13.2.1 Induced pluripotent stem cells

Induced pluripotent stem cells are differentiated cells that have been reprogrammed into an embryonic stem cell like state by the ectopic overexpression of four stem cell specific transcription factors, Oct3/4, Klf4, Sox2, and c-Myc, collectively referred to as OKSM. Induced pluripotent stem cells were first derived in a groundbreaking experiment by Yamanaka and Takashi in 2006 [77]. The team assessed the ability of 24 pluripotency associated candidate genes to covert primarily differentiated mouse tail tip fibroblasts into an embryonic stem cell state. Candidate genes were packaged into individual retroviruses and transduced into Fbx15geo/geo cells, which were grown in G418 containing media, an aminoglycoside antibiotic with conferred resistance to the neomycin gene. If the cells converted to an ESC like fate the embryonic stem cell specific locus Fbx15 containing a -galactosidase and neomycin fused reporter cassette would become activated, thereby inoculating the cells against neomycin. Transduction with all 24 factors proved to be successful in converting the fibroblasts into ESCs. Through the process of elimination, the team narrowed the list of factors down to just four factors needed to reprogram fibroblast cells to an ESC state [77]. Yamanaka and Takashi expanded their groundbreaking discovery to human cells a year later [78].

The discovery of induced pluripotent stem cells ignited the field with possibility. It was a new research tool that could be used to analyze development and cell specialization. Additionally, the possibility of deriving pluripotent stem cells was also a new therapeutic research tool that if harnessed and understood could be used for personalized cell therapy and disease modeling. Researchers quickly began differentiating iPSCs into different cell lineages.

Induced pluripotent stem cell derived-cardiomyocytes (iPSC-CMs) were generated similarly to established methods for differentiating embryonic stem cells into cardiomyocytes [7981] (Fig.13.1C). The cells were first differentiated into embryoid bodies and then exposed to serum-containing medium, which fostered a propensity to differentiate into cardiomyocytes. After 50 or more days in culture, cells derived under these conditions stained positive for sarcomeric myosin light and heavy chains, cardiac troponin T, and alpha-actinin. Additionally, the embryoid bodies demonstrated action potentials akin to atrial, ventricular, and nodal cells, and underwent rapid adaptive response to electrical stimulation and were cable of visible contractions. Despite well-established protocols the purity of cardiomyocytes derived using this technique are often times lower than 1% [8284]. However, the efficiency and purity of cardiomyocytes generated from embryoid body differentiation could be enhanced by following a step wise induction process similar to the naturally occurring cardiac differentiation process in the developing embryo [85].

To increase purity, and the usability for downstream applications monolayer culture methods were developed to facilitate a more controllable and reproducible environment to generate iPSC-CMs [86]. Monoculture conditions consist of growth on Matrigel-coated plates with mouse embryonic fibroblast conditioned media and gradual supplementation with activin A and BMP-4 growth factors. The combination of these conditions have been shown to yield greater than 50% beating iPSC-CMs [87,88]. A variation of this method, called the matrix sandwich method exists and boasts yields of up to 98% beating iPSC-CMs [89]. However, it should be noted that this method only works for some cell lines and requires growth factor batch optimizations to maintain high yields [90]. Alternatively, modifying Wnt/-catenin signaling using shRNA and small molecules has also been shown to increase iPSC-CM yield to approximately 85% [91,92].

The need for complex culture conditions to yield high iPSC-CM outputs makes identifying the biological underpinnings of iPSC-CM differentiation difficult to elucidate. One study claims to have reduced the complexity of iPSC-CM derivation to just three components, referred to as CDM3 [93]. When used in combination with lactate selection the study authors claim to achieve a yield of 80%90% troponin T positive iPSC-CMs [94]. The simplicity of the culture conditions used in this study allowed for the first time the identification of key signaling pathways implicated in iPSC-CM carcinogenesis.

The first and only case thus far of an autologous iPSC derived cell treatment making it to the clinic was reported in 2014. In a trial lead by Takahashi and colleagues, human iPSC derived retinal pigment epithelium cell sheets were transplanted into a human patient to resolve age related macular regeneration [95]. There have been no clinical trials testing iPSC-CM safety or efficacy in repairing the injured heart. However, iPSC-CMs derived using the previously mentioned matrix sandwich technique were transplanted in a non-human primate model, where they were shown to improve cardiac function after induced myocardial infraction. However, the transplanted iPSC-CMs also induced high rates of ventricular arrhythmia [96].

Despite the great hope for patient specific treatments, it is uncertain if autologous iPSC-CM treatments for myocardial infractions will make it to the clinic within the next few years. The production of patient-specific stem cells is expensive and variable. Specifically, iPSC-CM derivation efficiency still remains low and variable without the use of complex culture systems. Streamlining human iPSC cardiomyocyte differentiation to an effective simple differentiation process is key. Large numbers of iPSC-CM cells would be needed for human clinical trials, which would be impractical to accomplish using current culture systems and methods. Currently macaque trials require about 108109 reprogrammed iPSC-CM cells. The number of cells required for a human trial is projected to be a least a magnitude higher [5,97]. Additionally, like all iPSC derived cell therapies, and even embryonic stem cell therapies there is the concern that the transplanted stem cells could develop into tumor and/or cancer cells because of the possible carryover of few highly multi- or pluripotent cells in the transplanted pool [98]. Safety assessment is key before any iPSC-CM trial can make it to the clinical setting.

However, iPSC-CMs do have the potential to be somewhat useful for in vitro screening assays and drug development. iPSC-CMs have been used to improve the identification of false positive and negative data in electrophysiological assays [99]. They have also been shown to be responsive for research purposes to several cardiac and non-cardiac drugs, a prospect that might be of interest for drug screening purposes [100103]. Furthermore, disease-specific iPSC-CMs derived from people with pre-existing heart conditions have been shown to be more responsive to cardiotoxic drugs as measured by action potential duration and drug-induced arrhythmia, consistent with what would be expected naturally in the patient [104].

While iPSC-CMs might have some usefulness for drug screening, the results should be considered in light of the fact that iPSC-CMs are not equivalent to true CMs found in the adult heart. iPSC-CMs have lower conduction velocities and shorter action potential duration. They are altogether functionally immature, disorganized, fetal-like, and are not molecularly equivalent to true cardiomyocytes found in the adult heart [90,105107]. There is a need to understand cardiomyocyte maturation to facilitate regeneration and differentiation into cardiomyocytes capable of maintaining the functions of an adult heart.

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Induced Pluripotent Stem Cell - an overview - ScienceDirect

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