Archive for the ‘Bone Marrow Stem Cells’ Category

DUBLIN--(BUSINESS WIRE)--The "Global Autologous Cell Therapy Market: Growth, Trends and Forecasts (2020-2025)" report has been added to ResearchAndMarkets.com's offering.

The Global Autologous Cell Therapy market is anticipated to grow at a CAGR of 15.9% during the forecast period.

The major factors attributing to the growth of the autologous cell therapy market are the rising incidence of chronic diseases such as autoimmune diseases, cancer, blood disorder, and others.

A rise in the population suffering from chronic diseases is also propelling the demand for market growth. In 2018, as per the AARDA (American Autoimmune Related Diseases Association) statistics, around 50 million Americans have an autoimmune disease, and this number is expected to rise in the future.

As per the CDC (Centers for Disease Control and Prevention) estimates Sickel Cell Disease (SCD) affects around 100,000 Americans annually - and there are few more factors which are playing crucial roles in taking the autologous cell therapy market to the next level, among them one is on-going drug developments for new applications which are expected to further propel the growth of the autologous cell therapy market.

Key Market Trends

Bone Marrow Segment Expected to Hold the Largest Market Share

Bone marrow transplant is a technique for replacing damaged and destroyed cells with new stem cells in the bone marrow. Bone marrow is the most commonly used for autologous cell therapy as it can benefit individuals with a range of cancer (malignant) and non-cancer (benign) diseases and will drive the market during the forecast period.

As per the statistics from Globocan 2018, worldwide 18,078,957 individuals have cancer. Asia remains the leading contributor in the rising incidence of cancer with a reported share of 48.4% followed by Europe, North and Latin America, Africa, and Oceania with a share of 23.4%, 13.2% and 7.8%, 5.8%, and 1.4% respectively.

North America Dominates the Market and is Expected to do Same Over the Forecast Period

North America is expected to dominate the overall autologous cell therapy market, throughout the forecast period. This is owing to factors such as the rising incidence of chronic diseases such as cancer, blood disorder, autoimmune diseases, and other diseases and the availability of advanced healthcare infrastructure among the major factors.

In North America, the United States holds the largest market share owing to the factors such as increasing number of population suffering from cancer and other chronic diseases, along with the rising geriatric population and developments related to stem cell therapy and rising demand for biotechnological practices in the country, is anticipated to further drive the demand in this region.

Competitive Landscape

The autologous cell therapy market is moderately competitive and consists of several major players. In terms of market share, few of the major players are currently dominating the market. And some prominent players are vigorously making acquisitions and joint ventures with the other companies to consolidate their market positions across the globe.

Some of the companies which are currently dominating the market are Vericel Corporation, Pharmicell Co. Inc., Holostem Terapie Avanzate S.r.l., Lineage Cell Therapeutics Inc., and Opexa Therapeutics.

Key Topics Covered

1 INTRODUCTION

1.1 Study Deliverables

1.2 Study Assumptions

1.3 Scope of the Study

2 RESEARCH METHODOLOGY

3 EXECUTIVE SUMMARY

4 MARKET DYNAMICS

4.1 Market Overview

4.2 Market Drivers

4.2.1 Rising Incidence of Chronic Diseases

4.2.2 Emphasis Increasingly on Drug Development for New Applications

4.3 Market Restraints

4.3.1 Systemic Immunological Reactions Possibility

4.3.2 Expensive Practise, Product and High Capital Investment

4.4 Porter's Five Force Analysis

5 MARKET SEGMENTATION

5.1 By Therapy

5.1.1 Autologous Stem Cell Therapy

5.1.2 Autologous Cellular Immunotherapies

5.2 By Application

5.2.1 Oncology

5.2.2 Musculoskeletal Disorder

5.2.3 Blood Disorder

5.2.4 Autoimmune Disease

5.2.5 Others

5.3 By Source

5.3.1 Bone Marrow

5.3.2 Epidermis

5.3.3 Others

5.4 By End User

5.4.1 Hospitals

5.4.2 Research Centers

5.4.3 Others

5.5 Geography

5.5.1 North America

5.5.2 Europe

5.5.3 Asia-Pacific

5.5.4 Middle-East and Africa

5.5.5 South America

6 COMPETITIVE LANDSCAPE

6.1 Company Profiles

6.1.1 Vericel Corporation

6.1.2 Pharmicell Co. Inc.

6.1.3 Holostem Terapie Avanzate S.r.l.

6.1.4 Lineage Cell Therapeutics, Inc.

6.1.5 Opexa Therapeutics

6.1.6 BrainStorm Cell Therapeutics

6.1.7 Sangamo Therapeutics

7 MARKET OPPORTUNITIES AND FUTURE TRENDS

For more information about this report visit https://www.researchandmarkets.com/r/gydkh

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World Autologous Cell Therapy Industry 2020-2025 with Vericel, Pharmicell, Holostem Terapie Avanzate, Lineage Cell Therapeutics and Opexa Therapeutics...

New Jersey, United States,- Verified Market Researchhas recently published an extensive report on the Stem Cell Therapy Market to its ever-expanding research database. The report provides an in-depth analysis of the market size, growth, and share of the Stem Cell Therapy Market and the leading companies associated with it. The report also discusses technologies, product developments, key trends, market drivers and restraints, challenges, and opportunities. It provides an accurate forecast until 2027. The research report is examined and validated by industry professionals and experts.

The report also explores the impact of the COVID-19 pandemic on the segments of the Stem Cell Therapy market and its global scenario. The report analyzes the changing dynamics of the market owing to the pandemic and subsequent regulatory policies and social restrictions. The report also analyses the present and future impact of the pandemic and provides an insight into the post-COVID-19 scenario of the market.

Global Stem Cell Therapy Market was valued at USD 117.66 million in 2019 and is projected to reach USD 255.37 million by 2027, growing at a CAGR of 10.97% from 2020 to 2027.

The report further studies potential alliances such as mergers, acquisitions, joint ventures, product launches, collaborations, and partnerships of the key players and new entrants. The report also studies any development in products, R&D advancements, manufacturing updates, and product research undertaken by the companies.

Leading Key players of Stem Cell Therapy Market are:

Competitive Landscape of the Stem Cell Therapy Market:

The market for the Stem Cell Therapy industry is extremely competitive, with several major players and small scale industries. Adoption of advanced technology and development in production are expected to play a vital role in the growth of the industry. The report also covers their mergers and acquisitions, collaborations, joint ventures, partnerships, product launches, and agreements undertaken in order to gain a substantial market size and a global position.

1.Stem Cell Therapy Market, By Cell Source:

Adipose Tissue-Derived Mesenchymal Stem Cells Bone Marrow-Derived Mesenchymal Stem Cells Cord Blood/Embryonic Stem Cells Other Cell Sources

2.Stem Cell Therapy Market, By Therapeutic Application:

Musculoskeletal Disorders Wounds and Injuries Cardiovascular Diseases Surgeries Gastrointestinal Diseases Other Applications

3.Stem Cell Therapy Market, By Type:

Allogeneic Stem Cell Therapy Market, By Application Musculoskeletal Disorders Wounds and Injuries Surgeries Acute Graft-Versus-Host Disease (AGVHD) Other Applications Autologous Stem Cell Therapy Market, By Application Cardiovascular Diseases Wounds and Injuries Gastrointestinal Diseases Other Applications

Regional Analysis of Stem Cell Therapy Market:

A brief overview of the regional landscape:

From a geographical perspective, the Stem Cell Therapy Market is partitioned into

North Americao U.S.o Canadao MexicoEuropeo Germanyo UKo Franceo Rest of EuropeAsia Pacifico Chinao Japano Indiao Rest of Asia PacificRest of the World

Key coverage of the report:

Other important inclusions in Stem Cell Therapy Market:

About us:

Verified Market Research is a leading Global Research and Consulting firm servicing over 5000+ customers. Verified Market Research provides advanced analytical research solutions while offering information enriched research studies. We offer insight into strategic and growth analyses, Data necessary to achieve corporate goals, and critical revenue decisions.

Our 250 Analysts and SMEs offer a high level of expertise in data collection and governance use industrial techniques to collect and analyze data on more than 15,000 high impact and niche markets. Our analysts are trained to combine modern data collection techniques, superior research methodology, expertise, and years of collective experience to produce informative and accurate research.

Contact us:

Mr. Edwyne Fernandes

US: +1 (650)-781-4080UK: +44 (203)-411-9686APAC: +91 (902)-863-5784US Toll-Free: +1 (800)-7821768

Email: [emailprotected]

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Stem Cell Therapy Market Size by Top Companies, Regions, Types and Application, End Users and Forecast to 2027 - Bulletin Line

For many patients with multiple myeloma, a new generation of drugs and drug combinations is producing better outcomes and fewer side effects. In recent months, several novel therapies studied and tested by Dana-Farber scientists have gained approval from the U.S. Food and Drug Administration (FDA) or taken a step toward approval after posting solid results in clinical trials.

The drugs are the fruit of years of research into improving treatment for multiple myeloma, a cancer of white blood cells known as plasma cells in the bone marrow. Many of the new agents are biologically derived made from substances such as proteins and antibodies found in living things and target biological mechanisms in a very specific, targeted fashion. Dana-Farber researchers have played a key role in these efforts.

These are each powerful examples of how next-generation novel therapies translated here at Dana-Farber from bench to bedside are further improving outcomes for our patients, and at a remarkable pace, says Paul G. Richardson, MD, clinical program leader and director of clinical research at the Jerome Lipper Multiple Myeloma Center at Dana-Farber.

Option for relapsed or refractory (non-responsive) myeloma

Following a Dana-Farber-led clinical trial, the FDA recently approved the novel drug isatuximab in combination with pomalidomide and dexamethasone for adults with relapsed or refractory (non-responsive) myeloma who have received at least two prior therapies, including lenalidomide and drugs known as proteasome inhibitors. The drug went into trials after laboratory work by Dana-Farbers Yu-Tzu Tai, PhD, and Kenneth Anderson, MD, showed it was active against myeloma cells. In the clinical trial, the three-drug combination lowered the risk that the disease would progress by 40%, compared to pomalidome and dexamethasone alone.

A drug that doesnt cause hair loss

Dana-Farber investigators conducted laboratory research and led the first clinical trial of the drug melflufen plus dexamethasone in patients with relapsed or refractory myeloma. Melflufen is a peptide conjugate drug made of a stub of protein, or peptide, joined to a chemotherapy agent and delivers a toxic payload directly to myeloma cells in a selective, time-sparing approach.

Results from an early-phase clinical trial published in Lancet Oncology showed the drug is active in patients with myeloma and is safe at recommended doses. Unlike the previously used standard drug melphalan, it doesnt cause mucositis inflammation of membranes within the digestive tract or hair loss. The results prompted investigators to launch two larger trials, some of whose results are being processed and are due to be published soon.

Drug for patients eligible for stem cell transplant

In a major study published in Blood, Dana-Farber researchers and their associates found that in patients newly diagnosed with myeloma who are eligible for a stem cell transplant, adding the drug daratumumab to the standard three-drug regimen produced more responses, and deeper responses, than in patients receiving the three-drug therapy alone.

Targeting myeloma cells and cell division

Dana-Farber researchers were involved in the development and initial testing of the drug belantamab mafodotin, which has shown considerable promise in clinical trials and has been granted priority review for approval by the FDA.

An antibody conjugate drug consisting of an antibody that specifically targets myeloma cells and an agent that disrupts cell division, its use was informed by a preclinical trial at Dana-Farber involving Yu-Tzu Tai, PhD, and Kenneth Anderson, MD. Balantamab mafodotin was tested in studies led by Paul Richardson, MD, in patients with relapsed or refractory multiple myeloma whose disease continued to worsen after a stem cell transplant, chemotherapy, or other treatment. In the DREAMM-1 and -2 trials, the drug showed strong anti-myeloma activity with manageable side effects.

This article was originally published on August 4, 2020, by Dana-Farber Cancer Institute. It is republished with permission.

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Wave of New Therapies Improve Outcomes for Patients with Multiple Myeloma - Cancer Health Treatment News

New York, Aug. 13, 2020 (GLOBE NEWSWIRE) -- Reportlinker.com announces the release of the report "Cell Isolation/Cell Separation Market Research Report by Product, by Cell Type, by Cell Source, by Technique, by Application, by End User - Global Forecast to 2025 - Cumulative Impact of COVID-19" - https://www.reportlinker.com/p05913776/?utm_source=GNW

The Global Cell Isolation/Cell Separation Market is expected to grow from USD 6,356.88 Million in 2019 to USD 14,485.68 Million by the end of 2025 at a Compound Annual Growth Rate (CAGR) of 14.71%.

Market Segmentation & Coverage:This research report categorizes the Cell Isolation/Cell Separation to forecast the revenues and analyze the trends in each of the following sub-markets:

Based on Product, the Cell Isolation/Cell Separation Market studied across Consumables and Instruments. The Consumables further studied across Beads, Disposables, and Reagents, Kits, Media, and Sera. The Instruments further studied across Centrifuges, Filtration Systems, Flow Cytometers, and Magnetic-Activated Cell Separator Systems.

Based on Cell Type, the Cell Isolation/Cell Separation Market studied across Animal Cells and Human Cells. The Human Cells further studied across Differentiated Cells and Stem Cells.

Based on Cell Source, the Cell Isolation/Cell Separation Market studied across Adipose Tissue, Bone Marrow, and Cord Blood/Embryonic Stem Cells.

Based on Technique, the Cell Isolation/Cell Separation Market studied across Centrifugation-Based Cell Isolation, Filtration-Based Cell Isolation, and Surface Marker-Based Cell Isolation.

Based on Application, the Cell Isolation/Cell Separation Market studied across Biomolecule Isolation, Cancer Research, In Vitro Diagnostics, Stem Cell Research, and Tissue Regeneration & Regenerative Medicine.

Based on End User, the Cell Isolation/Cell Separation Market studied across Biotechnology & Biopharmaceutical Companies, Hospitals & Diagnostic Laboratories, and Research Laboratories & Institutes.

Based on Geography, the Cell Isolation/Cell Separation Market studied across Americas, Asia-Pacific, and Europe, Middle East & Africa. The Americas region surveyed across Argentina, Brazil, Canada, Mexico, and United States. The Asia-Pacific region surveyed across Australia, China, India, Indonesia, Japan, Malaysia, Philippines, South Korea, and Thailand. The Europe, Middle East & Africa region surveyed across France, Germany, Italy, Netherlands, Qatar, Russia, Saudi Arabia, South Africa, Spain, United Arab Emirates, and United Kingdom.

Company Usability Profiles:The report deeply explores the recent significant developments by the leading vendors and innovation profiles in the Global Cell Isolation/Cell Separation Market including Beckman Coulter Inc. (Subsidiary of Danaher Corporation), Becton, Dickinson and Company, Bio-Rad Laboratories, Inc., GE Healthcare, Merck KGaA, Miltenyi Biotec, Pluriselect Life Science Ug (Haftungsbeschrnkt) & Co. Kg, Stemcell Technologies, Inc., Terumo Bct, and Thermo Fisher Scientific, Inc..

FPNV Positioning Matrix:The FPNV Positioning Matrix evaluates and categorizes the vendors in the Cell Isolation/Cell Separation Market on the basis of Business Strategy (Business Growth, Industry Coverage, Financial Viability, and Channel Support) and Product Satisfaction (Value for Money, Ease of Use, Product Features, and Customer Support) that aids businesses in better decision making and understanding the competitive landscape.

Competitive Strategic Window:The Competitive Strategic Window analyses the competitive landscape in terms of markets, applications, and geographies. The Competitive Strategic Window helps the vendor define an alignment or fit between their capabilities and opportunities for future growth prospects. During a forecast period, it defines the optimal or favorable fit for the vendors to adopt successive merger and acquisition strategies, geography expansion, research & development, and new product introduction strategies to execute further business expansion and growth.

Cumulative Impact of COVID-19:COVID-19 is an incomparable global public health emergency that has affected almost every industry, so for and, the long-term effects projected to impact the industry growth during the forecast period. Our ongoing research amplifies our research framework to ensure the inclusion of underlaying COVID-19 issues and potential paths forward. The report is delivering insights on COVID-19 considering the changes in consumer behavior and demand, purchasing patterns, re-routing of the supply chain, dynamics of current market forces, and the significant interventions of governments. The updated study provides insights, analysis, estimations, and forecast, considering the COVID-19 impact on the market.

The report provides insights on the following pointers:1. Market Penetration: Provides comprehensive information on the market offered by the key players2. Market Development: Provides in-depth information about lucrative emerging markets and analyzes the markets3. Market Diversification: Provides detailed information about new product launches, untapped geographies, recent developments, and investments4. Competitive Assessment & Intelligence: Provides an exhaustive assessment of market shares, strategies, products, and manufacturing capabilities of the leading players5. Product Development & Innovation: Provides intelligent insights on future technologies, R&D activities, and new product developments

The report answers questions such as:1. What is the market size and forecast of the Global Cell Isolation/Cell Separation Market?2. What are the inhibiting factors and impact of COVID-19 shaping the Global Cell Isolation/Cell Separation Market during the forecast period?3. Which are the products/segments/applications/areas to invest in over the forecast period in the Global Cell Isolation/Cell Separation Market?4. What is the competitive strategic window for opportunities in the Global Cell Isolation/Cell Separation Market?5. What are the technology trends and regulatory frameworks in the Global Cell Isolation/Cell Separation Market?6. What are the modes and strategic moves considered suitable for entering the Global Cell Isolation/Cell Separation Market?Read the full report: https://www.reportlinker.com/p05913776/?utm_source=GNW

About ReportlinkerReportLinker is an award-winning market research solution. Reportlinker finds and organizes the latest industry data so you get all the market research you need - instantly, in one place.

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Cell Isolation/Cell Separation Market Research Report by Product, by Cell Type, by Cell Source, by Technique, by Application, by End User - Global...

Use of a novel anti-CD30 CAR T-cell therapy following treatment with fludarabine-based lymphodepletion induced a high rate of durable responses in patients with heavily pretreated relapsed or refractory Hodgkin lymphoma, according to data published in Journal of Clinical Oncology.

Results from the parallel phase 1 and phase 2 studies also demonstrated that the CAR T-cell therapy was safe and did not produce any serious or severe side effects.

Researchers from the UNC Lineberger Comprehensive Cancer Center and Baylor College of Medicine administered anti-CD30 CAR T cells to 41 patients with relapsed or refractory Hodgkin lymphoma. All patients underwent lymphodepletion with bendamustine alone, bendamustine and fludarabine, or cyclophosphamide and fludarabine prior to the anti-CD30 CAR T-cell therapy.

Measuring safety was the primary goal of the two parallel studies.

The overall response rate, or the percentage of partial or complete responses to therapy, among 37 evaluable patients was 62%. Thirty-four of the patients received fludarabine-based lymphodepletion 17 of which received it with bendamustine, and the other half received it with cyclophosphamide. Two of these patients were considered to be complete response at infusion and maintained the response, so they were not included in final analysis.

The overall response rate among the remaining patients was 72%, with 59% of patients achieving a complete response. After a median follow-up of 533 days, researchers identified the one-year progression free survival rate to be 36% and the one-year overall survival rate to be 94%.

This is particularly exciting because the majority of these patients had lymphomas that had not responded well to other powerful new therapies, said senior study author Dr. Barbara Savoldo, professor in the Department of Microbiology and Immunology at the UNC School of Medicine, in a press release.

Patients within the study had received a median of seven previous lines of therapy that included checkpoint inhibitors and autologous or allogeneic stem cell therapies, therapies known to be powerful but also tend to come with a host of side effects.

However, treatment with the anti-CD30 CART cells demonstrated a favorable safety profile. Although 10 patients developed cytokine release syndrome, all cases were considered minor.

Patients who received fludarabine-containing lymphodepletion were the only participants in the study to have a response to the anti-CD30 CAR T-cell therapy.

Although CD30 CAR T (cells) showed modest activity in (Hodgkin lymphoma) when infused without lymphodepletion, robust clinical responses were achieved when these cells were infused in hosts lymphodepleted with fludarabine-containing regimens, the authors wrote.

The activity of this new therapy is quite remarkable and while we need to confirm these findings in a larger study, this treatment potentially offers a new approach for patients who currently have very limited options to treat their cancer, said Dr. Jonathan Serody, director of the bone marrow transplant and cellular therapy program at UNC Lineberger Comprehensive Cancer Center, in the release. Additionally, unlike other CAR T-cell therapies, clinical success was not associated with significant complications from therapy. This means this treatment should be available to patients in a clinic setting and would not require patients to be hospitalized, which is critical in our current environment.

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Novel CAR T-Cell Therapy Shows Promise in Advanced Hodgkin Lymphoma - Curetoday.com

INTRODUCTION

In recent years, a number of growth factors have been tested in clinical trials for a variety of therapeutic applications including bone regeneration and neovascularization of ischemic tissues. Despite early promising results, the results obtained in larger phase 2 trials have often not shown the expected benefit to patients (1, 2), with some having marked adverse effects (35). The Infuse bone graft, which consists of recombinant human bone morphogenetic protein-2 (rhBMP-2) soaked onto a collagen sponge at a dosage of 1.5 mg/ml, has received Food and Drug Administration approval for certain spinal, dental, and trauma indications and is in widespread clinical use. However, major complications and adverse effects have increasingly been attributed because of the off-label use of the product (3, 4). Clinically, the current delivery vehicle for BMP-2 is a collagen powder or sponge that has been shown to result in a large initial burst release, which contrasts with the expression profile observed during normal fracture repair where BMP expression increases until day 21, suggesting a need for slower and more sustained growth factor release profile (6, 7). Furthermore, because of the short half-life of the growth factor and the harsh fracture environment (5), supraphysiological dosages of BMP-2 are being delivered to elicit bone regeneration, which has been linked to adverse effects such as heterotopic ossification. Therefore, there is a clear clinical need to develop alternative strategies to deliver single or multiple growth factors to the site of injury with sustainable and physiologically relevant dosages such that repair is induced without these adverse effects.

A number of growth factors have been shown to be expressed at different phases of fracture healing, including vascular endothelial growth factor (VEGF) and BMPs. The coupled relationship in bone healing, both physical and biochemical, between blood vessels and bone cells has long been recognized (8, 9). During fracture healing, VEGF is released directly after injury and predominately drives the formation of the fracture hematoma (9). Inhibition of VEGF has been shown to disrupt the repair of fractures and large bone defects (1012). Despite this, VEGF delivery alone is often not sufficient to heal critically sized bone defects, which may be due to suboptimal dosing or the timing of VEGF release. Furthermore, VEGF does not appear to drive progenitor cell differentiation toward the chondrogenic or osteogenic lineage; therefore, combination therapies with BMPs have been developed in an attempt to accelerate the regeneration of large bone defects (9, 1318). During normal fracture healing, VEGF expression peaks around day 5/10 (19, 20) and then decreases, whereas BMP-2 expression increases constantly until day 21, suggesting a need for delivery systems that support the early release of VEGF and the sustained release of BMP-2 (6, 7, 19, 20). To this end, composite polymer systems have been used to deliver VEGF and BMP-2 in a sequential fashion (1518). The timed release of VEGF/BMP-2 was found to enhance ectopic bone formation (1618); however, in an orthotopic defect, no significant benefit was observed (17, 18). This may be due to the high dose of VEGF used in these studies (18), which has previously been shown to disrupt osteogenesis as a result of abnormal angiogenesis and vascular structure (8), or due to suboptimal growth factor release profiles from these constructs. This suggests that novel strategies are required for delivering low-dosage VEGF and BMP-2, with tight temporal control, to enhance vascularization and subsequent bone formation in orthotopic defects. Nanoparticles such as hydroxyapatite (HA) and laponite are known to be osteoinductive and have previously been shown to facilitate the adsorption and immobilization of proteins such as VEGF and BMP-2 because of the strong attraction between the nanoparticles and the growth factor (2123). This motivates the integration of these nanoparticles into regenerative implants to enable tight temporal control over the rate at which encapsulated growth factors are released into damaged tissue.

Processes such as angiogenesis are regulated not only by the temporal presentation of growth factors but also by spatial gradients of morphogens that regulate chemotactic cell migration. Using microfluidic devices (24, 25) or three-dimensional (3D) culture models (26, 27), it has been demonstrated that endothelial cell migration is mediated by gradients in VEGF. However, it is unclear whether incorporating gradients of VEGF into tissue-engineered scaffolds will enhance angiogenesis in vivo. Here, we used emerging multiple-tool biofabrication techniques (28) to deliver VEGF and BMP-2 with distinct spatiotemporal release profiles to enhance the regeneration of critically sized bone defects. To tune the temporal release of these morphogens from 3D printed constructs, we functionalized alginate-based bioinks with different nanoparticles known to bind these regulatory factors. Both the spatial position and temporal release of growth factor from the 3D printed implant determined its therapeutic potential. By slowing the release of BMP-2, it was possible to enhance bone formation in vivo within predefined positions of the implant. Furthermore, introducing spatial gradients of VEGF into 3D printed implants enhanced vascularization in vivo compared to controls homogenously loaded with the same total amount of growth factor. We also demonstrate accelerated large bone defect healing, with minimal ectopic bone formation, using 3D printed implants containing a spatial gradient of VEGF and spatially localized BMP-2.

To produce a printable bioink, various weight concentrations of methylcellulose were first added to RGD -irradiated alginate. Print fidelity (as measured by the filament spreading ratio) improved by increasing the methylcellulose content [see fig. S1 (A and B)]; however, the capacity to print multiple layers of material worsens because of the overly adhesive nature of the ink. For these reasons, a weight concentration of 2:1 (w/w) alginate to methylcellulose was chosen for all bioinks, as it substantially increased the print fidelity while allowing multiple layers of material to be accurately deposited.

To tune the temporal release profile of growth factor (here, VEGF), clay nanoparticles (22, 23, 29) or hydroxyapatite nanoparticles (nHA) (21) were added to the alginate-methylcellulose bioink. Adding methylcellulose to the alginate to produce a printable ink significantly increased the release of VEGF compared to that observed from alginate only [see fig. S1 (C and D)]. The addition of laponite, a clay-based nanoparticle, markedly slowed the release of VEGF (see fig. S1C), while the incorporation of nHA only had a small effect on growth factor release, producing a slightly more gradual release profile (see fig. S1D). This blend (alginate, methylcellulose, and nHA) will hereafter be referred to as the vascular bioink, as it allowed for the near complete release of VEGF over 10 days, mimicking that observed during normal fracture healing (19, 20). No laponite was included in this vascular bioink.

To demonstrate the utility of this vascular bioink, two strategies were compared to print implants containing a spatial gradient of VEGF (see fig. S1E). In the first, VEGF (100 ng/ml) was printed into the central 5-mm core of constructs 8 mm in diameter and 4 mm high, with a VEGF-free bioink used to print the periphery of the construct. In the second, VEGF (80 ng/ml) was printed into the center of the construct, and VEGF (20 ng/ml) was printed around the periphery of the implant. Control constructs containing a homogenous distribution of VEGF were also printed. One hour after printing, clear spatial differences in VEGF localization were observed in both gradient constructs, while roughly the same amount of protein was detected in the core and periphery of the homogenous VEGF control (see fig. S1F). Fourteen days after printing, a spatial gradient still existed in the construct that initially had all VEGF loaded into its central region, with no gradient observed in the other groups (see fig. S1G). This demonstrates that spatial gradients of growth factor can be maintained within constructs for at least 14 days after printing.

We next sought to assess whether depositing spatial gradients of VEGF within 3D printed polycaprolactone (PCL) implants would accelerate vascularization of the constructs in vivo. To this end, Homogenous VEGF, Gradient VEGF, and No VEGF constructs were implanted subcutaneously in the back of mice (see Fig. 1A), where the total amount of growth factor (25 ng) within the two VEGF-containing implants was constant. Two weeks after implantation, histological analysis of hematoxylin and eosin (H&E)stained samples revealed the presence of vessels in the Homogenous VEGF and Gradient VEGF groups; however, there were no obvious vessels present in the No VEGF group (see Fig. 1B). These vessels appeared mature, complete with smooth muscle actin (-SMA) and von Willebrand factor (vWF)stained walls and perfused with erythrocytes (see fig. S2A). The Homogenous VEGF constructs had vessels predominantly located in the periphery of the scaffold, with little to none present within the center of the scaffold. On the other hand, vessels were present both in the periphery and in the center of the Gradient VEGF group. Four weeks after implantation, all three experimental groups had mature vessels present (see Fig. 1C and fig. S2B). Similar to the Homogeneous VEGF group, the No VEGF group had vessels predominantly located in the periphery of the constructs, with little to none present within the center of the construct. When quantified, at both 2 and 4 weeks, there were significantly more vessels present in the Gradient VEGF group compared to both the Homogenous VEGF and No VEGF group (see Fig. 1D). There was significantly more vessels present in the periphery of the Gradient VEGF constructs at both 2 and 4 weeks in vivo compared to the other two experimental groups [see Fig. 1 (E and F)]. There was also a trend toward a larger number of vessels present in the center of the Gradient VEGF construct at 4 weeks compared to No VEGF (P = 0.09) and Homogenous VEGF (P = 0.1) groups (see Fig. 1F).

(A) Schematic of the 3D printed scaffold and experimental groups. Construct design (4 mm in diameter, 5 mm in height). H&E-stained sections of the three experimental groups at (B) 2 and (C) 4 weeks in vivo. Images were taken at 20. Arrows denote vessels. (D) Total number of vessels of the experimental groups at 2 and 4 weeks in vivo. Number of vessels present in the center versus the periphery at (E) 2 and (F) 4 weeks in vivo. **P < 0.01. Error bars denote SDs (n = 8 animals; n = 5 slices per animal). FBS, fetal bovine serum; pen/strep, penicillin/streptomycin.

Recognizing that a slower and more sustained release of BMP-2 could be beneficial for promoting osteogenesis (6, 7), we next sought to compare bone formation in vivo within implants with temporally distinct growth factor release profiles. To the base alginate-methylcellulose bioink (here termed the Fast BMP-2 Release bioink), laponite at varying w/w ratios of laponite to alginate were compared to determine the optimum ratio to generate a Slow BMP-2 Release bioink (see fig. S3). As there was little difference in the growth factor release profile from the different groups, a 6:1 alginate:laponite w/w ratio was chosen to minimize the amount of laponite in the bioink. The addition of laponite markedly slowed the in vitro release of BMP-2 from the bioink, resulting in a reasonable constant release of growth factor from day 7 to day 35 (see Fig. 2C). The addition of laponite also had no significant effect on the degradation rate of the bioink (Fig. 2B).

(A) Schematic of the experimental groups. Construct design (4 mm in diameter, 5 mm in height). MEM, alpha minimum essential medium. (B) Degradation of the two bioinks. (C) Cumulative release of BMP-2 of the fast release bioink versus the slow release bioink. (D) 3D reconstructions of the CT data for each group at 8 weeks. (E) CT analysis on total mineral deposition of each of the groups after 8 weeks in vivo. (F) CT analysis on the location of mineral deposition of each of the groups after 8 weeks in vivo. ***P < 0.001; error bars denote SDs (n = 8 animals). (G) Goldners trichromestained sections of both groups after 8 weeks in vivo. Images were taken at 20. White arrows denote developing bone tissue, and black arrows denote blood vessels. (H) Quantification of the amount of new bone formation per total area. Error bars denote SDs; **P < 0.01 (n = 8 animals, n = 6 slices per animal).

To assess whether slow and sustained release of BMP-2 would enhance ectopic bone formation in vivo, Fast BMP-2 Release (laponite) and Slow BMP-2 Release (+laponite) bioinks were mixed with bone marrowderived mesenchymal stem cells (BMSCs), deposited within 3D printed scaffolds, and then implanted subcutaneously in the back of mice (see Fig. 2A). Seeding these bioinks with MSCs was used to test their potential for promoting osteogenesis in an ectopic location. BMP-2 was specifically localized around the periphery of the implant. This pattern of growth factor presentation was chosen to test the capacity of the printed implants to spatially localize bone formation in vivo (note that the geometry of the implant is the same as that which will be used in the segmental defect study below, with the BMP-2 localized to the periphery of the implant such that bone would only form along the cortical shaft of the damaged limb rather than throughout). Eight weeks after implantation, there was significantly more mineral within the Slow BMP-2 Release group compared to the Fast BMP-2 Release group [see Fig. 2 (D and E)]. Microcomputed tomography (CT) reconstructions revealed that the mineral was preferentially deposited around the periphery of the constructs where the BMP-2 was localized [see Fig. 2 (D and F)]. Histological staining further verified this finding, with positive staining for new bone seen predominantly in the periphery of both groups (see Fig. 2G, denoted by white arrows). Quantification revealed that the Slow BMP-2 Release constructs had significantly more new bone formation per total area of construct (see Fig. 2H).

We next sought to assess whether the delayed release of BMP-2 from printed constructs containing spatial gradients in VEGF would enhance angiogenesis and bone formation within critically sized bone defects. To this end, VEGF gradient only, BMP-2 gradient only, and Composite (VEGF+BMP-2 gradient) constructs were printed and implanted in a 5-mm rat femoral defect (see Fig. 3A) and compared to an empty defect.

(A) Schematic of the 3D printed experimental groups including key features of the developed bioinks and the segmental defect procedure. Construct design (4 mm in diameter, 5 mm in height). (B) CT angiography representative images of vessel diameter. Red arrows denote leaky blood vessels denoted by pools of contrast agent. Quantification on (C) total vessel volume, (D) average vessel diameter, and (E) connectivity for all groups after 2 weeks in vivo. *P < 0.05 and **P < 0.01; error bars denote SDs (n = 9 animals). (F) Immunohistochemical staining of nuclei (blue), vWF (red), and SMA (green) of the experimental groups at 2 weeks after implantation. Images were taken at 40 and 63. Yellow arrows denote vessels with SMA and vWF dual staining; white arrows denote slightly less mature vessels with only vWF positive staining.

Two weeks after implantation, CT angiography was used to quantify and visualize the early vascular network that had formed within the defect site. 3D reconstructions revealed that vascular networks had formed in all four experimental groups (see Fig. 3B). When quantified, there was a significant increase in vessel volume in the Composite group compared to the VEGF gradient group (see Fig. 3C). There was also a significant increase in average vessel thickness in the BMP-2 gradient and Composite groups compared to the VEGF gradient group (see Fig. 3D). Although there was no significant difference in the connectivity of the vessels, there was a trend (P = 0.1) toward increased connectivity in the Composite group compared to the VEGF gradient group (see Fig. 3E). 3D reconstructions also revealed the presence of primitive immature blood vessels depicted by large globules of contrast agent (denoted by the red arrows in Fig. 3B). There appeared to be fewer primitive blood vessels present in the Composite group than the other three experimental groups. This was further verified by SMA and vWF staining, which revealed a larger number of vessels with only positive vWF-stained walls in the Empty and VEGF gradient groups (see Fig. 3F, denoted by white arrows). On the other hand, there were predominately more mature vessels with SMA and vWF-stained walls in both the BMP-2 gradient and Composite groups (see Fig. 3F, denoted by yellow arrows). Note that the differences in angiogenesis seen between the VEGF gradient and Composite groups (same amount of VEGF in both groups) could at least partially be explained by looking at the VEGF release profile from both groups (see fig. S4). The addition of the osteoinductive ink around the implant periphery significantly reduced the VEGF release rate from construct into the media, with a more linear release of growth factor over time.

Two weeks after surgery, defects within the Empty group were filled with a fibrous tissue (see Fig. 4A). In contrast, positive staining for cartilage and new bone deposition was observed in the BMP-2 gradient and Composite groups, suggesting that new bone was forming at least partially via endochondral ossification. When quantified, there was a trend toward increased cartilage development (red staining in Safranin O images) in both the BMP-2 gradient (P = 0.12) and Composite (P = 0.18) groups compared to the Empty (see Fig. 4B). No significant differences in bone formation was observed between any of the groups at week 2; however, the CT reconstructions showed mineralized calluses beginning to form in the BMP-2 gradient and Composite groups, which was less evident in the Empty and VEGF gradient groups [see Fig. 4 (C and D)].

(A) H&E- and Safranin Ostained sections of all groups after 2 weeks in vivo. Images were taken at 20. DB denotes cartilage undergoing endochondral ossification to become developing bone, and B denotes positive new bone tissue. Quantification of the amount of (B) bone formation and (C) developing bone per total area. Error bars denote SDs (n = 9 animals). (D) CT reconstructed images of the defect site.

Next, CT analysis was used to visualize and quantify bone formation within the defects at 4, 8, 10, and 12 weeks after implantation. Compared to the Empty group, there were significantly higher levels of new bone formation in the Composite group as early as 8 weeks after implantation [see Fig. 5 (A and B)]. A consistent pattern of healing was observed in the Composite group, with bone forming down through the PCL scaffold framework (see Fig. 5A and fig. S5). After 10 weeks of implantation, significantly higher levels of bone formation was observed in the BMP-2 gradient and Composite groups compared to the Empty group. By 12 weeks, all three experimental groups contained significantly higher levels of new bone compared to the Empty group. Twelve weeks after implantation, bone density mapping revealed that the new bone formed in the experimental groups consisted of a dense cortical-like bone present around the periphery of defect, comparable to the adjacent native bone (1200 mg HA/cm3) (see Fig. 5C). Quantitative densitometry analysis revealed no significant difference in the average density (mg HA/cm3) of the new bone that did form between any of the groups over the 12 weeks (see Fig. 5D).

(A) Reconstructed in vivo CT analysis of bone formation in the defects. (B) Quantification of total bone volume (mm3) in the defects at each time point. (C) Representative images of CT bone densities in the defects at 12 weeks halfway through the defect (scale bar, 1 mm throughout). (D) Average bone density (mg HA/cm3) in the defects at each time point. (E) Outline of ROI bone volume analysis including definitions of core, annulus, and heterotopic regions. (F) Total bone volume (mm3) in each region at 12 weeks. **P < 0.01, ***P < 0.001, and ****P < 0.0001; error bars denote SDs (n = 9 animals).

To assess the levels of heterotopic bone formation, region of interest (ROI) bone volume analysis was performed on the week 12 reconstructions. The total bone volume was quantified in the core, annulus, and heterotopic regions of the defect (see Fig. 5E). In all three experimental groups, bone preferentially formed in the annulus of the defect, with little ectopic bone formation (see Fig. 5F). All three experimental groups had significantly higher total bone volume in the annulus of the defect compared to the Empty annulus, with the highest total bone volume present in the Composite group.

We next sought to assess the nature of new bone tissue being formed using histological staining. Goldners trichrome staining revealed predominantly fibrous tissue formation, similar to what was seen previously at 2 weeks, in the Empty group (see Fig. 6A). There was positive staining for new bone, complete with marrow cavities, in all three experimental groups at 12 weeks after implantation. When quantified, there was significantly more bone found in all three experimental groups compared to the Empty group (see Fig. 6B). There were also significantly higher amounts of bone marrow present in the Composite group compared to the Empty group (see Fig. 6C). As observed in the CT 3D reconstructions, it is clear that the bone is forming down through the PCL scaffold framework, specifically in the Composite group. Safranin O staining revealed the presence of cartilage in all three experimental groups after 12 weeks, demonstrating that bone is continuing to develop via endochondral ossification. When quantified, there was significantly more cartilage present in the Composite group compared to all other groups at this time point (see Fig. 6D).

(A) Goldners trichrome and Safranin Ostained sections of all groups after 12 weeks in vivo. Images were taken at 20. BM denotes bone marrow. PCL denotes areas where the PCL frame was. DB denotes cartilage undergoing endochondral ossification to become new bone, and B denotes positive bone tissue. Quantification of the amount of (B) bone formation, (C) bone marrow, and (D) developing bone per total area. Error bars denote SDs. *P < 0.05, **P < 0.01, and ****P < 0.0001 (n = 9 animals).

Despite the tremendous potential of growth factor delivery, the results obtained in larger clinical trials have not always shown the expected benefit to patients (2), with some studies reporting marked adverse effects (35). The reasons for this are multifaceted, from the delivery methods to the supraphysiological dosages needed to elicit a therapeutic effect and the costs and adverse effects attached to these high doses. This study presents a novel alternative approach for spatiotemporally controlled delivery of growth factors. We developed a range of nanoparticle-functionalized bioinks to precisely control the temporal release of growth factors from 3D printed implants. Using multiple tool biofabrication techniques, we were able to print constructs containing spatiotemporal gradients of growth factors, which allowed for controlled tissue regeneration without the need for supraphysiological dosages. Specifically, the appropriate patterning of VEGF enhanced angiogenesis in vivo and, when coupled with defined BMP-2 localization and release kinetics, enhanced large bone defect healing with little heterotopic bone formation.

Alginate hydrogels are commonly used for bone tissue engineering, with a number of studies demonstrating the bone regeneration potential of RGD functionalized and -irradiated alginate (3033), making it a promising base bioink for the 3D bioprinting of osteogenic implants. However, one drawback to using RGD -irradiated alginate as a bioink is its low viscosity. It is imperative when printing multilayered structures that the bioink have appropriate rheological properties to prevent collapsing or sagging of the printed structure. The addition of methylcellulose to alginate-based bioinks was found to have a significant effect on both printability and the rate of growth factor release. The addition of methylcellulose has previously been shown to substantially increase the print fidelity of an alginate base bioink (22, 34, 35), although typically using higher concentrations than the one used in this study. Adding methylcellulose also accelerated the rate of growth factor release. This was previously seen with albumin release from alginate-methylcellulose beads (36). Such a polymeric network is at least partially defined by physical entanglements between the alginate or methylcellulose chains. As methylcellulose is characterized by high swellability, when the alginate/methylcellulose bioink is exposed to the medium, it swells rapidly, resulting in accelerated growth factor release from the bioink. The addition of methylcellulose may also have neutralized the charge on the alginate, which would also influence growth factor release kinetics. In contrast, the addition of nanoparticles, and, in particular, laponite, slowed the release of growth factor from the inks. Both nHA and laponite have previously been shown to facilitate with the adsorption and immobilization of VEGF within a hydrogel due to the strong attraction between the nanoparticles and the growth factor (2123). The stronger association between growth factors and laponite can be linked to the physiochemical properties of these particles (22, 29). These disc-shaped particles [typically 25 nm in diameter and 1 nm in thickness (37)] are characterized by a highly negatively charged face and a positively charged rim (22), with a zeta potential of 61 mV (as determined by the manufacturer). This allowed the positively charged growth factors such as VEGF to form strong electrostatic bonds with the negatively charged face of the nanoparticles (22). In contrast, the nHA nanoparticles used in this study, which we have previously shown to have a zeta potential of around 5 mV (38), would form a slightly weaker electrostatic bond with the VEGF. The addition of laponite to bioinks has also previously been shown to influence their mechanical properties (37). While we did not directly assess whether the addition of laponite influenced the stiffness of our ink, we did observe that it had no effect on their degradability, and on the basis of w/w ratio used in this study, we do not believe it had marked effects on mechanical properties such as matrix stiffness. Previous studies have shown that when using high concentrations of alginate (similar to that used in this study), the addition of laponite does not markedly affect the rheological properties of the bioink (37). However, future studies should investigate the overall mechanical properties of a bioink, as this may also influence its osteogenic potential (39). A potential limitation of laponite is that the strong electrostatic bond can limit the amount of growth factor released from a delivery system in the short-medium term (22). In this study, by tuning the ratio of laponite to alginate, it was possible to engineer bioinks that released most of their loaded protein over 35 days. Therefore, using specifically selected nanoparticles, it is possible to develop bioinks that support growth factor release profiles spanning days to weeks.

Using multiple-tool biofabrication, we demonstrated that distinct growth factor gradients can be established and maintained over time and that incorporating these gradients into printed implants can enhance sprouting angiogenesis in vivo. The process of sprouting angiogenesis begins with the selection of a distinct site on the mother vessel where sprout formation is initiated. This distinct site is referred to as the tip cell, and as the new sprout elongates, branches, and connects with other sprouts, the selection process for the tip cell is constantly reiterated (40). Previous studies have shown in the early postnatal retinas that tip cell migration depends on a gradient of VEGF-A and its proliferation is regulated by its concentration (40, 41). Therefore, the increase in vessel infiltration observed in VEGF gradient implants can possibly be attributed to tip cell migration and proliferation toward the areas of high VEGF concentration (40, 41). In contrast, when VEGF was homogenously distributed within the implant, there was less of a chemotactic effect, resulting in lower levels of vessel infiltration into the center of the construct.

When this bioprinting strategy was used to deliver both growth factors within a large bone defect, there was a significant increase in vessel infiltration within implants containing both a VEGF gradient and BMP-2 compared to those containing VEGF alone. Although it has been shown that delivery of BMP-2 alone can enhance new blood vessel formation within bone defects (42, 43), previous studies have not reported a benefit to delivering both growth factors to the defect site (17, 18). The finding that the laponite-functionalized bioink around the periphery of the implant was slowing the release of VEGF from the implant may partially explain the higher levels of vessel infiltration observed within the composite implant, with the slower VEGF release profile being perhaps more conducive to angiogenesis within the orthotopic environment. Somewhat unexpectedly, despite enhancing overall levels of bone formation, VEGF delivery alone did not increase early vessel infiltration into the implant. Note that orthotopic hematomas, generated by the surgical procedure, would have provided all defects with a source of endogenous chemotactic, angiogenic, and mitogenic growth factors (17). This may have mitigated the effect that an implant containing a VEGF gradient alone had on early angiogenesis.

3D printed implants containing spatial gradients of VEGF, coupled with defined BMP-2 localization, enhanced large bone defect healing with little heterotopic bone formation. Critically, this increase in bone healing was achieved using very low concentrations of exogenous growth factors. The concentration of VEGF used in this study was substantially less (80 to 160 times less) than previous studies (17, 18). Achieving therapeutic benefits with these low concentrations of growth factors is important for multiple reasons, not least of which is the observation that high concentrations of VEGF have been previously shown to disrupt osteogenesis as the result of abnormal angiogenesis and vascular structure (8). Furthermore, the concentrations of BMP-2 used here are at least an order of magnitude lower than that used previously to repair similar sized defects in a rat femoral defect model (28, 31). Repair in these studies is typically associated with a substantial amount of heterotopic bone formation (28, 31). Directly comparing to previous work in our lab, which used a clinically relevant BMP-2 dose in the same defect model (28), the results from this study exhibited substantially less heterotrophic bone formation [10% versus 50% (28) of total bone volume]. Although we did not observe full bone bridging after 12 weeks, new bone was still being formed via the process of endochondral ossification at 12 weeks, suggesting that regeneration was still proceeding. Allowing some level of physiological loading earlier in the healing process would likely have further accelerated regeneration (44). Together, the results from this study demonstrate the potential of 3D printing morphogen gradients for controlled tissue regeneration (with minimal heterotopic bone formation) without the need of supraphysiological dosages.

The translation of tissue engineering concepts from bench to bedside is a challenging, expensive, and time-consuming process. Numerous products have not made it past phase 2 trials, as they have not shown the expected benefit in patients (1, 2), while others have been associated with marked adverse effects (35). Here, we describe a previously unidentified approach for spatiotemporally defined growth factor delivery and demonstrate a potential clinical utility in the regeneration of large bone defects or the increased vascularization of any 3D printed construct. Proof-of-concept studies in small animals established the potential of these growth factor loaded bioinks for inducing enhanced angiogenesis and bone regeneration without the need for supraphysiological dosages. The benefit of this precise localization of growth factors in both time and space is that it allows for tightly controlled angiogenesis and new tissue formation, thereby reducing off-target effects. It is envisioned that this platform technology could be applied to the controlled regeneration of numerous different tissue types.

This study was designed to test whether the delayed release of BMP-2 from bioprinted constructs containing spatial gradients in VEGF will first enhance vascularization and sequentially enhance orthotopic bone regeneration. All animal experiments were conducted in accordance with the recommendations and guidelines of The Health Products Regulatory Authority, the competent authority in Ireland responsible for the implementation of Directive 2010/63/EU on the protection of animals used for scientific purposes in accordance with the requirements of the Statutory Instrument no. 543 of 2012. Subcutaneous mouse experiments were carried out under license (AE 19136/P069), and the rat femoral defect experiments were carried out under license (AE19136/P087) approved by The Health Products Regulatory Authority and in accordance with protocols approved by the Trinity College Dublin Animal Research Ethics Committee. The n for rodent models were based on the predicted variance in the model and was powered to detect 0.05 significance. For the subcutaneous surgeries, constructs were implanted in a balanced manner, such that each group contained an implant placed at each of the subcutaneous locations and samples for both surgical procedures were randomly distributed across the operated animals. For the rat surgeries, three rats from the empty group died from unforeseen complications and so were removed from the n number at the 12-week time point. One rat from the BMP-2 gradient group at 12-week time point was also removed, as it was deemed a statistical outlier using the Grubbs test.

Lowmolecular weight sodium alginate (58,000 g/mol) was prepared by irradiating sodium alginate (196, 000 g/mol; Protanal LF 20/40, Pronova Biopolymers, Oslo, Norway) at a gamma dose of 50,000 gray, as previously described (45). RGD-modified alginate was prepared by coupling the GGGGRGDSP to the alginate using standard carbodiimide chemistry. All bioinks were prepared by dissolving the RGD -irradiated alginate in growth medium, which consisted of alpha minimum essential medium (MEM) (GlutaMAX; Gibco, Biosciences, Ireland), 10% fetal bovine serum (FBS) (EU Thermo Fisher Scientific), penicillin (100 U/ml; Sigma-Aldrich), and streptomycin (100 g/ml; Sigma-Aldrich) (pen-strep) to make up a final concentration of 3.5% (w/v).

3D bioplotter from RegenHU (3DDiscovery) was used to evaluate the printability of the generated bioinks. The printability of varying the w/w ratio (2:1, 1:1, and 1:2) of methylcellulose to alginate was evaluated by measuring the spreading ratio as previously described (39)Spreading Ratio=Printed Filament DiameterActual Needle Diameter

To establish whether increasing the viscosity of the bioink influences growth factor release, methylcellulose (Sigma-Aldrich) was also added at ratio of 1:2 (w/w) to a 3.5% alginate solution of RGD -irradiated alginate. To establish whether the addition of clay-based particles to the bioink could further tailor the growth factor release profile of the bioinks, a 3.5% RGD -irradiated alginate solution was made, and either methylcellulose (2:1) (w/w) or a combination of both methylcellulose and laponite (Laponite XLG, BYK Additives & Instruments, UK) (6:3:1) (w/w) was added.

To establish whether the addition of nHA to the alginate would facilitate the adsorption and immobilization of growth factors within the hydrogel due to their strong electrostatic attraction between nHAs, three bioinks were tested (21). nHAs were prepared following a previously described protocol (46). A 3.5% RGD -irradiated alginate solution was made, and either methylcellulose (1:2) (w/w) or a combination of methylcellulose and nHA (2:1:2) (w/w) particles was added.

For all the growth factor release studies, VEGF (100 ng/ml; Gibco Life Technologies, Gaithersburg, MD, USA) was added to the solutions using dual-syringe approach, before precross-linking with 60 mM CaSO4 to make the bioinks as previously described (39). All constructs were cultured in growth medium in normoxic conditions, and media from each sample were changed bi-weekly. For VEGF release study, medium samples were taken (days 0, 3, 5, and 10) and snap-frozen at 80C. Hydrogels were also snap-frozen at 80C on day 0 to quantify the concentration of growth factor present in the constructs directly after printing.

To demonstrate the utility of the vascular bioink, two strategies were compared to print implants containing a spatial gradient of VEGF. The vascular bioink was prepared, cross-linked with 60 mM CaSO4, and printed to generate three experimental groups: (i) Homogenous VEGF. Bioink loaded with VEGF (100 ng/ml) was used to print constructs 8 mm in diameter and 4 mm high. (ii) Gradient 1. Bioink loaded with VEGF (100 ng/ml) was used to print a central 5-mm core with a VEGF-free bioink printed around the periphery of the 8-mm-diameter construct. (iii) Gradient 2. VEGF (80 ng/ml) was printed into the core, and VEGF (20 ng/ml) was printed into the periphery. Postprinting constructs were cross-linked again in a bath of 100 mM CaCl2 for 1 min. Constructs were cultured in growth medium in normoxic conditions for 14 days in vitro. The center and periphery of each construct were separated by coring out the center from the periphery of the scaffold and then snap-frozen at 80C, 1 hour after printing, and after 14 days in vitro.

To investigate whether the addition of laponite can tailor the growth factor release profile over a long culture period, a base bioink (Fast BMP-2 Release) and a laponite bioink (Slow BMP-2 Release) were compared. For both growth factor release profiles, a dual-syringe approach was used to deliver BMP-2 (200 ng/ml; PeproTech, UK) to the solutions before precross-linking with 60 mM CaSO4 to make the bioinks. These were printed into a 100 mM CaCl2 soak agarose mold to generate final constructs of 6 mm by 6 mm high. In addition to comparing the growth factor release profile of the two bioinks, the degradation rate of the bioinks was also investigated. These scaffolds were cultured in normoxic conditions for up to 35 days and media from each sample were changed weekly. For BMP-2 release study, medium samples were taken (days 0, 5, 7, 14, 21, and 35) and snap-frozen at 80C. Printed hydrogels were also snap-frozen at 80C on day 0 to quantify the concentration of growth factor present in the constructs directly after printing. For the degradation study, samples were washed and snap-frozen at 80C and each time point (days 0, 5, 7, 14, and 21). Samples were lyophilized by placing the samples in a freeze dryer (FreeZone Triad, Labconco, Kansas City, USA). Each sample was then weighed using an analytical balance (Mettler Toledo, XS205).

An enzyme-linked immunosorbent assay was used to quantify the levels of VEGF and BMP-2 (Bio-Techne, MN, USA) released by the alginates. The alginate samples were depolymerized with 1 ml of citrate buffer (150 mM sodium chloride, 55 mM sodium citrate, and 20 mM EDTA in H2O) for 15 min at 37C. The cell culture media and depolymerized alginate samples were analyzed at the specific time points detailed above. Assays were carried out as per the manufacturers protocol and analyzed on a microplate reader at a wavelength of 450 nm.

BMSCs were obtained from the femur of a 4-month-old porcine donor as previously described (47). All expansion was conducted in normoxic conditions, expanded in growth medium where the medium was changed twice weekly. Cells were used at the end of passage 3.

A 3D bioplotter from RegenHU (3DDiscovery) was used to print all of the scaffolds. Using a 30-gauge needle, constructs of 4 mm 5 mm high with both lateral and horizontal porosity and a fiber spacing of 1.2 mm were printed with PCL (Cappa, Perstop). The printing parameters of the PCL were as follows: temperature of thermopolymer tank (69C), temperature of thermopolymer head (72C), pressure (1 bar), screw speed (30 rpm), and feed rate (3 mm/s). Scaffolds were sterilized using ethylene oxide sterilization before hydrogel printing.

For the VEGF gradient study, the vascular bioink was prepared, cross-linked with 60 mM CaSO4, and printed within the PCL framework to generate three experimental groups: (i) No VEGF, bioink not loaded with VEGF; (ii) Homogenous, bioink loaded with VEGF (100 ng/ml) deposited (25 ng per construct) throughout the construct; and (iii) Gradient, bioink loaded with VEGF (500 ng/ml) deposited in the center (25 ng per construct) and VEGF-free bioink deposited on the outside (see Fig. 1A). Postprinting constructs were cross-linked again in a bath of 100 mM CaCl2 for 1 min.

For the BMP-2 release study, both a fast and slow release bioink were prepared and using the dual syringe approach, porcine MSCs were (2 106/ml) mixed to both bioinks to have an overall seeding density of 500 105 porcine MSCs/construct before being cross-linked with 60 mM CaSO4. Both bioinks were printed within the PCL framework to generate two experimental groups: (i) Fast release, fast release bioink loaded with BMP-2 (2 g/ml; 0.5 g per construct) deposited only in the periphery with the fast release bioink not loaded with BMP-2 in the center; and (ii) Slow release, slow release bioink loaded with BMP-2 (2 g/ml; 0.5 g per construct) deposited only in the periphery with the fast release bioink not loaded with BMP-2 in the center (see Fig. 2A). Postprinting constructs were cross-linked again in a bath of 100 mM CaCl2 for 1 min.

For the rat femoral defect, the vascular bioink, the osteoinductive bioink, and a base bioink (3.5% RGD -irradiated alginate and 1.75% methylcellulose) were prepared, cross-linked with 60 mM CaSO4, and printed within the PCL framework to generate three experimental groups: (i) VEGF Gradient, the vascular bioink loaded with VEGF (500 ng/ml) in the center of the implant and base bioink in the periphery; (ii) BMP-2 gradient, the osteoinductive bioink loaded with BMP-2 (10 g/ml) in the implant periphery (2 g per construct), with the base bioink in the center; and (iii) Composite (VEGF+BMP-2), the osteoinductive bioink in the periphery with the vascular bioink in the center (see Fig. 3A). Postprinting constructs were cross-linked again in a bath of 100 mM CaCl2 for 1 min.

Subcutaneous surgeries were performed on 20 8-week-old female BALB/c OlaHsd-Foxn 1nu nude mice (12 mice for the VEGF gradient study and 8 for the BMP-2 gradient study) (Envigo, Oxon, UK) as previously described (47). Scaffolds were 3D printed the morning of surgeries and implanted that day. Constructs were implanted in a balanced manner, such that each group contained an implant placed at each of the two subcutaneous locations and samples were randomly distributed across the operated animals.

For the rat segmental surgery, 72 12-week-old F344 Fischer male rats (Envigo, Oxon, UK) were anesthetized in an induction box using a mix of isoflurane and oxygen, initially at a flow rate of isoflurane of 5 liters/min to induce, followed by ~3 liters/min to maintain anesthesia. Once anesthetized, the animal was transferred to a heating plate that was preheated to 37C and preoperative analgesia was provided by buprenorphine (0.03 mg/ml). Surgical access to the femur was achieved via an anterolateral longitudinal skin incision and separation of the hindlimb muscles, the vastus lateralis, and biceps femoris. The femoral diaphysis was exposed by circumferential elevation of attached muscles, and the periosteum was removed. Before the creation of the defect, a PEEK plate was fixed to the anterolateral femur and was held in position using a clamp. Holes were created in the femur with a surgical drill using the plate as a template. Screws were then inserted into the drill holes in the femur to maintain the fixation plate in position. A 5-mm segmental defect was created using an oscillating surgical saw under constant irrigation with sterile saline solution. In the test groups, a scaffold was placed in the defect after a thorough washout of the surgical site. In the case of the empty defect group, the gap between bone ends was left empty. Soft tissue was accurately readapted with absorbable suture material. Closure of the skin wound was achieved using suture material and tissue glue.

Eight weeks after surgery, the BMP-2 gradient scaffolds were extracted and incubated in paraformaldehyde for 24 hours before being imaged via CT scans on a MicroCT42 (Scanco Medical, Brttisellen, Switzerland) as previously described (47).

Two weeks after surgery, 24 rats underwent a vascular perfusion protocol developed by Daly et al. (28). Briefly, the rat was sacrificed using CO2 asphyxiation, and the thoracic cavity was opened to insert a 20-gauge needle through the left ventricle of the heart. The inferior cava was cut and solutions of heparin (25 U/ml), and then, phosphate-buffered saline (PBS) was perfused through the vasculature using a peristaltic pump (Masterflex, Cole-Parmer, Vernon Hills, IL, USA) until the vasculature system was completely flushed clear. A solution of 10% formalin was then perfused for 5 min. Animals received a final perfusion of 20- to 25-ml radiopaque contrast agent MICROFIL (Flow Tech, Carver, MA, USA) and were left at 4C overnight. Explants were extracted and incubated in PBS for 24 hours before being imaged via CT scans on a MicroCT42 (Scanco Medical, Brttisellen, Switzerland) at 70 kVp, 113 A, and a 10-m voxel size. The volume of interest (VOI) was determined by positioning a 5-mm circle around the cross section of the femur with an overall length of 6.26 mm. MICROFIL has the same threshold as bone mineral, and therefore, to segment perfused vasculature from mineralized tissue within each construct, two scans were analyzed: calcified construct versus decalcified construct. The calcified constructs were scanned and postprocessed using a threshold value that accurately depicted both the mineral content and the vessel volume by visual inspection of the 2D grayscale tomograms (Scanco Medical MicroCT42). Noise was removed using a low-pass Gaussian filter (sigma = 1.2, support = 2), and a global threshold of 210 was applied. Next, samples were decalcified in EDTA (15 weight %, pH 7.4) for 2 weeks with the decalcification solution replaced daily (decalcified constructs). After 2 weeks, these decalcified constructs were scanned using the same settings and postprocessed at the same threshold as the calcified constructs to determine mineral content. Mineralized tissue content was determined by subtracting the bone volume of the decalcified scans from the calcified scans. Next, the decalcified scans were postprocessed at a threshold of 99 that accurately depicted just the vessel volume upon visual inspection of the 2D grayscale tomograms.

CT scans were performed on the rats using a Scanco Medical vivaCT 80 system (Scanco Medical, Bassersdorf, Switzerland). Rats (n = 9) were scanned at 4, 8, 10, and 12 weeks after surgery to assess defect bridging and bone formation within the defect. First, anesthesia was induced in an induction box using a mix of isoflurane and oxygen, initially at a flow rate of isoflurane of 5 liters/min to induce, followed by ~3 liters/min to maintain anesthesia. Next, the rats were placed inside the vivaCT scanner, and anesthesia was maintained by isoflurane-oxygen throughout the scan. Next, a radiographic scan of the whole animal was used to isolate the rat femur. The animals femur was aligned parallel to the scanning field of view to simplify the bone volume assessments. Scans were performed using a voltage of 70 kVp and a current of 113 A. A Gaussian filter (sigma = 0.8, support = 1) was used to suppress noise, and a global threshold of 210 was applied. A voxel resolution of 35 m was used throughout. 3D evaluation was carried out on the segmented images to determine bone volume and density and to reconstruct a 3D image. Bone volume and bone density in the defects were quantified by measuring the total quantity of mineral in the central 130 slices of the defect. To differentiate regional differences in bone formation, three VOIs were created. Concentric 2 mm, 4 mm, and 10 mm were aligned with the defect and used to encompass bone formation. The VOIs were aligned using untreated native bone along the femur. The core bone volume was quantified from the inner 2-mm VOI. The annular bone volume was quantified by subtracting the 2-mm VOI from the 4-mm VOI. Ectopic bone volume was quantified by subtracting the 4-mm VOI from the 10-mm VOI. The bone volume percentages for each region were then calculated by dividing the corresponding bone volume (i.e., bone volume in the annulus) by the total bone volume in the defect. The bone volume and densities were then quantified using scripts provided by Scanco.

For segmental defect samples, all constructs that were not being processed for vascular-CT imaging, were decalcified in Decalcifying Solution-Lite (Sigma-Aldrich) for 1 week before tissue processing. Once decalcified, all samples were dehydrated and embedded in paraffin using an automatic tissue processor (Leica ASP300, Leica). All samples were sectioned with a thickness of 8 m using a rotary microtome (Leica Microtome RM2235, Leica). Sections were stained with H&E for vessel infiltration, Safranin O to assess sulphated glycosaminoglycans (sGAG) content, and Goldners trichrome for bone formation. Quantitative analysis was performed on multiple H&E-stained slices, whereby vessels (positive staining for endothelium and erythrocytes present within the lumen), were counted on separate sections taken throughout each construct and averaged for each construct. Safranin O sections were evaluated for new developing bone (positive sGAG content). Massons trichromestained sections were evaluated for new bone formation. The percentage of developing bone, new bone, and marrow per total area of construct was measured in separate sections with the Deconvolution ImageJ plugin.

Immunofluorescence analysis was used to detect -SMA and vWF as previously described (47). Briefly, following blocking step, sections were then incubated overnight at +4C with goat polyclonal -SMA (1:250; ab21027, Abcam) in PBS with 3% of donkey serum (w/v) and 1% bovine serum albumin (BSA). After three washing steps with PBS containing 1% w/v BSA, the sections were incubated with Alexa Fluor 488 donkey anti-goat secondary antibody (1:200; ab150129, Abcam) for 1 hour at room temperature in the dark. The samples were washed three times in PBS with 1% w/v BSA, and the slides were then incubated overnight at +4C with rabbit polyclonal vWF antibody (1:200; ab6994, Abcam) in PBS with 3% of donkey serum (w/v) and 1% BSA (all from Sigma-Aldrich). After three washing steps with PBS and 1% w/v BSA, the sections were incubated with Alexa Fluor 647 donkey anti-rabbit secondary antibody (1:200; ab150075, Abcam) for 1 hour at room temperature in the dark. Last, samples were washed three times with PBS and 1% w/v BSA, and the sections were mounted using 4,6-diamidino-2-phenylindole mounting media (Sigma-Aldrich). Fluorescence emission was detected using a confocal laser scanning microscopy (Olympus FluoView 1000).

Results were expressed as means SD. Statistics was performed using the following variables: (i) When there were two groups and one time point, a standard two-tailed t test was performed. (ii) When there were more than two groups and one time point, a one-way analysis of variance (ANOVA) was performed. (iii) When there were more than two groups and multiple time points, a two-way ANOVA was performed. All analyses were performed using GraphPad (GraphPad Software, La Jolla, CA, USA; http://www.graphpad.com). For all comparisons, the level of significance was P 0.05.

Acknowledgments: We thank the staff at the Bioresources Unit in Trinity College Dublin for veterinary assistance and technical support. Funding: This publication has emanated from research supported by a research grant from the European Research Council (ERC) under grant no. 647004, the Irish Research Council (GOIPD/2016/324), and NIHs NIAMS grant R01AR063194. Author contributions: F.E.F. was responsible for technical design, development of bioinks, performing all animal surgeries, performing vessel perfusion, all CT scans, data interpretation, histological analysis, and drafting the paper. P.P. assisted with the rat surgeries and assisted with the vessel perfusions. L.H.A.v.D. assisted with CT analyses and CT scans. J.N. and D.C.B. assisted with all animal surgeries. J.-Y.S. and E.A. developed the RGD -irradiated alginate. D.J.K. conceived and helped design the experiments, oversaw the collection of results and data interpretation, and finalized the paper. Competing interests: Research undertaken in the laboratory of D.J.K. at Trinity College Dublin is part-funded by Johnson & Johnson. The authors declare no other 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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3D bioprinting spatiotemporally defined patterns of growth factors to tightly control tissue regeneration - Science Advances

According to latest research report on Global Stem Cell Banking Market report provides information related to market size, production, CAGR, gross margin, growth rate, emerging trends, price, and other important factors. Focusing on the key momentum and restraining factors in this market, the report also provides a complete study of future trends and developments in the market.

The Stem Cell Banking report contains all the details of the expected market dynamics and new market opportunities due to the COVID-19 outbreak. Stratagem Market Insights tried to cover all the market analysis of annual economic growth in the latest report on the Stem Cell Banking market.

According to analysts, the growth of the Stem Cell Banking market will have a positive impact on the global platform and will witness gradual growth over the next few years. This report study incorporates all the market growth and restraining factors along with the important trends mentioned between 2020 and 2025.

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Market segmentation:

The Stem Cell Banking market has been segmented into a variety of essential industries including applications, types, and regions. In the report, each market segment is studied extensively, taking into account market acceptance, value, demand, and growth prospects. Segmentation analysis allows customers to customize their marketing approach to make better orders for each segment and identify the most potential customers.

Global Stem Cell Banking Market Segmentation by Application:

Global Stem Cell Banking Market Segmentation by Product:

Competitive Landscape

This section of the report identifies various major manufacturers in the market. It helps readers understand the strategies and collaborations players are focusing on fighting competition in the marketplace. The comprehensive report gives a microscopic view of the market. The reader can identify the manufacturers footprint by knowing about the manufacturers global revenue, the manufacturers global price, and the manufacturers production during the forecast period.

The major manufacturers covered in this report:

Regional Insights of Stem Cell Banking Market:

In terms of geography, this research report covers almost all major regions around the world such as North America, Europe, South America, Middle East, Africa, and the Asia Pacific. Europe and North America are expected to increase over the next few years. Stem Cell Banking markets in the Asia-Pacific region are expected to experience significant growth during the forecast period. Advanced technology and innovation are the most important characteristics of North America and the main reason why the United States dominates the world market. The Stem Cell Banking market in South America is also expected to expand in the near future.

Years considered for this report:

Important Facts about Stem Cell Banking Market Report:

Questions Answered by the Report:

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Stem Cell Banking Market Applications, Types and Future Outlook Report 2020-2025 - express-journal.com

METUCHEN, N.J., Aug. 10, 2020 /PRNewswire/ --Tevogen Bio announces a joint partnership with renowned bone-marrow transplant expertNeal Flomenberg, M.D., Professor and Chair of the Department of Medical Oncology at Thomas Jefferson University, with the intent to evaluate Tevogen' s proprietary antigen-specific T cell technology as a potential treatment for COVID-19 and influenza-A patients.

This collaboration aims to harness Tevogen's proprietary immunotherapy platform and Dr. Flomenberg's expertise and research prowess to investigate potential treatments for viral infections.

Dr. Flomenberg has been at the forefront of immunogenetics and immunology for more than four decades. "Tevogen's technology resonated with me as there have been several groups who have used T cells to treat patients after bone-marrow transplants. The idea of utilizing T cell therapies to potentially treat COVID-19 and other viruses is truly remarkable," Flomenberg said. "I'm enthusiastic about moving forward with an investigation of Tevogen's technologies."

Tevogen CEO Ryan Saadi, M.D., M.P.H., is leading the new biotech's efforts. "Our work has been to pioneer T cell therapies that can be abundantly and efficiently reproduced to develop an affordable and scalable cellular treatment for the biggest global health threats, including COVID-19, influenza, and a variety of cancers. We are very excited about Dr. Flomenberg's contribution to our efforts and hope to initiate our investigational study soon."

In addition to developing its potential therapies, Tevogen is committed to organizational and manufacturing efficiency. This should allow it to engage in affordable innovation to the benefit of all patients.

About Tevogen Bio

Tevogen Bio was formed after decades of research by its contributors to concentrate and leverage their expertise, spanning multiple sectors of the health care industry, to help address some of the most common and deadly illnesses known today. The company's mission is to provide curative and preventative treatments that are affordable and scalablein order to positively impact global public health.

About Dr. Neal Flomenberg

Dr. Neal Flomenberg is the Chairman of Medical Oncology at Jefferson University in Philadelphia and also heads the Hematologic Malignancies, Blood and Marrow Transplantation (BMT) Program. Throughout his more than four decades of practice, he has maintained a longstanding interest in the immunogenetics and immunology of stem cell transplantation, with the goal of making transplantation safer and more widely available. Dr. Flomenberg developed an approach to bone-marrow transplants that uses half-matched relatives as donors, a breakthrough that assures that the majority of blood and bone-marrow cancer patients can benefit from this potentially curative treatment.

Media Contacts:

Mark Irion[emailprotected]

Katelyn Petroka [emailprotected]

SOURCE Tevogen Bio

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Tevogen Bio Announces Partnership With Preeminent Scientist Professor Neal Flomenberg, MD, to Investigate Proprietary T-Cell Therapy for Treatment of...

TEL AVIV, Israel, Aug. 12, 2020 /PRNewswire/ -- Cellect Biotechnology Ltd. (NASDAQ: APOP), a developer of innovative technology which enables the functional selection of stem cells, today reported financial and operating results for the second quarter ended June 30, 2020. The Company's six-month progress includes the development of several strategic initiatives, including growth-oriented opportunities in pain management and COVID-19 related therapeutics.

"Despite the COVID-19 pandemic business disruptions and the near-term delays to completing and commencing our clinical programs in Israel and the U.S., respectively, we acted swiftly over the past few months to leverage our sought-after technology to create several long-term business initiatives to enhance our value," commented Dr. Shai Yarkoni, Chief Executive Officer. "In addition to pursuing a potential merger with a global leader in the high growth medical-grade cannabis market, which is being delayed due to COVID-19, we have either initiated or are contemplating other business development activities that will greatly benefit from our innovation, technology and know-how. I believe each of these opportunities represents meaningful catalysts for Cellect in multi-billion-dollar markets, subject to resolution of the COVID-19 pandemic and return to normal course of business."

Notwithstanding the continued delays due to COVID-19, the Company remains focused on the following operational and clinical objectives:

The Company's cash and cash equivalents totaled $7 million as of June 30, 2020, which includes the approximately $1.5 million (gross before expenses)resulting from several investors exercising certain warrants that were issued in February 2019.

SecondQuarter 2020 Financial Results:

*For the convenience of the reader, the amounts above have been translated from NIS into U.S. dollars, at the representative rate of exchange on June 30, 2020 (U.S. $1 = NIS 3.466).

About Cellect Biotechnology Ltd.

Cellect Biotechnology (APOP) has developed a breakthrough technology, for the selection of stem cells from any given tissue, that aims to improve a variety of stem cell-based therapies.

The Company's technology is expected to provide researchers, clinical community and pharma companies with the tools to rapidly isolate stem cells in quantity and quality allowing stem cell-based treatments and procedures in a wide variety of applications in regenerative medicine. The Company's current clinical trial is aimed at bone marrow transplantations in cancer treatment.

Forward Looking Statements

This press release contains forward-looking statements about the Company's expectations, beliefs and intentions. Forward-looking statements can be identified by the use of forward-looking words such as "believe", "expect", "intend", "plan", "may", "should", "could", "might", "seek", "target", "will", "project", "forecast", "continue" or "anticipate" or their negatives or variations of these words or other comparable words or by the fact that these statements do not relate strictly to historical matters. For example, forward-looking statements are used in this press release when we discuss Cellect's expectations regarding timing of the commencement of its planned U.S. clinical trial and its plan to reduce operating costs. These forward-looking statements and their implications are based on the current expectations of the management of the Company only and are subject to a number of factors and uncertainties that could cause actual results to differ materially from those described in the forward-looking statements. In addition, historical results or conclusions from scientific research and clinical studies do not guarantee that future results would suggest similar conclusions or that historical results referred to herein would be interpreted similarly in light of additional research or otherwise. The following factors, among others, could cause actual results to differ materially from those described in the forward-looking statements: the Company's history of losses and needs for additional capital to fund its operations and its inability to obtain additional capital on acceptable terms, or at all; the Company's ability to continue as a going concern; uncertainties of cash flows and inability to meet working capital needs; the Company's ability to obtain regulatory approvals; the Company's ability to obtain favorable pre-clinical and clinical trial results; the Company's technology may not be validated and its methods may not be accepted by the scientific community; difficulties enrolling patients in the Company's clinical trials; the ability to timely source adequate supply of FasL; risks resulting from unforeseen side effects; the Company's ability to establish and maintain strategic partnerships and other corporate collaborations; the scope of protection the Company is able to establish and maintain for intellectual property rights and its ability to operate its business without infringing the intellectual property rights of others; competitive companies, technologies and the Company's industry; unforeseen scientific difficulties may develop with the Company's technology; the Company's ability to retain or attract key employees whose knowledge is essential to the development of its products; and the Company's ability to pursue any strategic transaction or that any transaction, if pursued, will be completed. Any forward-looking statement in this press release speaks only as of the date of this press release. The Company undertakes no obligation to publicly update or review any forward-looking statement, whether as a result of new information, future developments or otherwise, except as may be required by any applicable securities laws. More detailed information about the risks and uncertainties affecting the Company is contained under the heading "Risk Factors" in Cellect Biotechnology Ltd.'s Annual Report on Form 20-F for the fiscal year ended December 31, 2019 filed with the U.S. Securities and Exchange Commission, or SEC, which is available on the SEC's website, http://www.sec.gov, and in the Company's periodic filings with the SEC.

Cellect Biotechnology Ltd.

Consolidated Statement of Operation

Convenience

translation

Six months

ended

Six months ended

Three months ended

June 30,

June 30,

June 30,

2020

2020

2019

2020

2019

Unaudited

Unaudited

U.S. dollars

NIS

(In thousands, except share and per

share data)

Research and development expenses

837

2,901

7,086

1,364

3,564

General and administrative expenses

1,356

4,703

5,064

2,116

2,709

Operating loss

2,193

7,604

12,150

3,480

6,273

Financial expenses (income) due to warrants exercisable into shares

1,098

3,807

(7,111)

4,697

(5,919)

Other financial expenses (income), net

(15)

(55)

880

627

462

Total comprehensive loss

3,276

11,356

5,919

8,804

816

Loss per share:

Basic and diluted loss per share

0.010

0.034

0.029

0.024

0.004

Weighted average number of shares outstanding used to compute basic and diluted loss per share

338,182,275

338,182,275

200,942,871

365,428,101

224,087,799

Cellect Biotechnology Ltd.

Consolidated Balance Sheet Data

Convenience

translation

June 30,

June 30,

December 31,

2020

2020

2019

Unaudited

Unaudited

Audited

U.S. dollars

NIS

(In thousands, except share and per

share data)

CURRENT ASSETS:

View post:
Cellect Biotechnology Reports Second Quarter Financial and Operating Results; First Half 2020 Strategic Developments Create Long-Term Revenue...

Poverty, Government apathy and Covid-19 induced-lockdown restricting travel proved fatal for little Kishan, a 11-year-old boy suffering from Aplastic anemia, a life-threatening blood disorder condition in which the bone marrow and stem cells do not produce enough blood cells

Facing severe financial constraints and waiting timely medical aid, first at Safdarjung Hospital and then AIIMS, both Government hospitals in Delhi, Kishans life was cut short in March this year amid Covid-19 pandemic.

However, Kishans is not a lone case. Dr Nita Radhakrishnan, paediatric haemato-oncologist at Super Speciality Paediatric Hospital, Noida, Uttar Pradesh says that as the deadly Coronavirus captured the attention of the nation in the most unprecedented manner, the non-Covid patients particularly those with the Aplastic anemia have suffered the most in the crisis.

She gave instances of her two teenage patients who succumbed to blood disorder in the Covid catastrophe. Manish (name change), a 17-year-old was suffering with on-and-off fever, gum bleeding, and melena for three months, he came to us in December last year just when Coronavirus had started spreading its tentacles from China to other parts of the world.

The boy was diagnosed with severe Aplastic anemia and was recommended requisite treatment like regular hospital visit for red cell transfusion before he could be given bone marrow transplant (BMT), a life saving treatment.

However, while the family was not able to visit our hospital in Noida due to the covid-lockdown, no blood products were available at the hospital near to the patients locality. In want of blood, Manish could not survive more days.

13-year-old Suresh (name change) too faced similar fate. While Government funds could not be sanctioned for his BMT in time the boy could not visit the Noida hospital for further follow-up due to travel restrictions. Two weeks later, Suresh died due to hemorrhage at his native place, lamented the doctor.

These are just two reported cases from the NCR hospital located near the countrys capital. Several have gone unreported. The Government has no policy nor any long-term plan for such patients.

The prognosis of severe aplastic anemia in our country is dismal. The incidence of 46 per million population of childhood aplastic anemia in India and other Asian countries is higher than what is observed in the West, explains Dr Radhakrishnan. The scenario is gloomy for the patients afflicted with the disease as they need blood transfusion almost every 20 days.

A significant proportion of patients of aplastic anemia (around 30 per cent) die before any definitive treatment is initiated. A study by AIIMS based on a recent series of patients follow-up showed that out of 1501 patients diagnosed over last seven years, only 303 ie 20 per cent received the definitive treatment modalities through either BMT or IST with ATG and cyclosporine, says Dr Radhakrishnan in her case report Aplastic anemia: Non-COVID casualties in the Covid-19 era, published in the latest edition of Indian Journal of Palliative Care.

The doctors have sought urgent intervention. Dr Radhakrishnan says that as we await the peak of Covid-19 in our country and possibly secondary and tertiary waves thereafter, patients with aplastic anemia who are the sickest among all hematological illnesses would benefit greatly from urgent intervention from the Government to ensure timely treatment.

Those suffering with Aplastic anemia, there is mostly delay in diagnosis, delay in initiation of treatment due to monetary constraints, non-inclusion of the disease under government schemes such as Ayushman Bharat and NHM and delay in sanction of money from other Government schemes such as Rashtriya Arogya Nidhi, Chief Minister and Prime Ministers relief fund often due to lack of proper documents, she added.

Delay means, risk of contracting fungal infections and increase in drug-resistant bacterial infections increase which further hamper the treatment, point out Dr Ravi Shankar and Dr Savitri Singh in the study.

Though the Union Health Ministry, after few days of lockdown period, issued directions for continuing treatment for essential health services including reproductive and maternal health services, newborn care, severe malnutrition, and NCDs including cancer care, palliative care, dialysis, and care of disabled, unfortunately those with Aplastic anemia got ignored.

This despite of the fact that these patients are at the highest risk of death following a break in the treatment of few weeks, notes Dr Radhakrishnan.

Because of the closure of offices and absence of staff, during the lockdown period, there was delay in sanction of usual grants due to the lockdown of offices and inability in generating documents such as income certificate from the tehsils.

For instance, Suresh and Manish, both our patients received the Government grant after around 34 months of applying for the same. But both had died before they could reach the hospital for treatment, lamented the hematologist.

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Covid-19 Impact: Patients with aplastic anemia at receiving end - Daily Pioneer

New Jersey, United States,- The Stem Cell Therapy Market research report added by Verified Market Research is an in-depth analysis of the latest trends, market size, status, upcoming technologies, industry drivers, challenges, regulatory policies, with key company profiles and strategies of players. The research study provides market introduction, Stem Cell Therapy market definition, regional market scope, sales and revenue by region, manufacturing cost analysis, Industrial Chain, market effect factors analysis, Stem Cell Therapy market size forecast, 100+ market data, Tables, Pie Chart, Graphs and Figures, and many more for business intelligence.

The Stem Cell Therapy Market report includes an in-depth analysis of the Stem Cell Therapy market for the present as well as the forecast period. The report encompasses the competition landscape entailing share analysis of the key players in the Stem Cell Therapy market based on their revenues and other significant factors. Further, it covers several developments made by the prominent players of the Stem Cell Therapy market. The Stem Cell Therapy Market report is a beneficial source of perceptive data for a business approach. It presents the market overview with growth analysis together with historical & futuristic costs. Further identifies the revenue, specifications, company profile, demand, and supply data.

Global Stem Cell Therapy Market was valued at USD 117.66 million in 2019 and is projected to reach USD 255.37 million by 2027, growing at a CAGR of 10.97% from 2020 to 2027.

Leading Key players of Stem Cell Therapy Market are:

Stem Cell Therapy Market: Competitive Landscape

This section of the report identifies various key manufacturers of the market. It helps the reader understand the strategies and collaborations that players are focusing on combat competition in the market. The comprehensive report provides a significant microscopic look at the market. The reader can identify the footprints of the manufacturers by knowing about the global revenue of manufacturers, the global price of manufacturers, and sales by manufacturers during the forecast period of 2015 to 2019.

Stem Cell Therapy Market: Segment Analysis

The research report includes specific segments by region (country), by company, by Type and by Application. This study provides information about the sales and revenue during the historic and forecasted period of 2019 to 2027. Understanding the segments helps in identifying the importance of different factors that aid the market growth.

1.Stem Cell Therapy Market, By Cell Source:

Adipose Tissue-Derived Mesenchymal Stem Cells Bone Marrow-Derived Mesenchymal Stem Cells Cord Blood/Embryonic Stem Cells Other Cell Sources

2.Stem Cell Therapy Market, By Therapeutic Application:

Musculoskeletal Disorders Wounds and Injuries Cardiovascular Diseases Surgeries Gastrointestinal Diseases Other Applications

3.Stem Cell Therapy Market, By Type:

Allogeneic Stem Cell Therapy Market, By Application Musculoskeletal Disorders Wounds and Injuries Surgeries Acute Graft-Versus-Host Disease (AGVHD) Other Applications Autologous Stem Cell Therapy Market, By Application Cardiovascular Diseases Wounds and Injuries Gastrointestinal Diseases Other Applications

Stem Cell Therapy Market: Regional Analysis

The research report includes a detailed study of regions of North America, Europe, Asia, and South America. The report has been curated after observing and studying various factors that determine regional growth such as economic, environmental, social, technological, and political status of the particular region. Analysts have studied the data of revenue, sales, and manufacturers of each region. This section analyses region-wise revenue and volume for the forecast period of 2015 to 2026. These analyses will help the reader to understand the potential worth of investment in a particular region.

North America(United States, Canada, and Mexico)Europe(Germany, France, UK, Russia, and Italy)Asia-Pacific(China, Japan, Korea, India, and Southeast Asia)South America(Brazil, Argentina, Colombia, etc.)The Middle East and Africa(Saudi Arabia, UAE, Egypt, Nigeria, and South Africa)

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Verified Market Research is a leading Global Research and Consulting firm servicing over 5000+ customers. Verified Market Research provides advanced analytical research solutions while offering information enriched research studies. We offer insight into strategic and growth analyses, Data necessary to achieve corporate goals, and critical revenue decisions.

Our 250 Analysts and SMEs offer a high level of expertise in data collection and governance use industrial techniques to collect and analyze data on more than 15,000 high impact and niche markets. Our analysts are trained to combine modern data collection techniques, superior research methodology, expertise, and years of collective experience to produce informative and accurate research.

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Stem Cell Therapy Market 2020 by Manufacturers, Regions, Type and Application, Forecast to 2027 - Owned

His name means brave in Hindi. And for four year-old Veer Gudhka that couldnt be more appropriate.

For while the bubbly little boy might look fighting fit, he actually has just months to live.

Veer suffers from a rare blood disorder called Fanconi anaemia, which results in a decreased production of all types of blood cells.

But a stem cell donor will save his life.

In a heartfelt video message, the plucky toddler asks Sunday Mirror readers: Please be my life-saver? Will you be my superhero?

And today his family are appealing to those from BAME communities to help by signing up to the Anthony Nolan stem cell register.

Mum Kirpa and dad Nirav know the odds are stacked against them getting that all-important call because they are of Indian descent.

While 69 per cent of Northern European patients find the best possible stem cell match from a stranger, this drops to just 20 per cent for those with black, Asian or ethnic minority backgrounds.

Currently only two per cent of the population is on the UK stem cell register.

And with Asians making up just six per cent of the UK population, there is a smaller pool of potential donors.

Veer was diagnosed with the blood disorder last August, after he started suffering from extreme fatigue, and was referred for tests.

Doctors said he would need a stem cell transplant within three years for a chance of survival.

They hoped to buy Veer some time by putting him on steroids to boost his blood counts. But his condition has deteriorated fast.

Recent tests at Great Ormond Street Hospital in London show he now has just three to four months to find a donor.

Kirpa and Nirav were both tested, along with Veers six-year-old sister Suhani, but none of them were a match.

A search on the global stem cell register also drew a blank.

And his dad has been trying to encourage his fellow countrymen and women in India to join the register.

They have even signed up a female battalion of the Indian Army.

Kirpa, 37, from Harrow, London, said: We just feel so scared were going to lose our cheeky, amazing little boy. To look at Veer you wouldnt know hes critically ill.

Like his name, hes been brave from the start. Hes undergone countless tests and hospital visits but has had a constant smile on his face.

"He knows he needs a superhero to step forward, but his optimism and enthusiasm are infectious and keep us all going.

She added: Going on the register is incredibly quick and donating cells if you match someone in need is painless.

You can join the Anthony Nolan stem cell register today.

Nine out of 10 people donate their stem cells through the bloodstream in a simple IV process called peripheral blood stem cell collection.

One in 10 will have their stem cells collected via the bone marrow itself, while under general anaesthetic. Doctors transplant the new, healthy cells via the patients bloodstream, where they begin to grow and create healthy red blood cells, white blood cells and platelets.

A perfect match from a donor can mean a lifelong cure.

Veers dad Nirav, 40, said: I only learned about the Anthony Nolan stem cell register two years ago and even then I assumed it would involve long and painful procedures.

We need to raise awareness to save lives in every community.

Continued here:
Boy, 4, may look fighting fit but only has months to live - unless you can save him - Mirror Online

INTRODUCTION

The ability to regenerate complex body parts varies considerably in the animal kingdom. While planarian and hydra are able to regenerate their entire bodies, many avian and mammalian species mostly stop at the wound healing stage without a reparative regeneration process (1). This disparity may result from complexity differences among organisms by nature, yet it leaves us the hope that we may learn from highly regenerative species to improve our own regenerative potential.

Zebrafish is known for its ability to regenerate multiple complex body structures (2). Among regenerable tissues, the caudal fin serves as a great model due to its faithful and rapid regeneration, ease of manipulation, and relatively low complexity. A key step in regeneration is the formation of the blastema, a layer of proliferative and undifferentiated cells that accumulates between the wound site and the wound epidermis following initial wound closure. This step occurs in response to appendage loss and is one of the key features that separates regenerative systems from nonregenerative systems. At later stages of regeneration, the blastema further proliferates and differentiates to regenerate the missing complex structures.

However, the molecular signatures of blastemal cell state transitions during regeneration in zebrafish remain elusive. The state of a cell can be represented by its collective gene expression profile, which has only been measured in bulk for all genes or in specific lineages of cells for a subset of genes during caudal fin regeneration. Prior work has shown that both proliferation of progenitors and dedifferentiation of adult lineage cells contribute to the blastema (38). Progenitors respond to injury cue and proliferate as in normal development. Cells derived from mature adult lineages, however, lose their lineage-specific markers while obtaining progenitor-like markers when they proliferate. Neither type of cell gains multipotency, but rather, they proliferate and regenerate with lineage restrictions. The limited resolution and throughput of these approaches have prevented a more systematic understanding of blastema cells. The advent of single-cell transcriptomic technologies promises to reveal signals masked at the bulk tissue level (9), granting us an opportunity to define and monitor cellular state transition in regenerating fin at an unprecedented resolution.

In this study, we generated single-cell transcriptomic maps of regenerating fin tissue. These maps allowed us to separate the contribution from different cell types and track the transcriptomic dynamics in cell state transitions during regeneration. By comparing with the profiles obtained from uninjured fin tissue, we identified cell types involved in regeneration. We demonstrated the activation of cell cyclerelated programs shared across cell types as well as cell typespecific programs. Furthermore, we defined the heterogeneity in both epithelial and blastemal populations and their functional relations to the regeneration process.

To better understand cell type involvement in fin regeneration, we characterized single-cell transcriptional landscapes for both preinjury and regenerating caudal fin tissues using the 10x Genomics platform (see Materials and Methods and table S1) (9). We sampled regenerating fins from 1, 2, and 4 days post-amputation (dpa) time points to interrogate the stages of blastema formation, outgrowth, and maintenance (Fig. 1A). Fin samples were collected from multiple fish to control for individual variation while at the same position along the proximal-distal axis to avoid positional effects. To establish the transcriptional ground states for each cell type in the fin tissue, we first focused on cells collected from the preinjury time point. Via an unsupervised clustering of 4134 cells, we identified epithelial cells (epcam and cdh1), hematopoietic cells (mpeg1.1 and cxcr3.2), and mesenchymal cells (msx1b and twist1a) (fig. S1, A and B) (1014). Epithelial cells are from three transcriptionally distinct subgroups, representing the superficial (krt4), intermediate (tp63), and basal layers (tp63 and krtt1c19e) of the epithelium (fig. S1, A and B) (15, 16).

(A) General experimental design. Zebrafish caudal fin tissues at preinjury and 1/2/4 dpa stages were collected. (B) Clustering assignments for caudal fin cells collected from each stage. Uniform Manifold Approximation and Projection (UMAP) axes were calculated from the integrated cells dataset as in (C). (C) Clustering assignments for caudal fin cells collected from both preinjury and regenerating stages. Cells were plotted on UMAP axes. Color coding is the same as in (E). (D) Percentage distribution of the major cell types captured in caudal fin, grouped by their stage of collection. Color coding is the same as in (E). (E) Differential expressions of the key marker genes by the identified major cell types. Color gradient: normalized relative expression level. Dot size: percentage of cells in the cluster that express the specified gene.

To determine whether the same cell types existed in the regenerating stages, we performed analysis using two different approaches: (i) Cells from each stage were clustered independently, and (ii) cells from both uninjured fins and injured fins were integrated through the anchoring approach (see Materials and Methods; Fig. 1, B, C, and E; and table S2) (17). For both approaches, we regressed out cell cycle effects before principal components analysis (PCA). Agreement between cluster assignments was measured using Hubert and Arabies adjusted Rand index (ARI). An average ARI of 0.86 (preinjury, 0.86; 1 dpa, 0.85; 2 dpa, 0.90; and 4 dpa, 0.83) indicated that clustering results generated using the two approaches were highly consistent. Cell types identified in the preinjury cells presented consistently across all regenerating stages, suggesting that regenerating fins contain the same cell types as the preinjury fins.

New regenerates are built up by the proliferation and migration of cells located at a number of fin segments away from the amputation plane (2). In response to injury cues, these cells gained the ability to detach from local tissue, enter cell cycle, and migrate toward the wound site while undergoing transcriptional reprogramming. We computationally separated S phase, G2-M phase, and G1-phase cells based on the expression level of cell cyclerelated genes and performed clustering analysis using only S phase cells (see Materials and Methods and fig. S2A). In this cycling cell population, we identified epithelial, mesenchymal, and hematopoietic cell groups as before (Fig. 2, A to C, and table S3). Our data support a model in which cells likely keep their original identities during proliferation.

(A) Cell type clustering of S phase cells plotted onto UMAP axes calculated by S phase cell only. Cells are colored by the general cell types merged from major cells types in Fig. 1B. (B) Stage distribution of S phase cells. Cells were plotted on the same UMAP axes as in (A) and colored by stage when the cells were collected. (C) Relative expression levels of the top 30 differentially expressed genes from each cluster of only S phase cells. (D) Venn diagrams of numbers of genes shared between the cell cycleactivated genetic programs. Left, included all genes; right, included only cell cyclerelated genes (see Materials and Methods).

Next, we asked whether different regenerating cell types exhibited similar or distinct cell cycle regulations. To this end, we identified genes up-regulated in S phase cells compared to G1 phase cells in each cell type, respectively (logFC, >0.25; minimum percentage, >10%). Of the 1098 differentially expressed genes, 161 were shared across all three groups of comparisons (Fig. 2D and table S4). Of these shared genes, at least 54 genes were related to cell cycle regulation, underscoring a shared program governing cell cycle reentry (criteria described in Materials and Methods). In contrast, hundreds of genes differentially highly expressed in S phase exhibited cell typespecific pattern, of which dozens were related to cell cycle (Fig. 2D). We next evaluated the degree of conservation of these enriched genes by asking what fraction did not have human orthologs that had been curated in the Metascape database (18). Twenty-five percent of genes in the epithelial cellspecific group had no human ortholog, whereas all shared groups had at most 15% genes without a human ortholog, suggesting that enriched genes shared by cell types were more evolutionarily conserved (fig. S2C).

Some cell typespecific S-G1 enriched genes were also expressed in a cell typespecific manner regardless of their cell cycle phases: For example, psmb8a and psmb9a shared similar epithelial-hematopoietic enrichments (fig. S2D). The human homologs of these genes (PSMB8 and PSMB9) encode 5i and 1i subunits of the immunoproteasome (19). Together with 2i and PA28 subunits of the proteasome, they turn the proteasome into immunoproteasome and take part in immune response (20). Immunoproteasome digests peptides more efficiently, promoting antigen presentation by a major histocompatibility complex (MHC) class I molecule. Although they did not pass the differential expression criteria in the S-G1 comparison, zebrafish psmb10, psme1, and psme2 shared a differential expression signature similar to that of psmb8a and psmb9a, suggesting that zebrafish might use the same group of subunits for the assembly of immunoproteasomes that might help increase immune responses during regeneration, especially in epithelial and hematopoietic cells (fig. S2, D and E). In addition, we found three genes that shared the same expression signature with the immunoproteasome subunits (psmb13a, psmb12, and psma6l) (fig. S2E) without known human or mouse homologs, suggesting that they might form zebrafish-specific proteasomes with functional relevance to regeneration (19).

Consistent with current knowledge, we observed three transcriptionally distinct subgroups in the preinjury epcam+ epithelial cells, representing the superficial, intermediate, and basal layers of the adult zebrafish epithelium (Fig. 3A and fig. S1B) (15, 16). By integrating cells from all stages during regeneration, we found clusters of cells that corresponded to all three layers of the epithelium after injury (Fig. 1, B and C). In addition, we captured a rare agr2+ population (referred to as mucosal-like epithelium herein) that was too small to be clustered by itself in the preinjury stage (Fig. 1E). These cells shared general epithelial features with the other epithelial layers but exhibited higher expression of a unique set of 200 genes. We examined the expression distribution of the orthologs of these genes in human tissues (The Human Protein Atlas, http://proteinatlas.org/) (21). Among the top 30 genes with human orthologs, 11 showed enriched expressions in proximal digestive or gastrointestinal tract and another 11 in bone marrow of blood lineages, suggesting that this population is analogous to cells in the mucosa in mammalian systems (table S2). In zebrafish, agr2 is required for the differentiation of the mucosal-producing goblet cells in the intestinal epithelium (22). To confirm the cell typespecific expression pattern of this gene in the fin tissue, we performed in situ hybridization on agr2 in both uninjured and regenerating fin tissues (see Materials and Methods). agr2 transcripts are scattered within the epithelium regardless of the sample collection stage and reflect a round morphology of the cell expressing it (fig. S3, A, C, E, and G to I). A proportion of agr2+ cells overlap with positive dark blue staining of Alcian blue in serial sections, suggesting that these cells are mucous cells that are known to exist in the caudal fin epithelium (fig. S3, B, D, and F) (23).

(A) Diagram of the stratified adult zebrafish epithelium. (B) Differential expressions of claudin family and keratin family genes in epithelial subgroups shown as a dot plot. Known epithelial markers krt4, fn1b, tp63, and krtt1c19e were included for comparison. Cells were first grouped by major cell types and then separated into preinjury and regenerating stages. Darkness of dot color: relative expression level. Dot size: percentage of cells in the cluster that express the specified gene. (C) In situ hybridization targeting krt1-19d, cldna, cldn1, and krt4 of 4-dpa fin tissues. Brown dots indicate positive RNA signals from target genes, while pale blue blocks represent hematoxylin-stained cell nuclei. Zoomed-in views are presented. Original images can be found in fig. S4. All epithelial layers are above the black dotted lines. (D) Clustering assignment of epithelial cells plotted on UMAP axes calculated with only epithelial cells. Cells are colored by their epithelial layer identity as in (A). (E) The same UMAP visualization as in (D), with cells colored by stage of collection. Arrows connect the groups of comparison, with a direction from preinjury stage to regenerating stages (1, 2, and 4 dpa). Numbers next to the green triangle: number of genes up-regulated in regenerating stage. Numbers next to the red triangle: number of genes down-regulated in regenerating stage. (F) Clustered GO enrichment for genes up-regulated in regenerating basal, intermediate, and superficial epithelial cells comparing to their preinjury counterparts. GTPase, guanosine triphosphatase; ER, endoplasmic reticulum; PKN, protein kinases N; snRNP, small nuclear ribonucleoprotein.

Although the same three-layer classification of epithelial cells could be defined when cells from regenerating stages were integrated with the preinjury cells, the expression of the commonly used layer-specific marker genes changed dramatically during regeneration: Superficial epithelial marker krt4 expanded into basal and intermediate layers of the epithelium, the intermediate layer marker fn1b was also highly expressed in the basal layer, and the basal epithelial marker krtt1c19e was barely detectable in the postinjury cell populations (Fig. 3B) (15, 16). To better understand the molecular features of the epithelial populations, we identified genes significantly more highly expressed in epithelial cells than in hematopoietic and mesenchymal cells and found that cell-cell junction genes ranked high in the list. Among these, genes from the claudin and keratin families were detected at a ratio 20-fold higher than that in randomly selected detectable genes (2 test, P value of <0.0001). We focused on expression patterns of all claudin and keratin genes in zebrafish and found that cldne, cldnf, krt1-19d, and krt17 labeled the superficial cluster; cldnh labeled the mucosal-like cluster; cldna, krt93, and krt94 labeled the intermediate cluster; and cldn1 and cldni labeled the basal cluster (Fig. 3B). Claudin genes are expressed in a tissue-specific manner in zebrafish and are generally considered to be the proteins responsible for regulating the paracellular permeability in the vertebrate epithelium (24). Their differential expression signature in both uninjured and regenerating tissues suggests that they might play important roles in maintaining the permeability in each epithelial population. On the other hand, the expression of keratin genes displayed less restriction across the three layers relative to claudin genes but stronger dependence on regenerating states (Fig. 3B). The differential expression signature suggests that they might perform epithelial subtyperelated function in regeneration. To verify their expression pattern, we performed RNA in situ hybridization targeting the known marker krt4 and new candidates, including krt1-19d, cldna, and cldn1 (Fig. 3C) as well as cldne, krt94, and cldni (fig. S4, A to H). Comparing with the known marker krt4, these genes exhibited more restricted expression patterns in epithelial layers, better representing the molecular signatures of different epithelial populations in the fin tissue regardless of regeneration status (Fig. 3, B and C).

The three epithelial layers were present across the regeneration stages albeit with varying proportions (Fig. 1D). The proportion of basal epithelial cells peaked at 2 dpa, reaching up to 42%, whereas the superficial layer epithelial cells decreased from 27 to 6% at 2 dpa (the coefficient of variations of cell proportions between biological replicates is below 15%). The observed compositional change of the two epithelial populations is consistent with a previous finding that the initial migration of superficial layer cells to the new regenerates is followed by replenishment by basal epithelial cells (25). This basal replenishment was also reflected in the two-dimensional Uniform Manifold Approximation and Projection (UMAP) calculation from only epithelial cells, in which preinjury cells were separated by their respective layers, whereas regenerating cells were closer in the projection space (Fig. 3, D and E). Superficial layer cells from before and after injury stages were in juxtaposition to each other, consistent with our knowledge that this layer of epithelial cells directly migrates to and covers the wound site (25). On the other hand, basal layer cells from before and after injury stages were more distantly separated, suggesting more dramatic changes between resting and regenerating basal epithelial cells.

To understand the mechanisms of epithelium regeneration, we compared the transcriptome between preinjury and regenerating cells for the three epithelial layers. Basal layer cells up-regulated 1271 genes and down-regulated 198 genes during regeneration; both were the highest numbers across all comparisons (numbers of differentially expressed genes were from Wilcoxon rank sum test, adjusted P value of < 0.01; Fig. 3E). We performed gene ontology (GO) enrichment analysis on genes up-regulated in the regenerating stage by layer and found both common and layer-specific programs associated with regeneration (18). All three layers were enriched for oxidative phosphorylation (dre00190), proteasome (dre03050), and cell redox homeostasis (GO:0045454). While basal and intermediate layer cells could be regulated by Rho guanosine triphosphatasemediated Wnt signaling for extracellular matrix organization and actin filament depolymerization, respectively (R-DRE-195258, R-DRE-5625740, R-DRE-195721, GO:0030198, and GO:0030042), superficial layer cells showed enrichment mainly for general transcriptional and translational regulations (Fig. 3F). When comparing the expression profiles between regenerating superficial epithelial and basal epithelial, we saw enrichment for antigen presentation and apoptosis features in the superficial layer (table S5). In addition, the superficial layer contained the lowest proportion of cells in S phase or G2-M phase, further supporting that superficial layer epithelium was most likely maintained by migration and proliferation from other layers (fig. S2B).

Subcluster identification within regenerating basal epithelial cells revealed two subgroups that represented different functionalities during regeneration, one labeled by distally distributed fgf24, while the other by proximally distributed lef1 (fig. S5, A to C) (26, 27). We compared expression profiles between group I (distal) and group II (proximal) cells and found that their suggested functionalities were consistent with their expected roles in regeneration: The distal subgroup (or distal wound epidermis) up-regulated genes associated with extracellular matrix degradation, and the proximal subgroup (or proximal wound epidermis) up-regulated genes associated with organization of extracellular matrix, skeletal system development, and negative regulation of locomotion (fig. S5, D and E). In addition, the increase of proximal cell proportion was accompanied by the decrease of distal cell proportion, suggesting that basal layer epithelium become gradually active in promoting blastema proliferation and differentiation during the initial regeneration process (fig. S5C). To confirm the distribution of these cells, we performed RNA in situ hybridization targeting two candidate genes, stmn1b and sema3b, one from each cluster. The expression of stmn1b was first observed at the basal layer of the wound epidermis at 1 dpa but diminished as regeneration proceeded (fig. S4, I to K). On the contrary, sema3b showed expression at later stages and was enriched in the relatively proximal portion of the basal layer epithelium (fig. S4, L to N). The expression dynamics of these two genes matched the predicted proportion changes of the two clusters (fig. S5C). While sema3b was more restricted to the basal layer, stmn1b showed low expression levels in the intermediate layer as well, potentially suggesting that this subpopulation could be labeling cells transitioning from the basal layer to the other layers of epithelium.

We next performed subcluster analysis within the hematopoietic cluster and found four subpopulations (Fig. 4, A to C and table S6). The first three populations were enriched for the macrophage marker mpeg1.1, with the cluster H1 being M1-like (lgals2+ and lcp1+) and the cluster H3 M2-like (ctsc+ and lgmn+) (Fig. 4D) (12). We speculated that the cluster H2 represented a transition state between M1-like and M2-like or a state before the macrophages differentiate toward M1-like or M2-like. From 1 to 4 dpa, the proportion of M1-like macrophages remained at a constant level, while that of M2-like macrophages expanded (Fig. 4B), potentially suggesting a shift in the function of macrophages in the new regenerates from pro-inflammatory toward anti-inflammatory as regeneration proceeded. Macrophages are important for proper blastema proliferation (28). The change in the proportions of M1/M2-like macrophage in our data matched with that observed in the larvae fin, suggesting that the adults followed a similar rule for immune cell recruitment after injury.

(A) Subcluster assignments of the hematopoietic cells. Cells were plotted on UMAP axes. The same color code is used for (B) to (D). (B) Proportion of subgroups of hematopoietic cells. (C) Expression enrichment of the top 30 differentially expressed genes in the four subclusters within hematopoietic cluster shown as a heatmap. (D) Expression distribution of genes associated with macrophage activation grouped by subclusters. Expression levels were log normalized by Seurat. y axis: cluster identity. z axis: cell density. (E) Expressions of pigment cell markers gch2 and mlpha in the hematopoietic population.

The cluster H4 enriched for genes including mlpha and gch2, both well-characterized markers for the chromatophore lineages in zebrafish (Fig. 4E) (29). Chromatophores are derived from neural crest lineage, yet here, they clustered with macrophages that were from hematopoietic lineage. One possibility is that this clustering result could be driven by features related to antigen presentation via MHC class II, a feature of pigment cells based on studies using human melanocytes (30). The proportion of this cluster decreased as regeneration started, agreeing with the known pattern of fin stripe recovery after amputation (Fig. 4B) (31).

To understand the component and function of the cells in the mesenchymal cell cluster before and during fin regeneration, we focused on genes enriched in this cluster and found previously identified blastema marker genes that are required for fin regeneration, including the muscle segment homeobox family members msx1b and msx3 and the insulin-like growth factor signaling ligand igf2b (logFC, >0.25; minimum percentage, >25%; and adjusted P value of <1 105, as listed in table S1) (2, 13). The mesenchymal cluster expressed these genes nearly exclusively, confirming their blastema identity in regenerating stages (fig. S6A). In addition, we found key genes involved in zebrafish bone development and regeneration: twist1a, the transcription factor that controls the skeletal development by regulating the expression of runx2 (14); cx43, the gap junction protein required for building the fin ray up to the right length; and hapln1a and serpinh1b, two genes downstream of cx43 (32, 33). By performing conserved marker analysis using Seurat, we found that msx1b and twist1a were also among the markers conserved across all stages, underscoring shared features that existed between regenerating and preinjury mesenchymal cells (maximum P values across stages: 4.72 1010 and 2.84 109 for msx1b and twist1a, respectively). This theme of building and supporting bone tissues in mesenchymal cells was not only reflected by a handful of genes: GO analysis of all the detected up-regulated genes in this cluster revealed significant enrichment of genes associated with GO terms, including fin regeneration (GO:0031101) and skeletal system development (GO:0001501) (fig. S6B). When more stringent criteria for differential expression were used, genes were also significantly enriched for GO terms, including skeletal system morphogenesis (GO:0048705) and extracellular matrix organization (GO:0030198) (fig. S6C).

Previous work has shown that blastema comprises bone cells and non-bone cells but has not defined the cell types and the regeneration process of each type (23, 34, 35). To better understand the regeneration process by cell type, we performed clustering analysis within the mesenchymal cluster and identified nine distinct subgroups (Fig. 5A and fig. S6D). Of the two preinjury subgroups, M-2 represented the mature bone lineage, which was enriched for expressions of bglap, mgp, and sost (fig. S6E) (36, 37). Comparing to M-2, cluster M-1 presented low expression levels of bglap, mgp, and sost and high expression levels of a group of other genes, including fhl1a, fhl2a, and tagln (fig. S6E). Mammalian orthologs of these genes are required for chondrogenesis and osteogenesis, leading us to speculate that cluster M-1 could represent the supporting non-bone cell lineage in the preinjury state (38, 39).

(A) Subclustering assignments of mesenchymal cells shown on UMAP axes. Cells are colored by their cluster assignments and connected by Slingshot-reconstructed trajectories. Lineage 1: 1-2-3-4; lineage 2: 1-2-3-5-6; lineage 3: 1-2-3-5-7-8; lineage 4: 1-2-3-5-9. (B) By-lineage highlighting of mesenchymal cells. Cells with colors other than gray represent the cells included in each corresponding lineage in (A). (C) Expression distribution of genes labeling cell lineages and cell states in mesenchymal cells. Gene feature plots were connected by estimated lineages using the same lineage color code as in (A). (D to G) In situ hybridization targeting the tnfaip6 gene in (D) preinjury, (E) 1-dpa, and [(F) and (G)] 4-dpa fin tissues. Brown dots indicate positive RNA signals from target genes, while pale blue blocks represent hematoxylin-stained cell nuclei. A zoomed-in view for the region inside the focused rectangle is provided within (D). (G) Zoomed-in view for the region highlighted by a rectangle in (F). Dotted lines indicate the amputation plane. All scale bars, 100 m.

The remaining seven populations came from regenerates. Pseudotime analysis via Slingshot (40) suggested that these subgroups formed four trajectories, all initiated from the tnfaip6+ cluster (M-3), which was composed mainly of 1-dpa cells (Fig. 5, B and C, and fig. S6D). tnfaip6 was ranked top by an adjusted P value in the differentially expressed genes labeling the regeneration initiation cluster and was also expressed exclusively in the mesenchymal cluster (Fig. 5C and fig. S6A). The mammalian ortholog of this gene is required for proliferation and proper differentiation of mesenchymal stem cells (MSCs) and balances the mineralization via osteogenesis inhibitions (41). The expression of tnfaip6 in the postinjury zebrafish fin suggested that it could also be required in the early stages of regeneration for promoting mesenchymal proliferation. To confirm the expression pattern of tnfaip6, we performed RNA in situ hybridization for uninjured and regenerating fin tissues targeting this gene (Fig. 5, D and E). In the uninjured fin, tnfaip6 was expressed in a segmental pattern, presumably enriching at joints between bone segments. At 1 dpa, tnfaip6 was expressed not only near the bony rays but also in the cavity, showing a general activation in the mesenchymal population. As regeneration proceeded from 1 to 4 dpa, mesenchymal cells divided into cdh11+ (M-4) and tph1b+ (M-5) branches, with the latter further divided into mmp13a+ (M-6), tagln+ (M-7), and vcanb+ (M-9) branches (Fig. 5C and fig. S6D). The mmp13+ (M-6) cluster maintained a high-level tnfaip6 expression, whereas all other branches had a lower but detectable tnfaip6 expression. This was consistent with the observation we made from in situ hybridization at 4 dpa targeting tnfaip6: the broad expression in the mesenchymal population and segmental enrichments similar to that in the uninjured fin (Fig. 5, F and G).

The four trajectories initiated from the tnfaip6+ cluster revealed four putative lineages representing bone and non-bone cells in the blastema. cdh11+ lineage 1 specifically expressed runx2 and osterix/sp7, which are the key transcription factors regulating osteogenesis (fig. S6E) (42). Mammalian ortholog of cdh11 could induce Sp7-dependent bone and cartilage formation in vivo, suggesting that the cdh11+ branch in the blastema represented the regenerating osteoblasts (43). Genes highly expressed at the end of this lineage (M-4) compared to the initiation point (M-3) were associated with bone mineralization and skeletal system development, further supporting their bone cell identity (table S7).

Mesenchymal cells outside the osteoblast branch shared enrichment for tph1b and aldh1a2 expressions at 2 dpa, followed by and1 expression at 4 dpa (Fig. 5C and fig. S6F). These three genes had been suggested to label joint fibroblasts, fibroblast-derived blastema cells, and actinotrichia-forming cells in the blastema, respectively (34, 35, 44). However, their expression signatures implied that instead of labeling separate populations in the blastema, they might be labeling different states of the same non-osteoblastic cells at the early stage of fin regeneration.

Upon 4 dpa, these non-osteoblastic cells diverged into three groups (Fig. 5C and fig. S6D). To understand this separation, we performed differential expression analysis for each branch between cells at the end of the lineage tree (lineage 1, M-4; lineage 2, M-6; lineage 3, M-7 and M-8; and lineage 4, M-9) and cells in the initiation cluster (M-3). Genes highly expressed at the lineage end points were included for GO analysis for functional predictions (logFC, >0.25; minimum percentage of >25%; and adjusted P value of <0.01). These three lineages were also associated with skeletal system development or extracellular matrix organizations as were the bone cell lineage; however, the association was driven by a nearly completely different set of genes (table S7). Unlike the osteoblast lineage, none of these three non-bone cell lineages showed enrichment for bone mineralization, suggesting that these cells might indirectly contribute to bone formation. In lineage 2, top differentially expressed genes mmp13a and ogn both have mammalian orthologs that are associated with bone formation (Fig. 5C and fig. S6F) (45, 46). In addition, this lineage presented up-regulation of DLX family genes, especially dlx5a, suggesting the reactivation of fin outgrowthrelated developmental programs during regeneration (fig. S6F and table S7) (47). Lineages 3 and 4 both enriched for estrogen response and expressed the retinoic acid (RA) synthesis gene aldh1a2. However, only lineage 3 displayed up-regulation of the RA-degrading enzyme cyp26b1 (fig. S6F and table S7). The cyp26b1high-aldh1a2low pattern helped to reduce RA levels in the blastema, promoting redifferentiation of the osteoblasts (44). The differentiation-promoting signature was also reflected in the enrichment of genes, including col6a1 and tagln, whose mammalian orthologs are essential for bone formation (fig. S6F and table S7) (39, 48). These genes were also enriched in the preinjury non-bone cell population, suggesting a connection between this subset of the non-bone cells and their preinjury counterparts (Fig. 5C and fig. S6F). Top up-regulated genes in lineage 4, on the other hand, were main contributors of the extracellular matrix, including and1/2, loxa, and vcanb (35, 49, 50). Enriched expression of these genes suggested that this lineage could be responsible for creating and organizing the fibrous environment. Together, the various non-osteoblastic cells could potentially work collaboratively with the osteoblasts in creating the environment for bone tissue regeneration.

Genes that had been suggested to label progenitors contributing to fin regeneration (mmp9 and cxcl12a) and several orthologs of known mammalian MSC markers (lrrc15, prrx1a/b, and pdgfra) (6, 7, 51, 52) were expressed almost exclusively in the mesenchymal cluster (fig. S6A). Consistent with the observations made in the lineage-tracing study, the mmp9 expression was associated with the regenerating bone cell lineage (lineage 1; Fig. 5B and fig. S6E) (7). However, mmp9 was detected only in a small portion of the mesenchymal cells and was highly expressed in the basal epithelium cells at similar proportions. On the other hand, we observed coenrichment of cxcl12a (previously known as sdf-1) and orthologs of the known mammalian MSC markers in the preinjury population (fig. S6E). cxcl12a-expressing cells in zebrafish were found to carry osteogenic, adipogenic, and chondrogenic characteristics in vitro like MSCs would do and contributed to the mesenchyme of the newly developing bony rays during fin regeneration (6, 53). The coenrichment pattern suggested that some of the preinjury cxcl12a-expressing cells could be MSCs in the fin tissue, which contribute to fin regeneration.

Zebrafish caudal fin is a unique regeneration system to model the injury response and regeneration of vertebrate appendages despite being a simple structure without muscular and adipose tissues. Major components of the regenerating caudal fin are epithelial cells covering the wound site and blastemal cells producing the connective tissue and bone matrices. Early studies established that actively proliferating blastema is the key to regeneration. Formed by cell migration and proliferation, this layer of cells continues in outgrowth and differentiation, rebuilding the complex body structure. Despite efforts in understanding its importance, basic questions regarding the formation of blastema remained: (i) Which type of cells contributes to the blastema and (ii) how do they shape the regeneration process?

Using single-cell transcriptomes, we defined cell types in both preinjury and postinjury fin tissues. Although regenerating cells were drastically different from their preinjury counterparts, both stage-specific and integrated clustering analysis revealed the same major cell type compositions in the fin tissues regardless of their time of collection. Common cell types detected include epithelial cells from all three layers, hematopoietic cells, and mesenchymal cells. Our data lay a foundation for lineage-targeted analysis to investigate the role of epithelial layers and subtypes in fin regeneration.

For each cell type to be a consistent component in the regenerated fin, cell cycle entry is required. We found that both common and unique cell cycle programs activated in the regenerating fin, with the shared ones appearing to be more evolutionarily conserved than the unique ones. Among the genes showing cell typespecific S phase enrichment, several immunoproteasome subunits also showed a clear cell typespecific expression. We speculated that the increasing level of immunoproteasome subunits in epithelial and hematopoietic cells specifically might accelerate antigen processing and presentation, which could be important for immune cell recruitment and tumor necrosis factorinduced blastemal proliferation (54).

Epithelial cells were the most abundant cell type in the profiled fins and could be clustered into four different subgroups, including the three layers in the adult fish epithelium and the mucosal-like cells within the intermediate layer. However, markers labeling these layers did not perform well in separating cell groups when only regenerating cells were considered. An unbiased differential expression test suggested that some members of the krt and cldn families were expressed in specific layers more consistently throughout regeneration. RNA in situ hybridization targeting cldne, krt1-19d, cldna, krt94, cldni, and cldn1 confirmed their exclusive layer-specific expression pattern, underscoring their potential to serve as markers for the distinct epithelial layers during regeneration. Our epithelium-specific analysis suggested that basal layer epithelial cells proliferate and could be the main source for replenishing the other two layers of the epithelium, similar to findings in a previous study based on genetic lineage tracing in zebrafish and echoing findings made using the axolotl limb regeneration model (25, 55). We observed higher apoptosis and lower proliferation features in the superficial epithelial layer compared to the other layers. At the same time, we observed transition patterns in gene expression, connecting the basal to the intermediate and the superficial layer during regeneration.

The behavior of mucosal-like cells during regeneration had been rarely reported for zebrafish in literature. We found in this study that this group of cells was an integral part of the regeneration process. Enrichment of foxp1b in this population and enrichment of foxp4 in basal and intermediate epithelial cells supported that zebrafish foxp homologs could be involved in regulating agr2 expression as does the Fox family in mice and, furthermore, the mucin production in the epithelium during regeneration (Fig. 1E) (56). The protein encoded by amphibian homologs of agr2, nAG (from newts) and aAG (from axolotl), are necessary and sufficient for salamander limb regeneration (57, 58). They are expressed in both dermal glands and the nerve sheaththe pattern of which has also been recovered from single-cell RNA sequencing (scRNA-seq) analysis (55). Regeneration deficiencies caused by denervation before amputation can be rescued by the ectopic expression of nAG. Although we do not have data supporting the nerve sheath expression pattern, as shown for the amphibian models, we hypothesize that agr2 could similarly mediate neuronal signals in zebrafish during regeneration.

Macrophages are critical players in the zebrafish caudal fin regeneration (28, 54). We observed subgroups of the mpeg1.1+ macrophage population in the regenerating fin tissue, resembling M1 and M2 macrophages in mammalian systems. However, we were not able to recover other immune cell population in the hematopoietic cells. This could potentially be due to the systematic bias against certain cell types during tissue dissociation and droplet incorporation in the microfluidic device. The same bias might also explain why we were not able to recover some other known players in the regenerating fin tissue, including neurons and endothelial cells (4). Increasing the number of cells sampled for scRNA-seq or performing scRNA-seq on sorted hematopoietic lineage cells would help to better understand the involvement of these populations in the regeneration process.

The expression profiles of mesenchymal cells captured from the postinjury stages resembled those of blastema in histology studies. We found four connected but distinct lineages representing both bone and non-bone cells in the blastema. All four lineages initiated from one cluster mostly consisted of 1-dpa cells and enriched for the tnfaip6 expression. A similar scenario has been observed in the axolotl limb regeneration model. By using scRNA-seq on a lineage-labeled axolotl model, Gerber et al. (58) found that connective tissue cells funnel into a progenitor state at initiation. Whether the cluster identified in our study represented a shared cell origin for the blastema or a shared state across mesenchymal cell types in the initial blastema-formation stage requires further investigation. High proportion of epithelial population in the fins could also hamper the discovery of relatively rare population with multipotency. Finer dissection before single-cell profiling might help in future study designs in capturing these populations.

While the bone cell lineage has been well studied in the regenerating fin, non-bone cells had been labeled by different markers and given different names and their intercorrelations left to be clarified. We found that tph1b, aldh1a2, and and1/and2 genes were shared among the non-bone cell lineages and could be labeling states instead of types of blastemal cells during regeneration. Meanwhile, differential analysis revealed similar enrichment for bone formation in all lineages yet distinct associations with reactivation of developmental programs, RA signaling, and collagen metabolism, underscoring their collaborative and complementary roles in the regeneration process.

Our scRNA-seq data also provided more details about the fish system we are working with. For all sample collections, we used the transgenic strain Tg(sp7:EGFP)b1212, which specifically labels osteoblast lineage in the fish (59). It was reported that green fluorescence signal could be detected in the fish skin after 72 hours post-fertilization. This ectopic expression, however, does not interfere with confocal imaging of skeletal structures of fish at any stage due to the fact that they lie in different planes of focus. What these cells are and why they expressed the transgene were unclear. In this study, we obtained a holistic view of the transgene expression pattern in the fin region regardless of whether that was associated with the cell type of interest, i.e., osteoblasts in this context. Unsupervised clustering on the expression profiles from single fin cells suggested that green fluorescent protein (GFP) is not only expressed in the mesenchymal but also highly enriched in the superficial layer epithelium (table S2). A closer examination of this classic reporter gene construct revealed that the regulatory region of sp7 used for the construction of the transgene did not exactly represent the endogenous sp7 regulatory region. Tg(sp7:EGFP)b1212 was generated from bacterial artificial chromosome transgenesis using CH73-243G6 as the backbone, which did not contain the first exon of sp7 according to the annotation of the current genome assembly (chr6:58630884-58720045 and GRCz10), leading to the usage of a regulatory sequence different from the endogenous version. Whether this usage difference contributed to the ectopic expression pattern of the transgene requires further study. This finding points to the potential of using single-cellbased approaches in reporter line validation and more thorough analysis of the transgene behavior.

All zebrafish were used in accordance with protocol no. 20190041 approved by the Washington University Institutional Animal Care and Use Committee. Wild-type and Tg(sp7:EGFP) strains are maintained under standard husbandry in the Washington University Fish Facility, with the system water temperature at 28.5C and a day-night cycle controlled as 14-hour light/10-hour dark. For fin amputation, we anesthetized 1-year-old fish with MS-222 (0.16 g/liter) in the system water and then removed the distal half of their caudal fin with sterilized razor blades. The fish were then sent back to circulating water system for recovery. We collected regenerating fin tissue from 39 fish by doing secondary fin amputation at the primary cutting plane with the same anesthesia and recovery procedures.

Collected fin tissues were digested by Accumax (Innovative Cell Technologies), filtered through 40-m cell strainers, and washed with 1 Dulbeccos phosphate-buffered saline (DPBS)0.04% bovine serum albumin to generate single-cell suspensions. Libraries were constructed from these cell suspensions following the instruction of the Chromium Single Cell Gene Expression Solution 3 v2 (10x Genomics) and were subsequently sequenced on HiSeq2500 (Illumina) with read lengths of 26 + 75 (Read1 + Read2). Raw reads were processed by Cell Ranger (10x Genomics) with default parameters for read tagging, alignment to zebrafish reference genome (GRCz10), and feature counting based on Ensembl release 91 (cellranger count). EGFP sequence was added into the reference genome as a separate chromosome for mapping reads from the reporter gene.

We performed unsupervised clustering using Seurat v3.0 following the procedure of normalization (SCTransform), highly variable gene detection, dimensional reduction (principal components analysis), and cells clustering (Louvain clustering at resolutions from 0.1 to 0.6) (17). For integrating the four stages in finding conserved cell types, we used the anchoring approach provided by Seurat v3. Cell clustering was based on the top principal components that account for most of the cell-cell variances. The same set of principal components was used in UMAP calculation for visualization as well.

We found differentially expressed genes in each cluster by comparing the expression profiles of them with those of the rest of the cells using Wilcoxon rank sumbased approach with the criteria of log fold change more than 0.25 and a minimum cell percentage of 0.25. The same criteria were applied to all pairwise comparisons, unless stated otherwise. We made functional connections between the list of differentially expressed genes and the type of cell that they most likely represent by testing for GO term enrichment (18) and manual curation by searching The Zebrafish Information Network database and PubMed. Certain cell clusters were taken as independent samples for secondary clustering following the same unsupervised clustering procedures.

We calculated the by-cell average expression level of a set of S phase or G2-M phase markers suggested by Seurat that are detected in our zebrafish dataset and normalized by subtracting aggregated expression of control genes. Although G1 phase cells are also within cell cycle, they are hardly separable from G0 cells. To avoid false-positive labeling for active cycling cells, we set stringent thresholds and only included cells with |S.score G2M.score| > 0.1 in the S or G2-M group, while cells with both S.score and G2M.score below zero as G1. Other cells were not included in this part of the analysis. Differentially expressed genes were also identified by Wilcoxon rank sumbased approach. These differentially expressed genes were considered to be cell cycle related if they were in the list of genes associated with R-DRE-1640170 Cell Cycle and/or cycling marker genes used for cell cycle phase score calculations.

We collected uninjured and regenerating fin tissues from casper (nacrew2/w2;roya9/a9) fish and fixed in 4% paraformaldehyde overnight (60). Fixed tissues were subsequently submerged in 10% sucrose in 1 PBS, 20% sucrose in 1 PBS, and 30% sucrose in 1 PBS for 4 hours each. After sucrose exchange, tissues were embedded in Optimal Cutting Temperature (O.C.T.) compound (Fisher Healthcare Tissue-Plus) and snap frozen on dry ice. The frozen tissue blocks were then processed into 15-m sections on a Leica CM1950 cryostat. We performed RNA in situ hybridization targeting krt4, cldne, krt1-19d, cldna, krt94, cldni, cldn1, agr2, sema3b, stmn1b, and tnfaip6 for mRNA detection using an RNAscope kit (Advanced Cell Diagnostics, Hayward, CA, USA). Alcian blue/periodic acidSchiff (PAS) staining was subsequently performed on the same section or separately on a consecutive serial section following the manufacturers protocol (Newcomer Supply). Microscopic images were taken by ZEISS Axio Observer.

Cell trajectories were constructed using Slingshot v1.3.1 (40). Through initial subclustering and cell type identifications, we found one subcluster with high epcam expression, potentially a doublet cell contamination from the major cell type classifications. We removed this group of cells from all downstream analysis within the mesenchymal cluster. We used UMAP embedding and subclustering assignments as input for the Slingshot calculation.

We performed nonparametric Wilcoxon rank sum test to identify differentially expressed genes across cell groups as implemented in Seurat. P values were adjusted by all features in the dataset using Bonferroni correction.

See more here:
Cellular diversity of the regenerating caudal fin - Science Advances

Organ Transplant Rejection Medications Market: Introduction

According to the report, the globalorgan transplant rejection medications marketwas valued atUS$ 4.7 Bn in 2018and is projected to expand at a CAGR of~3% from 2019 to 2027. Rise in healthcare spending and improvements in healthcare infrastructure, and increase in the global geriatric population are the major factors anticipated to drive the organ transplant rejection Medication market from2019 to 2027.

Rise in Healthcare Spending and Improvement in Healthcare Infrastructure to Drive Global Market

Increase in patient awareness about personal health boosts the demand for medical devices. Patients are more aware and proactive about their health and are willing to seek a physician's advice at an early stage. Increase in per capita disposable income is encouraging people to spend more on healthcare facilities, which, in turn, fuels the global organ transplant rejection medications market. Advertisements have increased public visibility of new technology, thereby generating interest among chronic patients for organ transplant rejection medications.

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Changing demographics in emerging economies such as China, India, Brazil, and South Africa are expected to provide significant opportunities for organ transplant rejection medications. Moreover, public and private healthcare expenditure is expected to increase in these countries, which is likely to drive the organ transplant rejection medications market.

Increase in healthcare expenditure, rise in global per capita income, and improvement in healthcare infrastructure and government reimbursement programs in developed as well as developing countries are likely to propel the organ transplant rejection medications market in the near future.

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Kidney Transplant to Offer Lucrative Opportunities

The report offers detailed segmentation of the global organ transplant rejection medications market based on drug class, transplant type, distribution channel, and region. In terms of drug class, the organ transplant rejection medications market has been segmented into calcineurin inhibitors, antiproliferative agents, mTOR inhibitors, antibodies, and steroids. Based on transplant type, the market has been segmented into kidney transplant, bone marrow transplant, liver transplant, heart transplant, lung transplant, and other transplants. Kidney transplant is a highly preferred treatment for end-stage renal disease (ESRD). It is comparatively more cost-effective than dialysis and is associated with a long-term mortality improvement.

Based on distribution channel, the global organ transplant rejection medications market has been classified into hospital pharmacies, retail pharmacies, and online pharmacies. Hospitals are major clinical settings wherein a large number of surgeries are conducted. This makes hospital pharmacies a prominent segment of the organ transplant rejection medications market.

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North America to Lead Organ Transplant Rejection Medications Market

North America was the largest market for organ transplant rejection medications in 2018, due to presence of the maximum number of living as well as deceased donors and better organ-preserving practices in the region. However, high costs and complex procedures would adversely affect the organ transplant rejection medications market during the forecast period. The organ transplant rejection medications market in Asia Pacific is projected to expand at a relatively high CAGR of4.1%during the forecast period. Transplantation procedures vary substantially from region to region and country to country, due to factors such as difference in the rate of end-organ damage, economic differences in terms of ability to provide transplants or other treatments, cultural differences that can support or hinder organ donation and transplant, and reporting of legal transplants across regions.

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Major Market Players

The report provides profiles of leading players operating in the global organ transplant rejection medications market. These includeGlaxoSmithKline plc, Novartis AG, F. Hoffmann-La Roche Ltd., Astellas Pharma, Inc., Pfizer, Inc., AbbVie, Inc., Allergan plc, Bristol-Myers Squibb Company (BMS), and Sanofi.

Novartis AG is a leading company that specializes in the development and manufacture of branded as well as generic pharmaceutical and biopharmaceutical drugs. It is evaluating the experimental Facilitating Cell Therapy (FCR001), which involves taking stem cells of a kidney donor and grafting them in the transplant recipients bone marrow. This combination would trick the recipients immune system by accepting the donated kidney as its own.

Pfizer, Inc. is a global pharmaceutical company that develops, manufactures, and markets prescription medicines in 11 therapeutic segments, including cardiovascular, oncology, neuroscience, pain, and infectious diseases. The company offers a range of medicines and vaccines as well as consumer healthcare products for the prevention and treatment of infectious and chronic diseases for all age groups.

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Organ Transplant Rejection Medications Market: Drug Companies Focus on Improving Long-term Outcome of New Drugs - BioSpace

The latest report published by Regal Intelligence on Hematopoietic Stem Cell Transplantation (HSCT) market provides crucial market insights along with detailed segmentation analysis. The report examines key driving factors that are expected to drive the growth of the market.

Global Hematopoietic Stem Cell Transplantation (HSCT) Market Research Report gives knowledgeable information on various market situations, for example, potential development factors, factors controlling the development, market opportunities and dangers to the worldwide market. Also, the report broadly centers around competitive analysis of Hematopoietic Stem Cell Transplantation (HSCT) Market. The competitive analysis segment incorporates key manufacturers, fresh players, providers, market strategies, potential chances, operation landscape and analysis of the trends of the Hematopoietic Stem Cell Transplantation (HSCT) market. The market results are centered around current market scenario. To gauge and predict the degree of competition in this market. This report will likewise support all the manufacturers and speculators to have a superior comprehension of the investments to know where the market is heading.

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Leading players of Hematopoietic Stem Cell Transplantation (HSCT) including:Regen Biopharma Inc, China Cord Blood Corp, CBR Systems Inc, Escape Therapeutics Inc, Cryo-Save AG, Lonza Group Ltd, Pluristem Therapeutics Inc, ViaCord Inc

For Product type segment the report listed main product type:

AllogeneicAutologous

For Application segment the report listed main types:

Peripheral Blood Stem Cells Transplant (PBSCT)Bone Marrow Transplant (BMT)Cord Blood Transplant (CBT)

Key Highlights of the Hematopoietic Stem Cell Transplantation (HSCT) Market

Key Strategies adopted by major players Global driving factors of the market Developed and emerging markets Comprehensive description of the international players Market dynamic factors affecting the global market Evaluation of niche business areas Driving and restraining factors of the market growth Market share analysis

Moreover, the report briefly studies the performance of both historical records along with the recent trends. It includes a complete analysis of different attributes such as manufacturing base, type, and size. This report evaluates the market segmentation along with the competitive landscape at global as well as regional level. The report also discusses about the rising need for Hematopoietic Stem Cell Transplantation (HSCT) market.

Reasons for Buying this Report

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Report on Global Hematopoietic Stem Cell Transplantation (HSCT) Market 2020 comprises of 10 Sections in Table as follows:

The Hematopoietic Stem Cell Transplantation (HSCT)-market report reads pin-direct analysis for changing serious dynamics with reference towards changing elements that drives or limits market development. The report is comprehensively visualized to forecast the market point of view and opportunities where it has an extension to develop in future. Basically, the report segregates the ability of market in the present and the future possibilities from various edges in detail.

About Us:We, Regal Intelligence, aim to change the dynamics of market research backed by quality data. Our analysts validate data with exclusive qualitative and analytics driven intelligence. We meticulously plan our research process and execute in order to explore the potential market for getting insightful details. Our prime focus is to provide reliable data based on public surveys using data analytics techniques. If you have come here, you might be interested in highly reliable data driven market insights for your product/service,reach us here 24/7.

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Hematopoietic Stem Cell Transplantation (HSCT) Market 2020: Regen Biopharma Inc, China Cord Blood Corp, CBR Systems Inc, Escape Therapeutics Inc,...

Detailed Analysis & SWOT analysis, Stem Cell Therapy Market Trends 2020, Stem Cell Therapy Market Growth 2020, Stem Cell Therapy Industry Share 2020, Stem Cell Therapy Industry Size, Stem Cell Therapy Market Research, Stem Cell Therapy Market Analysis, Stem Cell Therapy market Report speaks about the manufacturing process. The process is analyzed thoroughly with respect three points, viz. raw material and equipment suppliers, various manufacturing associated costs (material cost, labor cost, etc.) and the actual process of whole Enterprise Stem Cell Therapy Market.

Stem Cell Therapy market 2020 is a professional and in-intensity look at on the modern state of the key-word industry. The document provides a simple review of the key-word marketplace together with definitions, classifications, programs and chain shape. The key-word enterprise evaluation is supplied for the worldwide marketplace which include improvement records, competitive landscape evaluation, and principal local development popularity.

Ask for a Sample Report 2020

The Stem Cell Therapy marketplace file elaborates Stem Cell Therapy industry evaluation with various definitions and category, Product kinds & its packages and chain shape. Stem Cell Therapy market document presentations the manufacturing, sales, charge, and market proportion and boom rate of every type as following.

2020 Short Detail of this Stem Cell Therapy market report:

Stem-cell therapy is the use of stem cells to treat or prevent a disease or condition. Bone marrow transplant is the most widely used stem-cell therapy, but some therapies derived from umbilical cord blood are also in use.

In the last several years, global stem cell therapy market developed fast at a average growth rate of 46.81%.

Market Analysis and Insights: Global Stem Cell Therapy Market

In 2019, the global Stem Cell Therapy market size was USD 403.6 million and it is expected to reach USD 1439.9 million by the end of 2026, with a CAGR of 19.7% during 2021-2026.

Global Stem Cell Therapy Scope and Market Size

Stem Cell Therapy market is segmented by Type, and by Application. Players, stakeholders, and other participants in the global Stem Cell Therapy market will be able to gain the upper hand as they use the report as a powerful resource. The segmental analysis focuses on revenue and forecast by Type and by Application in terms of revenue and forecast for the period 2015-2026.

Segment by Type, the Stem Cell Therapy market is segmented into Autologous, Allogeneic, etc.

Segment by Application, the Stem Cell Therapy market is segmented into Musculoskeletal Disorder, Wounds & Injuries, Cornea, Cardiovascular Diseases, Others, etc.

Regional and Country-level Analysis

The Stem Cell Therapy market is analysed and market size information is provided by regions (countries).

The key regions covered in the Stem Cell Therapy market report are North America, Europe, China, Japan, Southeast Asia, India and Central & South America, etc.

The report includes country-wise and region-wise market size for the period 2015-2026. It also includes market size and forecast by Type, and by Application segment in terms of revenue for the period 2015-2026.

Competitive Landscape and Stem Cell Therapy Market Share Analysis

Stem Cell Therapy market competitive landscape provides details and data information by vendors. The report offers comprehensive analysis and accurate statistics on revenue by the player for the period 2015-2020. It also offers detailed analysis supported by reliable statistics on revenue (global and regional level) by player for the period 2015-2020. Details included are company description, major business, company total revenue and the revenue generated in Stem Cell Therapy business, the date to enter into the Stem Cell Therapy market, Stem Cell Therapy product introduction, recent developments, etc.

The major vendors include Osiris Therapeutics, NuVasive, Chiesi Pharmaceuticals, JCR Pharmaceutical, Pharmicell, Medi-post, Anterogen, Molmed, Takeda (TiGenix), etc.

This report focuses on the global Stem Cell Therapy status, future forecast, growth opportunity, key market and key players. The study objectives are to present the Stem Cell Therapy development in North America, Europe, China, Japan, Southeast Asia, India and Central & South America.

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Stem Cell Therapy Market by Product Type:

Stem Cell Therapy Market by Applications:

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Next part of the Stem Cell Therapy Market analysis report speaks about the manufacturing process. The process is analysed thoroughly with respect three points, viz. raw material and equipment suppliers, various manufacturing associated costs (material cost, labour cost, etc.) and the actual process. Stem Cell Therapy market competition by top manufacturers, with production, price, and revenue (value) and market share for each manufacturer as per following;

Top Manufacturer Included in Stem Cell Therapy Market:

And More

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After the basic information, the Stem Cell Therapy report sheds light on the production, production plants, their capacities, global production and revenue are studied. Also, the Stem Cell Therapy Market growth in various regions and R&D status are also covered.

Stem Cell Therapy Market Report by Key Region:

The global Stem Cell Therapy market is anticipated to rise at a considerable rate during the forecast period, between 2020 and 2026. In 2020, the market was growing at a mild rate and with the rising adoption of strategies by key players, the market is predicted to rise over the projected horizon. The report also tracks the most recent market dynamics, like driving factors, restraining factors, and industry news like mergers, acquisitions, and investments.

The report can help to know the market and strategize for business expansion accordingly. Within the strategy analysis, it gives insights from market positioning and marketing channel to potential growth strategies, providing in-depth analysis for brand fresh entrants or exists competitors within the Stem Cell Therapy industry. Global Stem Cell Therapy Market Report 2020 provides exclusive statistics, data, information, trends and competitive landscape details during this niche sector.

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Further in the report, Stem Cell Therapy Market is examined for price, cost and gross revenue. These three points are analysed for types, companies and regions. In prolongation with this data sale price for various types, applications and region is also included. The Stem Cell Therapy Industry consumption for major regions is given. Additionally, type wise and application wise consumption figures are also given.

To provide information on competitive landscape, this report includes detailed profiles of Stem Cell Therapy Market key players. For each player, product details, capacity, price, cost, gross and revenue numbers are given. Their contact information is provided for better understanding.

Other Major Topics Covered in Stem Cell Therapy market research report are as follows:

And another component .

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Stem Cell Therapy Market Report Aims To Outline and Forecast , Organization Sizes, Top Vendors, Industry Research and End User Analysis By 2026 -...

Q: What's the connection between the novel coronavirus and the thymus gland? A friend of ours who is a doctor says it's probably what keeps young kids from getting so sick. I've never even heard of the thymus. What does it have to do with coronavirus?

A: From the earliest days of the novel coronavirus pandemic, the data revealed a puzzling disparity. Older adults were at increased risk of grave illness when infected with the virus, but children seemed to have a certain level of protection. And while it has since become clear that children can indeed become seriously ill if they become infected, they do so at far lower rates than adults. The reasons for this are still being investigated, but some researchers have recently suggested the role of the thymus gland as a possible factor.

If you place your finger at the notch at the top of your breast bone and draw a vertical line downward a few inches, you've traced the location of your thymus. It's made up of two roughly triangular lobes, which sit behind the breastbone and between the lungs. The thymus has several functions, but perhaps its most important role is to help produce the cells that will become T-lymphocytes, or T-cells. (The "T" stands for thymus-derived.) These are white blood cells that protect the body from bacteria, fungi, viruses and other pathogens.

T-cells, which are the ninjas of the immune system, start out in the bone marrow as stem cells. The immature stem cells exit the marrow, move through the blood and enter a specific region of the thymus. There, they undergo a complex process that teaches them how to recognize a wide range of potentially dangerous and deadly invaders. As T-cells, their job is to circulate throughout the body and, when they encounter the molecular signature of the pathogen they've been trained to recognize, to attack. T-cells also activate other immune cells, produce proteins known as cytokines and have a role in regulating immune response.

The thymus is unique in that it reaches maturity in utero and is at its largest and most active in children. Starting at puberty, it gradually becomes less active, and the glandular tissue begins to shrink. This continues throughout a person's life. By the time someone has reached their mid-60s, the thymus is largely inactive. By their mid-70s, the gland has been mostly replaced with fat. This decrease in thymus function is believed to be one of the reasons that, in their later years, older adults become more susceptible to disease and infection.

Emerging research into COVID-19 has shown a marked decrease in the number of T-cells in some gravely ill patients. Scientists are now asking whether age-related thymus decline, which means T-cells aren't quickly replaced, may play a role in the severity of illness seen in older adults. The flip side of this is whether, due to their robust production of T-cells, children's immune systems are able to stay one step ahead of the novel coronavirus. It's only a working theory, but it shows promise, and research into how this may affect and inform treatment continues.

Dr. Eve Glazier is an internist and associate professor of medicine at UCLA Health. Dr. Elizabeth Ko is an internist and assistant professor of medicine at UCLA Health. Send your questions to askthedoctors@mednet.ucla.edu.

More:
Theory suggests thymus plays role in severity of COVID - Chicago Daily Herald

You could be Jadens cure

Jaden lives in Hilo, just turned 18 and has been diagnosed with a blood cancer. He is in dire need of a bone marrow or blood stem transplant. You could be Jadens cure.

If you are between the ages of 18-44 you can register online and add your name to the Hawaii marrow donor registry. A sample kit is mailed to you, you touch one swab in your mouth and send it back. Your tissue type is registered for worldwide use until you reach age 61.

I registered in Flint, Michigan, in 2006. I got the call in 2010 here in Kona at age 60. My donation of stems cells was done at St. Francis in Honolulu. It was totally painless and easy and the stem cells were sent to the mainland where the patient was cured of caner. The BeTheMatchHawaii health care professionals at St. Francis are very good, very friendly and like family to all the donors and patients.

Every three minutes there is a new blood or bone cancer diagnosis on our planet. Every one should go to http://www.BeTheMatchHawaii.org, where all of your questions can be answered and most importantly you can get tested and registered. You have the opportunity to change the world and save a life. How could anybody refuse to offer the gift of life when you learn that you are a match?

Since 1990, Hawaii citizens have donated to 468 cancer patients worldwide: The U.S., Brazil, Korea, Japan, Italy, Poland, actually 28 countries so far, including eight patients in Hawaii.

Please check it out, sign up and spread the word to all of your family, friends, coworkers, classmates, etc. Jaden is counting on us and you may never be match for any cancer patient, but if you are a match you can literally give someone the gift of life. How cool is that?

Larry Boucher

Kailua-Kona

Letters policy

Letters to the editor should be 300 words or less and will be edited for style and grammar. Longer viewpoint guest columns may not exceed 800 words. Submit online at http://www.westhawaiitoday.com/?p=118321, via email to letters@westhawaiitoday.com or address them to:

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Letters to the Editor: Aug. 3, 2020 - West Hawaii Today

DARZALEX SC reduces administration time from hours to minutes and demonstrates consistent efficacy with a reduction in administration-related reactions compared to intravenous DARZALEX (daratumumab)

TORONTO, Aug. 4, 2020 /CNW/ - The Janssen Pharmaceutical Companies of Johnson & Johnson announced today that Health Canada has approved DARZALEX SC (daratumumab), a new subcutaneous formulation of daratumumab.1 DARZALEX SC is approved in four regimens across five indications in patients with multiple myeloma, most notably newly diagnosed, transplant-ineligible patients as well as relapsed or refractory patients. As a fixed-dose formulation, DARZALEX SC can be administered over approximately three to five minutes, significantly less time than intravenous (IV) DARZALEX, which is administered over hours.2 DARZALEX SC is the only subcutaneous CD38-directed antibody approved in the treatment of multiple myeloma.

In the Phase 3 COLUMBA study published in The Lancet, DARZALEX SC demonstrated a consistent overall response rate (ORR) and pharmacokinetics and a similar safety profile compared with IV DARZALEXin patients with relapsed or refractory multiple myeloma. In addition, there was a nearly two-thirds reduction in systemic administration-related reactions (ARRs) for DARZALEX SC compared to IV DARZALEX(13 per cent vs. 34 per cent, respectively).3

"DARZALEX has become a backbone therapy in the treatment of multiple myeloma, supported by a robust body of evidence in both the frontline and relapsed and refractory settings," says Dr. Darrell White, Hematologist, Queen Elizabeth II Health Sciences Centre, Halifax. "With this new subcutaneous formulation, not only is treatment much more convenient for patients, but it will also play a very important role in reducing wait times and the burden on our busy healthcare system, especially during this time."

The approval is based on data from the Phase 3 COLUMBA and Phase 2 PLEIADES studies.4,5In the COLUMBA study, the ORR was non-inferior for patients taking DARZALEX SC as monotherapy compared to those taking IV DARZALEXas monotherapy (41 per cent vs. 37 per cent, respectively).6 Additionally, in the Phase 2 PLEIADES study evaluating the efficacy and safety of DARZALEXSC in combination therapies, objective responses were demonstrated in combination with bortezomib, melphalan and prednisone (D-VMP) in newly diagnosed transplant ineligible patients. In addition, objective responses were demonstrated in combination with lenalidomide and dexamethasone (D-Rd) in relapsed or refractory patients who received one prior line of therapy.7In a pooled safety population of 490 patients who received DARZALEXSC as monotherapy or in combination, the ARR rate was 11 per cent.8

DARZALEX SC is approved in all current IV indicationsincluding (1) in combination with bortezomib, melphalan and prednisone in newly diagnosed patients who are ineligible for autologous stem cell transplant, (2) in combination with lenalidomide and dexamethasone in newly diagnosed patients who are ineligible for autologous stem cell transplant and in patients with relapsed or refractory multiple myeloma who have received at least one prior therapy, (3) in combination with bortezomib and dexamethasone in patients who have received at least one prior therapy, and (4) as monotherapy, in patients who have received at least three prior lines of therapy including a proteasome inhibitor (PI) and an immunomodulatory agent or who are double-refractory to a PI and an immunomodulatory agent.9

Active discussions are ongoing with public insurers to determine how DARZALEX SC can be made accessible for both relapsed or refractory patients as well as newly diagnosed, transplant ineligible patients.

"This approval exemplifies Janssen's mission and commitment to bringing together passion, science and ingenuity to advance novel solutions for patients," said Mathai Mammen, M.D., Ph.D., Global Head, Janssen Research & Development, LLC.

About the COLUMBA Study The randomised, open-label, multicenter Phase 3 COLUMBA study included 522 patients (median age of 67 years) with multiple myeloma who had received at least three prior lines of therapy including a proteasome inhibitor (PI) and an immunomodulatory drug (IMiD), or whose disease was refractory to both a PI and an ImiD. In the arm that received DARZALEX SC (n=263), patients received a fixed dose of DARZALEX SC 1,800 milligrams (mg), co-formulated with recombinant human hyaluronidase PH20 (rHuPH20) 2,000 Units per milliliter (U/mL), subcutaneously weekly for Cycles 1 2, every two weeks for Cycles 3 6 and every four weeks for Cycle 7 and thereafter. In the IV DARZALEXarm (n=259), patients received DARZALEXfor IV infusion 16 milligrams per kilogram (mg/kg) weekly for Cycles 1 2, every two weeks for Cycles 3 6 and every four weeks for Cycle 7 and thereafter. Each cycle was 28 days. In the arm that received DARZALEX SC, it was given in a fixed volume of 15 mL over three to five minutes; the median injection time was five minutes. In the arm that received the IV administration, the median durations of the first, second and subsequent IV DARZALEXinfusions were 7.0, 4.3 and 3.4 hours, respectively. Patients in both arms continued treatment until disease progression or unacceptable toxicity.10,11

About the PLEIADES Study The non-randomised, open-label, parallel assignment Phase 2 PLEIADES study included adults with multiple myeloma, including 67 patients with newly diagnosed multiple myeloma who were treated with 1,800 mg of DARZALEX SC in combination with bortezomib, melphalan, and prednisone (D-VMP) and 65 patients with relapsed or refractory disease who were treated with 1,800 mg of DARZALEX SC plus lenalidomide and dexamethasone (D-Rd). The primary endpoint for the D-VMP and D- Rd cohorts was overall response rate.12

About DARZALEXand DARZALEX SCDARZALEX is the first CD38-directed monoclonal antibody (mAb) approved to treat multiple myeloma and in 2020, DARZALEX SC (daratumumab) follows as the only subcutaneous CD38-directed antibody approved to treat patients with multiple myeloma.13It binds to CD38,a surface protein highly expressed across multiple myeloma cells.14 DARZALEX induces tumor cell death through cell lysis via multiple immune-mediated mechanisms of action, including complement-dependent cytotoxicity (CDC), antibody-dependent cellular cytotoxicity (ADCC) and antibody-dependent cellular phagocytosis (ADCP).15DARZALEX has also demonstrated immunomodulatory effects such as increasing CD4+ and CD8+ T-cells counts, which may contribute to clinical response.16

In August 2012, Janssen Biotech, Inc. and Genmab A/S entered a worldwide agreement, which granted Janssen an exclusive license to develop, manufacture and commercialize DARZALEX. Janssen Inc. commercializes DARZALEX and DARZALEX SC in Canada. For full Prescribing Information and more information about DARZALEX and DARZALEX SC, please visit http://www.janssen.com/canada.

About Multiple MyelomaMultiple myeloma is an incurable blood cancer that affects a type of white blood cell called plasma cells, which are found in the bone marrow.17 When damaged, these plasma cells rapidly spread and replace normal cells with tumors in the bone marrow. In 2020, it is estimated that 3,400 Canadians will be diagnosed with multiple myeloma and there will be 1,600 deaths associated with the disease.18 While some patients with multiple myeloma have no symptoms in the early stages, patients are diagnosed due to symptoms that can include bone disease or pain, anemia, calcium elevation, and kidney problems.19

About the Janssen Pharmaceutical Companies of Johnson & Johnson At Janssen, we're creating a future where disease is a thing of the past. We're the Pharmaceutical Companies of Johnson & Johnson, working tirelessly to make that future a reality for patients everywhere by fighting sickness with science, improving access with ingenuity, and healing hopelessness with heart. We focus on areas of medicine where we can make the biggest difference: Cardiovascular & Metabolism, Immunology, Infectious Diseases & Vaccines, Neuroscience, Oncology, and Pulmonary Hypertension.

Learn more at http://www.janssen.com/canada. Follow us at @JanssenCanada. Janssen Inc. is a member of the Janssen Pharmaceutical Companies of Johnson & Johnson.

*All trademark rights used under license. **Dr. White was not compensated for any media work. He has been compensated as a consultant.

Cautions Concerning Forward-Looking StatementsThis press release contains "forward-looking statements" as defined in the Private Securities Litigation Reform Act of 1995 regarding DARZALEX SC. The reader is cautioned not to rely on these forward-looking statements. These statements are based on current expectations of future events. If underlying assumptions prove inaccurate or known or unknown risks or uncertainties materialize, actual results could vary materially from the expectations and projections of Janssen Inc., any of the other Janssen Pharmaceutical Companies and/or Johnson & Johnson. Risks and uncertainties include, but are not limited to: challenges and uncertainties inherent in product research and development, including the uncertainty of clinical success and of obtaining regulatory approvals; uncertainty of commercial success; manufacturing difficulties and delays; competition, including technological advances, new products and patents attained by competitors; challenges to patents; product efficacy or safety concerns resulting in product recalls or regulatory action; changes in behavior and spending patterns of purchasers of health care products and services; changes to applicable laws and regulations, including global health care reforms; and trends toward health care cost containment. A further list and descriptions of these risks, uncertainties and other factors can be found in Johnson & Johnson's Annual Report on Form 10-K for the fiscal year ended December 29, 2019, including in the sections captioned "Cautionary Note Regarding Forward-Looking Statements" and "Item 1A. Risk Factors," and in the company's most recently filed Quarterly Report on Form 10-Q, and the company's subsequent filings with the Securities and Exchange Commission. Copies of these filings are available online at http://www.sec.gov, http://www.jnj.com or on request from Johnson & Johnson. None of the Janssen Pharmaceutical Companies nor Johnson & Johnson undertakes to update any forward-looking statement as a result of new information or future events or developments.

References:

1

[DARZALEX SC Product Monograph, Janssen Inc., July 29, 2020]

2

[DARZALEX SC Product Monograph, Janssen Inc., July 29, 2020]

3

Mateos MV, et al. Subcutaneous versus intravenous daratumumab in patients with relapsed or refractory multiple myeloma (COLUMBA): a multicentre, open-label, non-inferiority, randomised, phase 3 trial [published online ahead of print March 23, 2020]. Lancet Haematol doi.org/10.1016/S2352-3026(20)30070-3.

4

Mateos M-V et al. Efficacy and Safety of the Randomized, Open-Label, Non-inferiority, Phase 3 Study of Subcutaneous (SC) Versus Intravenous (IV) Daratumumab (DARA) Administration in Patients (pts) With Relapsed or Refractory Multiple Myeloma (RRMM): COLUMBA. 2019 American Society of Clinical Oncology Annual Meeting. June 2019.

5

Janssen Research & Development, LLC. A Study to Evaluate Subcutaneous Daratumumab in Combination With Standard Multiple Myeloma Treatment Regimens. In: ClinicalTrials.gov [Internet]. Bethesda (MD): National Library of Medicine (US). 2000 [cited July 5, 2019]. Available at: https://clinicaltrials.gov/ct2/show/NCT03412565. Identifier: NCT03412565.

6

Mateos MV, et al. Subcutaneous versus intravenous daratumumab in patients with relapsed or refractory multiple myeloma (COLUMBA): a multicentre, open-label, non-inferiority, randomised, phase 3 trial [published online ahead of print March 23, 2020]. Lancet Haematol doi.org/10.1016/S2352-3026(20)30070-3.

7

Chari A, M. J., McCarthy H, et al Subcutaneous daratumumab plus standard treatment regimens in patients with multiple myeloma across lines of therapy: PLEIADES study update. Poster presented at: 61st American Society of Hematology (ASH) Annual Meeting. Orlando, FL.

8

[DARZALEX SC Product Monograph, Janssen Inc., July 29, 2020]

9

[DARZALEX SC Product Monograph, Janssen Inc.,July 29, 2020]

10

[DARZALEX SC Product Monograph, Janssen Inc., July 29, 2020]

11

Mateos MV, et al. Subcutaneous versus intravenous daratumumab in patients with relapsed or refractory multiple myeloma (COLUMBA): a multicentre, open-label, non-inferiority, randomised, phase 3 trial [published online ahead of print March 23, 2020]. Lancet Haematol doi.org/10.1016/S2352-3026(20)30070-3.

12

Janssen Research & Development, LLC. A Study to Evaluate Subcutaneous Daratumumab in Combination With Standard Multiple Myeloma Treatment Regimens. In: ClinicalTrials.gov [Internet]. Bethesda (MD): National Library of Medicine (US). 2000 [cited July 5, 2019]. Available at: https://clinicaltrials.gov/ct2/show/NCT03412565. Identifier: NCT03412565.

13

Janssen Research & Development, LLC. A Study to Evaluate Subcutaneous Daratumumab in Combination With Standard Multiple Myeloma Treatment Regimens. In: ClinicalTrials.gov [Internet]. Bethesda (MD): National Library of Medicine (US). 2000 [cited July 5, 2019]. Available at: https://clinicaltrials.gov/ct2/show/NCT03412565. Identifier: NCT03412565.

14

[DARZALEX SC Product Monograph, Janssen Inc., July 29, 2020]

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[DARZALEX SC Product Monograph, Janssen Inc., July 29, 2020]

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[DARZALEX SC Product Monograph, Janssen Inc., July 29, 2020]

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Kumar, SK et al. Risk of progression and survival in multiple myeloma relapsing after therapywith IMiDs and bortezomib: a multicenter international myeloma working group study. Leukemia. 2012 Jan; 26(1):149-57.

18

Canadian Cancer Society. "Signs and Symptoms of Multiple Myeloma." Available at: https://www.cancer.ca/en/cancer-information/cancer-type/multiple-myeloma/statistics/?region=on.Accessed June 2020.

19

Canadian Cancer Society. "Signs and Symptoms of Multiple Myeloma." Available at: http://www.cancer.ca/en/cancer-information/cancer-type/multiple-myeloma/signs-and-symptoms/?region=on.Accessed June 2020.

SOURCE Janssen Inc.

For further information: Media Contact: Janssen Inc., Jennifer McCormack, Office: (416) 382-5121; Investor Contact: Jennifer McIntyre, Office: (732) 524-3922

http://www.janssen.ca/

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Janssen Announces Health Canada Approval of DARZALEX* SC, a New Subcutaneous Formulation for the Treatment of Patients with Multiple Myeloma - Canada...

Adrenoleukodystrophy (ALD) is a rare genetic condition that causes the buildup of very-long-chain fatty acids (VLCFAs) in the brain. The defective gene in ALD, commonly referred to as a genetic mutation, can cause several different but related conditions: adrenomyelopathy (AMN), Addisons disease, and the most common and most devastating form cerebral ALD. Cerebral ALD strikes boys between ages 4 and 10, leading to permanent disability and death usually within four to eight years.

Signs and symptoms of the adrenomyeloneuropathy type appear between early adulthood and middle age. Affected individuals develop progressive stiffness and weakness in their legs (paraparesis), experience urinary and genital tract disorders, and often show changes in behavior and thinking ability. Most people with the adrenomyeloneuropathy type also have adrenocortical insufficiency. In some severely affected individuals, damage to the brain and nervous system can lead to an early death.

For detailed analysis on the symptoms of Global Adrenoleukodystrophy Pipeline Analysis browse through https://univdatos.com/report/global-adrenoleukodystrophy-pipeline-analysis-2019

The diagnosis of the disease includes genetic counseling, differential diagnosis, biochemical and molecular diagnosis. Genetic counseling must be offered to the parents of affected boys, adult males, and women with X-ALD and their family to detect: carriers who can be offered prenatal diagnosis, and asymptomatic or pre-symptomatic men or women who can benefit from therapeutic interventions. Regular follow-up in presymptomatic males can prevent serious morbidity and mortality.

Despite significant mortality risk, allogeneic HCT remains the only therapeutic intervention that can arrest the progression of cerebral demyelination in X-ALD, provided the procedure is performed very early, i.e., when affected boys or men have no or minor symptoms due to cerebral demyelinating disease. In the future, transplantation of autologous hematopoietic stem cells that have been genetically corrected with a lentiviral vector before re-infusion might become an alternative to autologous HCT, once the very encouraging results obtained in the first two treated patients would have been extended to a larger number of patients with cerebral X-ALD.

For detailed analysis on the management in Global Adrenoleukodystrophy Pipeline Analysis browse through https://univdatos.com/report/global-adrenoleukodystrophy-pipeline-analysis-2019

There are currently only two available treatments for childhood cerebral ALD: Lorenzos oil and stem cell transplantation, using either umbilical cord stem cells or bone marrow stem cells. Both treatment approaches have shown promise and been effective for some boys with ALD, but they also both have drawbacks. The therapeutic pipeline of Adrenoleukodystrophy consists of approximately 9+ products in different stages of development. Currently, 3+ drugs are in Phase III development and major drugs are in the late stage.

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Some of the key players include Applied Genetic Technologies Corporation; Bluebird bio; Magenta Therapeutics; MedDay Pharmaceuticals; Minoryx Therapeutics; NeuroVia; Orpheris; ReceptoPharm; SOM Biotech; and Viking Therapeutics. Several M&As along with partnerships have been undertaken by these players to facilitate costumers with hi-tech and innovative products.

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Global Adrenoleukodystrophy Treatment Trends and Highlights - Bulletin Line

For many patients with multiple myeloma, a new generation of drugs and drug combinations is producing better outcomes and fewer side effects. In recent months, several novel therapies studied and tested by Dana-Farber scientists have gained approval from the U.S. Food and Drug Administration (FDA) or taken a step toward approval after posting solid results in clinical trials.

The drugs are the fruit of years of research into improving treatment for multiple myeloma, a cancer of white blood cells known as plasma cells in the bone marrow. Many of the new agents are biologically derived made from substances such as proteins and antibodies found in living things and target biological mechanisms in a very specific, targeted fashion. Dana-Farber researchers have played a key role in these efforts.

These are each powerful examples of how next-generation novel therapies translated here at Dana-Farber from bench to bedside are further improving outcomes for our patients, and at a remarkable pace, says Paul G. Richardson, MD, clinical program leader and director of clinical research at the Jerome Lipper Multiple Myeloma Center at Dana-Farber.

Following a Dana-Farber-led clinical trial, the FDA recently approved the novel drug isatuximab in combination with pomalidomide and dexamethasone for adults with relapsed or refractory (non-responsive) myeloma who have received at least two prior therapies, including lenalidomide and drugs known as proteasome inhibitors. The drug went into trials after laboratory work by Dana-Farbers Yu-Tzu Tai, PhD, and Kenneth Anderson, MD, showed it was active against myeloma cells. In the clinical trial, the three-drug combination lowered the risk that the disease would progress by 40%, compared to pomalidome and dexamethasone alone.

Dana-Farber investigators conducted laboratory research and led the first clinical trial of the drug melflufen plus dexamethasone in patients with relapsed or refractory myeloma. Melflufen is a peptide conjugate drug made of a stub of protein, or peptide, joined to a chemotherapy agent and delivers a toxic payload directly to myeloma cells in a selective, time-sparing approach.

Results from an early-phase clinical trial published in Lancet Oncology showed the drug is active in patients with myeloma and is safe at recommended doses. Unlike the previously used standard drug melphalan, it doesnt cause mucositis inflammation of membranes within the digestive tract or hair loss. The results prompted investigators to launch two larger trials, some of whose results are being processed and are due to be published soon.

In a major study published in Blood, Dana-Farber researchers and their associates found that in patients newly diagnosed with myeloma who are eligible for a stem cell transplant, adding the drug daratumumab to the standard three-drug regimen produced more responses, and deeper responses, than in patients receiving the three-drug therapy alone.

Dana-Farber researchers were involved in the development and initial testing of the drug belantamab mafodotin, which has shown considerable promise in clinical trials and has been granted priority review for approval by the FDA.

An antibody conjugate drug consisting of an antibody that specifically targets myeloma cells and an agent that disrupts cell division, its use was informed by a preclinical trial at Dana-Farber involving Yu-Tzu Tai, PhD, and Kenneth Anderson, MD. Balantamab mafodotin was tested in studies led by Paul Richardson, MD, in patients with relapsed or refractory multiple myeloma whose disease continued to worsen after a stem cell transplant, chemotherapy, or other treatment. In the DREAMM-1 and -2 trials, the drug showed strong anti-myeloma activity with manageable side effects.

After certification in Internal Medicine, Hematology and Medical Oncology, as well as working in Cancer Pharmacology from 1994 onwards at Dana-Farber Cancer Institute (DFCI), Dr. Paul Richardson joined the Jerome Lipper Myeloma Center in 1999, was appointed Clinical Director in 2001, and led the development of several first-generation novel drugs including bortezomib, lenalidomide and pomalidomide for the treatment of multiple myeloma. Subsequent studies have focused on next-generation novel drugs including panobinostat and second-generation proteasome inhibitors including ixazomib. More recently, his clinical innovations have been in the development of the breakthrough monoclonal antibodies elotuzumab and daratumumab for the treatment of both untreated and relapsed myeloma, as well as isatuximab and more broadly, antibody drug conjugates including belantamab mafodotin, as well as other immunotherapeutic strategies. In addition to these agents, he is leading the development of melflufen, a targeted cytotoxic and an first-in-class small molecule inhibitor selinexor, which inhibits XPO-1, a key nuclear export protein, as well as first-in-human studies of cereblon E3 ligase modulators (so called CELMoDs) for the treatment of relapsed and refractory myeloma.

Over the last decade, his major effort has been focused on the development of lenalidomide, bortezomib and dexamethasone (so-called RVD), and its incorporation as part of the Intergroup Francophone Myelome (IFM)/DFCI clinical trial in newly diagnosed patients eligible for stem cell transplant treated with RVD. This regimen has generated an unprecedented response rate, leading to its adoption in this international study, as well as others in the United States and elsewhere. This particular trial incorporates genomic and proteomic evaluation to establish a future platform for tailored therapy and the optimal positioning of stem cell transplant, with results anticipated in 2021-22. Furthermore, RVD has been established as a backbone to which next-generation agents are being added, including elotuzumab, daratumumab and isatuximab, as well as panobinostat.

He has published extensively, having authored or co-authored over 400 original articles and 330 reviews, chapters, and editorials in peer-reviewed journals. In addition to holding positions on the Editorial Boards of leading journals, he is prior Chairman of the Multiple Myeloma Research Consortium (MMRC), Clinical Trials Core, a position held for 5 years as part of a rotating tenure, and for which he continues as a member of the Steering and Project Review Committee. He was also a member of ASCO Hematologic Malignancies Subcommittee for the required one-year term, and then for one year on the ASCO Internet Cancer Information Committee during 2017. He was appointed Chair of the Alliance Myeloma Committee in 2011 and continues in this role.

Honors include the George Canellos Award for Excellence in Clinical Research and Patient Care, and The Tisch Outstanding Achievement Award for Clinical Research, as well as an honorary Fellowship of the Royal College of Physicians (UK), given in recognition for international contributions in multiple Myeloma and stem cell transplantation. He was a co-recipient of the prestigious Warren Alpert Foundation Prize in recognition of the successful therapeutic targeting of the ubiquitin-proteasome pathway in 2012. He was also a co-recipient of the Accelerator Award for contributions to clinical research and patient enrollment in MMRC studies, as well as for the Research Center of the Year Award in 2009, followed by the second award for Center of the Year in 2017. He was ranked by Thomson Reuters Science Watch amongst the top 19 investigators at DFCI for the most highly cited research in 2016. He was the co-recipient of the ASH Ernest Beutler Prize for clinical science and translational research in the development of proteasome inhibition as an effective treatment strategy for multiple myeloma in 2015; the COMY Award for MM research (Paris, France) in 2016, and the prestigious IMF Robert A. Kyle Lifetime Achievement Award in 2017, and the Morse Research Award in 2019.

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Wave of New Therapies Improve Outcomes for Patients with Multiple Myeloma - Dana-Farber Cancer Institute

Dear Doctor: What's the connection between the novel coronavirus and the thymus gland? A friend of ours who is a doctor says it's probably what keeps young kids from getting so sick. I've never even heard of the thymus. What does it have to do with coronavirus?

Dear Reader: From the earliest days of the novel coronavirus pandemic, the data revealed a puzzling disparity. Older adults were at increased risk of grave illness when infected with the virus, but children seemed to have a certain level of protection. And while it has since become clear that children can indeed become seriously ill if they become infected, they do so at far lower rates than adults. The reasons for this are still being investigated, but some researchers have recently suggested the role of the thymus gland as a possible factor.

If you place your finger at the notch at the top of your breast bone and draw a vertical line downward a few inches, you've traced the location of your thymus. It's made up of two roughly triangular lobes, which sit behind the breastbone and between the lungs. The thymus has several functions, but perhaps its most important role is to help produce the cells that will become T-lymphocytes, or T-cells. (The "T" stands for thymus-derived.) These are white blood cells that protect the body from bacteria, fungi, viruses and other pathogens.

T-cells, which are the ninjas of the immune system, start out in the bone marrow as stem cells. The immature stem cells exit the marrow, move through the blood and enter a specific region of the thymus. There, they undergo a complex process that teaches them how to recognize a wide range of potentially dangerous and deadly invaders. As T-cells, their job is to circulate throughout the body and, when they encounter the molecular signature of the pathogen they've been trained to recognize, to attack. T-cells also activate other immune cells, produce proteins known as cytokines and have a role in regulating immune response.

The thymus is unique in that it reaches maturity in utero and is at its largest and most active in children. Starting at puberty, it gradually becomes less active, and the glandular tissue begins to shrink. This continues throughout a person's life. By the time someone has reached their mid-60s, the thymus is largely inactive. By their mid-70s, the gland has been mostly replaced with fat. This decrease in thymus function is believed to be one of the reasons that, in their later years, older adults become more susceptible to disease and infection.

Emerging research into COVID-19 has shown a marked decrease in the number of T-cells in some gravely ill patients. Scientists are now asking whether age-related thymus decline, which means T-cells aren't quickly replaced, may play a role in the severity of illness seen in older adults. The flip side of this is whether, due to their robust production of T-cells, children's immune systems are able to stay one step ahead of the novel coronavirus. It's only a working theory, but it shows promise, and research into how this may affect and inform treatment continues.

Eve Glazier, M.D., MBA, is an internist and associate professor of medicine at UCLA Health. Elizabeth Ko, M.D., is an internist and assistant professor of medicine at UCLA Health.

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Theory suggests thymus plays role in severity of COVID - Times Record

Companies, Organizations Needed to Host Events

MONTVALE, NJ Because of the current, nationwide surge in COVID-19 infections throughout the Sun Belt, along with the constant importance of bolstering the local blood supply, theres now significant need for residents to donate blood and, if possible, convalescent plasma.

The nonprofit, blood-collection organization Vitalantis offering an open-to-the-public Mark Spatola Memorial Drive on Tuesday, August 11 from 2 p.m. to9 p.m. at St. Agnes Parish Center, 55 South Avenue,Atlantic Highlands.

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Vitalant is also seeking companies and organizations throughoutMonmouth Countyto host blood donation events. Information on hosting a donation event is available by clicking here.

Individuals who have recovered from COVID-19 are urged to donate blood plasma. Known as convalescent plasma, this blood component contains antibodies that may provide seriously ill patients an extra boost in fighting the disease.

Vitalant is providing an antibody test which is authorized by the U.S. Food and Drug Administration to all donors; results will be available in private, online donor accounts, approximately two weeks after a donation.There is also a great need for blood platelets small cells in the blood that form clots to prevent bleeding, while also helping with anemia and low blood countsand type O-negative,the universal blood type.

To promote the increase of blood, convalescent plasma, and platelet donations,regular event host companies and organizations many of which put their events on pause due to the pandemic are asked toconsider returning to a consistent schedule of donation events.

FEMA has specifically identified blood donation as an essential and integral component of the emergency support function. Of note, coronavirus cannot be transferred through the blood. And, as always, the blood collection process is safe with noimpact on the donor's immune system. Vitalant staff follows rigorous safety and disinfection protocols at its blood drives and donation centers and have always required individuals to be in good health to donate blood.

Vitalant also maintains four New Jersey blood centers, with hours and street addresses as follows:

Healthy individuals age 16 or older, who weigh at least 110 pounds, may donate blood; 16- and 17-year-olds must have proof of birth date and signed consent forms, either in English or Spanish. Donors should eat a moderate meal prior to donating, and also bring identification featuring their signature.

On occasion, last-minute changes to scheduling for a donation event will occur. As a result, it is recommended that anyone planning to donate blood at a Vitalant donation event call 201-251-3703, toll free, to confirm timing and location details. Additional information about donating blood is also available by visitingwww.vitalant.org.

About Vitalant in New Jersey

A not-for-profit organization that supplies blood and blood products to hospitals in the New Jersey/New York region, Bergen County-based Vitalant (previously Community Blood Services) has been devoted to serving the communitys transfusion medicine needs since 1953. Donations of blood and blood products, umbilical cord blood, stem cells, and bone marrow help to join individuals, organizations, businesses, and entire communities together in partnership to help save lives.

About Vitalant

Arizona-based Vitalant is among the nations oldest and largest transfusion medical organizations in the U.S. Founded in 1943, its blood centers division serves some 700 hospitals across the United States. A founding member of Americas Blood Centers and the AABB (formerly the American Association of Blood Banks), Vitalant also operates biological products distribution services, a quality consulting group, and a world-renowned transfusion medicine research institute. It also is a partner in the operation of high-volume donor testing laboratories.

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Vitalant to Hold Blood Donation Drive on August 11 in Atlantic Highlands - TAPinto.net

CRISPR is a catchy acronym that originally described a naturally occurring gene editing tool, derived from a bacterial defense mechanism against viruses. Its the name on everybodys lips in the intersecting realms of science, medicine, ethics, and politics. From the moment of its discovery, CRISPR-Cas9 looked like a miraculous solution to all of the problems that gene editing efforts have experienced over decades of trial and error. This revolutionary new gene editing technique has opened the doors to both massive scientific progress and ethical controversy. Now more than ever, were seeing that CRISPR still has massive kinks to work out. Can we ever fully understand the social and scientific implications of gene editing, and should we use it in humans before we learn how to properly harness it?

What is gene editing?

The 20th century saw genetic scientists increasingly focus their pursuits on the sub-microscopic. As science delved deeper into the human body in an attempt to uncover the molecular minutiae of life, the possibility of reaching into the cell and manipulating its genetic material began to look more and more real. Even by the 1950s, evidence had been mounting for decades that deoxyribonucleic acid (DNA), an unassuming molecule residing in a central cellular compartment called the nucleus, was the physical genetic material that passed information from parent to child. Finally, in 1953, landmark work by Kings College biochemist Rosalind Franklin allowed Cambridge researchers to reveal the structure of DNA and confirm its role in heredity once and for all.

Starting from a hesitant foundation, molecular genetics exploded in both scope and popularity over subsequent decades. With the secrets of heredity increasingly out in the open, human ambition demanded that we try to bend DNA to our willand now we can. These days, targeted gene editing techniques revolve around artificially-engineered molecular tools known as nucleases, whose earliest use was in 1996not even 50 years after the discovery of DNAs structure. Engineered nucleases are often described as molecular scissors. Fundamentally, they have two main parts: one part that finds and grabs onto the target DNA within a cell, and one part that snips a piece out of that DNA.

How CRISPR works

CRISPR is similar to other directed nucleases, but its much better at its job. The CRISPR part is secondary to the systems gene editing applications; the truly important discovery, which Jennifer Doudna made in 2012, was a protein that she called CRISPR-associated protein 9, or Cas9. This protein is the nuclease tool, the pair of molecular scissors that finds, sticks to, and snips target DNAand its more accurate than anything weve ever seen before.

In bacteria, CRISPR is a section of the genome that acts as an immune memory, storing little snippets of different viruses genetic material as DNA after failed infections, like trophies. When a once-active virus attempts to invade a bacterium, the mobile helper Cas9 copies down the relevant snippet from CRISPR in the form of ribonucleic acid, or RNA. RNA is a molecule thats virtually identical to DNA, except for one extra oxygen atom. Because of this property, the RNA sequence that Cas9 holds can pair exactly, nucleotide by nucleotide, with the viral targets DNA, making it extremely efficient at finding that DNA. With a freshly transcribed RNA guide, the bacterium can deploy Cas9 to findand cut outthe corresponding section of viral genetic material, rendering the attacker harmless.

The existence of CRISPR in bacteria was old news by 2012, but Doudnas discovery of Cas9s function was revolutionary. With a little creativity and ingenuity, such a simple and accurate nuclease can be modified to be much more than just a pair of scissors. Using synthetic RNA guides and certain tweaks, Cas9 can be used to remove specific genes, cause new insertions to genomes, tag DNA sequences with fluorescent probes, and much more.

The possibilities seem endless.What if we could go into the body of a human affected by a hereditary disease and change that persons DNA to cure them? What if we could modify reproductive germ cells in human bodies (which give rise to sperm and eggs), or make targeted genetic edits in the very first cell of an embryo? Nine months of division and multiplication later, that cell would give rise to a human being whose very nature has been deliberately tweakedand their childrens nature, and their childrens. With the accuracy and accessibility of the CRISPR/Cas9 system, these ideas arent hypotheticals. In 2019, CRISPR edits in bone marrow stem cells were successfully used to cure sickle cell anemia in a Mississippi woman. Beta thalassaemia, another genetic disease of the blood, has also been treated this way. In 2018, Chinese scientist He Jiankui even claimed that he had conferred HIV immunity upon twin girls using embryonic editing.

CRISPRs complications

At first glance, CRISPR looks like a miraclebut it isnt perfect. What if some cells were affected by edits, but others werent, creating a strange genetic mosaic in a human body? What if, in trying to modify a specific gene, we accidentally hit a different section of DNA nearby? What if we got the right gene, but it also affected a different part of the body that we didnt know about?

These problems arent hypotheticals either. So-called mosaicism and off-target editing are huge concerns among CRISPR scientists. Mosaicism is of particular concern in embryonic editing. Though CRISPR injections are carried out when an embryo is single-celled, CRISPR doesnt always appear to work until after several rounds of cell divisionand it doesnt work in every cell. If not all the cells in the body are affected by gene editing that is intended to eliminate a genetic disease, the disease could remain in the body. It may be possible to combat mosaicism with faster gene editing (so that cells dont replicate before theyve had a chance to become CRISPR-modified), altering sperm and egg cells before they meet to form an embryo, and developing more precise CRISPR gene editing which is in itself a challenge, thanks to off-target editing.

In nature, a little bit of off-target editing could actually make the CRISPR-Cas9 defense system stronger with the principle of redundancy. Flexibility in the form of imprecision could allow a bacterium to neutralize viruses whose exact genetic sequences have not yet been encountered: viruses related to, but not identical to, previous attackers. In clinical and therapeutic applications, on the other hand, precision is everything. And unfortunately, as time passes, CRISPRs level of precision seems further and further off. Preprints released just this year reveal that the frequency and magnitude of CRISPRs off-target edits in human cells may be worse than we had previously known. Large proportions of cells with massive unwanted DNA deletions, losses of entire chromosomes in experimental embryos, and shuffling of genetic sequences were observed.

Of course, not only do scientists need to avoid off-target edits, but they also need to know when such undesired edits have occurred. Off-target effects can be detected by genome sequencing and computer prediction tools, but theres no perfect way to do it yetthere may still be editing misses that were, well, missing. Off-target edits themselves could be minimized by altering the RNA transcript that Cas9 carries to make it more accurate, altering Cas9 itself, or reducing the actual amount of Cas9 protein released into the cell (though this could also reduce on-target effects). Replacing Cas9 itself with other Cas variants, like smaller and more easily deliverable CasX and CasY proteins, is a promising possibility for more efficient editing, but these candidates still run into many of the same problems as Cas9. More strategies are constantly being discovered, proposed, and explored, but were still nowhere near perfect.

Perhaps most importantly, even barring any purely technical problems, is that humans remain in sheer ignorance of much of the extent and consequences of pleiotropy, a phenomenon where a genes presence or deletion has more than one effect in the human body. Even genes whose function we think we know well might have totally unexpected additional functions. On the other side of the coin, we dont have a comprehensive understanding of how many different genetic contributors there are to any given trait or disease, much less where they lie in the genome. We dont understand the way that thousands of variations across the entire genome contribute to appearance, personality, and health. Assuming that some genes are good and others are bad is morally dangerous, and scientifically reprehensible. In reality, we are not ready for genetic determinism, and may never be.

A great responsibility

Humanity has discovered a great power, but we all know what comes with great power. Questions of which edits are necessary for health (is mild Harlequin syndrome a disease or a cosmetic concern?), whether edits are ethical (should autism and homosexuality be considered curable conditions?), and the possibility of designer babies, among others, are pertinent and require thorough discussion. We also need to realize that making these types of changes isnt our decision until we can get CRISPR right, and understand the genome well enough to target particular phenotypes. Though most scientists are aware of the difficulties of CRISPR and its use is generally tightly regulated, some scientistsand laypeopleare less careful. He Jiankuis apparent miracle HIV cure led to his arrest and imprisonment for unapproved and unethical practice. Its no great surprise that his work likely fell prey to off-target effects and mosaicism; even if he got it right, his intended change could alter cognitive function, and who knows what else?

Non-scientists are getting involved too: in 2018, self-proclaimed biohacker Josiah Zayner publicly injected his own arm with what he claimed was muscle-enhancing CRISPR. Though Zayner is one of the most vocal, hes not the only one of his kind. Quieter biohackers, untrained people without a scientific background or a good understanding of how CRISPR can go wrong, are attempting to edit themselves and even their pets.

Laypeople have an unquestionable place in science: the scientific discipline needs fresh perspectives and creativity that stuffy academics cant offer. CRISPR is still in its infancy, though. Before we know much, much more about its capabilities and consequences, there can be no place for black market gene editing kits, rogue scientists altering human embryonic and germline DNA, or basement geneticists injecting Cas9 into their dogs. Who can say what effects these interventions might have, not just on edited individuals, but on the futures of entire species?

Some say that gene editing is an act of hubris, destined to backfire spectacularly and horrendously. Others believe that its our responsibility to use CRISPR to improve lives. Which of these opinions is true depends on how science walks a narrow tightrope, though Im inclined to agree with the latterand add that our responsibility is not just to master gene editing, but to make clear and public its many faults and failings. The truth, in all its complexity, needs to overcome pop sciences oversimplification and sensationalism. Promising new advances and techniques are on the horizon, but we have a long way to go. Gene editing is no joke; humanity is playing with fire. With an incredibly accurate and accessible nuclease making its way into labs and garages across the world (while its flaws continue to be uncovered year by year), it is more important than ever for the world to understand and discuss the long-reaching consequences and responsible use of gene editing technology. CRISPR is not a miracle, but gene editing may very well be the future of humanityand its on us to keep it under control.

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The Trouble With CRISPR - Strand

The report offers a systematic presentation of the existing trends, growth opportunities, market dynamics that are expected to shape the growth of the Orthopedic Regenerative Medicine Market. The various research methods and tools were involved in the market analysis, to uncover crucial information about the market such as current & future trends, opportunities, business strategies and more, which in turn will aid the business decision-makers to make the right decision in future.

Whats keeping Curasan, Inc., Carmell Therapeutics Corporation, Anika Therapeutics, Inc., Conatus Pharmaceuticals Inc., Histogen Inc., Royal Biologics, Ortho Regenerative Technologies, Inc., Swiss Biomed Orthopaedics AG, Osiris Therapeutics, Inc., and Octane Medical Inc. Ahead in the Market? Benchmark yourself with the strategic moves and findings recently released by CMI

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List of Companies Mentioned:Curasan, Inc., Carmell Therapeutics Corporation, Anika Therapeutics, Inc., Conatus Pharmaceuticals Inc., Histogen Inc., Royal Biologics, Ortho Regenerative Technologies, Inc., Swiss Biomed Orthopaedics AG, Osiris Therapeutics, Inc., and Octane Medical Inc.

1) Does Study provides Latest Impact on Market due to COVID & Slowdown?

Yes study have considered a chapter on Impact Analysis and this 2020 Edition of the report provides detailed analysis and its impact on growth trends and market sizing to better understand current scenario.

2) How companies are selected or profiled in the report?

List of some players that are profiled in the the report includes Curasan, Inc., Carmell Therapeutics Corporation, Anika Therapeutics, Inc., Conatus Pharmaceuticals Inc., Histogen Inc., Royal Biologics, Ortho Regenerative Technologies, Inc., Swiss Biomed Orthopaedics AG, Osiris Therapeutics, Inc., and Octane Medical Inc.. list is sorted to come up with a sample size of atleast 50 to 100 companies having greater topline value to get their segment revenue for market estimation.

** List of companies mentioned may vary in the final report subject to Name Change / Merger etc.

3) Is it possible to narrow down business segments by Application of this study?

Yes, depending upon the data availability and feasibility check by our Research Analyst, further breakdown in business segments by end use application in relation to type can be provided (If applicable) by Revenue Size or Volume*.

4) What is the base year of the study? What time frame is covered in the report?

Furthermore, the years considered for the study are as follows:

Historical year 2014 2018

Base year 2018

Forecast period** 2019 to 2027 [** unless otherwise stated]

**Moreover, it will also include the opportunities available in micro markets for stakeholders to invest, detailed analysis of competitive landscape and product services of key players.

Detailed Segmentation:

By Procedure Cell TherapyTissue EngineeringBy Cell TypeInduced Pluripotent Stem Cells (iPSCs)Adult Stem CellsTissue Specific Progenitor Stem Cells (TSPSCs),Mesenchymal Stem Cells (MSCs)Umbilical Cord Stem Cells (UCSCs)Bone Marrow Stem Cells (BMSCs)By SourceBone MarrowUmbilical Cord BloodAdipose TissueAllograftsAmniotic FluidBy ApplicationsTendons RepairCartilage RepairBone RepairLigament RepairSpine RepairOthers

Regions included:

o North America (United States, Canada, and Mexico)

o Europe (Germany, France, UK, Russia, and Italy)

o Asia-Pacific (China, Japan, Korea, India, and Southeast Asia)

o South America (Brazil, Argentina, Colombia)

o Middle East and Africa (Saudi Arabia, UAE, Egypt, Nigeria, and South Africa)

Global Orthopedic Regenerative Medicine Market What to expect from this report:

Focused Study on Niche Strategy and Market Development & penetration Scenario

Analysis of M&As, Partnership & JVs in Global Orthopedic Regenerative Medicine Industry in United States & Other Emerging Geographies

Top 10 Global Orthopedic Regenerative Medicine Companies in Global Market Share Analysis: Leaders and Laggards in 2017, 2019

Gain strategic insights on competitor information to formulate effective R&D moves

Identify emerging players and create effective counter-strategies to outpace competitive edge

Identify important and diverse product types/services offering carried by major players for market development

And many more .

TABLE OF CONTENTS

Report Overview:It includes the Orthopedic Regenerative Medicine market study scope, players covered, key market segments, market analysis by application, market analysis by type, and other chapters that give an overview of the research study.

Executive Summary:This section of the report gives information about Orthopedic Regenerative Medicine market trends and shares, market size analysis by region and analysis of global market size. Under market size analysis by region, analysis of market share and growth rate by region is provided.

Profiles of International Players:Here, key players of the Orthopedic Regenerative Medicine market are studied on the basis of gross margin, price, revenue, corporate sales, and production. This section gives a business overview of the players and shares their important company details.

Regional Study:All of the regions and countries analyzed in the Orthopedic Regenerative Medicine market report is studied on the basis of market size by application, the market size by product, key players, and market forecast.

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Orthopedic Regenerative Medicine Market Insights and Business Outlook By Top Players Curasan, Inc., Carmell Therapeutics Corporation, Anika...