The Challenges to Advancing Induced Pluripotent Stem Cell-Dependent Cell Replacement Therapy

Induced pluripotent stem cells (iPSC) represent a potentially exciting regenerative-medicine cell therapy for several chronic conditions such as macular degeneration, soft tissue and orthopedic conditions, cardiopulmonary disease, cancer, neurodegenerative disorders and metabolic disorders. The field of iPSC therapeutics currently exists at an early stage of development. There are several important stakeholders that include academia, industry, regulatory agencies, financial institutions and patients who are committed to advance the field. Yet, unlike more established therapeutic modalities like small and large molecules, iPSC therapies pose significant unique challenges with respect to safety, potency, genetic stability, immunogenicity, tumorgenicity, cell reproducibility, scalability and engraftment. The aim of this review article is to highlight the unique technical challenges that need to be addressed before iPSC technology can be fully realized as a cell replacement therapy. Additionally, this manuscript offers some potential solutions and identifies areas of focus that should be considered in order for the iPSC field to achieve its promise. The scope of this article covers the following areas: (1) the impact of different iPSC reprogramming methods on immunogenicity and tumorigenicity; (2) the effect of genetic instability on cell reproducibility and differentiation; (3) the role of growth factors and post-translational modification on differentiation and cell scalability; (4) the potential use of gene editing in improving iPSC differentiation; (5) the advantages and disadvantages between autologous and allogeneic cell therapy; (6) the regulatory considerations in developing a viable and reproducible cell product; and (7) the impact of local tissue inflammation on cell engraftment and cell viability.


Introduction
Chronic disease resulting from degenerative organ dysfunction accounts for the vast majority of the global healthcare costs 1 .While organ transplantation is a definitive treatment for several end-staged organ disorders, there is an insufficient supply of available organ donors 2,3 .Furthermore, the high cost of organ transplantation poses substantial financial strain on national healthcare costs.Stem cell therapy represents a potential alternative solution to the This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
The aim of this review article is to highlight the unique technical challenges that need to be addressed before iPSC technology can be fully realized as a cell replacement therapy.Additionally, this manuscript offers potential solutions and identifies areas of focus that should be considered in order for the iPSC field to achieve its promise.The scope of this article covers the following areas: (1) the impact of different iPSC reprogramming methods on immunogenicity and tumorigenicity; (2) the effect of genetic instability on cell reproducibility and differentiation; (3) the role of growth factors and post-translational modification on cell scalability; (4) the potential use of gene editing in improving iPSC differentiation; (5) the advantages and disadvantages between autologous and allogeneic cell therapy; (6) the regulatory considerations in developing and transporting a reproducible cell product to the point of patient administration; and (7) the impact of local tissue inflammation on cell engraftment and cell viability.This report will focus primarily on the potential use of iPSC technology as a cell replacement therapy for solid organ disease, rather than the application of iPSC in adoptive cell therapy (ACT) for treating cancer.There are several good review articles on iPSC applications in the field of ACT [9][10][11][12] .

Induced Pluripotent Stem Cell Reprogramming Methods
Induced pluripotent stem cells (iPSCs) represent a noncontroversial source of pluripotent stem cells that can satisfy the requirements of providing an unlimited supply of cGMP production of master cell line stocks.Takahashi et al. were the first to report the dedifferentiation of somatic fibroblasts into pluripotent stem cells by retroviral gene delivery of Oct3/4, Sox2, Klf4 and c-Myc 13,14 .Yu et al. also reported creating cultured iPSC from fetal and neonatal fibroblasts by retroviral delivery of Oct4, Sox2, Nanog and Lin28 15 .Both groups demonstrated that pluripotent stem cells had similar characteristics to those reported in human ESCs.Nakagawa et al. further observed that deletion of c-Myc from the reprogramming scheme still created pluripotent colonies but eliminated teratoma formation in the short-term 16 .Yet, the authors reported a significantly lower reprogramming efficiency under this condition even when retroviral gene delivery was deployed.Nakagawa et al. published a follow-up report demonstrating that replacement of c-Myc with l-Myc eliminated, or at the very least reduced, the neoplastic effects associated with c-Myc 17 .While l-Myc does not promote teratoma formation at the same frequency as c-Myc in short-term experiments involving murine models, l-Myc has been associated with several clinical malignancies [18][19][20] .Also, heterologous expression of c-Myc as described in this report led to a much lower fraction of fully reprogrammed colonies than those created from heterologous l-Myc expression 17 .Taken together, the data indicate that the oncogenes, c-Myc and Lin28, are the chief determinants of the neoplastic risk associated with current iPSC reprogramming methods.In addition to the oncogenes that are used in cellular reprogramming, retroviral gene transfer methods also pose an additional risk of genomic integration that could silence important tumor suppressor genes.This could again increase the neoplastic risk, as well as mediate a risk of viral-mediated immunogenicity.
To improve the safety risks associated with viral-mediated reprogramming approaches, subsequent non-viral and/or non-integrating approaches were developed to produce clinicalgrade iPSC lines.These included nonviral reprogramming methods such as piggyback 21 , DNA minicircles 22 and microRNA 23 .However, these methods proved to be extremely inefficient for reprogramming.More efficient non-integrating approaches emerged and the most popular methods include the use of Sendai vectors [24][25][26][27] , mRNA reprogramming [28][29][30] , self-replicating RNA 31 and episomal vectors 32,33 .Each of these methods have advantages and disadvantages.Self-replicating or replicon RNA relies on the Venezuelan equine encephalitis virus positive sense, single-stranded RNA backbone and its ability to mimic cellular mRNA without having a DNA intermediate.It is known that to get an appreciable level of reprogramming with self-replicating RNA, transfection with co-agents that suppress immune response are necessary 31 .Moreover, PCR studies have shown they retain the expression of viral RNA components for at least four passages downstream.This makes iPSC colony selection, especially for therapeutics, a lengthy process.mRNAbased reprogramming [28][29][30] requires repeated daily transfections for up to 17 days and is laborious and expensive.Also, this method must contend with interferon production in transfected cells, which impacts reprogramming efficiency and which could present downstream immunological concerns.Moreover, somatic suspension cells are resistant to repeated transfection processes.While Sendai virus reprogramming is a popular and robust iPSC reprogramming method, a far greater number of cell divisions are required to dilute the cell line free of contaminating viral proteins and the vector 24 .Ultimately, iPSC colonies would have to be carefully screened for viral proteins or the viral vector before being selected for a cell therapy.Thus, whatever reprogramming efficiency Sendai viral vectors provide at the beginning is offset by the downstream additional quality control measures that must be implemented to confirm safety.
Episomal reprogramming is an ideal method for creating clinical-grade, safer, nonviral and nonintegrating iPSC.According to the Global Alliance for iPSC Therapies, episomal reprogramming is the most common approach used to produce clinical-grade iPSC because of the rapidity in which the transgene is cleared from targeted cells 34 .Episomal vectors are only active, on average for 17-21 days, before reaching an undetectable level due to dilution and instability caused by cell division.However, episomal reprogramming efficiency is quite low compared with other nonintegrating methods.To compensate for the lower reprogramming efficiency, others have utilized c-Myc or a combination of l-Myc and Lin28 32,35,36 .Yu et al. previously reported no colony formation using an episomal reprogramming strategy that delivered Oct4, Sox2, c-Myc, Nanog, Lin28 and Klf4 33 .However, the authors reported colony formation at an efficiency of approximately 0.0006% only after the addition of an SV40 large T-antigen gene.In contrast, Okita et al. previously reported a similar quantitative reprogramming efficiency from episomal-derived IPSC colonies with p53 suppression combined with l-Myc and Lin28 heterologous expression 32 .
We first reported a method using episomal vectors without using oncogenes (defined as free of Lin28, Myc, Nanog and SV40) in iPSC reprogramming in adherent cultured human foreskin fibroblasts (HFF) by using a mixture of reprogramming small molecules 37 .The use of these reprogramming molecules produced fully reprogrammed iPSC colonies even in the absence of c-Myc or a combination of l-Myc/Lin28.Virtually, 100 percent of the colonies were fully reprogrammed based on SSEA4 expression, which exceeds the 70 percent threshold recommended by the Global Alliance in iPSC Therapy 34 .Moreover, our reprogramming method lowers the neoplastic risk and eliminates the viral immunogenicity risk.We subsequently reported how the same method with minor adjustments produced reproducible iPSC cells from suspension cells 38 .We documented iPSC from purified cord blood derived CD34+ cells, cord blood derived mononuclear cells and peripheral blood mononuclear cells from patients with cystic fibrosis and alpha 1 anti-trypsin deficiency by first pre-treatment of cells with thrombopoietin 38 .Induced pluripotent stem cell colonies formed in the absence of oncogenes and virtually 100 percent of the colonies were fully reprogrammed as defined by the expression of SSEA4.Thus, our iPSC reprogramming method is reproducible, relatively quick, universal for both adherent and suspension cells, free of the need to screen colonies for pluripotency, and meets a high criterion for safety.Pluripotent stem cell-based therapy requires purification of the differentiated cell from residual pluripotent stem cells.However, purification processes in bio-manufacturing rarely achieves 100 percent.Even if a final cell differentiation product could be purified from residual pluripotent stem cells at a 99 percent level, there would be 700,000-1,400,000 residual pluripotent stem cells at typical adult systemic dose of 1-2 million cells/kilogram of body weight.Lee previously reported that it only took 10,000 human pluripotent stem cells to form teratomas when injected into murine skeletal muscle and 100,000 cells to form teratomas when injected into mouse hearts 39 .The neoplastic risk could potentially increase with immune-tolerant allogeneic iPSC if the intrinsic neoplastic risk is not reduced because the resulting cell therapy could evade the recipient's ability to immunologicallyclear undifferentiated pluripotent stem cells.

Genetic Instability of Induced Pluripotent Stem Cells
Pluripotent stem cells exhibit significant genetic instability 5,6 .Genetic instability typically results from non-random mutations on chromosomes 1,12,17 and 20 5,6 .It is well reported that c-Myc leads to genetic instability in cultured iPSC 40 .Most of these genetic changes are due to gains rather than losses in genetic material 5 .Genetic modifications on chromosomes 12 and 17 result in increased clonal proliferation.This imposes selective pressure that promotes the expansion of genetic variants thereby increasing the heterogeneity of iPSC cultures and which leads to decreased iPSC reproducibility.Further, genetic instability of iPSC may impair cell differentiation 6 .Conversely, cell differentiation may further increase genetic instability because of the prerequisite for additional cell expansion of sufficient iPSC before subjecting cells for differentiation processes.Thus, iPSC with preexisting genetic instability may be vulnerable to further genetic instability upon differentiation.Induced pluripotent stem cell-associated genetic instability includes copy number variants and single nucleotide variants in which the latter introduces point mutations in coding and noncoding gene sequences 6 .Genetic instability is caused by a shift from oxidative respiration to oxidative glycolysis, which leads to increased reactive oxidative stress.Increased oxidative stress leads to impaired DNA repair, mutations and double stranded DNA breaks.Genetic instability is linked to genomic integrated reprogramming methods; prolonged in vitro cell passaging; and oncogene reprogramming factors (particularly c-Myc) 6,40,41 .While karyotyping is typically used to evaluate cytogenetic mosaicism in pluripotent stem cells, the method is insensitive to detecting genetic instability.Multi-gene microarrays, whole genomic sequencing and qPCR are more accurate in detecting genetic instability than karyotyping 5,6 .
More importantly, little is known about how large-scale production of iPSC and differentiated protocols can impact genetic instability, cell heterogeneity and reproducibility.Quantitative analytical and computational approaches will be required to better define genetic instability and define cell phenotype.The extent of cell culturing conditions and scale-up production variables that lead to genetic instability of iPSC have not been defined, which ultimately could impact the ability to produce a reproducible cell product.Taken together, the need for quality controls in iPSC and differentiated cell manufacturing to create reproducible, safe and potent cell lines that minimize deleterious cell mutations will require high quality tissue culture conditions, computational models and avoidance of oncogenes that are used in reprogramming schemes.

The Impact of Growth Factors in Induced Pluripotent Stem Cell Therapeutic Development and Cell Scalability
Growth factors represent a seminal role in iPSC reprogramming and ex vivo expansion.
Transforming growth factor -beta and fibroblast growth factor are typically used in these processes 37 .It is important to point out that iPSC transformation into terminallydifferentiated cells, which is the ultimate goal in developing a therapeutic cell line, will require multiple differentiation steps, with each step requiring multiple growth factors [42][43][44][45][46][47] .Since human cell differentiation under in utero conditions is regulated, in part, by native human growth factors, this raises the question of whether manufacturing processes that produce iPSC-differentiated cell therapies should ideally use growth factors that reflect the native chemical structure of these peptides that are operating under in utero conditions.Many current commercial growth factors are manufactured from bacteria.However, bacteria lack a post-translational modification (PTM) system that incorporate glycosylated moieties.To circumvent this dilemma, the bacterial-derived growth factors are truncated to eliminate the glycosylated peptide sequence in order to manufacture that product.Additionally, the PTM pattern produced from cells is species specific.There are differences in the PTM pattern between non-mammalian cells and human cells [48][49][50] .Glycosylation affects protein folding, stability, solubility, protein-protein interactions, bioavailability, bio-distribution, pharmacokinetics, immunogenicity and protein activity [51][52][53][54][55][56][57][58] .There are several reports that document the glycosylation of several growth factors [59][60][61] .Moreover, there is little comparative data on iPSC reprogramming and differentiation in the presence of growth factors produced from bacteria, yeast, mammalian cells and human cell lines.
While HEK293 is an established human cell line that manufactures human proteins 62 , HEK293 poses important shortcomings.Notwithstanding that the cell is ethically controversial because it was derived from an aborted fetus 63 , HEK 293 cells have an aneuploidy karyotype 64 .HEK 293 cells were transformed by transfection with adenovirus 5'DNA 65 .Subsequent analysis has shown that the transformation was formed by inserting 4.5 kilobases from the left arm of the viral genome that was incorporated into chromosome 19 66 .There are differences in glycosylation pattern in proteins between Chinese Hamster Ovary (CHO) cells, HEK293 cells and human plasma 67 .For example, the glycosylation pattern for factor VII when produced from HEK293 cells differs significantly from plasmaderived factor VII 68 .It not known whether growth factors that display a more native PTM profile, would produce a more potent iPSC-derived cell therapy because the chemistry may more accurately reflect the differentiating activity under in utero conditions.In summary, standardized manufacturing processes will not be achieved until a rigorous comparison of cell characterization with growth factors manufactured from different cell sources.
A challenge in developing iPSC-derived cell therapies that may not be fully recognized is the high cost of research and development for producing prototypes during large scale manufacturing.Before a final product is available for preclinical and clinical testing, it is anticipated that a workflow for scaling up a prototypic product must be first optimized.This will initially occur under a less expensive Good Laboratory Practice (GLP), before transferring that process to occur under cGMP conditions, as is required for preclinical and clinical testing.The highest consumable cost in GLP-specific cell product development is the high cost of growth factors from supply chain providers.
Consider the following example.Our group has developed a large-scale workflow of differentiating iPSC into neuroprogenitor cells (NPC), which require Activin A, Noggin, Wnt3a and brain derived neural growth factor (BDNF).We produced these neural growth factors from immortalized human somatic stem cells (PCT/US2023/065911) in order to create peptides that would best approximate the native human PTM system.The following table (Table 1) represents the minimum mass of each neural growth factor required to differentiate 100 million iPSC into approximately 70 million NPC.If commercial supply chains for these growth factors were instead used (which are typically produced from non-human cell lines), then the estimated cost for the mass of growth factors required to produce 70 million NPC would add up to a final cost of $211,655.Thus, the cost of growth factors to scale-up 700 million and 7 billion NPC would increase by a factor of 10 and 100 respectively.These costs are not sustainable for prototype development.For Parkinson's disease, the cell therapeutic dose is 4.8 million dopaminergic cells 69 where 70 million NPC would provide sufficient dosages for 14 subjects to carry out a Phase 1 clinical trial.In turn, the bioprocessing requirements for Phase 2 and Phase 3 clinical trials would increase from 8 milligrams per growth factor for a Phase 1 clinical trial product to 8 grams per growth factor for a Phase 3 clinical trial product.Additional growth factors will be necessary if the final objective is to further differentiate a NPC into a dopaminergic neuron 70 for treating Parkinson's disease, which would further increase the manufacturing cost.Moreover, the final cost of growth factors produced under cGMP conditions will be even higher than those manufactured under GLP conditions.Thus, there may be a need to not only provide higher quality growth factors that better reflect the native human PTM system, but those research costs have to be substantially reduced to remove financial barriers to develop prototypic iPSC-derived products.
Consistent with the notion that the development costs for cell therapies are challenging, the biology and potency of cell therapies may not be reproducible from small scale to large scale like small and large molecules.Cell behavior is unpredictable during scale-up, which may require cell manufacturers to have the capacity to scale-up and analyze enough cells for a Phase 3 clinical trial even if a Phase 1 clinical trial is the initial objective.The cell phenotype would have to be subjected to rigorous preclinical assays to provide ample evidence that the cell properties are reproducible during the entire scale-up spectrum from a Phase 1 to Phase 3 clinical trials.Industry would have to document that the manufacturing processes from small scale to large scale-up processes produced a consistent and reproducible cell product.Thus, the stakeholders of cell therapies may have to be prepared for significant upfront preclinical research development costs in manufacturing and testing than stakeholders developing small and large molecule therapeutics.Contract manufacturers and contract research service organizations would require more integrative capacity in cell manufacturing and bioprocessing; along with novel and complex cell assay expertise to quantify cell potency; capacity to document quality controls; and the ability to contain costs and provide sufficient services to their biopharmaceutical clients.Taken together, stakeholders that are pursuing cell therapy will be subjected to greater financial, scientific and regulatory risk than those pursuing traditional small and large molecule platforms.Thus, these challenges may restrict cell therapy development to a specialized industry sector because the prerequisite requirements are so great.

Quality Controls and Regulatory Hurdles in Induced Pluripotent Stem Cell Therapies
Given the complexity associated with cell therapies, iPSC-derived therapies require rigorous quality controls at multiple stages from patient recruitment, tissue procurement, cell isolation, ex vivo cell expansion and differentiation, cell phenotype characterization, cell cryopreservation, and finally cold chain management.In the United Sates cellular therapy products are regulated under 21 CFR Part 1271 (Human Cells, Tissues, and Cellular and Tissue-Based Products) 71 , 21 Part 600 (Biological Products: General) 72 and 21 CFR Part 610 (General Biological Products Standards) 73 .Drug manufacturing requirements are defined in 21 CFR Part 211 (Current Good Manufacturing Practice) 74 .In Europe cell therapy is regulated by the European Medicines Agency 75 .A careful medical history of donors is required to confirm that donors are healthy and free of transmissible disease.Procured tissue require testing to confirm that the cell product is free of adventitious disease such as HIV and hepatitis.The Global Alliance in iPSC Therapy has established standard guidelines for developing iPSC therapies 34 .These guidelines included recommendations for testing genetic fidelity and stability, cell potency and other cellular characteristics.Lastly, in vitro bioassays along with computational models are needed to evaluate cell potency to provide an important quality control in monitoring the scale-up process in cell manufacturing.
Once cell products are finally manufactured, additional challenges include implementing cryopreservation methods that provide on-demand access to biological materials, which must mitigate against cryopreservation-induced delayed-onset cell death [76][77][78][79] .Lastly, cold chain management is critical to maintain temperature-sensitive cell therapies during transit to its final destination for patient administration 80 .Failure in these latter processes can lead to a loss of viable cells that could affect the efficacy of a cell therapy.

The Inefficiency of Induced Pluripotent Stem Cell Differentiation and Potential Solutions with Gene Editing
Induced pluripotent stem cell differentiation requires multiple steps, and each step requires multiple growth factors.However, the transformation efficiency at each step is frequently inefficient to the extent that only a minority of cells successfully differentiate 42,43 .Transfection efficiency has improved by the use of gene editing methods by incorporating fluorescent tags or other biomarkers that can enrich differentiated cell species through fluorescence-activated cell sorting (FACS) or by magnetic cell sorting at critical steps.
Gene editing is a rapidly evolving field with multiple methods available to alter cellular DNA.The most widely adopted methods include Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) and Transcription Activator Like Effector Nucleases (TALENS).CRISPR Cas9 has been shown to be an effective tool for cellular reprogramming applications due to its multiplexing capabilities as well as its ability to target endogenous loci 81 .The CRISPR Cas 9 gene system makes use of an inactivated Cas9 protein which is recruited to specific stretches of the genome specified by short guide RNA (gRNA) molecules 82 .The ability of the Cas9 effectors to control the transcription of specific endogenous loci makes it useful to facilitate cellular reprogramming which is predicated on precise activation and silencing of endogenous genes which leads to the proper conversion of cells 81 .There are significant advantages to using CRISPR for iPSC reprogramming.First, this system has consistently shown high efficiency in making targeted gene edits 83 .The guide RNA sequences are simple to design and directly target specific genes 83 .This method is also relatively cost-effective compared to TALENS. 83However, there are currently limitations to CRISPR that must be considered.The current CRISPR Cas9 system limits site selection.In this regard, there are no barriers for gene knockouts, but it may present difficulties when trying to knock in genes at specific locations (e.g.genetic safe harbors) 84 .Additionally, the extent of off-target effects of CRISPR remains largely unknown.Adopting this technology for large scale reprogramming will require determining whether potential off-target effects will affect the efficiency or downstream efficacy of the reprogramed cells 84 .
In contrast to CRISPR which is based on site-specific RNA protein interactions, the TALEN gene editing system recognizes a target site based on DNA-protein interactions.Differing associations of the TALE proteins with numerous functional domains such as endonucleases, transcriptional activators and repressors as well as endonucleases allow them to function as both transcriptional modulators as well as gene editing tools 85 .TALENs have been shown to exhibit high degrees of specificity and low cytotoxicity in numerous cell types.This makes them an attractive complement to more recent CRISPR technologies which currently experience high rates of off-target effects 86 .However, unlike CRISPR, TALENs are significantly more difficult to design which has limited their widespread adoption in research settings 85 .Given their ubiquitous use for other genome editing applications, it is anticipated that these gene editing strategies will quickly be adapted to iPSC reprogramming technologies and will improve the differentiation efficiency and decrease the time and cost of producing iPSC-derived cell therapies.

Autologous vs Allogeneic Induced Pluripotent Stem Cell Considerations
Induced pluripotent stem cell therapies can be either autologous or allogeneic derived.As previously described, the regulatory requirements for allogeneic cell therapy is more demanding than autologous cell therapy.There may be clinical conditions where an autologous cell therapy could have an advantage over allogeneic cell therapies.For example, an autologous natural killer cell (NKC) therapy may have an advantage in treating cancer when cancer cells no longer express major histocompatibility complex (MHC) class 1 antigens 87 .In contrast, it may be challenging for an allogeneic iPSC-derived NKC to distinguish between a cancer cell that displays low MHC class 1 antigens and non-cancer cells that express a foreign surface antigen.Thus, there may be greater risk for off-target cell injury with an allogeneic-derived NKC therapy.
Autologous iPSC therapies have the advantage for achieving a HLA match between recipient and donor without the need for anti-rejection medication.However, there are important disadvantages.First, the cost for a personalized cell therapy may be more expensive for the healthcare system to pay for such treatment.Second, the target cell for an adult could acquire somatic mutations over the lifespan of a patient that could pose a neoplastic risk.Third, autologous cell replacement therapy for genetic disorders is more expensive and time consuming to develop because putative mutations require corrective gene editing.Lastly, there is greater statistical variability in iPSC phenotypes because of genetic differences between subjects 88 .Systematic review of hundreds of different iPSC lines showed that 5-46 percent of the cell phenotype differences were due to intersubject differences 89 .Consequently, there will be greater statistical variance because of inter-subject variability in cell phenotype for a personalized cell therapy.Unlike clinical trials that use small and large molecules, the only source of statistical variability is the test subject's clinical response to a drug.In contrast, there are two sources of statistical variability in autologous cell therapy -the treatment response and the test agent.A previous report identified genetic and non-genetic determinants as the source of these inter-subject differences in iPSC lines 90 .
Allogeneic iPSC offers several advantages.First, the healthcare cost per patient is much less expensive than for an autologous cell therapy.Second, the inter-subject statistical variable for the cell phenotype is absent compared to autologous cell therapy since it is derived from a single tissue source.Neonatal sources like human foreskin fibroblasts, umbilical cord blood, umbilical cord tissue and placental tissue represent ideal target sources of somatic cell and stem cells to reprogram because they pose a much lower risk of somatic mutations.Moreover, cell heterogeneity among postnatal cells would be lower than adult cells in which the latter had more time to change from age, illness and environmental effects.
The main disadvantage of allogeneic cell therapy is that there is a HLA-mismatch between recipient and donor.Under these conditions, immunosuppressant agents are required to avoid host vs graft rejection.Yet, immunosuppressant drugs carry adverse clinical risks that include an increased risk of infection 91,92 .To avoid the risk of immunosuppressant agents, there have been several efforts to use gene-editing techniques to knock-out MHC-1 and MHC-2 antigens to avoid T-cell-dependent adaptive immune responses 93 .Yet, knock-out of the MHC-1 antigen evokes an innate cytotoxic immune response from NKC.Several strategies to avoid NKC-dependent immune responses include gene-editing knock-in of HLA-E, HLA-G, CD47 or membrane-bound and secreted β2m-HLA-G fusion protein [92][93][94][95][96][97] .
However, universal allogeneic-iPSC therapies pose a potentially increased neoplastic risk than their original parental iPSC cell lines if target cells are reprogrammed with oncogenes.Host vs graft rejection represents a protected mechanism for immunological clearance of undesired foreign tissue.Yet, the neoplastic risk of an immune-tolerant universal iPSC therapy increases if undifferentiated cells remain in the cell product and could evade host immune clearance mechanisms.A possible solution would be to integrate into the genome an inducible gene switch that "turns off" cell therapy in the event a serious side effect emerges such as CAR-T-mediated cytokine storm 98 .However, such inducible safety switches may be less effective for cell replacement therapy for preventing tumor formation since there is a lag time between tumor formation and diagnosis, and a delay in diagnosis could reduce the effectiveness of a genetically engineered switch.

Engraftment Challenges of Induced Pluripotent Stem Cell-Dependent Cell Replacement Therapy
Induced pluripotent stem cell-derived cell replacement therapy is dependent on the ability of differentiated cells to engraft to previously diseased tissue environments.However, the therapeutic challenge for a successful cell replacement therapy is that the transplanted cells are subjected to the same inflammatory environment from chronic disease that caused the destruction of the original specialized cells [99][100][101][102][103] .That state of local inflammation still persists, and transplanted cells would be subjected to the same inflammatory environment, which could cause the transplanted cells to fail to engraft or that the engraftment is  3) NPC induction stage.The estimated cost reflects the cost of growth factors if commercial third-party supply chains were instead used.The total estimated cost to produce 700 and 7 billion NPC is calculated by increasing the growth factor costs by a factor of 10 and 100 respectively.

Table 1 :
Estimated Costs of Using Supply Chain Growth Factors to Manufacture Neuroprogenitor Cells for Different Levels of Scale-Up

70 million NPC 700 million NPC 7 billion NPC Growth Factor Peptide Mass (μg) Estimated Supply Chain Cost Estimated Supply Chain Cost Estimated Supply Chain Cost
Table represents the estimated third-party supply chain cost of neural growth factors required to produce 70 million, 700 million and 7 billion neuroprogenitor cells (NPC).The minimum mass of each growth factor was produced from immortalized human somatic stem cells to differentiate 100 million iPSC into an estimated 70 million NPC based on expression of Nestin.iPSC differentiation into NPC was accomplished in three separate phases: (1) first neural rosette stage; (2) second neural rosette stage and (