stem Cell Therapy Market: Key Trends

The key factors influencing the growth of the global stem cell therapy market are increasing funds in the development of new stem lines, the advent of advanced genomic procedures used in stem cell analysis, and greater emphasis on human embryonic stem cells. As the traditional organ transplantations are associated with limitations such as infection, rejection, and immunosuppression along with high reliance on organ donors, the demand for stem cell therapy is likely to soar. The growing deployment of stem cells in the treatment of wounds and damaged skin, scarring, and grafts is another prominent catalyst of the market.

On the contrary, inadequate infrastructural facilities coupled with ethical issues related to embryonic stem cells might impede the growth of the market. However, the ongoing research for the manipulation of stem cells from cord blood cells, bone marrow, and skin for the treatment of ailments including cardiovascular and diabetes will open up new doors for the advancement of the market.

Global Stem Cell Therapy Market: Market Potential

A number of new studies, research projects, and development of novel therapies have come forth in the global market for stem cell therapy. Several of these treatments are in the pipeline, while many others have received approvals by regulatory bodies.

In March 2017, Belgian biotech company TiGenix announced that its cardiac stem cell therapy, AlloCSC-01 has successfully reached its phase I/II with positive results. Subsequently, it has been approved by the U.S. FDA. If this therapy is well- received by the market, nearly 1.9 million AMI patients could be treated through this stem cell therapy.

Another significant development is the granting of a patent to Israel-based Kadimastem Ltd. for its novel stem-cell based technology to be used in the treatment of multiple sclerosis (MS) and other similar conditions of the nervous system. The companys technology used for producing supporting cells in the central nervous system, taken from human stem cells such as myelin-producing cells is also covered in the patent.

The regional analysis covers:

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Global Stem Cell Therapy Market: Regional Outlook

The global market for stem cell therapy can be segmented into Asia Pacific, North America, Latin America, Europe, and the Middle East and Africa. North America emerged as the leading regional market, triggered by the rising incidence of chronic health conditions and government support. Europe also displays significant growth potential, as the benefits of this therapy are increasingly acknowledged.

Asia Pacific is slated for maximum growth, thanks to the massive patient pool, bulk of investments in stem cell therapy projects, and the increasing recognition of growth opportunities in countries such as China, Japan, and India by the leading market players.

Global Stem Cell Therapy Market: Competitive Analysis

Several firms are adopting strategies such as mergers and acquisitions, collaborations, and partnerships, apart from product development with a view to attain a strong foothold in the global market for stem cell therapy.

Some of the major companies operating in the global market for stem cell therapy are RTI Surgical, Inc., MEDIPOST Co., Ltd., Osiris Therapeutics, Inc., NuVasive, Inc., Pharmicell Co., Ltd., Anterogen Co., Ltd., JCR Pharmaceuticals Co., Ltd., and Holostem Terapie Avanzate S.r.l.

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Stem Cell Therapy Market Analysis and Demand 2017 2025 - Cole of Duty

Mesenchymal Stem Cells Market Trends by Manufacturers, States, Type and Application, Forecast to 2019 2027

Global Mesenchymal Stem Cells Market: Snapshot

The increasing use of mesenchymal stem cells (MSCs) for the treatment of diseases and disabilities of the growing aging population is having a positive influence on the global mesenchymal stem cells market. Mesenchymal stem cells are adult stem cells that are of various types such as adipocytes, osteocytes, monocytes, and chondrocytes.

The main function of mesenchymal stem cells is to replace or repair damaged tissue.

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Mesenchymal stem cells are multipotent, i.e. they can produce more than one type of specialized cells.

These specialized cells have their own distinguishing shapes, structures, and functions, with each of them belonging to a particular tissue.

Mesenchymal stem cells are traditionally found in the bone marrow. However, these cells can also be separated from other tissues such as cord blood, fallopian tube, peripheral blood, and fetal liver and lung.

Mesenchymal stem cells have long thin cell bodies containing a large nucleus. MSCs have enormous capacity for renewal keeping multipotency.

Due to these virtues, mesenchymal stem cells have huge therapeutic capacity for tissue repair.

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Mesenchymal stem cells can differentiate into a number of cell types that belong to our skeletal tissues that include cartilage, bone, and fat. Research is underway to discover if mesenchymal stem cells can be used to treat bone and cartilage diseases.

Scientists are also exploring the possibility if mesenchymal stem cells differentiate into other type of cells apart from skeletal tissues. This includes nerve cells, liver cells, heart muscle cells, and endothelial cells.

This will lead to mesenchymal stem cells to be used to treat other diseases.

Stem cells are specialized cells which have the capability of renewing themselves through cell division and differentiate into multi-lineage cells. Mesenchymal stem cells (MSCs) are non- hematopoietic, multipotent adult stem cells which can be isolated from bone marrow, cord blood, fat tissue, peripheral blood, fallopian tube, and fetal liver and lung tissue.

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Mesenchymal stem cells have the capacity to differentiate into mesodermal lineages, such as chondrocytes, adipocytes, and osteocytes, and non-mesodermal lineages such as ectodermal (neurocytes) and endodermal lineages (hepatocytes). These stem cells have specific features such as multilineage potential, secretion of anti-inflammatory molecules, and immunomodulation.

These cells have emerged as promising therapeutic agents for regenerating skeletal tissues such as damaged bone and cartilage tissues and treatment of chronic diseases owing to their specific features.

The global mesenchymal stem cells market is expected to be driven by the increasing clinical application of mesenchymal stem cells for the treatment of chronic diseases, bone and cartilage diseases, and autoimmune diseases. Studies have shown that these stem cells enhance the angiogenesis in myocardium and allow the reduction of myocardial fibrotic area.

The pre-clinical studies for using mesenchymal stem cells in treatment of cardiovascular diseases, liver diseases, and cancer are projected to create new market opportunities for mesenchymal stem cells. Mesenchymal stem cells also produce anti-inflammatory molecules which modulate humoral and cellular immune responses.

Features of these stem cells such as ease of isolation, regenerative potential, and immunoregulatory, the mesenchymal stem cell therapy has emerged as a promising tool for the treatment of chronic diseases, degenerative, inflammatory, and autoimmune diseases.

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Clinical studies are exploring MSCs for various conditions such as orthopedic injuries, graft versus host disease following bone marrow transplantation, and genetic modification of MSCs to overexpress antitumor genes for use as anticancer therapy, which are exhibiting new opportunities in therapeutic area. However, the mesenchymal stem cell research studies are tedious, lengthy, and complex.

In some cases, due to some adverse effects transplanted mesenchymal stem cells rapidly removed from the body which limits use of stem cells in therapeutic treatments. The conflicting results and regulatory compliances for approvals may also hamper the growth of this market.

The global mesenchymal stem cells market is segmented on the basis of source of isolation, end-user, and region. Stem cells are isolated from the bone marrow, peripheral blood, lung tissue, umbilical cord blood, amniotic fluids, adipose tissues, and synovial tissues.

Traditionally the MSCs were isolated from bone marrow aspiration which is associated with risk of infection and painful for the patient. The MSCs from adipose tissues are usually isolated from the biological material generated during liposuction, lipectomy procedures by using collagenase enzymatic digestion followed by centrifugation and washing.

In terms of end-user, the market is segmented into clinical research organizations, biotechnological companies, medical research institutes, and hospitals.

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Geographically, the global mesenchymal stent cells market is distributed over North America, Europe, Asia Pacific, Latin America, and Middle East & Africa. North America dominated the global market and is projected to continue its dominance in terms of market share during the forecast period owing to high R&D expenditure, availability of advanced research facilities and skilled professionals, and government initiatives.

Europe is the second largest market after North America. The Asia Pacific market is projected to expand at a high CAGR during the forecast period due to increased R&D budgets in Japan, China, and India.

Key global players operating in the mesenchymal stem cells market include R&D Systems, Inc., Cell Applications, Inc., Axol Bioscience Ltd., Cyagen Biosciences Inc., Cytori Therapeutics Inc., Stemcelltechnologies Inc., BrainStorm Cell Therapeutics, Stemedica Cell Technologies, Inc., and Celprogen, Inc.

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Mesenchymal Stem Cells Market trends by manufacturers, states, type and application, forecast to 2019 2027 - WhaTech Technology and Markets News

The Phase 2 study will assess the safety and efficacy of Longeveron's stem cell treatment under Japan's accelerated regulatory pathway for regenerative medicine.

MIAMI, June 1, 2020 /PRNewswire/ --Longeveron LLC announced today that Japan's Pharmaceutical and Medical Devices Agency (PMDA) (the Japanese agency akin to the United States' Food & Drug Administration) approved a Clinical Trial Notification (CTN) application (akin to an Investigational New Drug Application or "IND" in the US regulatory system), approving the initiation of a Phase 2 clinical trial evaluating the safety and efficacy of Longeveron's Mesenchymal Stem Cells (LMSCs) for the treatment of Aging Frailty in Japanese patients. This is another key milestone for Longeveron's Aging Frailty program, which includes two ongoing Phase 2 clinical trials in the U.S.

"We are extremely pleased to achieve this significant milestone," said Geoff Green, President of Longeveron."This study is designed to determine whether the transplant of donor-derived mesenchymal stem cells can improve healthspan in mild to moderately frail patients, thereby improving functionality and potentially lowering their risk of disability, and dependence on others for care."

Aging Frailty is a common, but reversible, life-threatening geriatric condition affecting millions of Japanese over the age of 65.Frail individuals are vulnerable to adverse health outcomes compared to their age-matched peers despite sharing similar comorbidities and demographics.Clinically, frailty manifests as a combination of symptoms that may include loss of muscle and decreased strength, slowed walking (sarcopenia), lower activity and energy levels, poor endurance, nutritional deficiencies, weight loss and fatigue.Collectively, these lead to overall decline in functionality, and increased risk of disability, dependency, and death.

"The biology of frailty is complex, and includes diminished stem cell activity, reduced ability to repair and regenerate tissue, and immunosenescence (deterioration of the immune system) and chronic systemic inflammation," said Dr. Anthony Oliva, Senior Scientist at Longeveron. "LMSCs have multiple mechanisms of action that can potentially address all of these issues, and thus make them extremely attractive as a therapeutic candidate for the unmet medical need of Aging Frailty."

The planned study is an investigator-initiated, randomized, double-blind, placebo-controlled design,and will be conducted at Juntendo University Hospital (Tokyo) and Japan's National Center for Geriatrics and Gerontology (NCGG) in Nagoya.The study's Principal Investigator, Dr. Hidenori Arai, President of the NCGG, commented that "Japan has one of the oldest and fastest aging societies in the world, with nearly 30% of Japan's citizens over the age of 65.Preventing and reversing functional decline associated with frailty is one of the nation's top priorities, and Longeveron's regenerative medicine approach is an exciting and innovative potential therapeutic option.With the disproportionate infection and mortality rate of older people with COVID-19 and Influenza infection, it is critically important to rapidly test treatments that may be effective."

In Japan, the "Pharmaceutical and Medical Device Act" and the "Act on the Safety of Regenerative Medicine" came into effect in 2014. Under this system, a "Time-limited Conditional Approval" option exists, which allows a manufacturer to conditionally sell regenerative medicine products while proceeding with its Phase 3 clinical trial.

Longeveron's Aging Frailty Research Program

Longeveron sponsors the most extensive and advanced Aging Frailty clinical research program in the world, with more than 200 patients treated with LMSCs worldwide.In the U.S., two clinical trials are currently ongoing:

About LMSCs

Longeveron Allogeneic Mesenchymal Stem Cells (LMSCs) is a regenerative medicine product sourced from the bone marrow of young healthy adult donors.LMSCs are culture expanded under the FDA's current good manufacturing practices (cGMP) to high standards, and maintained as individual "off-the-shelf" doses.

About Longeveron LLC

Longeveron (www.longeveron.com) is a regenerative medicine therapy company founded in 2014. Longeveron's mission is to provide biological solutions for aging-related diseases and life-threatening conditions, and is dedicated to developing safe and effective cell-based therapeutics for unmet medical needs such as Aging Frailty, the Metabolic Syndrome, Alzheimer's Disease, Acute Respiratory Distress Syndrome (ARDS) from COVID-19 infection, and congenital heart defects in children (hypoplastic left heart syndrome).

For information please contact:

Paul Lehr, JDplehr@longeveron.com305-338-6257

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The Phase 2 study will assess the safety and efficacy of Longeveron's stem cell treatment under Japan's accelerated regulatory pathway for regenerative medicine.

MIAMI, FL, USA I June 1, 2020 I Longeveron LLC announced today that Japan's Pharmaceutical and Medical Devices Agency (PMDA) (the Japanese agency akin to the United States' Food & Drug Administration) approved a Clinical Trial Notification (CTN) application (akin to an Investigational New Drug Application or "IND" in the US regulatory system), approving the initiation of a Phase 2 clinical trial evaluating the safety and efficacy of Longeveron's Mesenchymal Stem Cells (LMSCs) for the treatment of Aging Frailty in Japanese patients. This is another key milestone for Longeveron's Aging Frailty program, which includes two ongoing Phase 2 clinical trials in the U.S.

"We are extremely pleased to achieve this significant milestone," said Geoff Green, President of Longeveron."This study is designed to determine whether the transplant of donor-derived mesenchymal stem cells can improve healthspan in mild to moderately frail patients, thereby improving functionality and potentially lowering their risk of disability, and dependence on others for care."

Aging Frailty is a common, but reversible, life-threatening geriatric condition affecting millions of Japanese over the age of 65.Frail individuals are vulnerable to adverse health outcomes compared to their age-matched peers despite sharing similar comorbidities and demographics.Clinically, frailty manifests as a combination of symptoms that may include loss of muscle and decreased strength, slowed walking (sarcopenia), lower activity and energy levels, poor endurance, nutritional deficiencies, weight loss and fatigue.Collectively, these lead to overall decline in functionality, and increased risk of disability, dependency, and death.

"The biology of frailty is complex, and includes diminished stem cell activity, reduced ability to repair and regenerate tissue, and immunosenescence (deterioration of the immune system) and chronic systemic inflammation," said Dr. Anthony Oliva, Senior Scientist at Longeveron. "LMSCs have multiple mechanisms of action that can potentially address all of these issues, and thus make them extremely attractive as a therapeutic candidate for the unmet medical need of Aging Frailty."

The planned study is an investigator-initiated, randomized, double-blind, placebo-controlled design,and will be conducted at Juntendo University Hospital (Tokyo) and Japan's National Center for Geriatrics and Gerontology (NCGG) in Nagoya.The study's Principal Investigator, Dr. Hidenori Arai, President of the NCGG, commented that "Japan has one of the oldest and fastest aging societies in the world, with nearly 30% of Japan's citizens over the age of 65.Preventing and reversing functional decline associated with frailty is one of the nation's top priorities, and Longeveron's regenerative medicine approach is an exciting and innovative potential therapeutic option.With the disproportionate infection and mortality rate of older people with COVID-19 and Influenza infection, it is critically important to rapidly test treatments that may be effective."

In Japan, the "Pharmaceutical and Medical Device Act" and the "Act on the Safety of Regenerative Medicine" came into effect in 2014. Under this system, a "Time-limited Conditional Approval" option exists, which allows a manufacturer to conditionally sell regenerative medicine products while proceeding with its Phase 3 clinical trial.

Longeveron's Aging Frailty Research Program

Longeveron sponsors the most extensive and advanced Aging Frailty clinical research program in the world, with more than 200 patients treated with LMSCs worldwide.In the U.S., two clinical trials are currently ongoing:

About LMSCs

Longeveron Allogeneic Mesenchymal Stem Cells (LMSCs) is a regenerative medicine product sourced from the bone marrow of young healthy adult donors.LMSCs are culture expanded under the FDA's current good manufacturing practices (cGMP) to high standards, and maintained as individual "off-the-shelf" doses.

About Longeveron LLC

Longeveron (www.longeveron.com) is a regenerative medicine therapy company founded in 2014. Longeveron's mission is to provide biological solutions for aging-related diseases and life-threatening conditions, and is dedicated to developing safe and effective cell-based therapeutics for unmet medical needs such as Aging Frailty, the Metabolic Syndrome, Alzheimer's Disease, Acute Respiratory Distress Syndrome (ARDS) from COVID-19 infection, and congenital heart defects in children (hypoplastic left heart syndrome).

SOURCE: Longeveron

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Longeveron Announces Japanese Approval of Clinical Trial for Treatment of Aging Frailty With Longeveron's Stem Cells | DNA RNA and Cells | News...

The art (and existing science) of regenerative medicine in equine practice, and whats to come

Regenerative therapy is an umbrellaterm encompassing any method that encourages the body to self- heal. Because it is drawing onits own properties, healing tissue more closely resembles native tissue than weak, disorganized scar tissue typically seen post-injury.

The goal is to allow restoration of normal function and structure of the injured tissue to allow horses to perform at their previous level, whatever that might be, with a reduced risk of reinjury, says Kyla Ortved, DVM, PhD, Dipl. ACVS, ACVSMR, assistant professor of large animal surgery at the University of Pennsylvanias New Bolton Center, in Kennett Square.

She says the three main components of regenerative medicine that help tissues self-heal include:

A specific therapy may incorporate some or all three of these components, says Ortved.

Due to the regenerative therapy industrys popularity and continued growth, many articles weve published review recent laboratory studies about stem cell production and data on efficacy andsafety (you can find them at TheHorse.com/topics/regenerative-medicine). Here, well review the basics of three regenerative modalities commonly used in equine medicine and when veterinarians and horse owners might consider each.

With this approach the practitioner collects blood from a horse and processes it using a commercial system that concentrates the platelets. When he or she injects that concentrated platelet product back into the horse, granules within the platelets release an array of growth factors that aim to facilitate and modulate the healing process. Specifically, granule-derived growth factors encourage target tissue cells at the injury site to migrate and proliferate, improve extracellular matrix synthesis, and stimulate blood vessel development.

Recently, leukocyte-reduced PRP hasbecome many equine veterinarians PRP product of choice. These preparations contain fewer white blood cells (leukocytes) and, reportedly, inflammatory mediators than normal PRP products do. These mediators break tissues down, effectively counteracting the anabolic (tissue-building) effects of the platelets and their granules.

Veterinarians can easily prepare ACS by collecting a blood sample from the patient, then incubating it with special commercially available glass beads to stimulate interleukin-1 receptor antago- nist protein (IRAP) production. Theythen inject the resultant IRAP-rich serumsample back into the patient at the target location or injury site. This protein blocks the action of interleukin-1, a powerful and damaging pro-inflammatory mediator. Additionally, glass bead incubation stimulates the production of anti-inflammatory mediators and growth factors similar to those found in PRP.

Ortved says its important to remember that all biologics, including PRP and IRAP, contain various concentrations of growth factors and bioactive protein.

Remember, they are made from your horses blood and, therefore, containall of the components in blood, just in varying concentrations, she says.

Regenerative therapies that contain highconcentrations of IRAP include IRAP II, autologous protein solution (APS), and bone marrow aspirate concentrate (BMAC).

In certain tissues, such as adipose (fat) and bone marrow, we can find specific cells that have the ability to self-renew and grow more than 200 types of body cells. Veterinarians can isolate these cells, called stem cells or progenitor cells, and either:

Perhaps more important than theirability to differentiate into other celltypes, stem cells have powerful anti-inflammatoryproperties and play acentral role in coordinating healing in alltypes of tissues through cell-to-cell signaling,Ortved says.

Which of these three modality typeswill provide the most benefit to yourhorse depends on a variety of factors thatyou and your veterinarian will consider.

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Regenerative Therapies: Helping Horses Self-Heal The Horse - TheHorse.com

Stem Cell Assay Market: Snapshot

Stem cell assay refers to the procedure of measuring the potency of antineoplastic drugs, on the basis of their capability of retarding the growth of human tumor cells. The assay consists of qualitative or quantitative analysis or testing of affected tissues andtumors, wherein their toxicity, impurity, and other aspects are studied.

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With the growing number of successfulstem cell therapytreatment cases, the global market for stem cell assays will gain substantial momentum. A number of research and development projects are lending a hand to the growth of the market. For instance, the University of Washingtons Institute for Stem Cell and Regenerative Medicine (ISCRM) has attempted to manipulate stem cells to heal eye, kidney, and heart injuries. A number of diseases such as Alzheimers, spinal cord injury, Parkinsons, diabetes, stroke, retinal disease, cancer, rheumatoid arthritis, and neurological diseases can be successfully treated via stem cell therapy. Therefore, stem cell assays will exhibit growing demand.

Another key development in the stem cell assay market is the development of innovative stem cell therapies. In April 2017, for instance, the first participant in an innovative clinical trial at the University of Wisconsin School of Medicine and Public Health was successfully treated with stem cell therapy. CardiAMP, the investigational therapy, has been designed to direct a large dose of the patients own bone-marrow cells to the point of cardiac injury, stimulating the natural healing response of the body.

Newer areas of application in medicine are being explored constantly. Consequently, stem cell assays are likely to play a key role in the formulation of treatments of a number of diseases.

Global Stem Cell Assay Market: Overview

The increasing investment in research and development of novel therapeutics owing to the rising incidence of chronic diseases has led to immense growth in the global stem cell assay market. In the next couple of years, the market is expected to spawn into a multi-billion dollar industry as healthcare sector and governments around the world increase their research spending.

The report analyzes the prevalent opportunities for the markets growth and those that companies should capitalize in the near future to strengthen their position in the market. It presents insights into the growth drivers and lists down the major restraints. Additionally, the report gauges the effect of Porters five forces on the overall stem cell assay market.

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Global Stem Cell Assay Market: Key Market Segments

For the purpose of the study, the report segments the global stem cell assay market based on various parameters. For instance, in terms of assay type, the market can be segmented into isolation and purification, viability, cell identification, differentiation, proliferation, apoptosis, and function. By kit, the market can be bifurcated into human embryonic stem cell kits and adult stem cell kits. Based on instruments, flow cytometer, cell imaging systems, automated cell counter, and micro electrode arrays could be the key market segments.

In terms of application, the market can be segmented into drug discovery and development, clinical research, and regenerative medicine and therapy. The growth witnessed across the aforementioned application segments will be influenced by the increasing incidence of chronic ailments which will translate into the rising demand for regenerative medicines. Finally, based on end users, research institutes and industry research constitute the key market segments.

The report includes a detailed assessment of the various factors influencing the markets expansion across its key segments. The ones holding the most lucrative prospects are analyzed, and the factors restraining its trajectory across key segments are also discussed at length.

Global Stem Cell Assay Market: Regional Analysis

Regionally, the market is expected to witness heightened demand in the developed countries across Europe and North America. The increasing incidence of chronic ailments and the subsequently expanding patient population are the chief drivers of the stem cell assay market in North America. Besides this, the market is also expected to witness lucrative opportunities in Asia Pacific and Rest of the World.

Global Stem Cell Assay Market: Vendor Landscape

A major inclusion in the report is the detailed assessment of the markets vendor landscape. For the purpose of the study the report therefore profiles some of the leading players having influence on the overall market dynamics. It also conducts SWOT analysis to study the strengths and weaknesses of the companies profiled and identify threats and opportunities that these enterprises are forecast to witness over the course of the reports forecast period.

Some of the most prominent enterprises operating in the global stem cell assay market are Bio-Rad Laboratories, Inc (U.S.), Thermo Fisher Scientific Inc. (U.S.), GE Healthcare (U.K.), Hemogenix Inc. (U.S.), Promega Corporation (U.S.), Bio-Techne Corporation (U.S.), Merck KGaA (Germany), STEMCELL Technologies Inc. (CA), Cell Biolabs, Inc. (U.S.), and Cellular Dynamics International, Inc. (U.S.).

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Stem Cell Assay Market to Witness Growth Acceleration During 2017-2025 - Cole of Duty

SOUTH SAN FRANCISCO--(BUSINESS WIRE)--Imago BioSciences, Inc. (Imago), a clinical stage biopharmaceutical company developing innovative treatments for myeloid diseases, today announced that positive Phase 2 data from its lead pipeline program bomedemstat (IMG-7289), will be presented at the Virtual Edition of the 25th EHA Annual Congress beginning June 12, 2020.

Title: A PHASE 2 STUDY OF BOMEDEMSTAT (IMG-7289), A LYSINE-SPECIFIC DEMETHYLASE-1 (LSD1) INHIBITOR, FOR THE TREATMENT OF LATER-STAGE MYELOFIBROSIS (MF)

Session Topic: 16. Myeloproliferative Neoplasms

Final Abstract Code: EP1080

The data demonstrates the potential of bomedemstat as a monotherapy in intermediate-2 and high-risk patients with myelofibrosis who have become intolerant of, or resistant to, or are ineligible for a Janus Kinase (JAK) inhibitor.

Imago is currently conducting a Phase 2 study of bomedemstat in five countries. Clinical endpoints include spleen volume reduction, reduction in total symptom scores, and improvement in circulating inflammatory cytokines, anemia, bone marrow fibrosis and blast count. For additional information, visit cliniciatrials.gov (NCT03136185).

About Bomedemstat (IMG-7289)

Bomedemstat is being evaluated in an open-label Phase 2 clinical trial for the treatment of advanced myelofibrosis (MF), a bone marrow cancer that interferes with the production of blood cells. The endpoints include spleen volume reduction and symptom improvement at 12 and 24 weeks of treatment. Bomedemstat is used as monotherapy in patients who are resistant to, intolerant of, or ineligible for a Janus Kinase (JAK) inhibitor.

Bomedemstat is a small molecule developed by Imago BioSciences that inhibits lysine-specific demethylase 1 (LSD1 or KDM1A), an enzyme shown to be vital in cancer stem/progenitor cells, particularly neoplastic bone marrow cells. In non-clinical studies, IMG-7289 demonstrated robust in vivo anti-tumor efficacy across a range of myeloid malignancies as a single agent and in combination with other chemotherapeutic agents. Bomedemstat (IMG-7289) is an investigational agent currently being evaluated in ongoing clinical trials (ClinicalTrials.gov Identifier: NCT03136185 and NCT02842827). Bomedemstat has FDA Orphan Drug and Fast Track Designation for the treatment of myelofibrosis and essential thrombocythemia, and Orphan Drug Designation for treatment of acute myeloid leukemia.

About Imago BioSciences

Imago BioSciences is a clinical-stage biopharmaceutical company focused on discovering and developing novel anti-cancer therapeutics targeting epigenetic enzymes. Imago has developed a series of compounds that inhibit LSD1, an epigenetic enzyme critical for cancer stem cell function and differentiation. Imago is advancing the clinical development of its first LSD1 inhibitor, bomedemstat, for the treatment of myeloid neoplasms including myelofibrosis and essential thrombocythemia. Imago BioSciences is backed by leading strategic and venture investors including a fund managed by Blackstone Life Sciences, Frazier Healthcare Partners, Omega Funds, Amgen Ventures, MRL Ventures Fund, HighLight Capital, Pharmaron, Greenspring Associates and Xeraya Capital. The company is based in South San Francisco, California. To learn more, visit http://www.imagobio.com.

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Imago BioSciences To Present Update on Phase 2 results of Bomedemstat (IMG-7289), a Lysine Specific Demethylase-1 (LSD1) Inhibitor for the Treatment...

INTRODUCTION

Idiopathic pulmonary fibrosis (IPF) is a rapidly progressive and fatal interstitial pulmonary disease with a dismal median survival time of just 3 years after diagnosis (1, 2). To date, the IPF therapies depend on blocking myofibroblast activation to inhibit collagen I deposition (3, 4). However, the clinical data showed that these therapies remained far from achieving IPF revision. The main reason is that the IPF therapeutics lack an effectively targeted carrier or ignore some of the other risk factors such as the instability and tolerability of type II alveolar epithelial cell (AEC II) (5, 6). The AEC II, which is considered as injured AEC II (7, 8) in the IPF tissues, releases excessive amounts of reactive oxygen species (ROS) that initiate an antifibrinolytic coagulation cascade and promote the overexpression of connective tissue growth factor (CTGF) to provoke myofibroblast overactivation and extracellular matrix (ECM) development and then destroy the lung architecture (911). This situation has inspired us to propose that the combination of modulating superoxide in injured AEC II and antimyofibroblast activation as weeding and uprooting strategy will be a potential therapeutic strategy for synergistic antifibrosis. Furthermore, another limitation is that current therapies are rarely distributed in the lungs, which cannot achieve full therapeutic effect for treating IPF (12). Thus, the development of an effective lung-targeting drug delivery carrier is highly desirable for IPF therapy.

Recently, local preferred therapeutic agents generated using endogenous cells have served as a strong and promising delivery platform for targeting in situ, achieving considerable progress in several diseases (1317). In the inflammatory phase of IPF, precursor circulating monocytes (PCMs) have been found to undergo notable proliferation (18). PCMs and injured AEC II release chemotactic factors that specifically recruit chemokine receptorpositive (CR+) cells including monocyte-derived multipotent cell (MOMC) and guide the MOMC to migrate to injured lung tissues through specific binding to chemokine receptors on cell membrane (19, 20). Furthermore, in addition to this migration characteristic, these MOMCs, which originate from hematopoietic stem cells in the bone marrow, still have multipotency to differentiate into a variety of functional cells, including AEC II and endothelial cell (21, 22), which demonstrates that MOMC has the potential to participate in reestablishing lung functions (23). In addition, chronic hypoxic exposure induces the recruitment of MOMC to the pulmonary circulation, and the cell contributes to improving lung functions by producing angiogenic factors (24). It has been reported that monocytes from patients with IPF also show preconditioned prorepair features (25). In general, MOMC as a precise lung-targeting delivery platform will exhibit encouraging therapeutic effects, leading to the repair or regeneration of injured AEC II for IPF treatment.

In this study, we constructed the programmed therapeutics composed of surface-engineered nanoparticles (PER NPs) loading dual drugs adhered to MOMC (named MOMC/PER) to solve the issues in IPF therapy by improving drug accumulation in injured lung sites and completely destroying the fibrotic signaling network in IPF (Fig. 1). The MOMC/PER delivery platform realized efficacy through programmed modules, which consisted of a homing moiety, responsive release moiety, and retargeting moiety. (i) The homing moiety is the native ability of MOMC/PER to migrate to injured lungs due to the homing characteristic of MOMC. (ii) The responsive release moiety of MOMC/PER is activated by matrix metalloproteinase-2 (MMP-2) overexpression in IPF tissues, resulting in pathology-responsive release of PER NPs with exposed cyclic RGDfc (Arg-Gly-Asp) [c(RGDfc)] from the MOMC. (iii) The retargeting moiety is that exposed c(RGDfc) on PER NPs can anchor to injured AEC II via an interaction between v6 and c(RGDfc) (26), allowing the cytoplasm of the injured AEC II internalize PER NPs. Subsequently, astaxanthin (AST) and trametinib (TRA) are released from PER NPs to achieve a weeding and uprooting therapeutic effect. In general, the sustained injury of epithelial cells and highly heterogeneous myofibroblasts is considered as the most critical variable in achieving complete IPF reversion (27). To validate the above hypothesis, in this study, AST was chosen as an antioxidant by neutralizing superoxide to repair injured AEC II (28), and TRA suppressed the activation of myofibroblast by inhibiting CTGF production for IPF therapy (29). MOMC also participates in treating IPF by repairing injured AEC II to promote regeneration of IPF lungs (21). Overall, MOMC/PER, which mimics the features of chimeric antigen receptor T cell immunotherapy, is a precise lung-targeting platform to reverse IPF by improving drug accumulation due to the outstanding homing ability of MOMC to injured lung sites, and the destruction of the fibrotic signaling network by inhibiting the activation of myofibroblast and repairing injured AEC II to promote the damaged lungs regeneration.

(A) Bioconjugated MOMC/PER was prepared by incubating PER NPs with MOMC. (B) MOMC/PER has multifunctional moieties including a homing moiety, responsive release moiety, and retargeting moiety to reverse IPF. Then, a weeding and uprooting strategy contributes to IPF reversion. (C) Schematic illustration of MOMC/PER for improved drugs accumulation and antifibrotic effect in IPF lung microenvironment.

The quantities of MOMC in serum and lung tissues were significantly increased in IPF mice compared with normal mice (Fig. 2A). The proliferation of MOMC was positively related to IPF progression, which might be because increasing numbers of MOMC would be recruited from the bone marrow to the lesion sites when IPF occurred (30). Motivated by the fact that MOMC has a homing ability, we considered MOMC to be a potential delivery carrier to improve delivery efficiency in IPF treatment under pathological conditions.

(A) The proliferation of Nanog+ cells in serum and lung tissues by ELISA assay. (B) The MOMC phenotypes. The level of TGF- (C) and hydroxyproline (D) in IPF lung tissues, respectively. (E) The level of TGF-/Smad in vitro. (F) Schematic showing the preparation of MOMC/PER. (G) Schematic showing the adhesion of PER NPs to MOMC. (H) SEM images of MOMC and MOMC/PER-DiI. (I) Fluorescent signals of MOMC and PER-DiI NPs by CLSM. (J) The adhesion between MOMC and PER-DiI NPs by flow cytometry. (K) In vitro migration model. The migration ability of MOMC and MOMC/PER in CXCL 12 (L) and CCL 19 (M), respectively. (N) Schematic showing sensitive release of MOMC/PER-DiI triggered by MMP-2. (O) Characterizations by TEM. MOMC is the triangle, and PER-DiI NPs are the arrows. (P) Fluorescent images of MOMC and released PER-DiI NPs by CLSM. (Q) The flow cytometry showed responsive release. (R) Schematic showing the retarget ability of released PER NPs. (S) Characterization of retargeting ability by TEM. (T) The fluorescent images by CLSM. (U) Cellular uptake in A549 by flow cytometry (n = 3). Statistical significance was calculated via one-way analysis of variance (ANOVA).

We first isolated MOMC from the peripheral blood of C57BL/6J male mice of IPF. The morphologies of the MOMC were fusiform (fig. S1). To identify the phenotypes of MOMC isolated from IPF mice, we first investigated the presence of specific markers for MOMC by immunofluorescence staining. The results showed that MOMC expressed CD11b and smooth muscle actin (-SMA) (Fig. 2B), which was consistent with the literature (24). In addition, the MOMC also expressed the stem cell markers CD14 and Nanog protein and the injured AEC IIs marker pro-surfactant protein C (SPC), as shown in Fig. 2B. These results indicated that MOMC was pluripotent cells with stem cell and epithelial celllike properties. It has been reported that MOMC was recruited to damaged lung areas and participated in recovering injured lung normalization through growth factor release to repair injured AEC II (30). In addition, to inspect the potential risk of injecting MOMC into mice, we further investigated the feasibility of using isolated MOMC as a delivery carrier, including measuring the levels of transforming growth factor (TGF-) and hydroxyproline, which are closely related to the development of IPF in vivo. The results displayed approximately onefold reduction in TGF- and hydroxyproline levels in IPF mice treated with MOMC compared with untreated bleomycin (BLM)induced mice, and these indexes were barely changed in normal mice, indicating that MOMC would not induce the occurrence of IPF and partly relieved established IPF (Fig. 2, C and D).

We next prepared PER NPs that contained two target peptides named peptide E5 and c(RGDfc). The poly(lactide-co-glycolide)-block-poly(ethylene glycol) methyl ether maleimide (PLGA-PEG-Mal) and PLGA-PEG-c(RGDfc) (mass ratio, 10:1) were self-assembled by noncovalent interactions of amphiphilic PLGA-PEG copolymer into nanoparticles (31), and then, peptide E5 was bound on the NPs by the Michael reaction (fig. S2A). As determined by 1H nuclear magnetic resonance spectroscopy (fig. S2B) and SDSpolyacrylamide gel electrophoresis (SDS-PAGE) (fig. S2C), we successfully prepared PER NPs, and the grafting rate of peptide E5 in the PER NPs was 43.7%. The PER NPs showed particle sizes of approximately 110 10.39 nm and the zeta potential of 23.37 mV (fig. S2, D and E). In addition, AST and TRA were encapsulated into PER NPs (fig. S2F). The drug loading content of the PER NPs was 1.98 weight % (wt %) for AST and 2.83 wt % for TRA. The sustained release of the loaded AST was 49.5 wt %, and the pH-dependent release of the loaded TRA (weak alkalinity) was 79.6 wt %, which were obtained at pH 5.0 within 72 hours (fig. S2G). Then, we investigated the capacity of MOMC to differentiate into myofibroblast after treatment with PER NPs in vitro. As shown in Fig. 2E, the expression of TGF-/small mother against decapentaplegic (TGF-/Smad), which is molecule in the crucial pathway for myofibroblast activation, was decreased, suggesting that the PER NPs could inhibit MOMC differentiation. The possible reason for the inhibition was that the PER NPs partly covered the TGF- receptor on the MOMC and reduced exogenous TGF- stimulation within 8 hours, and then, the PER NPs could be gradually internalized. The released drugs could reduce TGF- expression of MOMC after 8 hours (fig. S3, A and B).

We next constructed MOMC/PER as a delivery platform/therapeutic carrier (Fig. 2F), and PER NPs loaded with both drugs could specifically adhere to MOMC through the interaction between peptide E5 of the PER NPs and the CXCR4 on the MOMC by a temperature-dependent manner (32, 33). The formation of MOMC/PER was positively correlated with the incubation time within 2 hours (fig. S3, A and B). Moreover, the PER NPs could specifically stick to the surface of the MOMC without internalization by the MOMC within 8 hours (Fig. 2G). The reasons may be that peptide E5 conjugated on the surface of the PER NPs is a long-chain peptide that limits internalization into the MOMC and that CXCR4 is not an endocytic receptor (34). The MOMC/PER had a loading capacity of 4.75 g of TRA and 1.5 g of AST/1 105 cells (fig. S3, C and D). In addition, MOMC cell viability was above 80% with different concentrations of PER NPs and different incubation times (fig. S4, A and B).

To investigate the adhesion of MOMC and PER NPs, the 1,1-dioctadecyl-3,3,3,3-tetramethylindocarbocyanine perchlorate (DiI) was loaded into blank PER NPs (PER-DiI NPs) to evaluate adhesion behavior. After incubating with MOMC and PER-DiI NPs for 2 hours, the morphologies of the MOMC/PER-DiI were confirmed by scanning electron microscopy (SEM) (Fig. 2H) and confocal laser scanning microscopy (CLSM) (Fig. 2I). Flow cytometry detection also indicated the formation of MOMC/PER-DiI in that the MOMC labeling green and PER-DiI NPs were collected in the double-positive quadrant (Fig. 2J).

Sequentially, migration via the interaction between a receptor and ligand is the vital characteristic that needs to be retained by MOMC/PER to realize efficient delivery (Fig. 2K). The migratory capability of MOMC/PER was detected by a Transwell invasion assay. The results indicated that the migratory ability of the MOMC/PER was unaffected by PER NPs adhering to the surface of MOMC (Fig. 2, L and M) at all studied concentrations (fig. S4, C and D).

To establish the retargeting ability of PER NPs, MMP-2 overexpressed in IPF tissues was used as an activating trigger to release PER NPs from MOMC/PER. As depicted in Fig. 2N, the separation of PER-DiI NPs from MOMC was well evidenced by transmission electron microscopy (TEM) and CLSM (Fig. 2, O and P) and flow cytometry (Fig. 2Q). We also detected the phenomenon by SDS-PAGE and particle sizes changes (fig. S4, E and F). After PER NPs were released from MOMC/PER, the exposed peptide c(RGDfc) of the PR NPs could retarget v6, which is overexpressed on the surface of injured AEC II (Fig. 2R) (35). Then, we investigated the capacity of injured AEC II to uptake PLGA-PEG-c(RGDfc)coumarin 6 (PR-C6) compared with free C6 and PLGA-PEGC6 (PP-C6) by TEM and CLSM. The internalization of PR-C6 was better than other forms (C6 and PP-C6) (Fig. 2, S to U). In addition, PR-C6 also underwent lysosomal escape (fig. S4G).

The homing ability of MOMC/PER was investigated in IPF models in vivo (Fig. 3A). We first examined the lung accumulation of 1,1-dioctadecyl-3,3,3,3-tetramethylindotricarbocyanine iodide (DiR)loaded into blank PER NPs (PER-DiR NPs) adhere to MOMC (MOMC/PER-DiR) after intravenous administration. The DiR fluorescence accumulated in the lungs of the MOMC/PER-DiR group, which indicated that compared with MOMC-loading DiR (MOMC-DiR) and free-DiR, MOMC/PER-DiR had a superior ability to target IPF lungs (Fig. 3B). Then, we quantitatively analyzed the drug distribution in the tissues of each organ. The DiR fluorescence intensity in the lungs was 3.5- and 0.5-fold greater than that in the liver in the MOMC/PER-DiR and MOMC-DiR groups, respectively. In addition, there was little accumulation of free DiR in the lungs than that in the liver (Fig. 3C). MOMC loading of DiR could improve DiR accumulation in IPF lungs due to the homing ability of the MOMC.However, the accumulation of MOMC-DiR was weaker than that of MOMC/PER-DiR. This may be because the free dye carried by the MOMC was limited compared with that carried by the PER NPs, which suggested that MOMC/PER could solve the limitation of conventional drug loading of cells. In addition, we further evaluated the homing capacity of MOMC/PER, the responsive release ability of MOMC/PER mediated by MMP-2, the released PER NPs with exposed c(RGDfc), and the retargeting to injured AEC II by immunofluorescence staining. The DiI was chose to a tracer agent, labeling the PER-DiI NPs with red fluorescence, and then, the PER-DiI NPs adhered to MOMC to form MOMC/PER-DiI. Nanog and SPC, which represented MOMC and injured AEC II, respectively, were labeled in green fluorescence. Then, the MOMC/PER-DiI was administered to IPF mice by intravenous injection. As shown in Fig. 3D, the PER-DiI NPs labeled in red overlapped with the MOMC marked in green, generating a merged yellow signal, which revealed that the MOMC/PER-DiI notably accumulated in the lungs of IPF mice in stage 1 (homing to lungs). In stage 2 (releasing PER-DiI NPs), the PER-DiI NPs labeled in red were separated from the MOMC labeled in green, indicating that the PER-DiI NPs were released from the MOMC membrane surface and exposed c(RGDfc) at fibrotic foci as a result of the overexpression of MMP-2 in the IPF microenvironment. Then, the PER-DiI NPs labeled in red overlapped with injured AEC IISPC+ labeled in green, implying that the PER-DiI NPs retargeted to injured AEC II through the interaction between the exposed c(RGDfc) ligand and the v6 receptor on the surface of the injured AEC II in stage 3 (retarget injured AEC II) (Fig. 3D). Collectively, these results showed that MOMC/PER-DiI had the native ability to home to damaged lungs and then were activated by programmed procedures, confirming that MOMC could function as a vehicle to deliver PER NPs to injured lungs.

(A) Schematic of the targeting performance of MOMC/PER in the blood circulation to IPF lungs. (B) In vivo fluorescence images of IPF mice intravenous injection with MOMC-DiR, MOMC/PER-DiR, and DiR (n = 3). (C) Quantification of the in vivo retention profile (n = 3). (D) The different stages of MOMC/PER-DiI. (E) The whole lungs were imaged and investigated after 28 days. Lung morphologies (i) [Photo credit (i): Xin Chang, China Pharmaceutical University], H&E staining (ii), and Masson staining (iii). The morphologies of mitochondria by TEM (iv). The levels of TGF- (F), IL-1 (G), and IL-4 (H) by ELISA assay (n = 5). The levels of lymphocytes (I), white blood cells (J), and neutrophils (K) in whole blood (n = 5). The levels of GSH (L) and SOD (M), respectively (n = 5). (N) The expression of SPC. (O) Survival rate curves (n = 10). Statistical significance was calculated via one-way ANOVA.

To confirm the curative effect of MOMC/PER, we investigated lung morphologies after the administration of MOMC/PER or other treatments. As showed in Fig. 3E, MOMC/PER could greatly relieve IPF according to hematoxylin and eosin (H&E) and Masson staining. Images of lung morphologies showed obvious normalization after treatment with MOMC or MOMC/PER compared with no treatment (Fig. 3E, i). H&E staining showed that lung tissues in the MOMC/PER group were not destroyed and that the alveolar sizes were same as normal lung tissues (Fig. 3E, ii). In addition, compared with no treatment, MOMC also partly protected the lung architecture; however, there was a gap between the MOMC/PER and normal groups. Similarly, Masson staining also showed that the MOMC/PER group exhibited an excellent reduction in collagen I deposition (Fig. 3E, iii). IPF is also induced by mitochondrial oxidative stress in injured AEC II. Hence, we examined the capability of MOMC/PER to repair injured AEC II by maintaining mitochondrial morphologies (Fig. 3E, iv). The morphologies of mitochondria were close to normal in the MOMC/PER group compared with the MOMC group and BLM group, suggesting that MOMC/PER could repair injured AEC II to maintain normal lungs by improving mitochondrial function. Furthermore, we tested the expression of proinflammatory cytokines [TGF-, interleukin-1 (IL-1), and IL-4], which play major roles in excessive ECM formation during IPF progression. As shown in Fig. 3 (F to H), the expression of TGF- in the MOMC/PER treatment group was nearly threefold lower than that in the BLM group, and the expression of IL-1 and IL-4 also decreased by nearly 0.5- and 1-fold, respectively, in the MOMC/PER group compared with the BLM group, suggesting that MOMC/PER could block IPF progression by inhibiting the secretion of proinflammatory cytokines. In addition, the formulations of MOMC and MOMC/PER showed well biocompatibility in a hemolysis test (fig. S5). In addition, inflammatory cells were quantified in whole blood in these groups after treatment. Compared with the BLM group, the MOMC/PER group showed inhibited inflammatory cell proliferation (Fig. 3, I to K), which indicated that MOMC/PER had the ability to alleviate IPF progression in the inflammatory phase. In addition, the results implied that MOMC had a certain ability to inhibit the proliferation of inflammatory cells. Next, glutathione (GSH) and superoxide dismutase (SOD), which are significant inhibitors of ROS, were used to balance the ROS content of injured AEC II. Compared with no treatment, treatment with MOMC/PER increased the GSH level nearly onefold (Fig. 3L), and MOMC also enhanced the GSH level. Similarly, MOMC/PER increased the SOD level to a certain extent in lung tissues (Fig. 3M). We further explored the repair mechanism for injured AEC II in IPF lungs treated with MOMC or MOMC/PER. The expression of SPC was markedly increased in the MOMC/PER group compared with the BLM group; there was also an augmentation in the expression of SPC in the MOMC group, which showed that MOMC/PER could up-regulate AEC II proliferation or recover injured AEC II to normalize the lungs in IPF and demonstrated that MOMC/PER could promote IPF lungs regeneration (Fig. 3N). The survival time of the MOMC/PER group exceeded 60 days, which was longer than the survival time of the BLM group (Fig. 3O), and the MOMC/PER group did not exhibit any changes in body weight (fig. S6).

To investigate the targeting ability of PER NPs through reprogramming to form MOMC/PER in the blood circulation, we conducted the following experiments. The E5-mediated targeting ability of PER-DiI NPs was first evaluated in IPF mice (Fig. 4A). CLSM showed the adhesion of PER-DiI NPs to the surface of MOMC (Fig. 4B, bottom). The confocal images produced the same result as Fig. 2I. Furthermore, PER-DiI NPs were administered to IPF mice model by intravenous injection. The results demonstrated that the PER-DiI NPs adhered to the surface of MOMC (Fig. 4B, middle). More detailed results revealed that the PER-DiI NPs could bind to the MOMC surface and reprogram the MOMC to form MOMC/PER in the peripheral blood by SEM (Fig. 4B, top). In addition, immunofluorescence staining confirmed that the PER-DiI NPs homed to IPF lungs and accumulated in the injured AEC II area after intravenous injection (Fig. 4C). In addition, Nanog-labeled MOMC (green fluorescence) accumulated in higher numbers in IPF lungs than normal lungs, which was similar to previous results (Fig. 2I). We also investigated the targeting capacity of PER-DiR NPs at different time points by in vivo imaging system following intravenous injection, and PLGA-PEG-DiR (PP-DiR NPs), PLGA-PEG-c(RGDfc)DiR (PR-DiR NPs), and PLGA-PEG-E5-DiR (PE-DiR NPs) were used as controls. The PP-DiR NPs and PR-DiR NPs were mainly found in the liver, while the PER-DiR NPs and PE-DiR NPs mainly accumulated in IPF lungs (Fig. 4D and fig. S7A). The accumulation of the PER-DiR NPs in the lungs peaked at 8 hours, while the lung accumulation of the PE-DiR NPs quickly decreased. The primary reason may be that the PE NPs were delivered to the lungs via MOMC; however, they could not anchor on injured AEC II because they lacked c(RGDfc) and were therefore more rapidly cleared from the circulation than the PER-DiR NPs. Compared with the PE-DiR NPs, the PER-DiR NPs accumulated in IPF lungs for a long time (more than 8 hours), which is important for treating lung disease. A quantitative region of interest (ROI) analysis of PER-DiR NPs accumulation was performed by detecting DiR signal variation in the lungs and other organs (Fig. 4E). Moreover, we evaluated PER-DiI NPs behavior in lung tissues after administration at different times (Fig. 4F). After administration at 0.5 hours, increasing levels of overlapping yellow fluorescence in lung blood vessels were observed for PER-DiI NPs labeled in red and MOMC marked in green, indicating that the PER-DiI NPs arrived at IPF lung tissues through reprogramming to form MOMC/PER-DiI in the blood circulation. Then, the red and green signals were separated at the time point of 2 hours, indicating that the PER-DiI NPs were released from the reprogrammed MOMC/PER-DiI due to the overexpression of MMP-2 in the IPF mice. Furthermore, the released PER-DiI NPs showed a wide distribution in the lung tissues at 4 hours after intravenous injection, which is powerful for treating diseases. These data demonstrated that PER NPs could target IPF lungs by means of attaching to circulating MOMC quickly and could accumulate in lung tissues for a long time to achieve therapeutic efficacy.

(A) Schematic of PER NPs circulation in vivo, reprogramming of MOMC/PER, and recruitment to IPF tissue. (B) The targeting ability of PER-DiI NPs. (C) The accumulation of PER-DiI NPs in normal and IPF lungs. (D) Fluorescence IVIS imaging (n = 3). (E) Ex vivo fluorescence imaging and quantification of major organs (n = 3). (F) The accumulation PER-DiI NPs in the lungs at different times. Lung function indexes of GSH (G), SOD (H), and MDA (I). TGF- (J), IL-1 (K), and IL-4 (L) by ELISA assay (n = 5). (M) Proliferation of fibroblasts. (N) Expression of collagen I. Statistical significance was calculated via one-way ANOVA.

We further investigated the antifibrotic efficacy of PER NPs in vivo. With IPF progression, injured AEC II gradually died out due to oxidative stress, which leads to mouse suffocation. Hence, restoring normal lung function has important significance. Compared with the BLM group, the PER NPs group had the promising abilities to repair injured lungs and keep them normal. GSH and SOD levels in the PER NPs group were obviously improved with 0.3- and 1-fold, respectively, which could relieve the oxidative stress in injured AEC II to some extent. The level of malondialdehyde (MDA), a key indicator of oxidative stress, was reduced 0.3-fold in the PER NPs group compared with the BLM group (Fig. 4, G to I). Compared with controls, treatment with PER NPs reduced the production of the three cytokines (TGF-, IL-1, and IL-4) in the lungs by 1-, 0.85-, and 0.7-fold, respectively, which showed that the PER NPs effectively inhibited the inflammatory response in IPF lungs (Fig. 4, J to L). We also examined the levels of TGF-, IL-1, and IL-4 in the spleen tissues (fig. S7, B to D), which showed consistent results. These results indicated that PER NPs could treat IPF by inhibiting inflammatory responses in IPF lungs. As seen in Fig. 4M, immunofluorescence staining results revealed that the population levels of fibroblasts CD90+ labeled in green remained in a relatively stable range, while the population levels of activated fibroblasts indicated great proliferation in the BLM group, supporting the conclusion that compared with no treatment group, the PER NPs had an efficient ability to reverse IPF by inhibiting the activation of fibroblasts. Figure 4N showed that the expression of collagen I was notably decreased in the PER NPs group, which confirmed that PER NPs could achieve therapeutic effects by inhibiting ECM deposition.

We next investigated the antifibrosis mechanism based on the synergistic effect of TRA and AST. We firstly established the different formulations, including PLGA-PEG-TRA-AST (PPTA), PLGA-PEG-TRA (PPT), and PLGA-PEG-AST (PPA), and the morphologies of PPA, PPT, and PPTA were evaluated by TEM (fig S8). As shown in Fig. 5A, the results of immunofluorescence staining showed that the expression of the vimentin as cytoskeletal protein was increased after treatment with different formulations in human lung epithelial cell carcinoma (A549). In particular, compared with other treatments, the PER NPs significantly increased the expression of vimentin, indicating that PER NPs had the capacity to keep injured AEC II normal. To assess the repair mechanism induced by the drugs combination, the ROS were detected using ROS probe 2,7-dichlorofluorescin diacetate (DCFH-DA) via inverted fluorescence microscopy and flow cytometry. The ROS content was significant decreased in the PPTA group compared with the untreated and single-drug groups (PPT and PPA) (Fig. 5B). Although the PPA group exhibited some changes than PPT, this effect was not as strong as that in PPTA group, because the PPTA groups exhibited synergistic effect that relieved oxidative stress in injured AEC II than other control formulations. Furthermore, the PPTA group also showed a reduced mitochondrial membrane potential in TGF-induced cells (Fig. 5C), which supported the conclusion that the efficacy in PPTA group was the result of repairing mitochondrial function with relief of oxidative stress in the mitochondria. In the microenvironment of IPF lungs, myofibroblast can be derived from injured AEC II undergoing epithelial-mesenchymal transition (EMT), which aggravates the progression of IPF. As observed in a wound healing assay and invasion assay (Fig. 5D), PPTA effectively inhibited the occurrence of EMT. Furthermore, fibronectin is a structural protein in the ECM, which is a crucial indicator of IPF progression. The expression of fibronectin was obviously decreased in PPTA group than PPT and PPA groups, thus inhibiting the differentiation of injured AEC II into myofibroblast (Fig. 5E). Next, we also tested IPF-reversing efficacy by monitoring the recovery of the lung architecture and improvement in lung functions in vivo. As shown in Fig. 5F, collagen I deposition in the PPTA group returned to normal levels, as determined by H&E and Masson staining, demonstrating that the PPTA could recover the architecture of injured lungs compared with no treatment or single-drug groups (PPT and PPA). Similarly, the expressions of -SMA and collagen I were tested by immunohistochemistry (IHC), which also obtained the same results that the synergistic effect of PPTA could effectively inhibit myofibroblast activation and ECM deposition. In addition, the level of hydroxyproline, the main component of the ECM, was also decreased after treatment with PPTA compared with other treatments (Fig. 5G), suggesting that PPTA had the ability to diminish ECM deposition and retard IPF progression. Furthermore, we detected the expression of -SMA to evaluate the myofibroblasts activation by Western blotting. The lungs were collected after treatment with PPTA, PPT, or PPA for 28 days. The results showed that -SMA expression, as the major evaluation index for IPF, was significantly reduced in PPTA group (Fig. 5H). In addition, the results of real-time quantitative polymerase chain reaction (qPCR) showed that the relative mRNA expressions of CTGF (Ctgf) and -SMA (Acta2) significantly decreased in PPTA group, which indicated that the combination of AST and TRA can achieve efficient therapeutic efficacy by inhibiting myofibroblasts overactivation (Fig. 5, I and J). Moreover, MDA expression decreased, and SOD and GSH levels increased after treatment in PPTA group compared with the PPT and PPA groups. Together, these results implied that the combination of AST and TRA could recover IPF lung function through synergistic effect that was not observed with the other treatments (Fig. 5, K to M). The various formulations as mentioned above were safe by intravenous injection through H&E staining (fig. S9).

(A) Expression of the vimentin in vitro. (B) The ROS level in vitro. (C) The changes of mitochondrial membrane potential. (D) Invasion assay. (E) Fibronectin expression. (F) H&E, Masson, and IHC staining. (G) The level of hydroxyproline. (H) The -SMA and -actin by Western blotting. The mRNA expression of Acta2 (I) and Ctgf (J) by qPCR (n = 3). Contents of GSH (K), MDA (L), and SOD (M) (n = 5). Statistical significance was calculated via one-way ANOVA.

To further investigate the antifibrotic efficacy of MOMC/PER and pirfenidone as a conventional therapeutics for IPF, we evaluated the ability of these treatments to repair lung tissue and inhibit collagen I deposition through H&E and Masson staining, respectively, after 28 days of administration. As shown in Fig. 6A, the alveolar structure in the BLM group collapsed, and alveolar wall thickness increased notably, indicating that collagen I was accumulated and that alveolar heterogeneity was aggravated. Similarly, the alveolar morphologies in the pirfenidone group also showed collapse via H&E staining. In contrast, MOMC/PER could obviously repair the collapsed part of the alveolar space, narrow the spaces between the alveoli, and produce a thinner alveolar wall that tended to appear normal by H&E staining, which demonstrated that MOMC/PER had greater reparative effect on alveolar structure than pirfenidone. In addition, MOMC/PER group showed notable decrease compared with the pirfenidone group in inflammatory cell infiltration. The PER NPs were also more competent in restoring alveolar structure than the clinical drug pirfenidone. This effect was observed because the PER NPs could undergo reprogramming to form MOMC/PER in the blood circulation and then reach their destination, which was consistent with the above results. We further confirmed therapeutic efficacy in regard to ECM deposition by Masson staining (Fig. 6B). Compared with that in the pirfenidone group, the ECM accumulation in the lungs, which appeared as blue staining, was notably reduced in the MOMC/PER group. The results for Masson staining showed that MOMC/PER had greater power than pirfenidone to prevent IPF progression by inhibiting ECM deposition. The main reason for the limited therapeutic effect of pirfenidone was its low bioavailability as an oral drug, and onefold treatment target is the second therapeutic limitation of pirfenidone. Overall, the antifibrotic efficacy in the MOMC/PER group was the best efficacy observed through H&E and Masson staining, and PER NPs had better efficacy than pirfenidone or MOMC. In addition, the fibrosis score of different formulations showed the same trend in fig. S10. These data demonstrated that MOMC/PER showed a preferable combination efficacy over the U.S. Food and Drug Administration (FDA)approved therapeutic pirfenidone or using MOMC or PER NPs alone.

(A) H&E staining. (B) Masson staining. The levels of TGF- in the lungs (C) and spleen tissues (D). N.S., not significant. The levels of IL-1 in the lungs (E) and spleen tissues (F). BUN (G), ALT (H), and aspartate aminotransferase (I) in serum (n = 4). Statistical significance was calculated via one-way ANOVA.

Then, we further evaluated the antifibrotic effect in various groups by examining biochemical indexes of IPF. We first examined the expressions of TGF- and IL-1 in the lungs and spleen tissues, respectively. The results in the lungs showed that TGF- expression was reduced onefold in the MOMC/PER group (P = 0.015) compared with the BLM group and became close to normal (Fig. 6, C and D). However, there was no significant difference between the pirfenidone group and the BLM group, and the level of TGF- in the PER NPs group was lower than that in the pirfenidone group. In addition, the TGF- level in the spleen tissues was significantly decreased in the MOMC/PER group (P = 0.002) compared with the BLM group. However, the level of TGF- in the pirfenidone group was similar to that in the BLM group. The main reason is that pirfenidone is used to treat IPF by inhibiting the accumulation of collagen I, but it has no therapeutic effect on the simultaneous inflammatory response or cytokine expression. The results demonstrated that MOMC/PER had the best antifibrotic efficacy, which was superior to the efficacy achieved by pirfenidone and was mediated by inhibiting the expression of cytokines in the lungs. Furthermore, we detected the expression of IL-1 in the lungs and spleen tissues to investigate the antifibrotic effects of different formulations (Fig. 6, E and F). The trends in IL-1 expression were similar to TGF-; all the treatments could reduce the expression of IL-1, and MOMC/PER showed the best therapeutic efficacy (P = 0.001) in all the treatments. In addition, the IL-1 level in the pirfenidone group was maximal, indicating that compared with the other treatment groups, including the MOMC/PER and PER NPs alone groups, the pirfenidone group showed minimal anti-inflammatory effects. These results indicated that MOMC/PER could achieve a greater treatment effect on IPF than pirfenidone by inhibiting the expression of cytokines in the inflammatory phase; the efficacy of PER NPs was second only to MOMC/PER, and pirfenidone and MOMC were weaker than the PER NPs.

To assess the safety of the treatments in vivo, we then evaluated biological indexes for each formulation after treatment. The levels of blood urea nitrogen (BUN), alanine transaminase (ALT), and aspartate aminotransferase were detected to evaluate the function of the kidneys, liver, and heart, respectively (Fig. 6, G to I). The levels of BUN, ALT, and aspartate aminotransferase were not significantly different between the pirfenidone and other groups (the MOMC/PER, PER NPs, and MOMC groups). As an oral drug approved by the FDA for the treatment of IPF, pirfenidone is highly recognized for its safety in application. Similarly, our different formulations obtained results of safety equivalent to pirfenidone for a certain period of time, indicating that MOMC/PER, MOMC, and PER NPs could also be safely administered by intravenous injection and could be used clinically.

IPF is characterized by injured AEC II and activated myofibroblast, resulting in ECM deposition. To date, the FDA has approved only two drugs (pirfenidone and nintedanib) for IPF treatment. Unfortunately, curing end-stage IPF is inefficient due to the narrow therapeutic spectrums and insufficient accumulation of these drugs in the lungs (3). As a result, traditional therapies have done little to reverse IPF (4). To address this problem, we developed programmed therapeutics MOMC/PER to reverse IPF by efficient lung delivery, programmed modules, and double synergetic strategies.

Two synergetic strategies including drug/drug and cell/drug involved in reversing IPF were shown for the MOMC/PER here. First, the drug/drug as weeding and uprooting strategy could repair injured AEC II and inhibit myofibroblast activation, achieving first synergetic antifibrosis effect. In particular, one drug (AST) acted as the uprooting part of the treatment strategy, repairing injured AEC II by neutralizing oxidative stress. The other drug (TRA) acted as the weeding portion of the strategy, inhibiting the differentiation of fibroblasts into myofibroblast by suppressing CTGF production. Second, some studies have demonstrated that MOMC is multipotent cell that can be specifically recruited to injured lung tissues through interactions between chemokine receptors and chemotactic factors (3638) and contribute to lung tissue normalization and regeneration (39). In addition, MOMC also plays a vital role in regulating the population of immune cells during the inflammatory phase of disease progression (40). Similarly, our results also showed that MOMC could inhibit the proliferation of inflammatory cells, such as lymphocytes and white blood cells. This is another synergetic effect called cell/drug. PER NPs also exhibited greater antifibrotic effects than pirfenidone due to their efficient lung-targeting ability and combination of AST and TRA, as these PER NPs could target MOMC in the circulation, accumulate in the lungs effectively, and then reverse IPF collaboratively. The limited treatment efficacy of pirfenidone is mainly due to its low bioavailability, narrow therapeutic spectrum, and functions by inhibiting myofibroblast activation only.

In addition, MOMC/PER is strategically distinct from nanodelivery carrier (41) and drug-loaded cells carrier (14). The traditional nanodelivery system for IPF always presents dissatisfactory accumulation and unexpected drug release at the lesion site. Even these defects can be avoided for IPF therapy, the therapeutic efficacy is also limited to onefold treating target, and these shortcomings make IPF hard to reverse. In addition to nanodelivery systems, cell-mediated drug delivery has also received more attention in disease treatment. The classic cell-based delivery strategy for treating disease is reliant on drugs being loaded into cells by endocytosis (15). However, cells are difficult to load with large quantities of drugs, and chemotherapeutics may be highly toxic to cells undergoing loading. Hopefully, the PER NPs adhere to the MOMC surface in our study could surmount this challenge in conventional drug loading of cells. PER NPs were firstly attached to MOMC surface and then precisely delivered to the lungs via the homing ability of MOMC and activated for IPF reversion. However, there is still an unresolved point in our research, which is that the treatment mechanism of MOMC remains unclear. Our results indicated that MOMC might up-regulate AEC II proliferation or recover injured AEC II to normalize the lungs. The mechanism of MOMC differentiation for IPF treatment requires further exploration. In addition, some studies have indicated that MOMC could partly treat early IPF through regulating the immune response by inhibiting the proliferation of immune cells in vivo (42). It is unclear whether MOMC is effective for IPF therapy during different periods.

Compared with conventional antifibrotic strategies, our previous unknown programmed therapeutics MOMC/PER has showed accurate lung targeting and excellent therapeutic effects. The excellent antifibrotic efficacy of the MOMC/PER was achieved through the following features. (i) MOMC has the ability to backpack PER NPs, constructing programmed therapeutics MOMC/PER. (ii) MOMC/PER can precisely accumulate in IPF lung tissues due to the homing ability of the MOMC. (iii) PER NPs are sensitively released from MOMC/PER due to the overexpression of MMP-2 in the IPF microenvironment. (iv) Released PER NPs are able to retarget injured AEC II through c(RGDfc). (v) PER NPs can reduce the secretion of TGF- by occupying TGF-latent sites. (vi) Two drugs loaded into PER NPs are the key factors in achieving IPF reversion of drug/drug as weeding and uprooting. In addition, MOMC also participates in AEC II regeneration using cell/drug strategy. Specifically, MOMC-mediated delivery therapeutics is convenient, and the materials used in our PER NPs have all been approved by the FDA, which indicates certain advantages for further clinical development. Overall, we have proposed an innovative concept to cure IPF through using native cells as a delivery carrier and a dual-drug combination as therapeutic agents, and this strategy is likely to be applicable to other major diseases.

MMP-2, -SMA rabbit anti-mouse antibody, and DCFH-DA were purchased from Sigma-Aldrich (St. Louis, USA). SPC rabbit anti-mouse antibody was purchased from Millipore (St. Louis, USA). Lymphocyte isolation kit was purchased from Solarbio Science & Technology Co. Ltd. (Beijing, China). PLGA-PEG-Mal and PLGA-PEG-c(RGDfc) were purchased from Jinan Daigan Biomaterial Co. Ltd. (Jinan, China). RPMI 1640, fetal bovine serum (FBS), and bicinchoninic acid (BCA) protein assay kit were purchased from Jiangsu KeyGEN BioTECH Co. Ltd. (Nanjing, China). The peptide E5 (CGPLGIAGQCGGRSFFLLRRIQGCRFRNTVDD) was synthesized by Top Peptide Biotechnology Co. Ltd. (Shanghai, China). AST was purchased from Yuanye Bio-Technology Co. Ltd. (Shanghai, China). TRA was purchased from J&K Scientific Co. Ltd. (Beijing, China). DiI, 3,3-dioctadecyloxacarbocyanine perchlorate (DiO), and DiR were purchased from Fanbo Biochemicals Co. Ltd. (Beijing, China). DAPI (4,6-diamino-2-phenylindole) and mitochondrial membrane potential kit of JC-1 were purchased from Beyotime Biotechnology Co. Ltd. (Shanghai, China). CXC chemokine ligand 12 (CXCL 12) and CC chemokine ligand 19 (CCL 19) were purchased from Zoonbio Biotechnology Co. Ltd. (Beijing, China). BLM was purchased from Zhejiang Huahai Pharmaceutical Co. Ltd. (Linhai, China). TGF- was purchased from Multi Sciences Biotech Co. Ltd. (Hangzhou, China). C6 was purchased from Tokyo Chemical Industry Co. Ltd. (Tokyo, Japan). LysoTracker Red DND-99 kit was purchased from Thermo Fisher Scientific (Waltham, USA). Nanog, TGF-/Smad and collagen I rabbit anti-mouse antibodies, and H&E and Masson staining kits were purchased from Servicebio Co. Ltd. (Nanjing, China). GSH, MDA, SOD, and hydroxyproline were purchased from Jiancheng Biotech Co. Ltd. (Nanjing, China). IL-1 and IL-4 detection kits were purchased from eBioscience (Waltham, USA). TGF- detection kit was purchased from BioLegend (CA, USA). Polyvinylidene difluoride (PVDF) was purchased from PALL (NY, USA). Electrogenerated chemiluminescence (ECL) was purchased from Tanon Science & Technology Co. Ltd. (Shanghai, China).

MOMC was isolated from peripheral blood by a mouse lymphocyte isolation kit. To obtain the MOMC, peripheral blood was collected from the C57BL/6J mice of IPF with EDTA and diluted three times with phosphate-buffered saline (PBS). The isolated protocol of MOMC was as follows: The mouse Percoll was added into diluted peripheral blood solution, and MOMC was isolated from peripheral blood by centrifugation at 600 rpm for 30 min. The solution was divided into upper, middle, and lower layers, and MOMC existed in the middle layer. Then, the MOMC was taken into centrifuge tube of 15 ml, and cell washing buffer of 5 ml was added into the tube. The cell suspension was then centrifuged for another 30 min, and it needed to be repeated three times to obtain MOMC. Last, the cell deposits were resuspended, and the suspension was put into the culture dish in RPMI 1640 medium with 10% FBS at 37C and 5% CO2 (22, 23). The adherence time for dishes of MOMC was about 2 weeks. The MOMC was a kind of adherent cells, and the morphologies of cells were fusiform. After the cells adhere to the dish, the medium was changed once every 3 days.

PER NPs were prepared by antisolvent precipitation method. Peptide E5 modification was prepared as follows. Specifically, the PLGA-PEG-Mal and PLGA-PEG-c(RGDfc) (mass ratio, 10:1) and dual drugs of AST and/or TRA were dissolved in dimethyl sulfoxide (50 mg/ml, 2 ml), added dropwise into deionized water with 100 ml, and then stirred with 300 rpm for 2 hours. Next, the prepared nanoparticle solution (NPs) was centrifuged at 2800 rpm for 15 min to discard the large particles and free drug. The NPs were then condensed to concentration of 2 ml by ultrafiltration device for further use. Last, the peptide E5 was added into the solution of NPs to form PER NPs. The preparation process of other groups including PLGA-PEG-c(RGDfc) (PR NPs), the preparation of PLGA-PEG-E5 (PE NPs), and the preparation of PLGA-PEG (PP NPs) were similar to PER NPs.

The preparation of MOMC/PER was carried out by incubating MOMC with PER NPs. Briefly, the MOMC (2 105 cells/ml) was cultured in a petri dishes with a diameter of 100 mm. After incubated with the FBS-free media for 1 hour, PER NPs at a TRA concentration of 40 g/ml were added into MOMC medium and incubated for 2 hours at 37C and 5% CO2. At the same time, the CXCR4 receptor and ligand peptide E5 would undergo bioconjugate reaction.

The hydrodynamic diameters and potentials for PER NPs suspended in 1 PBS were measured by Brookhaven Instruments (NY, USA). The morphologies of PER NPs were characterized by TEM (Hitachi TEM system, Japan).

For MOMC/PER-DiI, characterization of adhesion between MOMC and PER-DiI NPs was imaged by CLSM (Carl Zeiss 700, Germany). Specifically, MOMC was cultured in 35-mm culture dishes and incubated with PER-DiI NPs for 2 hours, and the preparation of PER-DiI NPs was the same as mentioned above. The nucleus of MOMC was labeled with DAPI and MOMC membrane was labeled by DiO. For SEM characterization, MOMC/PER-DiI was coated with gold/palladium and examined by Hitachi-SU8020 (Japan).

The sensitive release properties of PER NPs from MOMC/PER in vitro was evaluated under the microenvironment of MMP-2 in vitro. MMP-2 enzyme was applied to release PER NPs by degrading the linker of GPLGIAGQ between PER NPs and MOMC. The characterization of released activity was investigated in vitro. First, MMP-2 (2 g/ml, 1 ml) was added into MOMC/PER-DiI medium for 30 min. Then, MOMC and released PER-DiI NPs were detected by flow cytometry (BD FACSCalibur, USA) or fixed by 4% paraformaldehyde (w/v) for CLSM and 2.5% glutaral for TEM. Besides, the nucleus of MOMC was labeled with DAPI for 30 min at 37C, and the MOMC membrane was labeled with DiO for 15 min at 37C. MMP-2 enzyme was dissolved in deionized water and free RPMI 1640 (volume ratio, 1:20) at a concentration of 2 g/ml. After that, the released PER NPs and MOMC in solution were prepared for the other testing assay and image.

The loading ability of MOMC was detected by the concentration of TRA and AST. First, MOMC was cultured in dishes with 100 mm. After MOMC adhered on the dishes, PER NPs were added into the culture dishes and incubated with MOMC for 2 hours in RPMI 1640 with free FBS, and then, MOMC/PER was washed thrice with PBS and digested with 0.25% trypsin-EDTA solution. Next, cells were harvested by centrifugation at 1000 rpm for 3 min and counted with a hemocytometer. Last, the cells were resuspended with PBS of 1 ml, and the absorbance was determined at 326 nm for TRA and 491 nm for AST by Multiskan GO (Thermo Fisher Scientific, USA).

The migration capacity of MOMC/PER was investigated by a Transwell device. First, the MOMC were cultured into the upper chambers with pore sizes of 8.0 m with 2 104 in 400 l of RPMI 1640 with FBS-free media for 24 hours. The cells were divided into three groups, including CXCL 12 () or CCL 19 () of MOMC, CXCL 12 (+) or CCL 19 (+) of MOMC, and CXCL 12 (+) or CCL 19 (+) of MOMC/PER. RPMI 1640 with 10% FBS and CXCL 12 (10 g/ml) or CCL 19 of 600 l was added to the lower chamber into the 24-well plates. Then, the MOMC in MOMC/PER group was added PER NPs at concentration of 40 g/ml and incubated for another 24 hours. At last, the Transwell chambers were stained with crystal violet and were dissolved with 33% acetic acid, and the absorbance of solution was tested at 570 nm.

C57BL/6J male mice were obtained from East China Normal University Laboratory Animal Technology Co. Ltd. (Shanghai, China) and housed with a 12-hour light/12-hour dark cycle at 25C. All the animal protocols and procedures were performed under the guidelines for human and responsible use of animals in research approved by the regional ethics committee of China Pharmaceutical University. After acclimatization for 7 days, mice were subjected to IPF model experiments. IPF mice models were established by inhalation of BLM through endotracheal intubation (2 U/kg, 40 l; Braintree Scientific, USA). Next, the mice were randomly assigned to the treatment. For the cell culture, A549 and MOMC were cultured in RPMI 1640 media containing 10% FBS and 1% penicillin and streptomycin at 37C and 5% CO2.

A549 cells were cultured in six-well plates at 37C and 5% CO2 for 24 hours and then incubated for another 24 hours with pure PPA, PPT, or PPTA at TRA concentration of 20 nM. The expression of vimentin and fibronectin was investigated by immunofluorescence staining.

The invasion ability of TGF-induced A549 cells was evaluated by wound healing tests after treated with various formulations. First, A549 cells were seeded in six-well plates at 15 104 cells per dish and incubated for 24 hours. Next, a 10-l pipette tip was used to scratch wells in the middle of the dishes, and then, A549 cells were washed three times with PBS to remove suspended cells. The cells from each group were imaged by inverted fluorescence microscope (Nikon, Japan) to observe the extents of wound healing after treated with PPT, PPA, PPTA, and PER NPs at 24 hours.

The migration capability of TGF-induced A549 cells was investigated by a Transwell device. The A549 cells of 5 104 in 400 l of RPMI 1640 with FBS-free media were added to the upper chambers with pore sizes of 8.0 m for 24 hours, and RPMI 1640 with 10% FBS media of 600 l was added to the lower chamber into the 24-well plates. Then, the cells were incubated with PPT, PPA, PPTA, and PER NPs (20 nM of TRA concentration) for another 24 hours. After incubation, the Transwell chambers were stained with crystal violet and were dissolved with 33% acetic acid, and the absorbance of solution was tested at 570 nm.

A549 cells were seeded on six-well plates (15 104 per well) and incubated overnight. First, the cells were activated by TGF-, and then, various treatment groups were incubated with A549 cells for 24 hours (TGF-, normal, PPT, PPA, PPTA, and PER NPs). Next, the ROS probe DCFH-DA (5 M) was added into the dishes and incubated with cells in RPMI 1640 media with free FBS at 37C under 5% CO2 in the dark for 15 min. Last, cells were digested, and the content of ROS was analyzed by flow cytometry (BD Accuri C6, USA) and imaged by inverted fluorescence microscope, respectively.

A549 cells were cultured on six-well plates (15 104 per well) overnight at 37C and 5% CO2. After treated with different formulations for 24 hours, 500 l of mitochondrial membrane potential reagent of JC-1 (1) solution was added into the dishes for 20 min. Then, the cells were stained by 4% paraformaldehyde (w/v) and imaged by inverted fluorescence microscope.

First, A549 cells were seeded on a 35-mm sterile glass bottom culture dishes (2 105 cells) and cultured overnight in RPMI 1640 with 10% FBS. The preparation of PR-C6 and PP-C6 was same as mentioned above for PER NPs. The PR-C6, PP-C6, and C6 were then incubated with A549 cells for 4 hours at 5 g/ml. Next, the cells were washed three times by PBS, and the nucleus was stained with DAPI. Images and data were acquired with CLSM and flow cytometry (BD Accuri C6, USA).

The A549 cells of 5 104 were cultured in a 35-mm sterile glass bottom culture dishes. After the cells were cultured overnight in RPMI 1640 with 10% FBS for 24 hours, PR-C6 and PP-C6 were incubated with A549 cells for 1 and 4 hours at 5 g/ml. Then, the cell dishes were washed three times by PBS and fix by 4% paraformaldehyde (w/v). Lysosome was stained with LysoTracker Red DND-99 kit (100 nM) for 15 min, and the dishes were washed three times with PBS. Then, the cell nucleus was stained by DAPI like above method. Images were acquired by CLSM.

MOMC/PER-DiR was prepared by the method as mentioned above for PER NPs. The homing ability of MOMC was detected in vivo by IVIS imaging system (Kodak, USA). C57BL/6J mice were induced IPF by inhalation of BLM. Then, the IPF mice were injected with MOMC/PER-DiR, MOMC-DiR, and free DiR by intravenous injection and tested by IVIS living system at different point times. Then, the mice are sacrificed, and the lungs and other organs were harvested for ex vivo imaging after 8 hours of intravenous injection. ROI was circled around the lungs and the other organs (liver, heart, spleen, and kidneys). The fluorescence intensity of the DiR was determined by living image software.

The PER NPs can be released from MOMC/PER-DiI by MMP-2, owing to responsive blocking the linker between PER NPs and MOMC. We have investigated the homing capability, responsive release, and retarget ability. We first administrated MOMC/PER-DiI by intravenous injection, and the preparation of MOMC/PER-DiI was applied by the methods as mentioned above. The mice were sacrificed, and lung tissues were harvested at different point time of 30 min, 1 hour, and 2 hours in a dark place. First, the lung tissues were fixed with 4% paraformaldehyde (w/v), and then, the tissues were embedded and dewaxed before slicing. Next, the lung slices were labeled by Nanog and SPC at 4C for 1 hour and washed with 0.2% Triton X-100 for three times. Then, they were incubated with relevant secondary antibodies for 2 hours. Thereafter, the slices were stained with DAPI and viewed under fluorescence microscope.

The IPF mice were injected with PER-DiR NPs, PE-DiR NPs, PR-DiR NPs, and PP-DiR NPs by intravenous injection at different point times for 1, 4, 8, 12, and 24 hours and tested by IVIS imaging system. The preparation of PER-DiR NPs, PE-DiR NPs, PR-DiR NPs, and PP-DiR NPs were the same method as mentioned above, and then, the mice were sacrificed, and the lungs and other organs were harvested to detect ex vivo imaging after intravenous injection at 24 hours. ROI was tested on the lungs and the other organs (liver, heart spleen, and kidneys). The fluorescence intensity of the DiR was determined by living image software.

The antifibrotic efficacy in MOMC/PER, MOMC, PER NPs, and pirfenidone for IPF treatment in vivo was evaluated on IPF male mice (C57BL/6J, age of 6 to 8 weeks). The preparations of MOMC/PER and PER NPs were the same method as mentioned above. Pirfenidone was administered by gastrointestinal because it is an oral medication, and other formulations were administered via intravenous injection.

The contents of MDA, SOD, and GSH were detected after treated with various treatments in lung tissues. The protocols are as follows: Solution 1 of 1.5 ml was added into the lung tissues solution (0.5 ml) and mixed thoroughly. Then, the samples were centrifuged for 10 min at 3500 to 4000 rpm. The sample supernatant was added into 3,3,5,5-tetramethyl benaidine (TMB substrate), and the absorbance of GSH was detected after 5 to 10 min at 420 nm. The contents of SOD and MDA were tested at 550 and 532 nm, respectively.

The lungs and spleen tissues were collected and diluted in precooled solution. The IL-1, TGF-, and IL-4 in lung tissues were assayed using enzyme-linked immunosorbent assay (ELISA) method as instructed by the manufacturer. First, the wells were washed three times with a washing buffer for 3 min each time. Next, the blocking solution of 200 l was added into each well and incubated for 1 to 2 hours at 37C. Then, the sealing film was removed carefully and putted it into the washing machine and washed three to five times. Furthermore, the sample of 100 l was added and should be tested diluted appropriately to the above coated reaction wells, and the diluted biotinylated antibody working solution was added with 100 l into each well. Then, the samples were sealed with a sealing membrane and incubated at 37C for 1 hour. The following step was that 100 l of diluted enzyme conjugate working solution was added into each well. Next, TMB substrate solution with 100 l was added into each well and should be avoided reaction with light for 10 to 30 min at 37C until a notable color gradient appears in the diluted standard well. Within 10 min, the absorbance of each well was measured on a microplate reader at 450 nm with zero adjustment of the blank control well.

The content of hydroxyproline was detected by a hydroxyproline detection kit. The lung tissues of 30 to 100 mg were mixed with hydrolysate to 1 ml and hydrolyzed in boiling water for 20 min. The sample solution was pH 6.0 to 6.8. Then, the serum hydrolysate was added activated carbon and mixed at 60C for 15 min. After cooling, the serum samples were centrifuged at 3000 rpm for 20 min, and the supernatant was detected by microplate reader at 550 nm.

The whole blood from mice was collected by anticoagulant tube with EDTA, and white blood cell counts (including lymphocytes and monocytes) were assayed using a standard blood analyzer (Mindray, China).

After treatment with different formulation, the lungs, heart, liver, spleen, and kidneys were harvested, and lungs were investigated by H&E, Masson, and IHC staining. Other organs were detected by H&E staining to evaluate the application security. First, the lung tissues were fixed with 4% paraformaldehyde (w/v) for more than 48 hours and embedded in paraffin. Then, the lung tissues were cut into 4-m sections for H&E staining and Masson staining. The levels of collagen I and -SMA were evaluated by IHC, and protocols are as follows: The slices were incubated with primary antibodies (collagen I and -SMA or fluorescently labeled CD90 and collagen I, Servicebio, China) and then incubated with corresponding secondary antibodies to detect the expression of relative proteins.

The qPCR analysis was evaluated RNA expression of Ctgf and Acta2. qPCR was conducted in ABI StepOnePlus (Thermo Fisher Scientific, USA). Lung tissues (100 mg) were homogenized to extract the total RNA according to the protocols. Complementary DNA (2 g) was prepared using the Reverse Transcription System, and then, the expression of related genes was determined using q-PCR. The primers used are Acta2 (NM_007392.3), Ctgf (43), and Gapdh (NM_008084.2).

The harvested lungs were homogenized in PBS buffer and then centrifuged at 3000 rpm for 30 min. The supernatant of total protein was taken for further experiments. Total protein concentration in the solution was determined with a BCA protein assay kit. After detecting in SDS-PAGE with protein samples in different treatment groups, the bands were transferred onto PVDF membrane. Next, the PVDF membranes were blocked with 5% milk at room temperature for 2 hours and incubated with primary antibodies (-SMA, TGF-/Smad, and -actin rabbit anti-mouse antibodies) at 4C overnight and then incubated with corresponding secondary antibodies for 2 hours at room temperature. Last, the bands were detected using ECL (Tanon, China) Western blotting substrate (Thermo Fisher Scientific, USA). The -actin was used as an endogenous control.

The mice were sacrificed, and the serum samples were detected in different treatment groups. The contents of ALT, aspartate aminotransferase, and BUN in the serum were determined using the relevant assay kits (Servicebio, China).

Statistical analyses were performed using GraphPad Prism software (GraphPad Software, USA). All error bars were means SEM; differences detection index between the treated groups and control groups were determined via one-way analysis of variance (ANOVA). P < 0.05 was considered significantly different.

Acknowledgments: We thank the Cellular and Molecular Biology Center of China Pharmaceutical University for assistance with confocal microscopy work. Funding: This work was supported by the National Key R&D Program of China (2017YFA0205400). We thank the National Natural Science Foundation of China (NSFC; grant nos. 81773667, 81573369, and 81430082) and NSFC Projects of International Cooperation and Exchanges (81811540416). This work was also supported by the Fundamental Research Funds for the Central Universities (2632018PT01 and 2632018ZD12), the 111 Project from the Ministry of Education of China and the State Administration of Foreign Experts Affairs of China (B16046), the Double First-Class Project (CPU2018GY06), and a project funded by the Priority Academic Program Development of Jiangsu Higher Education Institutions. Author contributions: H.-L.J., H.-P.H., X.C., and L.X. conceived the project, designed all the experiments, analyzed the data, and wrote the manuscript. X.C. and L.X. conducted the experiments. X.C. and Y.W. analyzed the data. X.C., Y.W., C.-X.Y., Y.-J.H., T.-J.Z., X.-D.G., and L.L. wrote the manuscript. All authors edited the manuscript. Competing interests: The authors declare that they have no competing interests. Data and materials availability: All data needed to evaluate the conclusions in the paper are present in the paper and/or the Supplementary Materials. Additional data related to this paper may be requested from the authors.

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Monocyte-derived multipotent cell delivered programmed therapeutics to reverse idiopathic pulmonary fibrosis - Science Advances

Acute lung injury (ALI) is a devastating disease process involving pulmonary edema and atelectasis caused by capillary membrane injury [1]. The main clinical manifestation is the acute onset of hypoxic respiratory failure, which can subsequently trigger a cascade of serious complications and even death [2]. Thus, ALI causes a considerable financial burden for health care systems throughout the world. ALI can result from various causes, including multiple traumas, large-volume blood transfusions, and bacterial and viral infections [2, 3]. A variety of viruses, including influenza virus, coronavirus (CoV), adenovirus, cytomegalovirus (CMV), and respiratory syncytial virus (RSV), are associated with ALI [4]. Importantly, most viruses, whose hosts are various animal species, can cause severe and rapidly spreading human infections. In the early 2000s, several outbreaks of influenza virus and CoV emerged, causing human respiratory and intestinal diseases worldwide, including the more recent SARS-CoV-2 infection [5,6,7]. To date, SARS-CoV-2 has affected more than 80,000 people, causing nearly 3300 deaths in China and more than 1,800,000 people, causing nearly 110,000 deaths all over the world (http://2019ncov.chinacdc.cn/2019-nCoV/).

Infectious respiratory diseases caused by different viruses are associated with similar respiratory symptoms ranging from the common cold to severe acute respiratory syndrome [8]. This makes the clinical distinction between different agents involved in infection very difficult [8, 9]. Currently, the clinical experience mainly includes antibacterial and antiviral drug treatment derived from handling several outbreaks of influenza virus and human CoVs. Numerous agents have been identified to inhibit the entry and/or replication of these viruses in cell culture or animal models [10]. Although these antiviral drugs can effectively prevent and eliminate the virus, the full recovery from pneumonia and ALI depends on the resistance of the patient. Recently, stem cell-based therapy has become a potential approved tool for the treatment of virus-induced lung injury [11,12,13]. Here, we will give a brief overview of influenza virus and CoVs and then present the cell-based therapeutic options for lung injury caused by different kinds of viruses.

Influenza virus and human CoV are the two most threatening viruses for infectious lung injury [14]. These pathogens can be transmitted through direct or indirect physical contact, droplets, or aerosols, with increasing evidence suggesting that airborne transmission, including via droplets or aerosols, enhances the efficiency of viral transmission among humans and causes uncontrolled infectious disease [15]. Throughout human history, outbreaks and occasional pandemics caused by influenza virus and CoV have led to approximately hundreds of millions of deaths worldwide [16].

Influenza virus is a well-known human pathogen that has a negative-sense RNA genome [17]. According to its distinct antigenic properties, the influenza virus can be divided into 4 subtypes, types A, B, C, and D. Influenza A virus (IAV) lineages in animal populations cause economically important respiratory disease. Little is known about the other human influenza virus types B, C, and D [18]. Further subtypes are characterized by the genetic and antigenic properties of the hemagglutinin (HA) and neuraminidase (NA) glycoproteins [19]. Sporadic and seasonal infections in swine with avian influenza viruses of various subtypes have been reported. The most recent human pandemic virusesH1N1 from swine and H5N1 from aviancause severe respiratory tract disease and lung injury in humans [20, 21].

CoVs, a large family of single-stranded RNA viruses, typically affect the respiratory tract of mammals, including humans. CoVs are further divided into four genera: alpha-, beta-, gamma-, and delta-CoVs. Alpha- and beta-CoVs can infect mammals, and gamma- and delta-CoVs tend to infect birds, but some of these viruses can also be transmitted to mammals [22]. Human CoVs were considered relatively harmless respiratory pathogens in the past. Infections with the human CoV strains 229E, OC43, NL63, and HKU1 usually result in mild respiratory illness, such as the common cold [23]. In contrast, the CoV responsible for the 2002 severe acute respiratory syndrome (SARS-CoV), the 2012 Middle East respiratory syndrome CoV (MERS-CoV), and, more recently, the SARS-CoV-2 have received global attention owing to their genetic variation and rapid spread in human populations [5,6,7].

Usually, the influenza virus can enter the columnar epithelial cells of the respiratory tract, such as the trachea, bronchi, and bronchioles. Subsequently, the influenza virus begins to replicate for an asymptomatic period of time and then migrate to the lung tissue to cause acute lung and respiratory injury [24]. Similar to those with influenza virus infection, patients with SARS, MERS, or SARS-CoV-2 present with various clinical features, ranging from asymptomatic or mild respiratory illness to severe ALI, even with multiple organ failure [5,6,7]. The pathogenesis of ALI caused by influenza virus and human CoV is often associated with rapid viral replication, marked inflammatory cell infiltration, and elevated proinflammatory cytokine/chemokine responses [25]. Interestingly, in IAV- and human CoV-infected individuals, the pulmonary pathology always involves diffuse alveolar damage, but viral RNA is present in only a subset of patients [26]. Some studies suggest that an overly exaggerated immune response, rather than uncontrolled viral spread, is the primary cause in fatal cases caused by virus infection [27]. Several immune cell types have been found to contribute to damaging host responses, providing novel approaches for therapeutic intervention [28].

IAV infection, the most common cause of viral pneumonia, causes substantial seasonal and pandemic morbidity and mortality [29]. Currently, antiviral drugs are the primary treatment strategy for influenza-induced pneumonia. However, antiviral drugs cannot repair damaged lung cells. Here, we summarize the present studies of stem cell therapy for influenza virus-induced lung injury.

Mesenchymal stem/stromal cells (MSCs) constitute a heterogeneous subset of stromal regenerative cells that can be harvested from several adult tissue types, including bone marrow, umbilical cord, adipose, and endometrium [30]. They retain the expression of the markers CD29, CD73, CD90, and CD105 and have a rapid proliferation rate, low immunogenicity, and low tumorigenicity [30]. MSCs also have self-renewal and multidifferentiation capabilities and exert immunomodulatory and tissue repair effects by secreting trophic factors, cytokines, and chemokines [31]. Due to these beneficial biological properties, MSCs and their derivatives are attractive as cellular therapies for various inflammatory diseases, including virus-induced lung injury.

Several studies on IAV-infected animal models have shown the beneficial effects of the administration of different tissue-derived MSCs [32,33,34,35]. H5N1 virus infection reduces alveolar fluid clearance (AFC) and enhances alveolar protein permeability (APP) in human alveolar epithelial cells, which can be inhibited by coculture with human bone marrow-derived MSCs (BMSCs) [32]. Mechanistically, this process can be mediated by human BMSC secreted angiopoietin-1 (Ang1) and keratinocyte growth factor (KGF) [32]. Moreover, in vivo experiments have shown that human BMSCs have a significant anti-inflammatory effect by increasing the number of M2 macrophages and releasing various cytokines and chemokines, such as interleukin (IL)-1, IL-4, IL-6, IL-8, and IL-17 [32]. Similar anti-inflammatory effects have been achieved in another virus-induced lung injury model. The intravenous injection of mouse BMSCs into H9N2 virus-infected mice significantly attenuates H9N2 virus-induced pulmonary inflammation by reducing chemokine (GM-CSF, MCP-1, KC, MIP-1, and MIG) and proinflammatory cytokine (IL-1, IL-6, TNF-, and IFN-) levels, as well as reducing inflammatory cell recruitment into the lungs [33]. Another study on human BMSCs cocultured with CD8+ T cells showed that MSCs inhibit the proliferation of virus-specific CD8+ T cells and the release of IFN- by specific CD8+ T cells [36].

In addition, human umbilical cord-derived MSCs (UC-MSCs) were found to have a similar effect as BMSCs on AFC, APP, and inflammation by secreting growth factors, including Ang1 and hepatocyte growth factor (HGF), in an in vitro lung injury model induced by H5N1 virus [34]. UC-MSCs also promote lung injury mouse survival, increase the body weight, and decreased the APP levels and inflammation in vivo [34]. Unlike Ang1, KGF, and HGF mentioned above, basic fibroblast growth factor 2 (FGF2) plays an important role in lung injury therapy via immunoregulation. The administration of the recombinant FGF2 protein improves H1N1-induced mouse lung injury and promotes the survival of infected mice by recruiting and activating neutrophils via the FGFR2-PI3K-AKT-NFB signaling pathway [37]. FGF2-overexpressing MSCs have an enhanced therapeutic effect on lipopolysaccharide-induced ALI, as assessed by the proinflammatory factor level, neutrophil quantity, and histopathological index of the lung [38].

MSCs secrete various soluble factors and extracellular vesicles (EVs), which carry lipids, proteins, DNA, mRNA, microRNAs, small RNAs, and organelles. These biologically active components can be transferred to recipient cells to exert anti-inflammatory, antiapoptotic, and tissue regeneration functions [39]. EVs isolated from conditioned medium of pig BMSCs have been demonstrated to have anti-apoptosis, anti-inflammation, and antiviral replication functions in H1N1-affected lung epithelial cells and alleviate H1N1-induced lung injury in pigs [35]. Moreover, the preincubation of EVs with RNase abrogates their anti-influenza activity, suggesting that the anti-influenza activity of EVs is due to the transfer of RNAs from EVs to epithelial cells [35]. Exosomes are a subset of EVs that are 50200nm in diameter and positive for CD63 and CD81 [40]. Exosomes isolated from the conditioned medium of UC-MSCs restore the impaired AFC and decreased APP in alveolar epithelial cells affected by H5N1 virus [34]. In addition, the ability of UC-MSCs to increase AFC is superior to that of exosomes, which indicates that other components secreted by UC-MSCs have synergistic effects with exosomes [34].

Despite accumulating evidence demonstrating the therapeutic effects of MSC administration in various preclinical models of lung injury, some studies have shown contrasting results. Darwish and colleagues proved that neither the prophylactic nor therapeutic administration of murine or human BMSCs could decrease pulmonary inflammation or prevent the progression of ALI in H1N1 virus-infected mice [41]. In addition, combining MSC administration with the antiviral agent oseltamivir was also found to be ineffective [41]. Similar negative results were obtained in another preclinical study. Murine or human BMSCs were administered intravenously to H1N1-induced ARDS mice [42]. Although murine BMSCs prevented influenza-induced thrombocytosis and caused a modest reduction in lung viral load, murine or human BMSCs failed to improve influenza-mediated lung injury as assessed by weight loss, the lung water content, and bronchoalveolar lavage inflammation and histology, which is consistent with Darwishs findings [42]. However, the mild reduction in viral load observed in response to murine BMSC treatment suggests that, on balance, MSCs are mildly immunostimulatory in this model [42]. Although there are some controversial incidents in preclinical research, the transplant of menstrual-blood-derived MSCs into patients with H7N9-induced ARDS was conducted at a single center through an open-label clinical trial (http://www.chictr.org.cn/). MSC transplantation significantly lowered the mortality and did not result in harmful effects in the bodies of the patients [43]. This clinic study suggests that MSCs significantly improve the survival rate of influenza virus-induced lung injury.

The effects of exogenous MSCs are exerted through their isolation and injection into test animals. There are also some stem/progenitor cells that can be activated to proliferate when various tissues are injured. Basal cells (BCs), distributed throughout the pseudostratified epithelium from the trachea to the bronchioles, are a class of multipotent tissue-specific stem cells from various organs, including the skin, esophagus, and olfactory and airway epithelia [44, 45]. Previously, TPR63+/KRT5+ BCs were shown to self-renew and divide into club cells and ciliated cells to maintain the pseudostratified epithelium of proximal airways [46]. Several studies have shown that TPR63+/KRT5+ BCs play a key role in lung repair and regeneration after influenza virus infection. When animals typically recover from H1N1 influenza infection, TPR63+/KRT5+ BCs accumulate robustly in the lung parenchyma and initiate an injury repair process to maintain normal lung function by differentiating into mature epithelium [47]. Lineage-negative epithelial stem/progenitor (LNEP) cells, present in the normal distal lung, can activate a TPR63+/KRT5+ remodeling program through Notch signaling after H1N1 influenza infection [48]. Moreover, a population of SOX2+/SCGB1A/KRT5 progenitor cells can generate nascent KRT5+ cells as an early response to airway injury upon H1N1 influenza virus infection [49]. In addition, a rare p63+Krt5 progenitor cell population also responds to H1N1 virus-induced severe injury [50]. This evidence suggests that these endogenous lung stem/progenitor cells (LSCs) play a critical role in the repopulation of damaged lung tissue following severe influenza virus infection (Table2).

Taken together, the present in vitro (Table1) and in vivo (Table2) results show that MSCs and LSCs are potential cell sources to treat influenza virus-induced lung injury.

Lung injury caused by SARS, MERS, or SARS-CoV-2 poses major clinical management challenges because there is no specific treatment that has been proven to be effective for each infection. Currently, virus- and host-based therapies are the main methods of treatment for spreading CoV infections. Virus- and host-based therapies include monoclonal antibodies and antiviral drugs that target the key proteins and pathways that mediate viral entry and replication [51].The major challenges in the clinical development of novel drugs include a limited number of suitable animal models for SARS-CoV, MERS-CoV, and SARS-CoV-2 infections and the current absence of new SARS and MERS cases [51]. Although the number of cases of SARS-CoV-2-induced pneumonia patients is continuously increasing, antibiotic and antiviral drugs are the primary methods to treat SARS-CoV-2-infected patients. Similar to that of IAV, human CoV-mediated damage to the respiratory epithelium results from both intrinsic viral pathogenicity and a robust host immune response. The excessive immune response contributes to viral clearance and can also worsen the severity of lung injury, including the demise of lung cells [52]. However, the present treatment approaches have a limited effect on lung inflammation and regeneration.

Stem cell therapy for influenza virus-induced lung injury shows promise in preclinical models. Although it is difficult to establish preclinical models of CoV-induced lung injury, we consider stem cell therapies to be effective approaches to improve human CoV-induced lung injury. Acute inflammatory responses are one of the major underlying mechanisms for virus-induced lung injury. Innate immune cells, including neutrophils and inflammatory monocytes-macrophages (IMMs), are major innate leukocyte subsets that protect against viral lung infections [53]. Both neutrophils and IMMs are rapidly recruited to the site of infection and play crucial roles in the host defense against viruses. Neutrophils and IMMs can activate Toll-like receptors (TLRs) and produce interferons (IFNs) and other cytokines/chemokines [54]. There are two functional effects produced by the recruitment of neutrophils and IMMs: the orchestration of effective adaptive T cell responses and the secretion of inflammatory cytokines/chemokines [55]. However, excessive inflammatory cytokine and chemokine secretion impairs antiviral T cell responses, leading to ineffective viral clearance and reduced survival [56].

MSCs are known to suppress both innate and adaptive immune responses. MSCs have been suggested to inhibit many kinds of immune cells, including T cells, B cells, dendritic cells (DCs), and natural killer (NK) cells in vitro and in vivo [57] (Fig.1). Several molecules, including IL-1, TNF-, and INF-, most of which are produced by inflammatory cells, are reported to be involved in MSC-mediated immunosuppression [58]. Furthermore, MSCs can produce numerous immunosuppressive molecules, such as IL-6, PGE2, IDO, and IL-10, in response to inflammatory stimuli. PGE2 has been reported to mediate the MSC-mediated suppression of T cells, NK cells, and macrophages. Moreover, PGE2 has been found to act with IDO to alter the proliferation of T cells and NK cells [59]. In contrast, MSCs have come to be recognized as one type of adult stem cell actively participating in tissue repair by closely interacting with inflammatory cells and various other cell types [60]. Numerous reports have demonstrated that MSCs can release an array of growth and inhibitory factors, such as EGF, FGF, PDGF, and VEGF, and express several leukocyte chemokines, such as CXCL9, CCL2, CXCL10, and CXCL11. These factors provide an important microenvironment to activate adaptive immunity for lung repair [61]. Thus, the dual functions of MSCs may improve lung recovery after human CoV-induced ALI. Recently, MSCs was transplanted intravenously to enrolled patients with COVID-19 pneumonia. After treatment, the pulmonary function and symptoms of these patients were significantly improved. Meanwhile, the peripheral lymphocytes were increased, the C-reactive protein decreased, the level of TNF- was significantly decreased, and the overactivated cytokine-secreting immune cells disappeared. In addition, a group of regulatory DC cell population dramatically increased. Thus, the intravenous transplantation of MSCs was effective for treatment in patients with COVID-19 pneumonia [62, 63].

Stem cell therapies for treatment of influenza virus and coronavirus-induced lung injury. CoVs, coronavirus; MSCs, mesenchymal stem/stromal cells; LSCs, lung stem/progenitor cells; NK cells, natural killer cells; DC cells, dendritic cells

In addition, endogenous LSCs also play an important role in lung cell reconstitution after virus-induced ALI. In particular, TPR63+/KRT5+ airway BCs comprise approximately equal numbers of stem cells and committed precursors and give rise to differentiated luminal cells during steady state and epithelial repair after lung injury [44, 64]. Research has shown that KRT5+ cells repopulate damaged alveolar parenchyma following influenza virus infection [47]. However, there is still little evidence for the role of altered TPR63+/KRT5+ stem cells during lung injury repair caused by human CoVs.

In summary, exogenous MSCs may modulate human CoV-induced lung injury repair and regeneration through their immunoregulatory properties. These cells are capable of interacting with various types of immune cell, including neutrophils, macrophages, T cells, B cells, NK cells, and DCs. Furthermore, viral infections can activate endogenous LSCs to produce new lung cells and maintain lung function (Fig.1). Thus, we propose that MSCs and LSCs are two potential cell sources for treating human CoV-induced lung injury.

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Stem cell therapy: a potential approach for treatment of influenza virus and coronavirus-induced acute lung injury - BMC Blogs Network

SHANGHAI, May27, 2020 /PRNewswire/ -- Shanghai Cell Therapy Group (SHCell) recently entered intoa six-year research collaborative project with Professor Qi-Long Ying from the University of Southern California (USC). Through the project, sponsored by $3.6 million from the Baize Plan Fund, the Ying laboratory aims to develop conditions for the long-term ex vivo expansion of mouse and human hematopoietic stem and progenitor cells.

"Hematopoietic stem cells, or HSCs, are found in the bone marrow of adults," said Professor Qijun Qian, CEO of Shanghai Cell Therapy Group. "HSCs have the ability for long-term self-renewal and differentiation into various types of mature blood cells, and for rebuilding normal hematopoiesis and immune function in patients. They also have enormous potential to treat diseases, including tumors, autoimmune diseases, severe infectious disease, and inherited blood diseases, and to combat the effects of aging."

This research project will be conducted and supervised by Professor Qi-Long Ying, a Professor of Stem Cell Biology and Regenerative Medicine at the Keck School of Medicine of USC. Professor Ying's pioneering stem cell research has won international acclaim, including the 2016 McEwen Award for Innovation, the highest honor in the field.

"We'll develop and optimize culture conditions for the long-term ex vivo expansion of HSCs," said Professor Ying. "We'll also test combinations of basal media, small molecules, cytokines and growth factors, and characterize ex vivo expanded hematopoietic stem and progenitor cells. These cells will then be genetically modified and tested for their potential to treat different diseases, including blood disorders and cancers."

Professor Andrew P. McMahon, Director of Eli and Edythe Broad Center for Regenerative Medicine and Stem Cell Research of USC, added: "Stem cell biology represents an exciting area in medicine with great therapeutic potential. I am delighted SHCell is supporting Professor Ying. A breakthrough in the ability to propagate and manipulate HSCs will have lasting clinical significance."

The project also plans to build animal models of different blood diseases and cancers and test the safety and effectiveness of genetically modified hematopoietic stem and progenitor cells before clinical translation. SHCell will actively explore clinical applications of hematopoietic stem and progenitor cells in the treatment of cancers or blood diseases.

As SHCell's first overseas collaboration, this project aims to advance the goals of the Baize Plan: to provide first-class cell treatments and cell therapies at an affordable price to cure cancer and increase life expectancy. SHCell hopes that this project will also accelerate original scientific breakthroughs in the stem cell field.

Shanghai Cell Therapy Group

Founded in 2013, Shanghai Cell Therapeutics Group Co., Ltd is located at the Shanghai Municipal Engineering and Technology Research Center, which was established by the Shanghai Science and Technology Commission. With a mission of "changing the length and abundance of life with cell therapy", SHCell has created a closed-loop industrial chain and an integrated platform for cell treatment and cell therapy. It comprises cell storage, cell drug research and cell clinical transformation with cell therapy as its core business.

The Baize Plan was proposed in 2016 by Wu Mengchao, an Academician of the Chinese Academy of Sciences (CAS) and initiated by Professor Qian, aiming to provide first-class cell treatments and cell therapies at an affordable price with the goal of curing cancers and increasing life expectancy. The Baize Plan Fund was created by the Shanghai Cell Therapy Group to realize the vision of the Baize Plan.

University of Southern California (USC)

Founded in 1880, the University of Southern California is one of the world's leading educational and research institutions, and also the oldest private research university in California. Located in the heart of Los Angeles, the University of Southern California comprises 23 schools and units, and students are encouraged to explore different fields of study. The University of Southern California ranked #22 in National Universities in the 2020 edition of Best Colleges, published by U.S. News & World Report.

For more information, visit http://www.shcell.com/

SOURCE Shanghai Cell Therapy Group

http://www.shcell.com

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Shanghai Cell Therapy Group Launches Collaboration with USC researcher to Improve the ex vivo Expansion of Hematopoietic Stem Cells for Clinical...

She just wanted to be somewhere safe, somewhere familiar, where people looked and spoke like her and she could stand to eat the food. Midnight Robber by Nalo Hopkinson

Midnight Robber (2000) is about a woman, divided. Raised on the high-tech utopian planet of Touissant, Tan-Tan grows up on a planet populated by the descendants of a Caribbean diaspora, where all labor is performed by an all-seeing AI. But when she is exiled to Touissants parallel universe twin planet, the no-tech New Half-Way Tree, with her sexually abusive father, she becomes divided between good and evil Tan-Tans. To make herself and New Half-Way Tree whole, she adopts the persona of the legendary Robber Queen and becomes a legend herself. It is a wondrous blend of science fictional tropes and Caribbean mythology written in a Caribbean vernacular which vividly recalls the history of slavery and imperialism that shaped Touissant and its people, published at a time when diverse voices and perspectives within science fiction were blossoming.

Science fiction has long been dominated by white, Western perspectives. Vernes tech-forward adventures and Wells sociological allegories established two distinctive styles, but still centered on white imperialism and class struggle. Subsequent futures depicted in Verne-like pulp and Golden Age stories, where lone white heroes conquered evil powers or alien planets, mirrored colonialist history and the subjugation of non-white races. The civil rights era saw the incorporation of more Wellsian sociological concerns, and an increase in the number of non-white faces in the future, but they were often tokensparts of a dominant white monoculture. Important figures that presaged modern diversity included Star Treks Lieutenant Uhura, played by Nichelle Nichols. Nichols was the first black woman to play a non-servant character on TV; though her glorified secretary role frustrated Nichols, her presence was a political act, showing there was space for black people in the future.

Another key figure was the musician and poet Sun Ra, who laid the aesthetic foundation for what would become known as the Afrofuturist movement (the term coined by Mark Dery in a 1994 essay), which showed pride in black history and imagined the future through a black cultural lens. Within science fiction, the foundational work of Samuel Delany and Octavia Butler painted realistic futures in which the histories and cultural differences of people of color had a place. Finally, an important modern figure in the decentralization of the dominant Western perspective is Nalo Hopkinson.

A similarly long-standing paradigm lies at the heart of biology, extending back to Darwins theoretical and Mendels practical frameworks for the evolution of genetic traits via natural selection. Our natures werent determined by experience, as Lamarck posited, but by genes. Therefore, genes determine our reproductive fitness, and if we can understand genes, we might take our futures into our own hands to better treat disease and ease human suffering. This theory was tragically over-applied, even by Darwin, who in Descent of Man (1871) conflated culture with biology, assuming the Wests conquest of indigenous cultures meant white people were genetically superior. After the Nazis committed genocide in the name of an all-white future, ideas and practices based in eugenics declined, as biological understanding of genes matured. The Central Dogma of the 60s maintained the idea of a mechanistic meaning of life, as advances in genetic engineering and the age of genomics enabled our greatest understanding yet of how genes and disease work. The last major barrier between us and our transhumanist future therefore involved understanding how genes determine cellular identity, and as well see, key figures in answering that question are stem cells.

***

Hopkinson was born December 20, 1960 in Kingston, Jamaica. Her mother was a library technician and her father wrote, taught, and acted. Growing up, Hopkinson was immersed in the Caribbean literary scene, fed on a steady diet of theater, dance, readings, and visual arts exhibitions. She loved to readfrom folklore, to classical literature, to Kurt Vonnegutand loved science fiction, from Spock and Uhura on Star Trek, to Le Guin, James Tiptree Jr., and Delany. Despite being surrounded by a vibrant writing community, it didnt occur to her to become a writer herself. What they were writing was poetry and mimetic fiction, Hopkinson said, whereas I was reading science fiction and fantasy. It wasnt until I was 16 and stumbled upon an anthology of stories written at the Clarion Science Fiction Workshop that I realized there were places where you could be taught how to write fiction. Growing up, her family moved often, from Jamaica to Guyana to Trinidad and back, but in 1977, they moved to Toronto to get treatment for her fathers chronic kidney disease, and Hopkinson suddenly became a minority, thousands of miles from home.

Development can be described as an orderly alienation. In mammals, zygotes divide and subsets of cells become functionally specialized into, say, neurons or liver cells. Following the discovery of DNA as the genetic material in the 1950s, a question arose: did dividing cells retain all genes from the zygote, or were genes lost as it specialized? British embryologist John Gurdon addressed this question in a series of experiments in the 60s using frogs. Gurdon transplanted nuclei from varyingly differentiated cells into oocytes stripped of their genetic material to see if a new frog was made. He found the more differentiated a cell was, the lower the chance of success, but the successes confirmed that no genetic material was lost. Meanwhile, Canadian biologists Ernest McCulloch and James Till were transplanting bone marrow to treat irradiated mice when they noticed it caused lumps in the mices spleens, and the number of lumps correlated with the cellular dosage. Their lab subsequently demonstrated that each lump was a clonal colony from a single donor cell, and a subset of those cells was self-renewing and could form further colonies of any blood cell type. They had discovered hematopoietic stem cells. In 1981 the first embryonic stem cells (ESCs) from mice were successfully propagated in culture by British biologist Martin Evans, winning him the Nobel Prize in 2007. This breakthrough allowed biologists to alter genes in ESCs, then use Gurdons technique to create transgenic mice with that alteration in every cellcreating the first animal models of disease.

In 1982, one year after Evans discovery, Hopkinson graduated with honors from York University. She worked in the arts, as a library clerk, government culture research officer, and grants officer for the Toronto Arts Council, but wouldnt begin publishing her own fiction until she was 34. [I had been] politicized by feminist and Caribbean literature into valuing writing that spoke of particular cultural experiences of living under colonialism/patriarchy, and also of writing in ones own vernacular speech, Hopkinson said. In other words, I had models for strong fiction, and I knew intimately the body of work to which I would be responding. Then I discovered that Delany was a black man, which opened up a space for me in SF/F that I hadnt known I needed. She sought out more science fiction by black authors and found Butler, Charles Saunders, and Steven Barnes. Then the famous feminist science fiction author and editor Judy Merril offered an evening course in writing science fiction through a Toronto college, Hopkinson said. The course never ran, but it prompted me to write my first adult attempt at a science fiction story. Judy met once with the handful of us she would have accepted into the course and showed us how to run our own writing workshop without her. Hopkinsons dream of attending Clarion came true in 1995, with Delany as an instructor. Her early short stories channeled her love of myth and folklore, and her first book, written in Caribbean dialect, married Caribbean myth to the science fictional trappings of black market organ harvesting. Brown Girl in the Ring (1998) follows a young single mother as shes torn between her ancestral culture and modern life in a post-economic collapse Toronto. It won the Aspect and Locus Awards for Best First Novel, and Hopkinson was awarded the John W. Campbell Award for Best New Writer.

In 1996, Dolly the Sheep was created using Gurdons technique to determine if mammalian cells also could revert to more a more primitive, pluripotent state. Widespread animal cloning attempts soon followed, (something Hopkinson used as a science fictional element in Brown Girl) but it was inefficient, and often produced abnormal animals. Ideas of human cloning captured the public imagination as stem cell research exploded onto the scene. One ready source for human ESC (hESC) materials was from embryos which would otherwise be destroyed following in vitro fertilization (IVF) but the U.S. passed the Dickey-Wicker Amendment prohibited federal funding of research that destroyed such embryos. Nevertheless, in 1998 Wisconsin researcher James Thomson, using private funding, successfully isolated and cultured hESCs. Soon after, researchers around the world figured out how to nudge cells down different lineages, with ideas that transplant rejection and genetic disease would soon become things of the past, sliding neatly into the hole that the failure of genetic engineering techniques had left behind. But another blow to the stem cell research community came in 2001, when President Bushs stem cell ban limited research in the U.S. to nineteen existing cell lines.

In the late 1990s, another piece of technology capturing the public imagination was the internet, which promised to bring the world together in unprecedented ways. One such way was through private listservs, the kind used by writer and academic Alondra Nelson to create a space for students and artists to explore Afrofuturist ideas about technology, space, freedom, culture and art with science fiction at the center. It was wonderful, Hopkinson said. It gave me a place to talk and debate with like-minded people about the conjunction of blackness and science fiction without being shouted down by white men or having to teach Racism 101. Connections create communities, which in turn create movements, and in 1999, Delanys essay, Racism and Science Fiction, prompted a call for more meaningful discussions around race in the SF community. In response, Hopkinson became a co-founder of the Carl Brandon society, which works to increase awareness and representation of people of color in the community.

Hopkinsons second novel, Midnight Robber, was a breakthrough success and was nominated for Hugo, Nebula, and Tiptree Awards. She would also release Skin Folk (2001), a collection of stories in which mythical figures of West African and Afro-Caribbean culture walk among us, which would win the World Fantasy Award and was selected as one ofThe New York Times Best Books of the Year. Hopkinson also obtained masters degree in fiction writing (which helped alleviate U.S. border hassles when traveling for speaking engagements) during which she wrote The Salt Roads (2003). I knew it would take a level of research, focus and concentration I was struggling to maintain, Hopkinson said. I figured it would help to have a mentor to coach me through it. That turned out to be James Morrow, and he did so admirably. Roads is a masterful work of slipstream literary fantasy that follows the lives of women scattered through time, bound together by the salt uniting all black life. It was nominated for a Nebula and won the Gaylactic Spectrum Award. Hopkinson also edited anthologies centering around different cultures and perspectives, including Whispers from the Cotton Tree Root: Caribbean Fabulist Fiction (2000), Mojo: Conjure Stories (2003), and So Long, Been Dreaming: Postcolonial Science Fiction & Fantasy (2004). She also came out with the award-winning novelThe New Moons Arms in 2007, in which a peri-menopausal woman in a fictional Caribbean town is confronted by her past and the changes she must make to keep her family in her life.

While the stem cell ban hamstrung hESC work, Gurdons research facilitated yet another scientific breakthrough. Researchers began untangling how gene expression changed as stem cells differentiated, and in 2006, Shinya Yamanaka of Kyoto University reported the successful creation of mouse stem cells from differentiated cells. Using a list of 24 pluripotency-associated genes, Yamanaka systematically tested different gene combinations on terminally differentiated cells. He found four genesthereafter known as Yamanaka factorsthat could turn them into induced-pluripotent stem cells (iPSCs), and he and Gurdon would share a 2012 Nobel prize. In 2009, President Obama lifted restrictions on hESC research, and the first clinical trial involving products made using stem cells happened that year. The first human trials using hESCs to treat spinal injuries happened in 2014, and the first iPSC clinical trials for blindness began this past December.

Hopkinson, too, encountered complications and delays at points in her career. For years, Hopkinson suffered escalating symptoms from fibromyalgia, a chronic disease that runs in her family, which interfered with her writing, causing Hopkinson and her partner to struggle with poverty and homelessness. But in 2011, Hopkinson applied to become a professor of Creative Writing at the University of California, Riverside. It seemed in many ways tailor-made for me, Hopkinson said. They specifically wanted a science fiction writer (unheard of in North American Creative Writing departments); they wanted someone with expertise working with a diverse range of people; they were willing to hire someone without a PhD, if their publications were sufficient; they were offering the security of tenure. She got the job, and thanks to a steady paycheck and the benefits of the mild California climate, she got back to writing. Her YA novel, The Chaos (2012), coming-of-age novelSister Mine (2013), and another short story collection, Falling in Love with Hominids (2015) soon followed. Her recent work includes House of Whispers (2018-present), a series in DC Comics Sandman Universe, the final collected volume of which is due out this June. Hopkinson also received an honorary doctorate in 2016 from Anglia Ruskin University in the U.K., and was Guest of Honor at 2017 Worldcon, a year in which women and people of color dominated the historically white, male ballot.

While the Yamanaka factors meant that iPSCs became a standard lab technique, iPSCs are not identical to hESCs. Fascinatingly, two of these factors act together to maintain the silencing of large swaths of DNA. Back in the 1980s, researchers discovered that some regions of DNA are modified by small methyl groups, which can be passed down through cell division. Different cell types have different DNA methylation patterns, and their distribution is far from random; they accumulate in the promoter regions just upstream of genes where their on/off switches are, and the greater the number of methyl groups, the lesser the genes expression. Furthermore, epigenetic modifications, like methylation, can be laid down by our environments (via diet, or stress) which can also be passed down through generations. Even some diseases, like fibromyalgia, have recently been implicated as such an epigenetic disease. Turns out that the long-standing biological paradigm that rejected Lamarck also missed the bigger picture: Nature is, in fact, intimately informed by nurture and environment.

In the past 150 years, we have seen ideas of community grow and expand as the world became more connected, so that they now encompass the globe. The histories of science fiction and biology are full of stories of pioneers opening new doorsbe they doors of greater representation or greater understanding, or bothand others following. If evolution has taught us anything, its that nature abhors a monoculture, and the universe tends towards diversification; healthy communities are ones which understand that we are not apart from the world, but of it, and that diversity of types, be they cells or perspectives, is a strength.

Kelly Lagor is a scientist by day and a science fiction writer by night. Her work has appeared at Tor.com and other places, and you can find her tweeting about all kinds of nonsense @klagor

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On the Origins of Modern Biology and the Fantastic: Part 18 Nalo Hopkinson and Stem Cell Research - tor.com

SUZHOU, China and SHANGHAI, May 29, 2020 /PRNewswire/ -- Gracell Biotechnologies Co., Ltd. ("Gracell"), a clinical-stage immune cell and gene therapy company, today announced that two presentations were accepted at the 2020 American Society of Clinical Oncology (ASCO) Virtual Scientific Program.

Both presentations can be found in the Development Therapeutics Immunotherapy session, central on Gracell's TruUCAR GC027 in relapsed or refractory T-cell acute lymphoblastic leukemia (r/r T-ALL) patients and EnhancedCAR GC008t in patients with advanced mesothelin-positive solid tumors.

"We are delighted to report on both TruUCAR GC027 in T-ALL and EnhancedCAR GC008t in solid tumors" said Dr. Martina Sersch, CMO of Gracell. "and glad to share safety and preliminary efficacy data on two of our exciting new CAR-T platform therapies with the scientific community at the ASCO annual meeting." Dr. William CAO, CEO of Gracell, added "Thanks to our highly efficient gene editing capability, CAR-T cells with PD-1 gene edited are generated to have enhanced capability of tumor control in inhibitory tumor microenvironment. We believe this strategy will improve CAR-T/TCR-T's potency against solid tumors.Gracell carried out this strategy as early as 2017, upon our foundation. With two years' preclinical and clinical investigations, we are very glad to see it showing first encouraging results in an effort to enhance CAR-T cells to combat solid tumors".

Session type: poster discussionAbstract Title: Safety and efficacy results of GC027: The first-in-human, universal CAR-T cell therapy for adult relapsed/refractory T-cell acute lymphoblastic leukemia (r/r T-ALL)Abstract ID: 3013Link: https://meetinglibrary.asco.org/record/185068/poster

Session type: posterAbstract Title: Phase I study of CRISPR-engineered CAR-T cells with PD-1 inactivation in treating mesothelin-positive solid tumorsAbstract ID: 3038Link:https://meetinglibrary.asco.org/record/189057/poster

About TruUCAR

TruUCAR is Gracell's proprietary and patented platform technology, with selected genes being edited to avoid GvHD and immune rejection without using strong immunosuppressive drugs. In addition to T-ALL antigen, the platform technology can also be implemented for other targets of hematological malignancies.

About GC027

GC027is an investigational, off-the-shelf CAR-T cell therapy, redirected to CD7 for the treatment of T cell malignancies. GC027 was manufactured from T cells of human leukocyte antigen (HLA) unmatched healthy donors using TruUCAR technology, which is expected to improve efficacy and reduce production time, available for off-the-shelf use in a timely manner.

About EnhancedCAR

EnhancedCAR is Gracell's proprietary and patented platform technology, with selected genes edited to enhance immune cell performance in terms of killing efficiency, in vivo persistence, including selected PD-1 and TCR mediations. The technology can be implemented to many other targets with high editing precision and efficiency.

About GC008t

GC008t is an investigational, autologous CAR-T cell therapy, redirected to mesothelin with PD-1 disruption for the treatment of mesothelin-positive solid tumors. With PD-1 knocking out, GC008t is expected improve persistence and clinical efficacy.

About T-ALL

T - Lymphoblastic Leukemia (T-ALL) is an aggressive form of acute lymphoblastic leukemia, with a diffuse invasion of bone marrow and peripheral blood. In 2015, T-ALL affected around 876,000 people globally and resulted in 110,000 deaths worldwide. T-ALL compromises about 15%-20% of all children and adult acute lymphoblastic leukemia[1].Current standard of care therapies for T-ALL are chemotherapy and stem cell transplantation. 40-50% of patients will experience relapse within two years following front line therapy with limited treatment options available[2][3]. Treatment of relapsed and refractory T-ALL remains a high unmet medical need.

About Mesothelin-positive Solid Tumors

Mesothelin, a cell surface antigen, has high expression to a broad spectrum of solid tumors while express low levels on normal cells. Mesothelin is believed as a good target for multiple solid tumors. The GC008t study enrolled patients with advanced solid tumors, including pancreatic cancer, ovarian cancer, and colorectal cancer, of which clinical outcome of standard of care remains poor.

About Gracell

Gracell Biotechnologies Co., Ltd. ("Gracell") is a clinical-stage biotech company, committed to developing highly reliable and affordable cell gene therapies for cancer. Gracell is dedicated to resolving the remaining challenges in CAR-T, such as high production costs, lengthy manufacturing process, lack of off-the-shelf products, and inefficacy against solid tumors. Led by a group of world-class scientists, Gracell is advancing FasTCAR, TruUCAR (off-the-shelf CAR), DualCAR and EnhancedCAR-T cell therapies for leukemia, lymphoma, myeloma, and solid tumors.

[1]Pediatric hematologic Malignancies: T-cell acute lymphoblastic Leukemia, Hematology 2016

[2]Progress and innovations in the management JAMA Oncol 2018

[3]Defining the course and prognosis of adults with acute lymphoblastic leukemia, Cancer 2010

SOURCE Gracell

http://www.gracellbio.com

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Gracell Announces Two Presentations at the Annual Meeting of American Society of Clinical Oncology (ASCO) - PRNewswire

How myeloma promotes bone loss

Multiple myeloma can lead to bone loss by reducing the differentiation of mesenchymal stem cells (MSCs) into osteoblasts. Using a combination of single-cell RNA sequencing, in vitro coculture, and experiments with human myeloma cells and MSCs in mice, Liu et al. demonstrated how direct contact between myeloma cells and MSCs shifted the balance of MSC differentiation to favor adipogenesis over osteoblastogenesis. Integrin 4 on the surface of myeloma cells activated the adhesion molecule VCAM1 on MSCs, leading to protein kinase C 1 (PKC1)dependent repression of the E3 ubiquitin ligase MURF1 and subsequent stabilization of the adipocyte transcription factor PPAR2. These findings suggest a possible avenue for preventing or treating myeloma-induced bone loss in patients.

The suppression of bone formation is a hallmark of multiple myeloma. Myeloma cells inhibit osteoblastogenesis from mesenchymal stem cells (MSCs), which can also differentiate into adipocytes. We investigated myeloma-MSC interactions and the effects of such interactions on the differentiation of MSCs into adipocytes or osteoblasts using single-cell RNA sequencing, in vitro coculture, and subcutaneous injection of MSCs and myeloma cells into mice. Our results revealed that the 4 integrin subunit on myeloma cells stimulated vascular cell adhesion molecule1 (VCAM1) on MSCs, leading to the activation of protein kinase C 1 (PKC1) signaling and repression of the muscle ring-finger protein-1 (MURF1)mediated ubiquitylation of peroxisome proliferatoractivated receptor 2 (PPAR2). Stabilized PPAR2 proteins enhanced adipogenesis and consequently reduced osteoblastogenesis from MSCs, thus suppressing bone formation in vitro and in vivo. These findings reveal that suppressed bone formation is a direct consequence of myeloma-MSC contact that promotes the differentiation of MSCs into adipocytes at the expense of osteoblasts. Thus, this study provides a potential strategy for treating bone resorption in patients with myeloma by counteracting tumor-MSC interactions.

More than 80% of patients with multiple myeloma suffer from bone destruction, which greatly reduces their quality of life and has a severe negative impact on survival (1). New bone formation, which usually occurs at sites of previously resorbed bone, is strongly suppressed in patients with myeloma, and bone destruction rarely heals in these patients (2). Therefore, prevention of bone disease is a priority in myeloma treatment, and understanding the mechanisms by which myeloma cells disturb the bone marrow (BM) is fundamental to myeloma-associated bone diseases.

Osteoblasts originate from mesenchymal stem cells (MSCs) and are responsible for bone formation. It has been reported that myeloma cells inhibit MSC differentiation into mature osteoblasts (35). Osteoblasts and adipocytes arise from a common MSC-derived progenitor and exhibit lineage plasticity, which further complicates the relationship between these two cell types in myeloma cellinfiltrated BM (6). Traditionally, initiation of adipogenesis and osteogenesis has been widely regarded as mutually exclusive, and factors that inhibit osteoblastogenesis activate adipogenesis and vice versa (7). Previous studies have demonstrated that MSCs differentiate into either adipocytes or osteoblasts depending on the stimulator (8), and adipocytes transdifferentiate into osteoblasts in patients with several benign diseases (9). However, the underlying effects of myeloma cells on the activation of adipogenic transcriptional factors and the molecular mechanisms involved are still obscure.

Peroxisome proliferatoractivated receptor 2 (PPAR2) is a key transcription factor for the regulation of fatty acid storage and glucose metabolism (10), and it activates genes important for adipocyte differentiation and function (11). Previous findings have demonstrated that PPAR2 plays important roles not only in the activation of adipogenesis but also in the suppression of osteoblastogenesis (12, 13). In vitro coculture of MSCs from multiple myeloma patients with malignant plasma cell lines enhances adipocyte differentiation of the MSCs due to increased PPAR2 in the MSCs (14), suggesting that PPAR2 mediates myeloma-induced adipogenesis. However, the mechanism by which myeloma cells activate PPAR2 in MSCs, thereby causing MSCs to differentiate into adipocytes rather than osteoblasts, remains unclear.

In the present study, we demonstrated that myeloma cells enhanced the differentiation of human MSCs into adipocytes rather than osteoblasts by stabilizing PPAR2 protein through an integrin 4protein kinase C 1 (PKC1)muscle ring-finger protein-1 (MURF1) signaling pathway in MSCs. Our study thus provides a potential therapeutic strategy for myeloma-associated bone disease.

To determine whether myeloma cells affect MSC fate, we characterized the heterogeneity of human BMderived MSCs after exposure to myeloma cells. We cultured MSCs alone (controls) or cocultured them with myeloma cells in a 1:1 mixture of adipocyte:osteoblast (1:1 AD:OB) medium (Fig. 1A). An aliquot of cells was cultured for 48 hours and then subjected to single-cell RNA sequencing (scRNA-seq). We cultured another aliquot of cells for 2 weeks, removed the myeloma cells, and assessed the ability of the MSCs to differentiate into mature osteoblasts or adipocytes using Alizarin red-S, which stains calcium deposits, and Oil red O, which stains lipids (Fig. 1A). Trajectory analysis indicated the dynamic cellular transition processes of MSCs in vitro, in line with the in vivo MSC fates, reported by Wolock et al. (15). We observed a fate shift in MSC differentiation when MSCs were cocultured with myeloma cells (Fig. 1B). T-distributed stochastic neighbor embedding cluster analysis based on the entire transcriptome gene signature showed that both control and cocultured MSCs had specific transcriptome characteristics (Fig. 1C). After identification of genes with highly variable expression across the dataset, clusters were identified in each of the control and coculture groups (Fig. 1C). Enrichment analysis demonstrated that the adipokine signaling pathway and the mineral absorption pathway were among the 20 pathways most significantly changed in MSCs cocultured with myeloma cells (Fig. 1D). We identified clusters 0, 1, 6, and 8 in the MSCs cocultured with myeloma cells as being of adipogenic lineage because their expression of the specific markers of adipogenesis, the ADD1 and PPAR genes, were markedly higher than that of other clusters (Fig. 1E). These results demonstrated that myeloma cells at least partially increase MSC transformation into adipocytes.

(A) System for coculturing of human MSCs with the human multiple myeloma cell (MM) line MM.1S in a 1:1 mixture of adipocyte (AD) and osteoblast (OB) medium. Cells were cocultured for 48 hours and then MSC-derived cells were subjected to single-cell RNA sequencing (scRNA-seq). As a control, scRNA-seq was also performed on MSCs cultured alone in 1:1 AD:OB medium. (B) The single-cell trajectory reconstructed by Monocle in the control (Ctrl) and coculture (Coculture) groups. Each point represents a cell, and colors indicate their respective group. n = 2 independent experiments. The trajectory constructed by Monocle is in black. (C) T-distributed stochastic neighbor embedding (t-SNE) plot depicting clusters of MSCs cultured alone (Ctrl) or cocultured with MM cells. The first two dimensions are shown. Each cluster represents individual cells with similar transcriptional profiles of MSCs or different MSC lineages, with total of 10 clusters from aggregated samples of two biologically independent experiments. (D) Enrichment analysis showing the 20 most significantly changed pathways in the MSCs cocultured with MM cells. Red indicates activated pathways, and green indicates repressed pathways. (E) Distributions of unique transcripts per cell and PPARG and CEBPB gene expression in all cell clusters. The red frame shows the highest expression among the clusters. TGF-, transforming growth factor.

The coculture of MSCs and myeloma cells resulted in lower Alizarin red-S staining and higher Oil red O staining in MSCs, indicating an increase in the generation of adipocytes, compared to culture of MSCs alone (Fig. 2A). We further labeled cocultured MSCs with antibodies recognizing the osteoblast marker osteocalcin or the adipocyte marker fatty acid binding protein 4 (FABP4) and analyzed them using flow cytometry. We observed that culturing MSCs in osteoblast medium increased the osteocalcin+ population and that coculturing MSCs with myeloma cells inhibited this increase. Also, culturing MSCs in adipocyte medium increased the FABP4+ population, and coculturing them with myeloma cells further increased it. When we cultured MSCs alone in the 1:1 AD:OB medium, both the osteocalcin+ and FABP4+ populations increased, whereas coculturing MSCs with myeloma cells reduced the osteocalcin+ population but increased the FABP4+ population (Fig. 2, B and C). We obtained similar effects on osteoblastogenesis (Fig. 2D) and adipogenesis (Fig. 2E) when we cocultured MSCs with six other myeloma cell lines or with CD138+ primary myeloma cells isolated from BM aspirates from five patients with myeloma, but not with plasma cells from healthy donors (Fig. 2, F and G). Real-time polymerase chain reaction (PCR) analysis further showed lower expression of the osteoblast differentiationassociated genes alkaline phosphatase (ALP), secreted phosphoprotein 1 (SPP1), collagen type I alpha 1 chain (COL1A1), and bone gamma-carboxyglutamate protein (BGLAP; Fig. 2H) and higher expression of the adipocyte differentiationassociated genes delta-like noncanonical Notch ligand 1 (DLK1), diacylglycerol O-acyltransferase 1 (DGAT1), FABP4, and fatty acid synthase (FASN; Fig. 2I) in MSCs cocultured with ARP-1 or MM.1S myeloma cells than in MSCs cultured alone. These results demonstrate that myeloma cells directed the differentiation of MSCs preferentially toward adipocytes than to osteoblasts.

(A) Representative images of Alizarin red-S and Oil red O staining (whole wells and enlarged views) of MSCs cultured alone or cocultured with ARP-1 or MM.1S myeloma cell lines in MSC medium, adipocyte (AD) medium, osteoblast (OB) medium, or mixed 1:1 AD:OB medium as indicated. n = 3 independent experiments. Scale bars, 5 mm (whole wells) and 20 m (enlargements). (B and C) Flow cytometric analysis showing the percentage of osteocalcin+ (B) and FABP4+ (C) cells in cultures of MSCs alone or in direct contact with ARP-1 cells in the indicated medium. Data are representative of three independent experiments with each sample analyzed in triplicate. (D and E) Quantification of Alizarin red-S (D) and Oil red O (E) staining of MSCs cultured alone (No MM) or cocultured with the six indicated myeloma cell lines. Combined data are from three biologically independent experiments. (F and G) Quantification of Alizarin red-S (F) and Oil red O (G) staining of MSCs cultured alone or cocultured with primary myeloma cells isolated from BM aspirates of five patients with myeloma (P1 to P5) or normal plasma cells from the BM of two healthy donors (PC1 and PC2). Combined data are from n = 3 experiments using the same donor source material. (H and I) Quantitative reverse transcription PCR showing the expression of the osteoblast differentiationassociated genes ALP, SPP1, COL1A1, and BGLAP (H) and the adipocyte differentiationassociated genes DLK1, DGAT1, FABP4, and FASN (I) in cells generated by coculture of MSCs with myeloma cells relative to expression of each gene in MSCs cultured alone. Combined data are from n = 3 independent experiments. All data are means SD. *P 0.05 and **P 0.01. P values were determined using one-way ANOVA with Tukeys multiple comparisons test.

We next investigated the mechanism of myeloma-induced shifting of MSCs from osteoblastogenesis to adipogenesis. We focused on PPAR2 because it is a key transcriptional factor for the activation of adipogenesis. scRNA-seq showed higher PPAR2 mRNA expression in MSCs cocultured with myeloma cells compared to MSCs cultured alone (Fig. 1E). Using the coculture system with MSCs and myeloma cells in a 1:1 mixture of adipocyte and osteoblast medium, we again observed the transformation of osteoblastogenesis into adipogenesis in MSCs cocultured with myeloma cells (Fig. 3A), as well as an increase in the abundance of PPAR2 in MSCs cultured with myeloma cells (Fig. 3B and fig. S1). To determine the importance of PPAR2 in MSC transformation, we added the PPAR2 antagonist G3335 to cocultures. G3335 inhibited the myeloma cellinduced increase in PPAR2 protein (Fig. 3B and fig. S1). Consistent with the Western blot results, G3335 treatment decreased Oil red O staining (Fig. 3C) and adipocyte gene expression (Fig. 3D) and increased Alizarin red-S staining (Fig. 3E) and osteoblast gene expression (Fig. 3F). These results suggest that PPAR2 mediated myeloma-induced MSC transformation into adipocytes.

(A) Representative images of Oil red O or Alizarin red-S staining of MSCs cultured alone or cocultured with ARP-1 or MM.1S myeloma cells in 1:1 OB:AD medium and treated with the PPAR2 antagonist G3335 as indicated. Scale bar, 5 mm. (B) Representative Western blot for PPAR2 in cells treated as in (A). Quantitation is presented in fig. S1. Actin is a loading control. (C to F) Quantitative analysis of Oil red O staining (C), adipocyte differentiationassociated gene expression (D), Alizarin red-S staining (E), and osteoblast differentiationassociated gene expression (F) in cells treated as in (A). Data are means SD from n = 3 independent experiments. *P 0.05 and **P 0.01. P values were determined using Students t test for paired samples (D and F) and one-way ANOVA with Tukeys multiple comparisons test (C and E).

To determine whether myeloma cells distort MSC transformation through myeloma-secreted soluble factors or cell-to-cell contact, we cocultured MSCs with ARP-1 or MM.1S myeloma cells in 1:1 AD:OB medium either together or separated by transwell inserts. We observed that the transwell coculture had a slight effect on increased Oil red O staining, whereas cell-to-cell contact coculture in the mixed medium produced much more significant boost of this staining, suggesting that direct interaction between MSCs and myeloma cells was needed for enhancing adipogenesis from MSCs (Fig. 4A). When we added supernatants collected from 24-hour cultures of ARP-1 or MM.1S cells to MSC cultures, we obtained results similar to those for the transwell coculture (Fig. 4A), reaffirming the importance of direct contact of MSCs with myeloma cells.

(A) Oil red O staining in MSCs cultured alone (No MM) or cocultured with ARP-1 or MM.1S myeloma cells in 1:1 AD:OB medium directly (cell-cell) or separated by transwell inserts (Trans) or in myeloma cell culture media (sup). Staining was quantified relative to the No MM condition. Representative data are from three independent experiments. (B to D) Relative Oil red O staining (B) and the relative expression of the indicated osteoblast (C) and adipocyte (D) marker genes in MSCs cultured alone (No MM) or cocultured with ARP-1 or MM.1S cells with or without neutralizing antibodies against integrin subunits 4, 5, V, or L. Combined data are from three independent experiments. (E) Western blot showing integrin 4 and integrin 1 in ARP-1 and MM.1S cells expressing shRNA targeting integrin 4 (4 KD) or nontargeted control shRNAs (NT Ctrl). Actin is a loading control. (Blot is a representative of three independent experiments, and blot quantitation data are presented in fig. S2C. (F to J) PPAR2 protein (F), Alizarin red-S staining (G), Oil red O staining (H), osteoblast marker gene expression (I), and adipocyte marker gene expression (J) in MSCs cultured alone or cocultured with ARP-1 or MM.1S cells expressing NT Ctrl or 4 KD shRNA. Blots in (E) and (F) are representative of three independent experiments, and blot quantitation is presented in fig. S2 (A and D). Data in (G) to (J) are means SD from n = 3 independent experiments using MSCs derived from BM aspirates of three healthy donors. Data are **P 0.01. P values were determined using one-way ANOVA with Tukeys multiple comparisons test.

To identify the specific molecules involved in adipocyte differentiation, we tested the effect of blocking antibodies against various integrins, which are highly expressed in myeloma cells, in cocultures of MSCs with ARP-1 or MM.1S cells in 1:1 AD:OB medium. The addition of an antibody against integrin 4but not antibodies against integrins 5, V, or L or a control immunoglobulin G (IgG)markedly reduced Oil red O staining in cocultures with both myeloma cell lines (Fig. 4B). The addition of the antibody recognizing integrin 4 to cocultures of MSCs and ARP-1 cells in the mixed medium also increased osteoblast gene expression (Fig. 4C) and decreased adipocyte gene expression (Fig. 4D) substantially more than did the addition of the control IgG. To determine whether integrin 4 affected PPAR2 production in MSCs, we infected ARP-1 and MM.1S cells with a lentivirus carrying short hairpin RNAs (shRNAs) targeting integrin 4 (fig. S2A). Integrin 4 knockdown (4 KD) reduced integrin 4 production without changing the cell viability or proliferation, whereas integrin 1 remained unchanged in ARP-1 and MM.1S cells (Fig. 4E and fig. S2, A to C). We also cocultured MSCs with control or 4 KD myeloma cells in the mixed medium. Western blot analysis demonstrated that 4 KD in myeloma cells reduced PPAR2 protein production in MSCs more than did myeloma cells expressing a nontargeting control shRNA (Fig. 4F and fig. S2D). In addition, coculture of MSCs with 4 KD myeloma cells induced higher Alizarin red-S staining (Fig. 4G) and osteoblast gene expression (Fig. 4H) but lower Oil red O staining (Fig. 4I) and adipocyte gene expression (Fig. 4J) compared to MSCs cocultured with myeloma cells expressing the control shRNA.

Because vascular cell adhesion molecule1 (VCAM1) is a major ligand of integrin 4, we investigated whether it mediated myeloma-induced MSC transformation by adding a blocking antibody against VCAM1 or control IgG to MSC and myeloma cell cocultures. Addition of the antibody, but not IgG, increased Alizarin red-S staining (Fig. 5A) and osteoblast gene expression (Fig. 5B) but decreased Oil red O staining (Fig. 5C) and adipocyte gene expression (Fig. 5D) in MSCs. To determine whether binding of integrin 4 to VCAM1 induced an increase in PPAR2, we constructed MSCs with reduced expression of VCAM1 using a lentivirus carrying VCAM1 shRNAs (VCAM1 KD) (Fig. 5E and fig. S3A) and cocultured myeloma cells with control or VCAM1 KD MSCs. Western blot analysis showed that cocultured VCAM1 KD MSCs had reduced PPAR2 protein production compared to cocultured MSCs expressing nontargeting control shRNA (Fig. 5F and fig. S3B). We also found that VCAM1 KD in MSCs considerably abrogated myeloma-induced suppression of osteoblastogenesis and activation of adipogenesis, because Oil red O staining and adipocyte gene expression decreased significantly (Fig. 5, G and H), whereas Alizarin red-S staining and osteoblast gene expression both increased (Fig. 5, I and J).

(A to D) Alizarin red-S staining (A), Oil red O staining (B), and real-time PCR analysis of the expression of osteoblast (C) and adipocyte (D) marker genes in MSCs cultured alone (No MM) or cocultured with ARP-1 or MM.1S myeloma cells in the presence of a neutralizing antibody against VCAM1 or IgG (control). Data are from n = 3 independent experiments. (E) Western blotting analysis showing VCAM1 in the MSCs infected with a lentivirus carrying nontargeted control shRNAs (NT Ctrl-MSCs) or human VCAM1 shRNAs (VCAM1 KD-MSCs). Actin is a loading control. Blot is a representative of three independent experiments, and blot quantitation is presented in fig. S3A. (F to J) PPAR2 protein (F), adipocyte gene expression (G), Oil red O staining (H), Alizarin red-S staining (I), and osteoblast gene expression (J) in MSCs expressing NT Ctrl or VCAM1 shRNAs cocultured with ARP-1 or MM.1S cells in 1:1 OB:AD medium. Blot in (F) is a representative of three independent experiments, and blot quantitation is presented in fig. S3B. Data are means SD from n = 3 independent experiments. *P 0.05 and **P 0.01. P values were determined using one-way ANOVA with Tukeys multiple comparisons test except in (G) and (J), where Students t test for paired samples were used.

Because VCAM1 stimulates intracellular signaling that results in the activation of protein kinase C (PKC), we examined PKC activation in cocultures. Coculture of myeloma cells and MSCs enhanced the phosphorylation of PKC1 but did not affect phosphorylation of the PKC isoforms PKC, PKC, or PKC/ or the abundance of total PKC and reduced the phosphorylation of PKC and PKC (Fig. 6, A and B). Addition of the PKC inhibitor Go6976 to the cocultures markedly reduced PKC1 phosphorylation and PPAR2 protein in MSC cells cocultured with ARP-1 or MM.1S cells (Fig. 6C and fig. S4). Functionally, treatment of cocultures with Go6976 reduced Oil red O staining and increased Alizarin red-S staining (Fig. 6, D to F). Together, these results demonstrate that myeloma cells activated PPAR2 in MSCs and induced MSC differentiation into adipocytes rather than osteoblasts through the integrin 4-VCAM1-PKC1 pathway.

(A) Western blotting for all phosphorylated PKCs (p-PKC pan), the indicated phosphorylated PKC isoforms, and total PKC in MSCs cultured alone or cocultured with ARP-1 or MM.1S myeloma cells. The abundances of total PKC served as protein loading controls. (B) Quantification of the phosphorylation of PKC isoforms in MSCs cocultured with myeloma cells in (A) relative to the MSC-only control. The cutoff values are fold change more than twofold or less than 0.5-fold. (C) Western blotting for phosphorylated PKC1, total PKC, and PPAR2 in MSCs cocultured with ARP-1 or MM.1S cells in the presence of the PKC inhibitor Go6976 or DMSO (control). Actin is a loading control. Blot is a representative of three independent experiments, and blot quantitation is presented in fig. S4. (D) Representative images of Oil red O staining and Alizarin red-S staining of MSCs cultured alone or cocultured with ARP-1 or MM.1S myeloma cells in the presence of the PKC inhibitor Go6976 or DMSO (control). Scale bar, 5 mm. (E and F) Quantification of Oil red O staining (E) and Alizarin red-S staining (F), in cells treated as in (D). Data are means SD from n = 3 independent experiments. *P 0.05 and **P 0.01. P values were determined using one-way ANOVA with Tukeys multiple comparisons test.

Because a key mechanism of regulation of PPAR2 is its ubiquitylation-dependent proteasome-mediated degradation (16), we added the proteasome inhibitor MG132 to cultures of MSCs. We found that treatment with MG132 increased the presence of PPAR2 protein in MSCs in a time- and dose-dependent manner (Fig. 7A and fig. S5A). MG132 treatment causes the accumulation of ubiquitylated PPAR2 in MSCs, and coculturing these cells with myeloma cells reduced PPAR2 ubiquitylation (Fig. 7B and fig. S5B). However, the addition of a neutralizing antibody against VCAM1 to the cocultures restored ubiquitylation of PPAR2 (Fig. 7C and fig. S5C). These results suggested that myeloma cells activate PPAR2 in MSCs through inhibition of its ubiquitylation.

(A) Western blotting analysis for PPAR2 in MSCs cultured in 1:1 OB:AD medium and treated with the proteasome inhibitor MG132 for the indicated amounts of time. Actin is a loading control. (B) Immunoblotting (IB) for ubiquitin in PPAR2 immunoprecipitates (IP) from MSCs cultured alone or cocultured with ARP-1 or MM.1S myeloma cells in the presence of MG132. (C) Western blotting for ubiquitin in PPAR2 immunoprecipitates from MSCs cocultured with ARP-1 or MM.1S cells in the presence of MG132 and an antibody against VCAM1 or IgG (control). (D) Expression of the E3 ligaseencoding genes USP7, MURF1, MKRN1, CRBN, CRL4B, and TRIM23 in MSCs cocultured with myeloma cells relative to the expression in MSCs cultured alone (No MM). Data are means SD from n = 3 independent experiments. **P 0.01. P values were determined using one-way ANOVA with Tukeys multiple comparisons test. (E) Western blotting for USP7, MURF1, and MKRN1 in MSCs cultured alone or cocultured with myeloma cells. (F) Western blotting for MURF1 in MSCs cocultured with ARP-1 or MM.1S myeloma cells and treated with Go6976 or DMSO (control) as indicated. (G) Immunoblotting for MURF1 or PPAR2 in PPAR2 or MURF1 immunoprecipitates, respectively, from MSCs. IgG immunoprecipitates and whole-cell lysate (input) were used as controls. (H) Immunoblotting for ubiquitin in PPAR2 immunoprecipitates from MSCs expressing nontarget control (NT Ctrl) or MURF1 shRNAs in the presence of MG132. Each blot is representative of n = 3 independent experiments, and blot quantitation is presented in fig. S5.

To investigate the mechanism by which myeloma cells inhibited PPAR2 ubiquitylation, we examined the E3 ubiquitin ligases known to induce ubiquitylation of PPARs (17). Among the tested ligases, we found that MURF1 mRNA (Fig. 7D) and MURF1 protein (Fig. 7E and fig. S5D) were reduced in MSCs cocultured with myeloma cells. Addition of the PKC inhibitor Go6976 to the cocultures increased MURF1 protein in MSCs (Fig. 7F and fig. S5E), indicating that myeloma cells inhibited MURF1 production in MSCs through the PKC signaling pathway. Because the effects of MURF1 on PPAR2 ubiquitylation are unclear, we examined the interaction of these two proteins in MSCs. Co-immunoprecipitation of PPAR2 from MSCs demonstrated an interaction between MURF1 and PPAR2 (Fig. 7G), and knockdown of MURF1 in MSCs reduced the ubiquitylation of PPAR2 (Fig. 7H and fig. S5, F and G). These results demonstrate that myeloma cells activated PPAR2 in MSCs by reducing MURF1-mediated ubiquitylation of PPAR2.

To test the influence of myeloma cells on MSC differentiation in vivo, we established an extramedullary bone formation model in mice. Matrigel containing MSCs and Matrigel containing MSCs plus -irradiated ARP-1 cells were subcutaneously implanted into the right and left flanks of nonobese diabetic/severe combined immunodeficiency/interleukin-2rnull mice, respectively (Fig. 8A). Each sample also included human endothelial colony-forming cells (ECFCs) to stimulate blood vessel formation in the implant. In line with results of a previous study (18), we observed lower bone density in the extramedullary bones that formed in the left flanks, which were implanted with MSCs plus irradiated myeloma cells, compared to the extramedullary bones that formed on the right side, which were implanted with MSCs alone (Fig. 8A). Furthermore, we examined subcutaneous tissues on both sides of mice using histologic or immunohistochemical staining with antibodies against the mature osteoblast marker osteocalcin, the adipocyte marker perilipin, the myeloma marker CD138, and human MURF1. We observed lower numbers of new bones and osteocalcin+ osteoblasts and higher numbers of perilipin+ adipocytes in tissues on the sides of mice implanted with both MSCs and myeloma cells, reduction of MURF1 abundance in tissues on the sides of mice implanted with MSCs alone, and CD138+ cells only in tissues on the sides of mice implanted with myeloma cells (Fig. 8B).

(A) Representative images of subcutaneous tissues and bone density in mice implanted with human MSCs plus ECFCs in the right flank and MSCs plus ECFCs mixed with ARP-1 myeloma cells in the left flank. The arrows indicate bone formation in subcutaneous tissue, and the bars indicate bone density. (B) Representative hematoxylin and eosin (H&E) and immunohistochemical staining for the osteoblast marker osteocalcin, the adipocyte marker perilipin, the myeloma cell marker CD138+, and MURF1 of the subcutaneous tissues from (A). Scale bar, 20 m. Data represent n = 3 independent experiments with five mice each. (C) Expression of MURF1 in MSCs from BM aspirates from 12 patients with myeloma and 12 age-matched healthy donors relative to expression in a randomly selected sample from healthy donor. Data are from n = 3 experiments using the same donor source material. *P 0.05. P values were determined using Students t test. (D and E) Western blotting for MURF1 and PPAR2 (D) and Alizarin red-S and Oil red O staining (E) in MSCs from BM aspirates from three healthy donors and three patients with myeloma. Blots and images are representative of three experiments using the same donor materials, and blot quantitation is presented in fig. S6. Scale bars, 5 mm (whole wells) and 100 m (enlargements). (F) Quantitation of Alizarin red-S and Oil red O staining in the cultures of MSCs from BM aspirates from healthy donors and patients with myeloma in (C). Data are from n = 3 experiments using the same donor source material. P values were determined using Students t test. OD490, optical density at 490 nm.

We also isolated MSCs from the BM of 12 healthy human donors and 12 age-matched patients with myeloma and found markedly lower MURF1 mRNA expression in patient-derived MSCs compared to healthy donor MSCs (Fig. 8C). Western blotting validated the negative correlation between MURF1 and PPAR2 at the protein level in MSCs isolated from 3 of 12 samples in both groups (Fig. 8D and fig. S6). When we cultured these primary MSCs in 1:1 AD:OB medium, we found lower Alizarin red-S staining and higher Oil red O staining in cultures of patient-derived MSCs than in cultures of healthy donor MSCs (Fig. 8, E and F). These findings demonstrate that myeloma cells reduced MURF1 in MSCs and skewed MSC differentiation to favor adipogenesis, resulting in the suppression of osteoblast-mediated new bone formation in myeloma-bearing mice and in cells from patients with myeloma.

Using scRNA-seq, an in vitro coculture system, and mouse models, we demonstrated that myeloma cells shift the differentiation of MSCs into adipocytes rather than osteoblasts. Mechanistic studies revealed that integrin 4 on myeloma cells bound to VCAM1 on MSCs and inhibited ubiquitylation of PPAR2 through PKC-MURF1 signaling. The resulting increase in PPAR2 enhanced adipogenesis and suppressed osteoblastogenesis from MSCs. Thus, our study elucidates a previously unknown mechanism underlying myeloma-induced suppression of osteoblast-mediated bone formation and provides a potential approach for treating bone resorption in patients with myeloma.

Suppressed differentiation of osteoblasts is well known to be a key reason for bone loss and skeleton-related events in patients with myeloma (19). The molecules and pathways involved in myeloma-induced suppression of osteoblastogenesis include the Wnt signaling inhibitor Dickkopf-related protein 1 (DKK-1) (2, 20). However, antibody-mediated blocking of DKK-1 function cannot restore new bone formation completely or heal myeloma-induced resorbed bone, suggesting that additional factors expressed by myeloma cells critically affect bone formation. In the present study, we demonstrated that the 4 subunit of integrin, which is highly abundant in myeloma cells, promoted MSC differentiation into adipocytes, demonstrating that adhesion moleculesbut not soluble factorsproduced by myeloma cells primarily mediated the shift from osteoblastogenesis to adipogenesis. Integrin 41, also known as very late antigen-4, is a cell surface heterodimer present on malignant cells in patients with many types of cancer, including myeloma (21). It is a key adhesion molecule that acts as a receptor for the extracellular matrix protein fibronectin and the cellular receptor VCAM1. Interaction between integrin 41 and VCAM1 can activate mature osteoclast formation in patients with bone-metastatic breast cancer (22). In patients with multiple myeloma, this interaction promotes the secretion of interleukin-7 by tumor cells, which inhibits the expression of RUNX-2, which encodes a transcription factor that is essential for osteoblast differentiation, and RUNX-2 transcriptional regulatory activity in MSCs (23). This interaction also increases DKK-1 secretion by myeloma cells. Adding to these known mechanisms, we revealed that binding of myeloma cell integrin 41 to VCAM1 on the MSC surface activated the PKC signaling pathway. We also identified activation of PKC1, suppression of the downstream mediator MURF1, and the fundamental roles of such signaling pathways in the promotion of the MSC-derived adipocyte lineage. PKCs are also reportedly associated with Jagged-Notch signaling pathways, and they can regulate the transition of embryonic stem cells differentiating into postmitotic neurons (24). Some immunomodulatory drugs, such as lenalidomide, may affect osteoblast differentiation through this pathway (25), indicating the important role of Jagged-Notch in osteoblast differentiation from MSCs. We may further investigate their impacts and mechanisms on myeloma-induced the shift of MSC fates in our next studies.

BM adipocytes are recognized as important regulators of bone remodeling rather than just being inert filler cells (26, 27). Normal BM adipocytes have been shown to be reprogrammed by myeloma cells and gain the ability to resorb bone in myeloma patients in remission (13). Focusing on the determination of MSC fate in this study, we investigated the molecular mechanism underlying the shift from osteoblastogenesis to adipogenesis induced by myeloma cells. Lineage-tracing experiments have revealed that adipocytes can also originate from osterix-positive cells and are closely related to osteoblasts (28). Chan et al. (29) reported that BM adipocytes were derived from a progenitor cell that was also the progenitor for osteoblasts. In addition, Gao et al. (30) reported plasticity between BM adipocytes and osteoblasts and potential transdifferentiation and transformation between these two identities after initiating differentiation. Despite this new knowledge about the balance between osteoblastogenesis and adipogenesis, how myeloma cells regulate this balance and transformation of MSCs is still unclear.

scRNA-seq can identify subpopulations using the transcriptome to avoid the complicated isolation procedures after cell-cell contact culture (15). We found that MSCs could be naturally divided into two populations by transcriptomic data, and at least one cluster of MSCs cocultured with myeloma cells highly expressed adipocyte marker genes. Coculture of myeloma cells pushed MSC differentiation toward adipocytes rather than osteoblasts, resulting in the suppression of bone formation in the in vivo extramedullary bone assay. Because MSCs are pluripotent stem cells capable of differentiation into other cell types, such as chondrocytes and skeletal muscle cells (31), whether myeloma cells affect MSC transformation into these cell types instead of osteoblasts remains unclear. It is possible that the observed differentiation from MSC to adipocyte in the presence of myeloma cells might have been rather the result of a differentiation of MSCs into osteoblasts followed by a transdifferentiation from osteoblast into adipocyte. Further investigation is needed to address this possibility.

Like other transcription factors and coregulators, PPAR2 can undergo posttranslational modifications, such as phosphorylation, acetylation, and SUMOylation (32). Researchers have identified the key enzymes and target amino acid sites involved in these modifications, but modification of PPAR2 by ubiquitylation, especially that induced by myeloma cells, is still unclear. Many E3 ligases, such as MURF1 and makorin ring finger protein 1 (MKRN1), are reported to be regulators of ubiquitylation of PPAR proteins (17, 3335), whereas investigators have identified only polyubiquitylation at Lys184 and Lys185 (K184/185) mediated by MKRN1 (16). In the present study, we demonstrated that the E3 ligase MURF1 contributed to PPAR2 ubiquitylation, and inhibition of MURF1 by myeloma cells reduced PPAR2 ubiquitylation, leading to enhanced protein stability in MSCs. MURF1 contains a canonical N-terminal RING-containing E3 ligase that is required for its ubiquitin ligase activity (36). Others have reported dysregulation of MURF1 in experimental models of fasting, diabetes, cancer, denervation, and immobilization (37). However, none have reported the substrate proteins, such as PPAR2, that are targeted for proteasomal degradation by MURF1 in patients with myeloma bone disease. Although the amino acids in PPAR2 that MURF1 targets remain to be identified, we demonstrated that the reduced MURF1 production in MSCs induced by myeloma cells was critical for the inhibition of PPAR2 ubiquitylation and thus stabilization of the PPAR2 protein. Other posttranslational modifications may also regulate PPAR2 protein, especially SUMOylation, which was not addressed in the current study. For example, the transcriptional activity of PPAR2 can be inhibited by SUMOylation at Lys107 to regulate insulin sensitivity (38), and growth differentiation factor 11 promotes osteoblastogenesis through enhancement of PPAR2 SUMOylation (39). A logical next step could be the investigation of the role of SUMOylation in myeloma-induced MSC transformation and how it interplays with the mechanisms described here.

In summary, our results shed light on the cross-talk between myeloma cells and MSCs and the impact of this interaction on the determination of the MSC-derived adipocyte lineage and the suppression of osteoblastogenesis from MSCs. Myeloma cell integrin 4 promoted phosphorylation of PKC1 through VCAM1, and the activated PKC1 reduced the production of MURF1 in MSCs, leading to reduced PPAR2 ubiquitylation. Therefore, counteracting 4-VCAM1-MURF1mediated adipogenesis from MSCs may be a promising strategy to heal myeloma-induced bone resorption.

Myeloma cell lines ARP-1 and ARK were provided by University of Arkansas for Medical Sciences (Little Rock, AR, USA), and others were purchased from American Type Culture Collection. Primary myeloma cells or normal plasma cells were isolated from the BM aspirates of patients with myeloma or healthy donors using antibody-coated magnetic beads against CD138, respectively (Miltenyi Biotec Inc.) (40). The cells were maintained in RPMI 1640 medium with 10% fetal bovine serum (FBS). MSCs from BM of healthy donors or patients with myeloma were maintained and augmented in Dulbeccos modified Eagles medium (DMEM) with 10% FBS (13). Information of healthy donors and patients were listed in table S1. The study was approved by the Institutional Review Board at The University of Texas MD Anderson Cancer Center.

Human MSCs were generated from BM mononuclear cells from fetal bones of healthy human donors, characterized using flow cytometry, and labeled with antibodies against MSC markers (CD44, CD90, and CD166) (41). Mature adipocytes were generated from MSCs using an adipocyte medium, which was formulated of DMEM medium with 10% FBS, 1 M dexamethasone, 0.2 mM indomethacin, insulin (10 g/ml), and 0.5 mM 3-isobutyl-l-methylxanthine (41). Mature adipocytes were fixed with 4% paraformaldehyde, stained with Oil red O for 1 hour, and observed under a light microscope. Mature osteoblasts were generated from MSCs using an osteoblast medium, which was formulated of alpha MEM medium with 10% FBS, 100 nM dexamethasone, 10 mM -glycerophosphate, and 0.05 mM l-ascorbic acid 2-phosphate (42). The bone-forming activity of osteoblasts was determined using Alizarin red-S staining (43, 44). Human MSCs were cultured alone or cocultured with myeloma cells at a ratio of 5:1 in MSC medium, osteoblast medium, adipocyte medium, or 1:1 mixed of osteoblast and adipocyte medium with or without inhibitors (G3335 or Go6976) or neutralizing antibodies for 2 weeks. Addition of dimethyl sulfoxide (DMSO) served as vehicle control for inhibitor-treatment experiments, and addition of IgG served as control for antibody-neutralizing experiments. In the transwell nondirect contact model, adipocytes were seeded onto the bottom of culture wells and cocultured with the myeloma cells on the insert. In direct contact coculture system, MSCs were seeded together with the myeloma cells in the culture wells to allow direct cell-cell contact. Supernatants collected from 24-hour cultures of myeloma cells were added to the MSCs in mixed osteoblast and adipocyte medium at a ratio of 1:5. In the experiments with primary cells, MSCs were cultured in the mixed medium for a week (45) and then cocultured with primary myeloma cells isolated from BM aspirates from patients with myeloma or normal plasma cells from BM of healthy donors for another week. Medium, inhibitors, and antibodies were refreshed every 3 days. After culture, the myeloma cells were removed, and the residual cells were stained with Alizarin red-S to assess osteoblast differentiation and with Oil red O to assess adipocyte differentiation. Culture of MSCs alone served as a control.

Single-cell preparation, complementary DNA (cDNA) library synthesis, RNA sequencing, and data analysis were performed by Gene Denovo Inc. Briefly, 1 106 MSCs were plated for 6 hours, 5 106 myeloma cells were added to the MSCs directly, and the cells were cocultured in mixed culture media for 48 hours; control MSC cells were cultured alone at the same media and then mixed with myeloma cells at the same ratio just before preparation for analysis. After removal of dead cells, the cells in these groups were counted using a Countess II Automated Cell Counter, and the concentration was adjusted to 1000 cells/l. The single-cell suspensions were bar-coded labeled and reverse-transcribed into scRNA-seq library using the Chromium Single Cell 3 GEM, Library and Gel Bead Kit (10X Genomics). The cDNA libraries from two independent experiments were sequenced on the Illumina HiSeq X-Ten platform, and data were pooled for the analysis. Myeloma cells were excluded using CD138 markers. The raw scRNA-seq data were aligned, filtered, and normalized using Cell Ranger (10X Genomics) software (tables S2 to S6). The cell subpopulation was grouped by graph-based clustering based on the gene expression profile of each cells in Seurat (tables S7 and S8). Subsequent data analysis including standardization, cell subpopulation, difference of gene expression, and marker gene screening were achieved by Seurat software.

MSCs were cultured alone or cocultured with myeloma cells with or without G3335 or neutralizing antibodies for 48 hours. In some experiments, MG132 was added to the cultures 6 hours before the cell collection. Addition of DMSO served as vehicle control for inhibitor experiment; addition of IgG served as neutralizing antibody control.

Quantitative real-time PCR was performed as described (46). The primers are listed in table S9. For Western blotting, cells were lysed with 1 lysis buffer (Cell Signaling Technology), subjected to 4 to 20% gradient gel electrophoresis, transferred to, and immunoblotted with antibodies against integrin 4 (R&D Systems), integrin 1, VCAM1, PKC, MURF1, and phosphorylated isoforms of PKC along with p-PKC-pan (Cell Signaling Technology) and PPAR2 (Santa Cruz Biotechnology). The membrane was stripped and reprobed with an antibody against -actin to ensure equal protein loading, and last, signals were detected using peroxidase-conjugated secondary antibody followed by enhanced chemiluminescence system (Millipore) in the MiniChem system (Saizhi Biotech), and quantitative analysis of blots were performed using the Fiji-based ImageJ software (version 1.51n, National Institutes of Health, Bethesda, MA, USA).

Viral particles were produced by human embryonic kidney 293T cells transfected with PMD2G and PSPAX2 packaging plasmids (Addgene) together with lentivirus-expressing shRNA vectors targeting 4, MURF1, or VCAM1 (Sigma-Aldrich). Nontargeted shRNA control (Sigma-Aldrich) was used as control. Sequences for knocking down specific genes are the following: 4, 5-CCGGGCTCCGTGTTATCAAGATTATCTCGAGATAATCTTGATAACACGGAGCTTTTT-3; VCAM1, 5-CCGGGGAATTAATTATCCAAGTTACCTCGAGGTAACTTGGATAATTAATTCCTTTTTTG-3; MURF1, 5-CCGGGAAGAGGAAGAGTCCACAGAACTCGAGTTCTGTGGACTCTTCCTCTTCTTTTTG-3 or 5-CCGGGTATAATAATGCCTGGTCATTCTCGAGAATGACCAGGCATTATTATACTTTTTG-3. Supernatants carrying the viral particles were harvested 48 hours later and concentrated to a 100 volume using polyethylene glycol 8000 (Sigma-Aldrich). MSCs (1 106 cells) were seeded 6 hours before the infection. Concentrated viral particles were added to MSCs or myeloma cells, respectively, in the presence of polybrene (8 g/ml) for 12 hours. The medium was then changed, and cells were cultured for another 48 hours until further management.

Cells were harvested and lysed using NP-40 lysis buffer supplemented with complete protease inhibitors, and the supernatant was precleaned with protein G beads (Thermo Fisher Scientific) and incubated with a mouse antibody against MURF1 (Santa Cruz Biotechnology) or monoclonal rabbit antibody against PPAR2 antibody (Santa Cruz Biotechnology) at 4C overnight with protein A/G agarose beads (Thermo Fisher Scientific). The next day, the pellet was washed four times with lysis buffer and then subjected to Western blot analysis using the antibodies against PPAR2 or MURF1. IgG was used as a control and total cell lysates (5%) were used as input controls.

For a ubiquitylation assay, diluted lysates were incubated with an antibody against PPAR2 at 4C overnight after precleaning with protein G beads (Thermo Fisher Scientific). Protein G beads were added to the washed lysate/antibody mixture at 4C for 4 hours. The resin was washed and applied to Western blot analysis using an antibody against ubiquitin.

MSCs were cultured alone or cocultured with myeloma cells for 2 weeks. Abundance of FABP4 and osteocalcin was assessed by immunofluorescence using fluorescein isothiocyanate or allophycocyanin-conjugated antibodies (BD Biosciences). After staining, cells were resuspended in phosphate-buffered saline with 1% FBS and analyzed using a BD LSR Fortessa flow cytometer.

The animal experiments in the present study were approved by the MD Anderson Institutional Animal Care and Use Committee. In vivo extramedullary bone formation in nonobese diabetic/severe combined immunodeficiency/interleukin-2rnull mice was established and examined (18). Briefly, MSCs alone or a mixture of human MSCs (1.5 106) and human ECFCs (1.5 106) in 0.2 ml of Matrigel (Corning Inc.) was subcutaneously injected into the right flanks of mice. This mixture and an additional 2 105 -irradiated (5000 centigrays) myeloma cells were injected into the left flanks of the mice. At 8 weeks after implantation, subcutaneous tissues were established, and the mice were intraperitoneally injected with OsteoSense 750 to assess new bone formation in those tissues. The subcutaneous tissues were collected after the mice were sacrificed and subjected to hematoxylin and eosin or immunohistochemical staining of cells labeled with an antibody against osteocalcin (a marker of mature osteoblasts), an antibody against perilipin (a marker of mature adipocytes), or an antibody against CD138 (a marker of myeloma cells).

The subcutaneous tissues were extracted from the mice and then formalin-fixed and paraffin-embedded. Tissue sections were deparaffinized with xylene and rehydrated to water through a graded alcohol series. Endogenous peroxidase activity was quenched with 3% hydrogen peroxide. The presence of CD138 (R&D Systems), osteocalcin, perilipin, and MURF1 (Abcam) in tissues was detected using specific antibodies. Signals were detected using secondary biotinylated antibodies and streptavidin/horseradish peroxidase. Chromagen 3,3-diaminobenzidine/H2O2 (Dako) was used, and slides were counterstained with hematoxylin. All slides were observed under a light microscope, and images were captured using a SPOT RT camera (Diagnostic Instruments).

Experimental values were expressed as means SD unless indicated otherwise. Statistical significance was analyzed using the GraphPad Prism v7.0 with two-tailed unpaired Students t tests for comparison of two groups and one-way analysis of variance (ANOVA) with Tukeys multiple comparisons test for comparison of more than two groups. P values less than 0.05 were considered statistically significant. All results were reproduced in at least three independent experiments.

stke.sciencemag.org/cgi/content/full/13/633/eaay8203/DC1

Fig. S1. G3335 inhibits PPAR2 accumulation in MSCs cocultured with myeloma cells.

Fig. S2. 4 KD in myeloma cells.

Fig. S3. VCAM1 knockdown in MSCs.

Fig. S4. PKC inhibition reduces PKC1 phosphorylation and PPAR2 abundance in MSCs cocultured with myeloma cells.

Fig. S5. Coculture with myeloma cells reduces ubiquitylation of PPAR2 in MSCs.

Fig. S6. MSCs from patients with myeloma show decreased MURF1 and increased PPAR2.

Table S1. Characteristics of patients with myeloma and healthy donors.

Table S2. Read quality control of the samples for scRNA-seq.

Table S3. Mapping quality control of aligned scRNA-seq data.

Table S4. Basic information of the aggregated samples for scRNA-seq before and after normalization.

Table S5. Information of each sample after aggregation.

Table S6. Cell quality control showing the cell numbers before and after the filtration.

Table S7. Number of cells in each subpopulation.

Table S8. Number of cells in each subpopulation of control and cocultured samples.

Table S9. Primers used in the quantitative reverse transcription PCR analysis.

Data file S1. scRNA-seq data from control sample.

Data file S2. scRNA-seq data from coculture sample.

Acknowledgments: We thank M. J. Li from Department of Genetics, Tianjin Medical University for the evaluation of our statistical analysis. Funding: This work was supported by R01 grants from NCI (CA190863 and CA193362 to J.Y.) and by the Research Scholar Grant from the American Cancer Society (127337-RSG-15-069-01-TBG to J.Y.). It was also supported by NIH/NCI (Core Labs at UT MD Anderson Cancer Center, P30CA016672) for the Small Animal Imaging and Research Histopathology Facilities. Author contributions: Z.L. and J.Y. designed all experiments and wrote the manuscript. H.L., Z.L., and J.H performed all experiments and statistical analysis. P.L. provided and interpreted patient samples. Q.T. provided critical suggestions. Conflict of interests: The authors declare that they have no competing interests. Data and materials availability: All of the data needed to evaluate the conclusions in the paper are provided in the main text or the Supplementary Materials. Stable cell lines carrying targeted shRNA are available with a materials transfer agreement between Houston Methodist Research Institute and the requesting institution.

See the original post:
Myeloma cells shift osteoblastogenesis to adipogenesis by inhibiting the ubiquitin ligase MURF1 in mesenchymal stem cells - Science

IN A stark hospital room, Damian Cross waits for his 14-year-old daughter to save his life.

Shauna is less than 10km away at the Queensland Children's Hospital having her bone marrow extracted.

Despite only being a half match for her father, it was the best solution during a time when full match bone marrow was difficult to come by due to COVID-19 travel restrictions.

The family are a long way from their Coraki home where for a year Damian has been in remission from leukaemia after five rounds of chemotherapy.

"Leukaemia has come back and my only hope for cure now is my 14-year-old daughter," he said.

At Royal Brisbane Hospital with his partner Amy Rolfe by his side, the 33-year-old was under sedation for a bone marrow biopsy.

Shauna's bone marrow will be collected through a needle in her neck.

"Shauna has a fear of needles but hasn't batted an eye at the catheter in her neck," Amy said.

Coraki's Damian Cross in hospital in Brisbane waiting for a bone marrow transplant from his 14 year old daughter. PIC: AMY ROLFE Amy Rolfe

In preparation to receive his daughter's bone marrow, Damian will undergo three days of chemotherapy and four days of radiation to wipe out his cells.

"Then he gets her cells," Amy said.

Donor cells, especially when they are a half match, could attack Damian's cells.

"He'll be here for 100 days after the transplant," Amy said.

"Three to four weeks in hospital and then we have to stay in Brisbane for three months."

Damian will be on anti-rejection drugs and the procedure can fail within a three-year period.

The family is hopeful though and urge Australians to consider registering for bone marrow donation through the Australian Bone Marrow Donor Registry.

The World Marrow Donor Association operates a global database to find the best stem cell

source with a database of 36,214,535 donors from 98 different registries in 53 different countries.

Amy said Germany had the best bone marrow donor rate.

The WMDA said COVID-19 infection had the potential to impact and interfere with the timely provision of cells across international borders.

It is currently uncertain whether COVID-19 is transmissible parenterally, and it seems prudent to defer donors from countries with a high rate of COVID-19 infection, WMDA said.

Support the family through their crowdfunding campaign.

The rest is here:
14-year-old girl is only chance to save dad's life - Chinchilla News

Key points:

NEW YORK, May 25, 2020 (GLOBE NEWSWIRE) -- Mesoblast Limited (Nasdaq:MESO; ASX:MSB), global leader in cellular medicines for inflammatory diseases, today announced that clinical outcomes of its allogeneic mesenchymal stem cell (MSC) medicine RYONCIL (remestemcel-L) in children and adults with steroid-refractory acute graft versus host disease (GVHD) have been published in three peer-reviewed articles and an accompanying editorial in the May issue of Biology of Blood and Marrow Transplantation, the official publication of the American Society for Transplantation and Cellular Therapy.

Mesoblast Chief Medical Officer Dr Fred Grossman said: Results from these three trials show a consistent pattern of safety and efficacy for RYONCIL (remestemcel-L) in patients with the greatest levels of inflammation and the most severe grades of acute GVHD. These clinical outcomes provide a compelling rationale for use of remestemcel-L in children and adults with other conditions associated with severe inflammation and cytokine release, including acute respiratory distress syndrome (ARDS) and systemic vascular manifestations of COVID-19 infection.

In the accompanying editorial, Dr Jacques Galipeau, Professor and Assistant Dean of Medicine at the Stem Cell & Regenerative Medicine Center at the University of WisconsinMadison and Chair of the International Society of Cell and Gene Therapy (ISCT) MSC Committee, concluded that after more than a decade of clinical study involving three distinct advanced trials, it appears that remestemcel-L might well have finally met the regulatory requirements for marketing approval in the United States for steroid refractory acute GVHD in children, and it is to be determined whether this industrial MSC product will find utility for adults afflicted by acute GVHD or other indications.

The trials highlighted in the three articles all evaluated the same treatment regimen of RYONCIL, with patients receiving twice weekly intravenous infusions of 2 million cells per kg body weight over a four-week period. RYONCIL was well-tolerated in all studies with no identified safety concerns. The three trials were:

1. Study 275: An Expanded Access Program in 241 children across 50 centers in eight countries where RYONCIL was used as salvage therapy for steroid-refractory acute GVHD in patients who failed to respond to steroid therapy as well as multiple other agents.

2. Study GVHD001/002: A Phase 3 single-arm trial in 55 children across 20 centers in the United States where RYONCIL was used as the first line of treatment for children who failed to respond to steroids for acute GVHD.

3. Study 280: A Phase 3 randomized placebo-controlled trial in 260 patients, including 28 children, across 72 centers in seven countries where RYONCIL or placebo were added to second line therapy in patients with steroid-refractory acute GVHD who failed to respond to steroid treatment.

About Acute Graft Versus Host DiseaseAcute GVHD occurs in approximately 50% of patients who receive an allogeneic bone marrow transplant (BMT). Over 30,000 patients worldwide undergo an allogeneic BMT annually, primarily during treatment for blood cancers, and these numbers are increasing.1 In patients with the most severe form of acute GVHD (Grade C/D or III/IV) mortality is as high as 90% despite optimal institutional standard of care.2,3 There are currently no FDA-approved treatments in the United States for children under 12 with steroid-refractory acute GVHD.

About RYONCILTMMesoblasts lead product candidate, RYONCIL (remestemcel-L), is an investigational therapy comprising culture-expanded mesenchymal stem cells derived from the bone marrow of an unrelated donor. It is administered to patients in a series of intravenous infusions. RYONCIL is believed to have immunomodulatory properties to counteract the inflammatory processes that are implicated in SR-aGVHD by down-regulating the production of pro-inflammatory cytokines, increasing production of anti-inflammatory cytokines, and enabling recruitment of naturally occurring anti-inflammatory cells to involved tissues.

References1. Niederwieser D, Baldomero H, Szer J. Hematopoietic stem cell transplantation activity worldwide in 2012 and a SWOT analysis of the Worldwide Network for Blood and Marrow Transplantation Group including the global survey.Bone Marrow Transplant 2016; 51(6):778-85.2. Westin, J., Saliba, RM., Lima, M. (2011) Steroid-refractory acute GVHD: predictors and outcomes. Advances in Hematology 2011;2011:601953.3. Axt L, Naumann A, Toennies J (2019) Retrospective single center analysis of outcome, risk factors and therapy in steroid refractory graft-versus-host disease after allogeneic hematopoietic cell transplantation. Bone Marrow Transplantation 2019;54(11):1805-1814.

Story continues

About MesoblastMesoblast Limited (Nasdaq:MESO; ASX:MSB) is a world leader in developing allogeneic (off-the-shelf) cellular medicines. The Company has leveraged its proprietary mesenchymal lineage cell therapy technology platform to establish a broad portfolio of commercial products and late-stage product candidates. The Companys proprietary manufacturing processes yield industrial-scale, cryopreserved, off-the-shelf, cellular medicines. These cell therapies, with defined pharmaceutical release criteria, are planned to be readily available to patients worldwide.

Mesoblasts Biologics License Application to seek approval of its product candidate RYONCIL (remestemcel-L) for pediatric steroid-refractory acute graft versus host disease (acute GVHD) has been accepted for priority review by the United States Food and Drug Administration (FDA), and if approved, product launch in the United States is expected in 2020. Remestemcel-L is also being developed for other inflammatory diseases in children and adults including moderate to severe acute respiratory distress syndrome. Mesoblast is completing Phase 3 trials for its product candidates for advanced heart failure and chronic low back pain. Two products have been commercialized in Japan and Europe by Mesoblasts licensees, and the Company has established commercial partnerships in Europe and China for certain Phase 3 assets.

Mesoblast has a strong and extensive global intellectual property (IP) portfolio with protection extending through to at least 2040 in all major markets. This IP position is expected to provide the Company with substantial commercial advantages as it develops its product candidates for these conditions.

Mesoblast has locations in Australia, the United States and Singapore and is listed on the Australian Securities Exchange (MSB) and on the Nasdaq (MESO). For more information, please see http://www.mesoblast.com, LinkedIn: Mesoblast Limited and Twitter: @Mesoblast

Forward-Looking StatementsThis announcement includes forward-looking statements that relate to future events or our future financial performance and involve known and unknown risks, uncertainties and other factors that may cause our actual results, levels of activity, performance or achievements to differ materially from any future results, levels of activity, performance or achievements expressed or implied by these forward-looking statements. We make such forward-looking statements pursuant to the safe harbor provisions of the Private Securities Litigation Reform Act of 1995 and other federal securities laws.Forward-looking statements include, but are not limited to, statements about the initiation, timing, progress and results of Mesoblast and its collaborators clinical studies; Mesoblast and its collaborators ability to advance product candidates into, enroll and successfully complete, clinical studies; the timing or likelihood of regulatory filings and approvals; and the pricing and reimbursement of Mesoblasts product candidates, if approved;the potential benefits of strategic collaboration agreements and Mesoblasts ability to maintain established strategic collaborations; Mesoblasts ability to establish and maintain intellectual property on its product candidates and Mesoblasts ability to successfully defend these in cases of alleged infringement; the scope of protection Mesoblast is able to establish and maintain for intellectual property rights covering its product candidates and technology.You should read this press release together with our risk factors, in our most recently filed reports with the SEC or on our website. Uncertainties and risks that may cause Mesoblasts actual results, performance or achievements to be materially different from those which may be expressed or implied by such statements, and accordingly, you should not place undue reliance on these forward-looking statements. We do not undertake any obligations to publicly update or revise any forward-looking statements, whether as a result of new information, future developments or otherwise.

Release authorized by the Chief Executive.

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Clinical Outcomes Using RYONCIL (remestemcel-L) in Children and Adults With Severe Inflammatory Graft Versus Host Disease Published in Three Articles...

In the current situation of restricted movement and reduced workforce, (due to COVID-19 Pandemic) new technologies have been developed to provide end-to-end automation in different sectors such as food processing. Automated systems are hired by the companies to ensure continued supply and manufacturing of products with the least manual interference

The advent of Health Information Technology (HIT) components such as electronic health records (EHR), hospital information systems (HIS), picture archiving and communication systems (PACS), and vendor neutral archives (VNA) has had just as transformational an impact on the overall healthcare sector as the concerns regarding security and privacy. Data theft, undue access to personal health records, and cyber-attacks are very real threats that the healthcare sector faces today.

Myelofibrosis or osteomyelofibrosis is a myeloproliferative disorder which is characterized by proliferation of abnormal clone of hematopoietic stem cells. Myelofibrosis is a rare type of chronic leukemia which affects the blood forming function of the bone marrow tissue. National Institute of Health (NIH) has listed it as a rare disease as the prevalence of myelofibrosis in UK is as low as 0.5 cases per 100,000 population. The cause of myelofibrosis is the genetic mutation in bone marrow stem cells. The disorder is found to occur mainly in the people of age 50 or more and shows no symptoms at an early stage. The common symptoms associated with myelofibrosis include weakness, fatigue, anemia, splenomegaly (spleen enlargement) and gout. However, the disease progresses very slowly and 10% of the patients eventually develop acute myeloid leukemia. Treatment options for myelofibrosis are mainly to prevent the complications associated with low blood count and splenomegaly.

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The global market for myelofibrosis treatment is expected to grow moderately due to low incidence of a disease. However, increasing incidence of genetic disorders, lifestyle up-gradation and rise in smoking population are the factors which can boost the growth of global myelofibrosis treatment market. The high cost of therapy will the growth of global myelofibrosis treatment market.

The global market for myelofibrosis treatment is segmented on basis of treatment type, end user and geography:

As myelofibrosis is considered as non-curable disease treatment options mainly depend on visible symptoms of a disease. Primary stages of the myelofibrosis are treated with supportive therapies such as chemotherapy and radiation therapy. However, there are serious unmet needs in myelofibrosis treatment market due to lack of disease modifying agents. Approval of JAK1/JAK2 inhibitor Ruxolitinib in 2011 is considered as a breakthrough in myelofibrosis treatment. Stem cell transplantation for the treatment of myelofibrosis also holds tremendous potential for market growth but high cost of therapy is foreseen to limits the growth of the segment.

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On the basis of treatment type, the global myelofibrosis treatment market has been segmented into blood transfusion, chemotherapy, androgen therapy and stem cell or bone marrow transplantation. Chemotherapy segment is expected to contribute major share due to easy availability of chemotherapeutic agents. Ruxolitinib is the only chemotherapeutic agent approved by the USFDA specifically for the treatment of myelofibrosis, which will drive the global myelofibrosis treatment market over the forecast period.

Geographically, global myelofibrosis treatment market is segmented into five regions viz. North America, Latin America, Europe, Asia Pacific and Middle East & Africa. Northe America is anticipated to lead the global myelofibrosis treatment market due to comparatively high prevalence of the disease in the region.

Some of the key market players in the global myelofibrosis treatment market are Incyte Corporation, Novartis AG, Celgene Corporation, Mylan Pharmaceuticals Ulc., Bristol-Myers Squibb Company, Eli Lilly and Company, Taro Pharmaceuticals Inc., AllCells LLC, Lonza Group Ltd., ATCC Inc. and others.

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Regional analysis includes

Report Highlights:

Our unmatched research methodologies set us apart from our competitors. Heres why:PMRs set of research methodologies adhere to the latest industry standards and are based on sound surveys.We are committed to preserving the objectivity of our research.Our analysts customize the research methodology according to the market in question in order to take into account the unique dynamics that shape the industry.Our proprietary research methodologies are designed to accurately predict the trajectory of a particular market based on past and present data.PMRs typical operational model comprises elements such as distribution model, forecast of market trends, contracting and expanding technology applications, pricing and transaction model, market segmentation, and vendor business and revenue model.

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Global Myelofibrosis Treatment Market to Register Growth in Incremental Opportunity During the Forecast Period 2016 2022 - Cole of Duty

THE world's top sports stars are preferring to use stem cell treatment, instead of undergoing major surgery that could leave them out for months.

Cristiano Ronaldo, Rafael Nadal, and most recently Mike Tyson have all tried the therapy, which can cost anywhere from 4,000 to 15,000, when they've suffered injury.

Ailments that can be treated, include tendon inflammation, muscle strain, arthritis, degenerative disc disease, and even bone fractures.

And sportsman who have undergone stem cell therapy are benefitting from improved results, as well as a faster recovery time.

Collected from the blood from a newborn babys umbilical cord, the bone marrow or from body fat, stem cells are injected into an athletes' affected area.

They get to work by replenishing damaged cells from an injury or through wear and tear.

Stem cells also help reduce pain and inflammation, increase blood flow, and promote soft-tissue growth.

It helps the body to heal naturally, and means sports stars can potentially avoid going under the surgeon's knife.

When you're a top sports star, if you get injured the first thing you want to do is get back into the thick of action as quick as possible.

Unfortunately, many injuries can take a long time to heal, and will never allow the sportsman in question to return to the same level he/she was at before the injury.

That's where stem cell treatment is a game-changer.

Forget surgery, steroid injections, and lengthy physiotherapy, which don't always repair the issue at hand.

Stem cell treatments offer an alternative, albeit at a price, to have a non-surgical therapy that's non-evasive and, more importantly, heals the problem fast cutting out the need of rehabilitation.

Better still, some patients have reported that the therapy has not only reversed existing damage, but has strengthened cells against further damage.

Juventus star Ronaldo and Spanish tennis hero Nadal are all fans of stem cell treatment.

Back in 2016, when the Portuguese forward was playing for Real Madrid, he suffered a hamstring tear that threatened to keep him out of action of an important Champions League game against Manchester City.

Although he missed the first leg, he was back for the second - less than three weeks after suffering the problem.

That same year, Ronaldo tore a collateral ligament in his knee during Portugals Euro 2016 final against France.

Again, he turned to stem cell treatment and was back in training with Los Blancos just a month after his knee complaint.

Nadal's chronic knee problems forced him to take seven months off from tennis in 2013.

But stem cell treatment allowed the cartilage to repair. In the seven years since he's won six Grand Slams, there's been no setbacks from his troublesome knee and he appears as mobile as ever.

The Spaniard also cured a long-standing back problem with the therapy.

The former heavyweight champion, who is considering making a comeback, is the latest name to have tried stem cell treatment.

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It is not known what Tyson, 53, was suffering from - but he was happy to reveal all in an Instagram chat with basketball legend Shaquille O'Neal.

Iron Mike said: "You know what I had done? I had stem-cell research therapy.

"I feel like a different person but I can't comprehend why I feel this way. It's really wild what scientists can do."

See more here:
Tyson, Ronaldo, and more sports stars who use stem cell treatment costing up to 15,000 to speed up healing - The Sun

Before he knew Anuroop, Vihaan (right) addressed him as the Superhero.

Vihaan is a bright and active 4-year-old boy. He is a thalassemia survivor. And just over a year ago, he desperately needed a bone marrow transplant.

Anuroop, is a young man from Kerala. He doesn't know Vihaan's family. He just felt that donating bone marrow for someone in need was the right thing to do after he got a call from Datri, a non-profit agency coordinating such donations.

Anuroop told NDTV, "Actually, it was a matter of choice. I got a call from Datri about one year ago and they discussed with me this matter. They said, 'A 4 year old child, he is suffering from Thalassemia. Maybe only you can save him.' But at that point, I was not sure that I would do it. But later, with the support of my family and people from Datri, I decided to do it."

Anuroop and Vihaan came face to face - on camera - for the first time ever on NDTV. Anuroop was clearly emotional as he saw images of an active Vihaan, flanked by his parents, grinning and waving at the screen. Bhavana, Vihaan's mother was emotional too as she set eyes for the first time on Anuroop, who has given the family life and hope.

She told NDTV, "He is the answer to all our prayers. When Vihaan was diagnosed when he was 6 months old, we didn't know how Vihaan is going to be. We didn't know what to do. And then we went to Dr Sunil Bhatt and we registered with Datri. They told us that the procedure of finding a match is very difficult. And then we found a donor so we just couldn't believe that we were blessed to find a donor. Those were anxious days. But yeah, glad now."

Asked how her son was doing, Bhavana said, "Young Vihaan is doing great, thanks to Dr Sunil, thanks to Anuroop and thanks to God's grace. Vihaan is doing well."

Looking at the screen in front of her which showed, Anuroop, Dr Bhatta and Gayathri Shenoy of Datri, she told her son '"Just say hi, Vihaan!" He did, with a cheery wave.

Anuroop was moved by the response. He said, "I'm super excited - I waited for too long. I waited for one year. From that day of donation, the whole family, he was always in my prayers. I'm super excited now. That's all."

Finding a matching donor in a case like this is a very difficult task. Vihaan's doctor, Dr Sunil Bhatt, is HoD, Paediatric Haematology, Oncology, Bone Marrow Transplantation at the Mazumdar Shaw Cancer Centre in Bengaluru. And this professional medical man admitted to the deep emotions he feels at such times when a donor meets a recipient. "It gives me goosebumps," he told NDTV.

"You do so many times, again and again, but every time when an unrelated donor meets a patient - it is always an emotional moment for all of us," he said.

"Vihaan was diagnosed with a disease called Thalassemia at six months of age. What happens in this disease is that they don't make their own blood. So they have to be given blood transfusions from outside every few weeks to sustain life and that is life-long. But what blood does is it brings its own complications along with it and many of those and unfortunately most of these children do not live more than second or third decade of life. So the only cure for this is bone marrow transplantation and as we all know for Bone Marrow Transplantation we need someone to donate for them. There has to be a healthy donor who can donate," he said.

To find a matching donor is far from easy. Dr Bhatt said, "Sometimes you'll find that in the families - the chances of that being 25-30 per cent. But 70 per cent of the patients who require transplants will not have anyone in their families to donate for them. So here comes the role of unrelated donor transplantations that means someone else in the same country, in the world who matches the patient. And the chances of that being one in 20,000 to one in a million. So it depends on what ethnic background you're from - South Indian is going to match South Indian, North Indian going to match North Indian - chances will be higher in your own ethnic community. And hence the registries play a huge role because they enrol these unrelated healthy donors, put them on their database and when patients like Vihaan require such transplantation we approach these registries and ask them if there is any donor in the registry who is matching our patient. If there is one, that person is requested to donate and they donate stem cells to save someone's life."

Datri helped coordinate this life saving procedure with its all-important database. Gayathri Shenoy, Head-Patient Relations of Datri told NDTV, "I represent Datri which is India's largest blood stem cell registry. We are 10 years old and we have about 4.4 lakh registered donors and 712 donations of that. But as you can imagine that is a very small number compared to the population of our country because there are so many patients who have blood cancer who are waiting for their Anuroop to show up."

Asked if it had been physically difficult to donate bone marrow, Anuroop said, "Physically not that hard - like I need some rest but it is not that hard. Anyone can do it anytime if they find a match. I didn't find it very difficult and all. I heard some cases like people will be hesitant to do something like this - but I didn't find anything that should people hesitate. It is an easy process and you would be given a general anaesthesia. You won't be knowing anything."

Bhavana said, We just wanted to say that everyday in our prayers all of these people have been there. We didn't know the donor - so he was just addressed as the Superhero Donor, because it was very difficult to make Vihaan understand. Dr. Sunil, Datri - I don't know what we would have done if it was not for Datri. So just feeling blessed.

Original post:
When A Bone Marrow Donor Met His 4-Year-Old Recipient For The First Time - NDTV

Stem cell banking or preservation is a combined process of extraction, processing and storage of stem cells, so that they may be used for treatment of various medical conditions in the future, when required. Stem cells have the amazing power to get transformed into any tissue or organ in the body. In recent days, stem cells are used to treat variety of life-threatening diseases such as blood and bone marrow diseases, blood cancers, and immune disorders among others.

The market of stem cell banking is anticipated to grow with a significant rate in the coming years, owing to factors such as, development of novel technologies for stem cell preservation and processing, and storage; growing awareness on the potential of stem cells for various therapeutic conditions. Moreover, increasing investments in stem cell research is also expected to propel the growth of the stem cell banking market across the globe. On other hand rising burden of major diseases and emerging economies are expected to offer significant growth opportunities for the players operating in stem cell banking market.

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Market Segmentation:

The source segment includes, placental stem cells (PSCS), dental pulp-derived stem cells (DPSCS), bone marrow-derived stem cells (BMSCS), adipose tissue-derived stem cells (ADSCS), human embryo-derived stem cells (HESCS), and other stem cell sources. Based on service type the market is segmented into, sample processing, sample analysis, sample preservation and storage, sample collection and transportation. Based on application, the market is segmented as, clinical applications, research applications, and personalized banking applications.

Company Coverage:

1. Cordlife2. ViaCord (A Subsidiary of PerkinElmer)3. Cryo-Save AG4. StemCyte India Therapeutics Pvt. Ltd.5. Cryo-Cell International, Inc.6. SMART CELLS PLUS.7. Vita 348. LifeCell9. Global Cord Blood Corporation10. CBR Systems, Inc.

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Region Coverage:

Asia-Pacific(Vietnam, China, Malaysia, Japan, Philippines, Korea, Thailand, India, Indonesia, and Australia)

Europe(Turkey, Germany, Russia UK, Italy, France, etc.)

North America(the United States, Mexico, and Canada.)

South America(Brazil etc.)

The Middle East and Africa(GCC Countries and Egypt.)

Stem Cell Banking Market: Competitive Landscape-

Analysts have thoroughly evaluated the competitive landscape in the Stem Cell Banking Market. The report includes the study of key players in the Stem Cell Banking Market. It also outlines the strategic initiatives companies have taken in recent years to keep pace with increasing competition. It also includes an assessment of the financial perspectives of these companies, their research and development plans, and their future business strategies.

Stem Cell Banking Market: Drivers and Restraints-

The comprehensive market assessment of Stem Cell Banking contains a complete explanation of the controls available on the market. Analysts have studied investment in research and development, the impact of changing economies, and consumer behaviour to determine the factors that will drive the market in general. In addition, analysts have attempted to take into account changes in manufacturing and industrial operations that determine product sales in the Stem Cell Banking Market.

This chapter also explains the possible restrictions on the Stem Cell Banking Market. Assess the reasons that could hinder market growth. Analysts have assessed growing environmental concerns and fluctuating raw material costs, which are predicted to dampen the spirit of the Stem Cell Banking Market. However, analysts have also identified potential opportunities that players in the Stem Cell Banking Market can rely on. The chapter on controls, restrictions, threats and opportunities offers a holistic view of the Stem Cell Banking Market.

Key Questions Answered

Answering these types of questions can be very useful for gamers to clear up their doubts as they implement their strategies to grow in the global Stem Cell Banking Market. The report provides a transparent picture of the actual situation in the global Stem Cell Banking Market so that companies can work more effectively. It can be tailored to the needs of readers to better understand the global market for Stem Cell Banking.

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Expected A Drastic Growth In Stem Cell Banking Market Key Insights Based On Source, Service Type, Application - 3rd Watch News

Leukaemia is the cancer of blood cells. Blood cells originate from HSCs, hematopoietic stem cells, in the bone marrow. Thereafter they undergo maturation process called hematopoiesis. Multipotent hematopoietic stem cells often undergo a process of differentiation while in maturation stage to give rise to progenitor cells of myeloid and lymphoid origin. These Myeloid cells include neutrophils, basophils, monocytes, macrophages, erythrocytes, dendritic cells, eosinophils, and megakaryocytes or platelets. While, Lymphoid cells include B cells, T cells and natural killer cells.

Recently in 2016, Global Leukaemia Therapeutics Market was valued at nearly USD 9.44 billion and is expected to grow at a CAGR of 4.1% between 2017 and 2022, accounting to market worth USD 11.97 billion by end of 2022.

The Final Report will cover the impact analysis of COVID-19 on this industry.

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Normally, the blood forming cells in the bone marrow produce leukocytes, that protects against viruses and bacteria. If these leukocytes get damaged and if they are left untreated they get accumulated in the body and invade in other parts like liver, spleen and central nervous system, hence damaging the entire body. The main reasons causing leukaemia are ionizing radiation, smoking, prior chemotherapy and Down syndrome.

Market Dynamics

Recently in 2016, Global Leukaemia Therapeutics Market was valued at nearly USD 9.44 billion and is expected to grow at a CAGR of 4.1% between 2017 and 2022, accounting to market worth USD 11.97 billion by end of 2022.

Global Leukaemia Therapeutics market is majorly driven by the growing number of incidences of target disease across the globe. Also, development of novel agents, advancements in technology and combination therapy with reduced side effects and better survival conditions are some other key factors that drives the Leukaemia Therapeutics Market.

However, the high cost of combination therapies and clinical trials coupled with post-treatment complications, adverse events and side effects are the major constraints that limit the growth of the market.Nevertheless, initiatives like increasing focus on healthcare and personalized medicine along with huge govt. investment & R&D in anti-leukaemia therapeutics research are sure short to boost the market growth in the near future.

Market SegmentationGlobal Leukaemia Therapeutics Market can be segmented as follows :Segmentation by TypeChronic leukaemiaChronic myeloid leukaemiaChronic lymphatic leukaemiaAcute leukaemiaAcute myeloid leukaemiaAcute lymphatic leukaemia

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Segmentation by TherapyBiological TherapyRadiation therapyChemotherapyTargeted therapy

Regional/Geographic AnalysisEurope, North America, Latin America, Asia-Pacific, Middle East & Africa are key market segments of global Leukaemia Therapeutics. North America is the leading region and is anticipated to remain one in the near future, over the forecast period. Demand for leukaemia therapeutics was highest in North America especially in the U.S attributing to increasing geriatric population and increased number of cases. While, Asia Pacific region along with Middle East, Africa and Latin America is expected to grow at moderate pace.

Key Players

The key players in global leukaemia therapeutics market includeF. Hoffmann-La Roche Ltd., Bristol-Myers Squibb, Amgen, Pfizer, Teva Pharmaceuticals, Novartis International AG., GlaxoSmithKline plc., Genzyme Corporation, AbbVie Inc. and others.

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Leukaemia Therapeutics Market is expected to grow at a CAGR of 4.1% between 2017 and 2022 - WaterCloud News

The Netherlands is busy adapting in all kinds of ways in order to deal with the corona pandemic. But are our corona measures effective at all? What are the benefits of testing? And why does a vaccine take so long to develop? Biomedical chemist Willem Mulder offers answers to these questions. And he explains how his research is contributing to a solution to the pandemic.

For the past fifteen years, Mulder (43) has been conducting research into nanomaterials that can regulate how the immune system functions. His main focus is on the development of new treatment methods for cardiovascular diseases, cancer, and post-transplant rejection. Although his work can also be applied to many other diseases, including the novel coronavirus. But how can research into new treatments for combatting cancer and carrying out transplants be of any use in the current pandemic? In order to understand this, we need to explain Mulders work method.

Normally Mulder travels back and forth between New York and Eindhoven. In New York, he is Professor of Radiology and Professor of Oncological Sciences at the BioMedical Engineering and Imaging Institute at Icahn School of Medicine at Mount Sinai. In Eindhoven, he works part-time as a professor of Precision Medicine at the Eindhoven University of Technology (TU/e). Furthermore, he is co-founder of Trained Therapeutix Discovery, a company that develops immune therapies which are based on nanomaterials.

On both sides of the ocean, the work of Mulder and his fellow scientists focuses on guiding our immune system to fight diseases. We use nanotechnology to regulate the immune response. Immune cells are produced by stem and precursor cells in the bone marrow. We take control of this production process, so to speak. This allows us to ensure that the immune system achieves whats called a tolerant immune status when the immune system is suppressed. This is very important in organ transplants, for example, so that a patients body will not consequently reject a transplanted organ. The opposite is true for cancer. Then the immune system needs to be intolerant towards a tumor. In these situations, we actually want to trigger immunity, Mulder explains.

Mulders work is best understood when you keep these two scenarios in mind. In one case, you want a patients immune system to have a specific level of tolerance for a transplanted organ. In the other, you want to induce an aggressive immune response against tumor cells. Both scenarios can be applied to many different pathologies, including the novel coronavirus. The regulation of the immune response is crucial when dealing with SARS-CoV-2 infections. A properly functioning immune system can prevent or in case of infection swiftly eradicate the infection. On the other hand, the infection can cause COVID-19 disease in infected patients whose immune system is not functioning adequately. With potentially disastrous consequences. There are plenty of parallels with COVID-19. We see many similarities between hyperinflammation in COVID-19 patients and the immune response after transplants, Mulder continues.

Now, about our immune system. It comprises two parts. The congenital (or non-specific) part is mainly made up of phagocytes. These are cells that can, as it were, eat bacteria, viruses, and fungi. This part of the immune system is our first line of defense and is ready to fight off an invasion of our bodies. When someone has mild symptoms after becoming infected with the coronavirus, that persons natural immune system is perfectly capable of getting rid of the virus.

When the congenital part of the immune system is unable to get rid of an infection, the adaptive part of the immune system takes over the defense task. A virus is made up of a genetic code (RNA) that is packaged in tiny globules of lipids and proteins. Certain types of phagocytes referred to as antigen-presenting cells break down a virus into small molecular fragments called antigens. Cells of the adaptive immune system (lymphocytes) recognize the antigens and are thereby activated. This triggers a cascade of processes that generate a specific immunological memory where antibodies play an important role.

Consequently, those people who experience few issues after infection with SARS-CoV-2 may not be able to build up a high enough level of immunity. When the natural immune system is able to clear the infection itself, there is no strong adaptive immune response needed in order to achieve immunity.

When asked why the elderly in particular are not resistant to COVID-19, Mulder answers: Although it does happen, the amount of young people dying from this virus is statistically negligible. That may be because the immune system doesnt function as well as it should since the number of lymphocytes in the blood declines with age. This is also often the case with people with underlying conditions. For example, the immune systems of people who are overweight, diabetic, or have cardiovascular diseases tend to age much faster. Thats why we suspect that COVID-19 patients with underlying conditions are more susceptible to the disease process spiraling out of control.

Mulder says that it is especially important now to use tests as a means of gaining insight into whether herd immunity is being built up or not. In order to do this, it is essential to know how many people among the population have antibodies. Mulder: Because the only people who have been tested in The Netherlands are those who have had the disease get out of hand, you tend to get a distorted picture. Now it seems as if it is mainly the elderly who are infected. We want to know exactly how the disease progresses in people who have no noticeable symptoms. So far, our policy is based on one-sided data.

In countries where a lot of testing has been done, we see that lots of young people have been infected. From the outset of the crisis, I couldnt understand why no investment was being made into testing on a large scale. It is only then that its actually possible to gain proper insights into how immunity works when it comes to SARS-CoV-2.

Mulder is concerned about the future course of the corona crisis. He emphasizes that the pandemic has just begun. And that we will experience the consequences of the subsequent (economic) damage for a long time to come. Its now just a matter of waiting for a vaccine before we can fully get back to the normal order of the day. As well as a definitively stamp out any new infections. Mulder points out that there are viral infections such as HIV where its never been possible to produce a vaccine. However, he is hopeful that this will succeed for SARS-CoV-2 because it has now been proven that laboratory animals can be vaccinated against the virus.

Making a vaccine is a very complicated and time-consuming process. Mulder explains: A vaccine ensures that you build up immunity and produce antibodies yourself. These antibodies can also be obtained in other ways. One way to do this is to take plasma from people who have been infected and who, as a result, have developed antibodies. Or you could develop antibodies in a lab. The latter has been done by research teams at the Erasmus University in Rotterdam and Utrecht University here in The Netherlands. This type of antibody therapy can certainly provide some relief. However, it is laborious, costly, and difficult to implement on a large scale.

The development of a vaccine takes a long time because it requires a method of getting a pathogen into a person without making that person sick. The pathogen has to be recognized by the immune system in order to trigger an immune response that ultimately provides immunity. Various strategies are possible for achieving this. A vaccine can be based on weakened strains of the pathogen, e.g. by using harmless viruses, by using the genetic code of antigens or by producing the antigens themselves.

The complexity of our immune system makes it extremely difficult to predict which strategy is most likely to succeed. In any event, a considerable amount of time is needed to test the vaccines and produce them on a large scale. Normally, it can take up to 10 years to develop a successful vaccine. Hopefully, that will now happen faster. At the moment, there are about a hundred serious initiatives underway for this at major pharmaceutical companies such as Johnson & Johnson as well as at start-ups and universities, Mulder adds.

Reports have appeared in the media about the use of the malaria medication hydroxychloroquine in the treatment of COVID-19. The immune response to COVID-19 can get out of control with hyperinflammation as a result. Drugs such as hydroxychloroquine may help. Nephrologist Raphal Duivenvoorden of the Radboud University Medical Center (Radboudumc) is researching the effects of this drug on the immune system.

Willem Mulder is also participating in that study. It is a cheap drug with relatively few side effects. However, the timing of the treatment is very important because the immune systems response can worsen the disease. We expect to complete our study this month. Incidentally, there are plenty of medications that are undergoing testing at the moment. Take, for example, those immune therapies that specifically render immune-regulating molecules harmless.

Since the start of the lockdown, The Netherlands has been working hard to make the one-and-a-half-meter society part and parcel of daily life. Some have been critical of the measures introduced by the government and prefer a Swedish corona policy whereby the economy is kept going for the most part.

Mulder understands the decisions made by the Dutch government: Its new territory. We didnt know how the virus would behave or what the long-term effects would be. When you get the flu once in a while, your immune system is quite capable of maintaining a certain level of immunity against new flu strains.

We are born in a situation where both the flu and a certain degree of immunity to it already exists. In contrast, SARS-CoV-2 is completely new. Anyone can get infected. Then it is only logical that strict measures are introduced even when the mortality rate is relatively low. However, its quite difficult to compare countries and regions. Sweden is a sparsely populated country. The Netherlands is not. Population density is also the reason that there are so many infections and deaths in a huge city like New York.

Mulder goes on to add that he finds the introduction of corona measures somewhat worrying: A lockdown was necessary, but I hope it doesnt become the new normal. Governments in the West are now assuming a great deal of power. Freedoms are being taken away from young people while statistically, the problem does not rest with this group. We did what was necessary during the lockdown. I think the quid pro quo answer to this should be that this should not be abused. I hope that people are keeping a watchful eye on this.

More information about nanotechnology can be found here.

Also, check out these animations made by Willem Mulder:

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Leading scientist Willem Mulder: 'Frequent testing is crucial for predicting immune reactions' - Innovation Origins

MIKE TYSON has revealed he was injected with nearly-translucent blood in his bid to make a comeback... and the former heavyweight champ said it made him feel "weird".

The 53-year-old - who retired from boxing in 2005 - has announced his intention to dust off the gloves and return to compete in exhibition bouts.

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His return to action has been aided by stem-cell research therapy, that has left him feeling like a "different person".

He said: "You know what I had done? I had stem-cell research therapy.

"I feel like a different person but I can't comprehend why I feel this way. It's really wild what scientists can do."

Stem-cell therapy is the use of stem cells to treat or prevent a disease or condition that usually takes the form of a bone marrow transplantation.

In a recent interview with rapper LL Cool J on the Rock the Bells Radio show on SiriusXM, Tyson opened up on the effects the treatment has had on him.

Commenting on the mental aspect of training for a fight for the first time in 15 years, he said: "My mind wouldnt belong to me.

"My mind would belong to somebody that disliked me enough to break my soul, and I would give them my mind for that period of time.

"Six weeks of this and Id be in the best shape Ive ever dreamed of being in. As a matter of fact, Im going through that process right now. And you know what else I did, I did stem-cell research."

Tyson was then asked whether that meant if his white blood had been spun and then put back in, to which he replied: "Yes. As they took the blood it was red and when it came back it was almost transfluid (sic).

"I could almost see through the blood, and then they injected it in me.

"And Ive been weird ever since, Ive got to get balanced now."

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BACK IN GLOVEHolyfield CONFIRMS Tyson talks over epic trilogy with pair close to deal

IF LOOKS COULD KILLMayweather training because he is 'very conscious about how he looks'

GO WITH THE FLOMayweather 'passionate about becoming a trainer' despite comeback rumours

MAY DAYNew footage of Floyd's win vs McGregor makes legend look even more impressive

PUSH AND PULPulev plotting Fury fight after Joshua having already beat up cousin Hughie

WHAT IS STEM CELL TREATMENT USED FOR?

Stem cell transplants are carried out when bone marrow is damaged or isnt able to produce healthy blood cells.

It can also be used to replace damaged blood cells as the result of intensive cancer treatment.

Here are conditions that stem cell transplants can be used to treat:

Iron Mike has been called out by former rival Evander Holyfield to complete their trilogy following their two meetings in 1990s.

And his unusual methods for getting back in shape seem to be working.

Tyson is looking in incredible condition as he uploaded a clip of himself that showed off his ferocious power and speed.

See more here:
Mike Tyson reveals doctors injected him with translucent blood that left him feeling weird during stem cell - The Sun