Hurdles to clinical translation of human induced pluripotent stem cells

Human pluripotent stem cells are known to have the capacity to renew indefinitely, being intrinsically able to differentiate into many different cell types. These characteristics have generated tremendous enthusiasm about the potential applications of these cells in regenerative medicine. However, major challenges remain with the development and testing of novel experimental stem cell therapeutics in the field. In this Review, we focus on the nature of the preclinical challenges and discuss potential solutions that could help overcome them. Furthermore, we discuss the use of allogeneic versus autologous stem cell products, including a review of their respective advantages and disadvantages, major clinical requirements, quality standards, time lines, and costs of clinical grade development.

As stem cell technology becomes a reality, one major goal is the establishment of the best methods to develop tissues for clinical application. Effective planning for commercialization requires anticipation of clinical requirements, production demands, and the resultant costs. Advance planning is essential for stem cell technologies because of their time intensive nature and high development costs. PSCs, such as ESCs or iPSCs, are highly versatile and readily produced in very large numbers. These valuable features make them ideal starting materials for developing scalable commercial cell products (17). However, manufacturing clinical-grade stem cell products for a clinical trial presents a number of difficult challenges that are not present in a research setting. For instance, reproducible manual handling in good manufacturing practice (GMP) clean room facilities needs to be established throughout the product’s life cycle (18–20). For the best outcome, GMPs should be instituted as early as possible in the process. Doing so can avoid additional problems while ensuring that the manufacturing process complies with quality control standards of regulatory agencies to create a product that is cost effective, robust, scalable, safe, and reproducible, with the maximum chance of achieving commercial success.

With PSCs, the use of defined cultured systems should be implemented (21, 22). It is preferable to avoid using chemically undefined media or materials of animal origin, such as fetal bovine serum and mouse embryonic fibroblasts, as support systems, because they carry a risk of transmitting xenopathogens to the recipient patients. Rigorous quality controls, documentation, and adherence to current GMP (cGMP) for each manufactured cell batch are essential. Furthermore, it is crucial to ensure that products are consistently manufactured and meet all necessary criteria in terms of viability, function, purity, and sterility during the differentiation. Finally, once the required specifications are confirmed, certificates of analysis should be generated for all product lots to certify them for clinical use (Figure 1).

Figure 1

Flow chart of pluripotent stem cell banking. Human PSCs are ideal candidates for developing cell therapies from either a tissue biopsy or blood collection (i). Allogeneic and autologous stem cell banking requires establishment and expansion of the starting pluripotent material (ii). Human ESCs and iPSCs are capable of differentiating into any adult cell type (iii). Therapeutically relevant cell products must undergo preclinical studies required by the FDA prior to an IND application (iv). TSE, transmissible spongiform encephalopathy. Cells must undergo processing prior to secure cryostorage (v). The cryopreserved products may either be used immediately or be held for future use as allografts in order to regenerate damaged organs (vi).

For preclinical animal studies, it is crucial that the PSC products studied are manufactured using processes comparable to those intended for the final GMP product. This is an important step, because these studies may be used to support future investigational new drug (IND) applications filed with the FDA. Although robust differentiation efficiencies can be achieved, undifferentiated cells could still remain within the cell product. Preclinical studies are intended to assess product safety, off-target effects, and the potential for teratoma formation from undifferentiated cells within the transplanted cell product.

Traditional histopathological techniques for preclinical studies cannot pinpoint the underlying mechanisms of stem cell biodistribution, engraftment, and migration in real time. Therefore, the risk of ectopic engraftment is unclear. It is also unknown where the nonengrafted cells go immediately after transplantation and what hazards they might present. Recent advances in positron emission tomography (PET) imaging, magnetic resonance imaging (MRI), fluorescence imaging (FLI), bioluminescence imaging (BLI), and other techniques have improved monitoring of transplanted grafts tremendously (23, 24). This has led to great progress in understanding cell behavior in vivo within tissues of interest, enabling the spatiotemporal mapping of transplanted cells for both long-term and short-term safety studies required by the FDA. For example, both BLI and MRI have been used successfully in small and large animal studies to track reporter gene or iron-labeled cells for long periods of time (25, 26). Although some of the aforementioned technologies have not yet become common in preclinical or clinical studies or are unlikely to do so because they are more suited for small animal work (e.g., FLI and BLI), such technologies are of growing importance and have already been used to study microenvironmental factors that regulate cell fate decisions (e.g., physical contact, cell-to-cell interactions, and adhesive properties of the cells in their interactions with the native tissue).

The most significant posttransplantation safety concern is the formation of either benign teratoma or malignant teratocarcinoma (27, 28). Because stem cell–derived clinical products may consist of a heterogeneous population of cells, it is crucial to avoid inadvertently introducing neoplasms. Heterogeneity seen during the differentiation process usually is a result of the product being contaminated with undifferentiated cells or the use of a differentiation process that yields cells of multiple lineages. In addition, the cell product might dedifferentiate into cells capable of producing neoplasms. Karyotype stability studies of the undifferentiated starting material and efforts to remove residual undifferentiated cells are necessary to ensure that the transplanted product presents a minimal tumor risk. Following cell transplantation, the toxicity and tumorigenicity of these cells can also be monitored by histologic assessment or human Alu quantitative PCR analysis (29, 30). Although human Alu quantitative PCR assay sensitivity is up to 0.1% compared to host genomic DNA, even greater sensitivity is desired. Newer methods such as digital PCR can improve the sensitivity and are currently being tested. Furthermore, strategies for tumor surveillance now exist that can provide cell death on demand, which are attractive fail-safe options to deal with possible tumor formation (31). For example, PET reporter genes, such as herpes simplex virus truncated thymidine kinase (HSV-tk), can be used for tumor surveillance because they allow the transplanted cells to take up substrates for imaging studies. In addition, these reporters act as suicide genes for tumor ablation therapy by inducing sensitivity to antiviral drugs such as ganciclovir (32, 33). In addition, several small molecules have been identified to eliminate human PSCs and prevent teratoma formation; use of these compounds may soon increase the safety profile of stem cell–based treatments (34, 35). Ultimately, only long-term studies of the transplanted cell products will be able to accurately assess the tumorigenesis risk associated with such therapies.

The FDA requires that all stem cell clinical products undergo safety/toxicity studies before obtaining IND approval (36). These studies are typically designed to demonstrate that stem cell derivatives are nontoxic and otherwise safe in preclinical animal models. The models used depend on the study and indication of product use. Preclinical studies are usually done in normal animals in acute, subacute, and chronic models for varying periods varying from 3, 9, 12, and 24 months. For these studies, the FDA requires investigators to look for major organ toxicity and blood counts of animals after cell transplantation. In addition, cell products must be tested rigorously for acute infusion toxicity that might result from damage to the site of transplantation as well as collateral damage to adjacent tissues stemming from an immune response against the cell product.

Poor stem cell survival and engraftment after delivery is partly due to cellular rejection triggered by the host’s immune response (37). The lack of an effective method of inducing immune tolerance to maintain graft survival is a major roadblock for cell-based therapies. PSCs were once considered immune privileged due to their lack of MHC class I, MHC class II, and costimulatory molecules (38). Although undifferentiated ESCs might be immune privileged, it has been shown that their differentiated derivatives can trigger cellular and humoral immune responses (39). By contrast, autologous iPSCs may avoid the costs and side effects associated with lifelong immunosuppression required for allogeneic cell transplantation (16). Despite some controversy over the immunogenicity of undifferentiated iPSCs (40), recent work has demonstrated that the differentiation of iPSCs results in the loss of immunogenicity (41). Pending further validation, this is a potentially important finding, because it could lead to induction of tolerance similar to the tolerance elicited by a corresponding self-somatic cell.

Autologous tissues have the advantage of not requiring immunosuppression and are likely exempt from immunologic rejection. Although the recipient can tolerate autologous iPSC transplantation, the high costs involved in autologous therapy compared with allogeneic cell banking are a major concern. In addition, the lack of immune rejection in the autologous setting raises the question of whether the risk of teratoma formation might be heightened, as any residual undifferentiated cells might not be detected by the recipient’s immune system. Studies using syngeneic xenotransplantation animal models might shed some light on this issue; however, it would still be very difficult to predict problems with human PSCs based on mouse-to-mouse studies. The rapid advancement of iPSC technology makes these questions immediately relevant, as medical communities are starting to build tissue banks that could offer groundbreaking treatments (42).

There are three options for tissue sourcing when developing iPSC-derived products: (a) autologous iPSCs, (b) allogeneic iPSCs or ESCs, and (c) HLA-matched allogeneic iPSCs. While autologous cells would be developed on an individual patient basis, allogeneic cells could be banked as a single product en masse. Currently, most groups with products nearing or in clinical testing are developing single allogeneic ESC lines, but as scientific understanding of successful tissue transplantation and immune rejection evolve, an HLA-matched allogeneic iPSC tissue bank may prove valuable. HLA-matched human iPSC banks have a number of potential advantages, which include (a) providing researchers with a tissue source to help tailor therapeutic development to address immunogenicity concerns for a specific product, and (b) making HLA-matched and even individualized iPSC products a clinical reality. However, the genetic complexity required of such a bank in order to be useful across ethnic barriers is an important aspect to understand before undertaking such development.

Experience with solid organ and bone marrow transplantation has been used to help estimate the scale of iPSC banking that would be required to provide adequately matched tissues in a population. The number of cell lines theoretically required is highly dependent on the demographics and genetic heterogeneity of a region. On the more favorable end of the spectrum, Nakajima et al. have shown that relatively small numbers of cell lines can be used to provide adequate coverage to a Japanese population (43). Using 2,578 donors from a hematopoietic stem cell registry, they were able to estimate the HLA-matching rates of human ESC lines from randomly donated embryos. Nakatsuji et al. were able to replicate this study for iPSCs by estimating that screening a database of 24,000 individuals would lead to 50 homozygous cell lines generated to provide a haplotype match for 90.7% of the Japanese population at HLA-A, HLA-B, and HLA-DR loci with two-digit specification (44). To extend these results to four-digit specifications, approximately 160,000 individuals would have to be screened to generate 140 homozygous cell lines for similar coverage (45). These findings were confirmed in subsequent studies, which showed that 150 homozygous cell lines could provide a haplotype match for 93% of the population of the United Kingdom (46, 47).

Other researchers have shown that generating a master cell bank for more diverse populations would be far more challenging (48). Gourraud et al. used a probabilistic model to estimate the rate of haplotype matching of a carefully selected homozygous HLA-type iPSC bank for a North American population (48). Their work demonstrates disparities for screening a diverse population. For example, they estimate that 22,000 individuals of European descent would have to be screened to generate 17 iPSC lines to offer a haplotype match to approximately 50% of that patient population. A screen of 100,000 random individuals in the same North American population would only offer a haplotype match to 45% of Hispanics, 35% of Asian Americans, and 22% of African Americans (Figure 2). Similarly, an iPSC bank of the 100 most common HLA types population wide would offer a haplotype match to 78% of individuals of European descent, 63% of Asians, 52% of Hispanics, and 45% of African Americans (48). These results suggest that customized banking for each ethnic group does not necessarily solve this problem. An iPSC bank of homozygous cell lines containing the 20 most common HLA types in an ethnic population could be built by screening 26,000 individuals of European descent but would require screening of 110,000 African Americans. This bank would offer a haplotype match for >50% of individuals of European heritage but only approximately 22% of African Americans. Hence, an allogeneic cell bank in genetically homogenous countries like Japan or Iceland could be a more viable option, whereas a similar bank in the US may be cost prohibitive.

Figure 2

Considerations for iPSC biobanking. (A) The fraction of cited populations that would theoretically find an HLA haplotype match in an iPSC bank that maintains a certain number of lines. (B) The number of individuals in four different ethnic groups that would have to be screened in a US population to develop a haplotype-matched iPSC bank to offer potential matches to a given fraction of each group. This figure consists of data compiled from papers cited within this Review (31, 34, 35). Pop, population.

There are also risks for health delivery disparities that must be considered, because groups of lower socioeconomic standing might require more extensive outreach in terms of tissue donation and storage, to offer treatment options to a significant portion of the population. Tissue storage is also complicated for groups with rare haplotypes, as regulations prohibit use of tissues stored for longer than five years (49). Finally, it should be noted that even highly matched cells could still trigger rejection. A recent study showed that ESCs made via nuclear transfer are still rejected when transplanted into the nuclear donor, presumably because of differences in mitochondrial antigens alone (50). Accordingly, some degree of immunosuppression may be required, even in highly homogeneous populations, although this remains speculative.

As described above, the scale of iPSC banking necessary to provide tissue for a diverse population is large. While start-up costs and time investment would be high, there are potential production benefits to banking allogeneic tissue over an autologous product approach. The most cost-prohibitive component of autologous, allogeneic, and HLA-matched allogeneic cell banking for clinical application is compliance with cGMP standards. cGMP standards require that the product be consistent in both safety and efficacy, that it be screened for the presence of certain pathogens, and that it be “well characterized” in order to exclude any contaminants. While PSC products are subject to existing FDA regulations and do not constitute a new class of biologic products, pioneering studies using human ESC-derived therapies (e.g., Geron’s spinal cord injury trial, Advanced Cell Technology’s macular degeneration trial, and ViaCyte’s type 1 diabetes trial; refs. 9, 10, 13) have helped to establish a preclinical and clinical development pathway for these products.

A primary concern for autologous iPSC-derived tissue generation is that variability among different stem cell lines can be significant, making the establishment of standard production processes that satisfy FDA regulatory requirements difficult (49). Autologous stem cell–derived tissues could be particularly difficult to standardize. Differences in the donor cell sources, collection methods, and subsequent reprogramming techniques among various laboratories all introduce significant product variability. Product variability can create safety concerns and may delay therapeutic production for conditions that require time-sensitive treatment (e.g., acute myocardial infarction or cerebral vascular accident). An HLA-matched allogeneic cell bank would help alleviate some of these concerns as production and tissue source could be standardized (49, 51).

iPSCs have inherent genetic and epigenetic variations that have been shown to exist among different iPSC lines and among different passages of the same iPSC line as well as between iPSC and ESC lines (52). This can lead to a natural variability in cellular purity and yield in the final product. Existing biologic therapies that have similar problems use reference materials to determine meaningful versus acceptable product variability. For example, small molecules like erythropoietin can be compared to quality-assured reference stock. This will be more difficult with iPSCs, because meaningful genetic mutations, phenotypic variability, and differences in culture purity are poorly understood. Extensive safety testing of every iPSC line of a large HLA-matched bank to determine safety would be costly. For this reason, allogeneic cell banking would allow easier development of reference materials to improve product safety for iPSC product development.

For regulatory reasons, the speed of clinical delivery may be dramatically increased by an allogeneic cell bank. Regional trade restrictions on biologics could limit clinical delivery of iPSC-based technology if hurdles to distribution are not anticipated. For example, countries such as Brazil and Sweden do not permit the sale of human-derived tissues, products containing genetic material may not be shipped out of India, and many different countries have varying barriers for stem cell–based technologies (53). An allogeneic cell bank could more easily implement production standards that satisfy diverse regulatory requirements and ethical standards simultaneously.

The standardization of production methods and regulatory licensure has business implications that could attract additional investment as well as innovation to stem cell technologies. Currently, there are two “valleys of death” that are well described in the life cycle of technologies making their way from the lab to clinical application. These two valleys are largely shaped by the costs and risks in translating novel therapies from the lab to the preclinical development phase and, ultimately, to the clinical development phase. An allogeneic stem cell bank could offer an attractive source of PSCs on which to build innovative technologies (54). In the first “valley of death,” companies may be reluctant to invest in stem cell technology if the product derivative has not been scientifically validated. In the second “valley of death,” companies may be averse to initiating clinical trials without fully comprehending production and licensing costs or validating product safety profiles. Hence, institutions or agencies such as the California Institute for Regenerative Medicine (CIRM) provide valuable impetus to the field, as they invest in initial developmental efforts, especially in this era of tight federal budget constraints (55). However, given that funding sources like CIRM are impermanent and intended mostly to launch stem cell technologies, an allogeneic cell bank could be a durable means for mitigating further development costs for future therapies.

One of the primary obstacles to the development of iPSC-based therapies is the high production cost. Generating an HLA-allogeneic cell bank based on iPSCs is labor intensive, which drives the majority of the cost and makes the cost of production difficult to reduce. We estimate that each line requires approximately two to four months to develop, starting from collection of primary cells for reprogramming to expansion of a meaningful iPSC population. Based on our experience, for an HLA-matched iPSC bank, companies should aim to store approximately 200 to 300 vials at approximately 2 × 10⁶ cells per vial. This should represent a sufficient number of PSCs from which a master and working banks of lines intended to produce allogeneic products can be established. Excluding high start-up costs, each line costs approximately $10,000 to $20,000 to produce and validate (49). The development of products that meet cGMP requirements can increase this cost to $50,000 to $100,000 per line (56). Development costs are even higher (~$800,000) for generating an iPSC-derived tissue product that is suitable for clinical use (49). Although cell product development from autologous sources may be more cost effective in the short run based on these estimates, production of autologous tissues is not as easily scalable. By comparison, while start-up costs for an allogeneic cell bank are higher, there are also many opportunities for cost savings downstream. A sizable fraction of the development cost comes from cGMP requirements. For example, in addition to genetic testing, it is necessary to screen for many infectious agents, including mycoplasma, intracellular bacteria, and viral contaminants (57). Some of the screenings require cultures as long as 28 days and transmission electron microscopy for testing. Nonspecific viral contaminant screening uses embryonated chicken eggs and costs ~$12,000 per cell line. Devito et al. have shown that samples from a single donor of human ESCs can be batched to reliably screen for infectious agents (57). They estimate that a bank pooled from 100 donors with three cell lines per donor could save approximately $18 million dollars in the clinical validation process. Although these techniques have yet to receive FDA approval, in the future they could be appropriately developed, scaled, and employed in an allogeneic iPSC bank to offset costs, whereas autologous production would require individualized screening.

Once all the preclinical and commercialization hurdles have been cleared, investigators or companies have to submit their new stem cell–derived therapy portfolio to the regulatory agencies for independent review and approval. The European Medicines Agency in Europe and the FDA in the US have jurisdiction over the commercialization of stem cell therapies in their respective territories. As discussed earlier, stem cell–based products are considered highly processed biologic tissues and require the submission of an IND application to the FDA prior to embarking on any clinical studies. The goal of these agencies is to review and provide oversight of the stem cell–based clinical trials to ensure their safety and efficacy, and to that end, the studies submitted to the FDA must demonstrate data with scientific merit and credibility. The best practice is to approach and establish a dialogue with the FDA early in the process in order to achieve consensus in the development of criteria for the cell products. An optimal execution of the required deliverables that fulfills safety and efficacy goals will ensure that the product is brought to the market in a timely manner.

Ongoing advances in stem cell therapeutics have the potential to radically improve current treatment approaches for a variety of diseases. Stem cell researchers who are trying to bring cell therapies to the clinic face significant translational challenges that by necessity involve a long and costly process. The FDA and other agencies have substantial regulatory requirements that stem cell therapies must meet. These include carrying out biodistribution, immunogenicity, tumorigenicity, dose toxicity, and pharmacodynamic proof-of-concept studies. To fully realize the tremendous potential of stem cell therapies, careful planning and proper resources must be devoted to meeting regulatory and scientific requirements alike to clearly demonstrate their safety and efficacy.

This work was supported by the American Heart Association (12POST12050363 and 14GNT18970018 to E. Neofytou); NIH grants R01 HL123968 and NIH R24 HL117756 (to J.C. Wu); and the California Institute for Regenerative Medicine (IT1-06596, TR3-05556, and DR2A-05394 to J.C. Wu).

Address correspondence to: Joseph C. Wu, 265 Campus Drive, G1120B, Stanford, California 94305-5454, USA. Phone: 650.736.2246; E-mail: joewu@stanford.edu.

Conflict of interest: J.C. Wu is a cofounder of Stem Cell Theranostics.

Reference information: J Clin Invest. 2015;125(7):2551–2557. doi:10.1172/JCI80575.

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