Induced pluripotent stem cell technology: a decade of progress

Nat Rev Drug Discov. Author manuscript; available in PMC 2019 Mar 14.

Published in final edited form as:

PMCID: PMC6416143

NIHMSID: NIHMS1013963

1Division of Stem Cell Biology Research, Department of Developmental and Stem Cell Biology, Beckman Research Institute of City of Hope, 1500 E. Duarte Rd, Duarte, CA 91010.

2Center for iPS Cell Research and Application (CiRA), Kyoto University, 53 Kawahara-cho Shogoin Sakyo-ku Kyoto 606-8507, Japan.

3Stanford Cardiovascular Institute, 265 Campus Drive, Rm G1120B, Stanford, CA 94305-5454.

4Center for iPS Cell Research and Application (CiRA), Kyoto University, 53 Kawahara-cho Shogoin Sakyo-ku Kyoto 606-8507, Japan.

5Gladstone Institute of Cardiovascular Disease, San Francisco, CA 94158, USA

1Division of Stem Cell Biology Research, Department of Developmental and Stem Cell Biology, Beckman Research Institute of City of Hope, 1500 E. Duarte Rd, Duarte, CA 91010.

2Center for iPS Cell Research and Application (CiRA), Kyoto University, 53 Kawahara-cho Shogoin Sakyo-ku Kyoto 606-8507, Japan.

3Stanford Cardiovascular Institute, 265 Campus Drive, Rm G1120B, Stanford, CA 94305-5454.

4Center for iPS Cell Research and Application (CiRA), Kyoto University, 53 Kawahara-cho Shogoin Sakyo-ku Kyoto 606-8507, Japan.

5Gladstone Institute of Cardiovascular Disease, San Francisco, CA 94158, USA

Since the advent of induced pluripotent stem cell (iPSC) technology a decade ago, enormous progress has been made in stem cell biology and regenerative medicine. Human iPSCs have been widely used for disease modeling, drug discovery, and cell therapy development. Novel pathological mechanisms have been elucidated, new drugs originating from iPSC screens are in the pipeline, and the first clinical trial using human iPSC-derived products has been initiated. In particular, the combination of human iPSC technology with recent developments in gene editing and three-dimensional organoids makes iPSC-based platforms even more powerful in each area of their application, including precision medicine. In this overview, we will discuss the progress in applications of iPSC technology that are particularly relevant to drug discovery and regenerative medicine, in light of the remaining challenges and the emerging opportunities in the field.

In 2006, a major technological breakthrough in science and medicine was made with the report that cells with gene expression/epigenetic profile and developmental potential that are similar to embryonic stem cells (ESCs) can be generated from somatic cells (such as fibroblasts) in mice by using a cocktail of four transcriptional factors1. These cells were termed induced pluripotent stem cells (iPSCs) and the four factors Oct4, Sox2, Klf4 and c-Myc were named Yamanaka factors. Just one year later, the generation of iPSCs from human fibroblasts was reported from two laboratories simultaneously2,3.

Human iPSC technology, which has evolved rapidly since 2007 (Box 1), has ushered in an exciting new era for the fields of stem cell biology and regenerative medicine, as well as disease modeling and drug discovery. Soon after the development of the technology, human iPSCs were rapidly applied to generate human disease-in-a-dish models and used for drug screening for both efficacy and potential toxicities. Such approaches are now becoming increasingly popular, given the surge of interest in phenotypic screening and the advantages of human iPSCs in disease modeling, compared with traditional cellular screens. These advantages include their human origin, easy accessibility, expandability, ability to give rise to almost any cell types desired, avoidance of ethical concerns associated with human ESCs, and the potential to develop personalized medicine using patient-specific iPSCs. Furthermore, recent advances with gene-editing technologies in particular the CRISPR/Cas9 technology are enabling the rapid generation of genetically defined human iPSC-based disease models. iPSCs are also a key component of an emerging generation of more physiologically representative cellular platforms incorporating three dimensional (3D) architectures and multiple cell types.

Since its beginning in 2006, iPSC technology has evolved rapidly. Because iPSCs were initially generated by introducing reprogramming factors using integrating viral vectors, such as retrovirus or lentivirus, there is a concern about clinical application of these iPSCs due to potential insertional mutagenesis that might be caused by integration of transgenes into the genome of host cells204. To make iPSCs clinically applicable, a variety of non-integrating methods have been developed to circumvent the risk of insertional mutagenesis and genetic alterations associated with retroviral and lentiviral transduction-mediated introduction of reprogramming factors205. These non-integrating methods include reprogramming using episomal DNAs206,207, adenovirus208, Sendai virus209, PiggyBac transposons210, minicircles211, recombinant proteins212, synthetic modified mRNAs213, microRNAs214,215, and small molecules216, although the small molecule approach is not applicable to human iPSC derivation yet. Among these approaches, episomal DNAs, synthetic mRNAs and sendai virus are commonly applied to derive integration-free iPSCs due to their relative simplicity and high efficiency185. The use of non-viral methods or non-integrating viruses could avoid genomic insertions, thus reducing the risk for translational application of iPSCs. Human iPSCs derived using these non-integrating approaches provide a cellular resource that is more relevant for clinical applications.

iPSC technology has also attracted considerable interest in its potential applicability for regenerative medicine. The first clinical study using human iPSC-derived cells was initiated in 2014, which used human iPSC-derived retinal pigment epithelial (RPE) cells to treat macular degeneration4, and was reported to have improved the patients vision5. Although the clinical study was subsequently put on hold due to the identification of two genetic variants in iPSCs of the patient, the trial is expected to resume6.

Clearly, human iPSC technology holds great promise for human disease modeling, drug discovery, and stem cell-based therapy, and this potential is only beginning to be realized. In this article, we overview the progress in each of the main applications of iPSCs in the decade since the discovery of the technology, featuring key illustrative examples, discussing remaining limitations and approaches to address them, and highlighting emerging opportunities.

Identifying pathological mechanisms underlying human diseases has a key role in discovering novel therapeutic strategies. Animal models have provided valuable tools for modeling human diseases, allowing the identification of pathological mechanisms at distinct developmental stages and in specific cell types in an in vivo setting. Moreover, in mice it is possible to develop in vitro iPSC-based disease models and the corresponding in vivo models in parallel. Comparing the phenotypes observed with corresponding in vitro and in vivo mouse models could provide a better understanding of the strength and limitations of in vitro human iPSC-based models.

However, significant species differences could prevent the recapitulation of full human disease phenotypes in animals such as mice, which are the most commonly used animal models. For example, although many transgenic mouse models have been created for Alzheimers disease, none has captured the entire spectrum of the human disease pathology, including considerable neuronal loss7,8. This is likely due to fundamental species differences between mouse and human neural cells. Thus, there is an urgent need to establish human disease modeling platforms to complement studies in animal models for biomedical research.

Disease modeling using primary patient-derived cells is helpful for studying the etiology of human diseases and developing therapeutic strategies for these diseases. However, the unavailability of expandable sources of primary cells from patients, especially hard-to-access cells such as brain cells and heart cells, is a critical limitation. Human iPSCs are therefore an attractive alternative because of the ease with which human diseases (particularly those with defined genetic causes) could in principle be modeled using iPSCs derived from easily accessible cell types, such as skin fibroblasts and blood cells from diverse patients. Because of their intrinsic properties of self-renewal and potential to differentiate into nearly any cell types in the body, patient-specific iPSCs could provide large quantities of disease-relevant cells and a variety of different cell types that were previously inaccessible, such as neurons and cardiomyocytes. Furthermore, because iPSCs can be derived from the relevant patients themselves, they could enable personalized disease modeling that would be a central part of precision medicine.

Both human ESCs and iPSCs have been used for modeling human genetic diseases. The earlier models were developed using ESCs9, but following the advent of human iPSC technology, human iPSCs have become the preferred option because of their availability and lack of potential ethical concerns associated with human ESCs. Human iPSCs are very similar to human ESCs. Both types of cells express human pluripotent factors and ESC surface markers, and exhibit developmental potential to differentiate into three germ layers2,3. Residual epigenetic memory of somatic cells could occur in iPSCs1012, which may affect the differentiation potential of these cells13. Although the persistence of epigenetic memory of parental cells has been reported in iPSCs1012, similar phenotypes have been reported in disease modeling using human ESCs and iPSCs in most cases13, validating the effectiveness of disease modeling using patient-derived iPSCs.

Disease modeling using human iPSCs starts by deriving iPSCs containing the disease-causing mutation(s) (). These cells are then differentiated into disease-relevant cell types. The resultant cells are used to reveal disease etiology and identify pathological mechanisms. In early studies of iPSC-based disease modeling, iPSCs derived from non-disease-affected individuals were used as controls for patient-derived iPSCs. However, like other cells, iPSCs have exhibited line-to-line variations, which complicates data interpretation because one has to distinguish the line-to-line variation from the true disease-relevant phenotypes.

A schematic for human iPSC-based disease modeling. Human iPSCs are derived from individual patients and differentiated into specific cell types. To develop new therapies, the resultant cells are used to observe disease-specific phenotypes and identify novel pathological mechanisms,. Human iPSC-based disease modeling with patient-specific cells now provides an exciting new approach for the development of personalized diagnosis and medicine.

Rapidly developing genome editing technologies now enable the introduction of genetic changes into iPSCs in a site-specific manner, including correction of disease-causing gene mutations in patient-derived iPSCs and introduction of specific mutations into non-disease affected wild type (WT) iPSCs. These approaches allow the generation of genetically matched, isogenic iPSC lines with the introduced mutation as the sole variable, ensuring the reliable identification of the true pathology while avoiding the confusion with any disparities in genetic background or epiphenomena resulting from possible line-to-line variations. The isogenic iPSC controls will be especially important when modeling sporadic or polygenic diseases, in which phenotypic differences are expected to be small14.

The development of programmable site-specific nucleases, including the zinc-finger nuclease (ZFN)15,16, transcription activator-like effector nucleases (TALENs)1719, and the CRISPR/Cas9 system2022,23 (), has improved gene editing efficiency in human ESCs and iPSCs substantially by inducing DNA double-strand breaks at the site of gene modification. The CRISPR/Cas9 technology in particular has attracted much attention and gained wide usage in gene editing of human ESCs and iPSCs due to its simplicity in design and ease of use. This gene editing technology allows researchers to introduce disease-causing mutations to WT iPSCs and eliminate such mutations in patient iPSCs to create isogenic controls for iPSC-based disease modeling ().

Technology for gene editing of human ESCs and iPSCs

However, a major challenge in applications using the CRISPR/Cas9 technology is the possibility of off-target effects. Nevertheless, although relatively high levels of off-target gene modifications by CRISPR/Cas9 have been described in cancer cell lines24, recent studies from multiple laboratories using whole genome sequencing (WGS) indicate that off-target gene modifications are rare in normal human cells, including human iPSCs and ESCs2529. WGS using genomic DNAs isolated from the original iPSCs and corresponding gene-edited iPSCs, coupled with comprehensive bioinformatic analysis25,2729, is useful for detecting off-target effects such as single nucleotide variants (SNVs) and insertions or deletions (indels), especially for cells that will be used for clinical applications. At present, WGS is expensive, but it is expected that the price will go down with continuous development of the technology. Alternative approaches for detecting off-target effects include exosome sequencing30 and targeted deep sequencing29. For targeted deep sequencing, one can search for potential off-target sites that are different from the on-target sites in the human genome using Cas-OFFinder (http://www.rgenome.net)31, an algorithm for identifying off-target sites, including off-target SNVs or indels.

Gene editing tools are also being continuously improved and refined, which may help address the issue of off-target effects. Originally CRISPR/Cas9 edits a genomic locus by inducing DNA double-strand breaks using a single guide RNA-directed wild type Cas9 nuclease. The nickase version of Cas9 (D10A mutant) directed by paired guide RNAs or the engineered Cas9 nuclease variants with enhanced specificity (eSpCas9) is now being used increasingly for genome editing3234, because both have been shown to reduce off-target effects substantially while retaining rigorous on-target cleavage34,35. Furthermore, catalytically dead Cas9 (dCas9) fused with transcriptional activator or suppressor has been used to modulate transcription of endogenous genes (so-called CRISPRi or CRISPRa) or image genomic loci by fusing with a fluorescent protein3234,36. Modifications of the CRISPR/Cas9 system also enable explicit introduction of DNA sequence changes in a precise mono-allelic or bi-allelic manner with high efficiency37. A recent development in base editing takes advantage of the fusion of CRISPR/Cas9 and a cytidine deaminase enzyme to allow direct conversion of cytidine to uridine without the need of double strand DNA break38. This new approach enhances gene editing efficiency and will further facilitate gene editing in human ESCs and iPSCs.

iPSC-based disease modeling is widely used for studying disorders caused by a single gene mutation (monogenic disorders) that have an early onset39,40, as the approach is ideally suited to such disorders because iPSCs can be easily derived from patients with these disorders and differentiated into disease-relevant cells, such as neurons. Furthermore, given the relative immaturity of cells differentiated from iPSCs41, there is greater confidence that the phenotypes of cells differentiated from iPSCs provide a good model for diseases with an early onset versus late onset, for which cellular aging may be important in disease pathology41. For example, neurons differentiated from patient iPSCs were used to model spinal muscular atrophy (SMA), an early-onset disease caused by mutations in the gene encoding the survival motor neuron 1 (SMN1)39. Mutations in the SMN1 gene led to degeneration of motor neurons and subsequent muscular atrophy. Type 1 SMA patients usually show symptoms at 6 months from birth, with a rapid disease progression that kills them by the age of two42. In an initial iPSC-based disease modeling study39, iPSCs were derived from fibroblasts of a type 1 SMA patient and differentiated into a disease-relevant cell type, motor neurons. Reduced survival of motor neurons differentiated from patient iPSCs was observed, compared to that of motor neurons derived from an unaffected control. Moreover, the SMA patient-derived iPSCs were able to respond to valproic acid and tobramycin, two compounds known to induce SMN protein levels, by increasing SMN protein levels and SMN protein-containing gems39. This study provides a proof-of-principle that patient-derived iPSCs could be used to model early-onset genetic diseases and serve as potential drug-screening platforms.

Modeling diseases that have a late onset is more challenging, because cells differentiated from human iPSCs in general exhibit fetal-like properties41. However, induced cellular aging has been used to aid in successful modeling of late-onset diseases4346. One way to induce aging in cells differentiated from human iPSCs is to treat cells with cellular stressors, including compounds that target mitochondrial function or protein degradation, such as pyraclostrobin and MG-13243,44,46,47. Another way to induce cellular aging is to ectopically express gene products that induce premature aging, such as progerin45. However, whether cellular stressors or progerin expression can elicit cellular aging through a mechanism that is similar to normal aging remains to be determined41. Moreover, recent studies indicate that cellular maturation and aging may be distinct events41,48. It remains unclear whether the cellular aging inducers can promote both cellular maturation and aging, as opposed to triggering cellular aging in immature cells48. Alternatively, the direct reprogramming approach that involves direct conversion of human fibroblasts into other lineage-specific cells, such as neurons, does not erase cellular aging markers49. Indeed, neurons derived from aged fibroblasts through direct reprogramming have been shown to maintain cellular age50, therefore offering an alternative cellular model to study age-related disorders. It is worth noting that there has also been success in promoting cellular maturation, such as by using improved formulation of cultured medium51 and neuron-astrocyte co-culture system52,53.

iPSCs also offer a new way to study sporadic diseases (the causes of which have not been identified in patients family histories or genetic mutations), which is important as the majority of patients with many diseases have sporadic forms of the disease. For example, in Alzheimers disease, 95% of patients fall under the sporadic category. Interestingly, analysis of iPSC-derived nerve cells from patients with sporadic Alzheimers disease identified several sporadic cases that exhibited the same phenotypes as familial Alzheimers disease with a specific gene mutation54 , which indicated that it may be possible to re-classify the sporadic condition using iPSCs. However, modeling sporadic diseases using iPSCs is generally more difficult than monogenic disorders because the phenotypic changes in such diseases are often thought to be induced by multiple small-effect genetic risk variants, in combination with environmental factors. Although iPSCs derived from patients with such diseases would contain disease-relevant risk variants, using iPSCs to model such diseases is complicated by line-to-line variation in genetic and epigenetic background. Such variation is more problematic for modeling sporadic diseases, because the phenotypes of the sporadic disease iPSC-derived cells are expected to be more subtle than for those derived from monogenic diease iPSCs.

Thus, a key question for human iPSC-based modeling of sporadic diseases is how to generate paired isogenic cell lines that only differ at relevant risk variants14. Recently, the CRISPR/Cas9 gene editing approach has been used to generate isogenic iPSC lines that differ at a PD-associated risk variant55. The ability to generate genetically controlled isogenic iPSC lines in which specific disease-associated genetic risk variants are the sole variable creates a well-controlled system, which in combination with an allele-specific assay has enabled robust dissection of a genetic risk variant for Parkinsons disease55. This experimental paradigm could be applied to studying genetic risk factors associated with other diseases.

To date, many diseases have been studied using a single disease-relevant cell type derived from iPSCs. For example, iPSC-derived neurons have been used to model Alzheimers disease54,5668 () and Parkinsons disease ()4345,55,6984. However, more than one cell type may be required to effectively model some diseases. Indeed, comparable efforts have been devoted to model schizophrenia using patient iPSC-derived neurons8587 and neural progenitor cells8892. To better recapitulate disease phenotypes, co-culture of more than one cell types may also be needed to study the interaction of different cell types. For example, astrocyte/neuron co-cultures have been used to model the pathology of amyotrophic lateral sclerosis (ALS)9396. The co-culture system allowed the investigation of non-cell-autonomous aspects of disease pathology, which would otherwise be impossible with single cell types, such as neurons. Moreover, these studies enabled the identification of astrocytes as a critical cellular component contributing to motor neuron degeneration in ALS and provided a drug screening platform for ALS using patient iPSC-derived astrocytes9396.

Patient iPSC-based modeling of Alzheimers disease (AD)

Patient iPSC-based modeling of Parkinsons disease (PD)

The interactions between different cell types can be better modeled using 3D organoids. Organoids have been generated for multiple organs, including the brain, retina, intestine, kidney, liver, lung, and stomach, using both tissue stem cells and pluripotent stem cells from mice and humans97. Human iPSC-derived organoids have been developed for a variety of applications due to their resemblance to endogenous cell organization and organ structure, and are particularly useful because they allow the possibility to study cell-cell interactions in a cellular context that mimics human physiology and development. The 3D organoids have been used in modeling human organ development and diseases, testing therapeutic compounds, and cell transplantation98114 (). Multiple cell types that are physiologically relevant can be generated in organoids following a spatial-temporal order. Moreover, cells generated in organoids can be functionally more mature than cells derived using directed differentiation protocols, due to the interaction of different cell types, such as neurons and astrocytes, in the 3D structure. Therefore, 3D organoids allow dissection of disease pathology in a developmentally relevant spatial-temporal context and have the potential to offer a drug response at the level of an organ, rather than at the level of individual cells.

human iPSC-derived organoids for modeling development and disease

While 3D organoids provide highly promising tools for iPSC-based disease modeling, the organoid technology has limitations. One challenge is to create an organoid platform with increased efficiency and reproducibility as compared to traditional two dimensional cultures115. The recent application of miniaturized spinning bioreactors with 3D design has allowed the generation of forebrain organoids with high reproducibility110. The development of more standardized organoid culture medium, together with a more defined extracellular matrix, would further facilitate the generation of a highly reproducible organoid system that is more applicable for accurate disease modeling, drug discovery, and therapeutic development116. Another challenge is the lack of vascularization in the current organoid system97. Accordingly, organoids exhibit limited growth and maturation due to the lack of continuous nutrient supply. Spinning bioreactors and shaking culture platforms have been shown to provide better nutrient supply and improve the growth of organoids110,117. Co-culture with endothelial cells has allowed generation of vascular-like network in organoids99. Moreover, transplantation of in vitro generated human organoids into relevant sites of animal hosts facilitates vascularization and maturation of organoids. This transplantation approach may be applied when organoids with increased size and improved maturation are needed for the study.

Many drug screens are based on targets that are considered to be relevant to the disease mechanisms. However, the low success rates of compounds originating from target-based screening have led to greater interest in phenotypic screening118. This revival in phenotypic screening has been aided by the discovery of iPSCs for numerous reasons, including the scalability of iPSC production, which facilitates assay development, and their pluripotency, which allows differentiation into multiple disease-relevant cell types (especially those that are otherwise hard to access, such as neurons)119. Patient-derived iPSC models make it possible to recapitulate disease phenotypes and pathologies in a culture dish. Cells differentiated from patient-derived iPSCs could present molecular and cellular phenotypes. Whether the phenotype that is selected as readout for drug screen is truly relevant to the disease can be confirmed by gene editing approach if the gene responsible for disease phenotypes is known, and can be further validated in patient samples and/or animal models120. In addition to phenotypic screening, iPSCs can also be used for target-based screening. Using human iPSC models, many drug screens have been conducted and potential drug candidates have been identified using either phenotypic or target-based screening.

To obtain target cells with high purity on a large scale, purification and enrichment technologies using specific cell surface markers121,122, cell-specific promoters123 and microRNAs124 have been established. In the first report of large-scale drug screening using an iPSC-based disease model, neural crest precursors for autonomic neurons were sorted and purified from iPSCs derived from patients with familial dysautonomia, a monogenic early-onset disease that is characterized by degeneration of neurons in the sensory and autonomic nervous systems121. It is caused by mutations in the gene coding for the IkB kinase complex-associated protein (IKBKAP) that result in a splicing defect and production of a dysfunctional truncated protein. The screening was conducted using 6,912 compounds, and a compound known as SKF-86466 was found to improve disease-specific aberrant splicing. Interestingly, SKF-86466 was not effective in non-target cells, including iPSCs, fibroblasts, and lymphocytes. These results illustrated the advantage of iPSC-based drug screening to explore cell type-specific pathogenesis.

Burkhardt et al. performed disease modeling and drug screening using sporadic ALS patient-derived iPSCs125. The authors identified de novo aggregation of TAR DNA-binding protein 43 (TDP-43) in motor neurons of sporadic ALS patients, using TDP-43 aggregation as readout for a high-content drug screen to identify compounds that reduce TDP-43 aggregation125. The same research team also made effective use of patient-derived iPSC model of Alzheimers disease126. The authors identified a disease-relevant protein, extracellular tau (eTau), in the conditioned medium of cortical neurons derived from the iPSCs of an Alzheimers disease patient, generating a therapeutic antibody against eTau126. This disease-relevant protein would not have been discovered without using the human iPSC model. eTau causes neuronal hyperactivity and increases amyloid beta (A) production. Using human iPSC models as a tool to identify disease-relevant targets could be a critical component for future drug development. Naryshkin et al.127 found that an SMA patient-derived iPSC model could be used to validate human- and disease-specific drug responsiveness after initial screening using a HEK293 cell line127. These compounds were then validated in patient-specific fibroblasts, and in motor neurons differentiated from patient-derived iPSCs that serve as a patient-specific and disease-relevant cellular model127. Finally, the hit compound was evaluated in a mouse model for in vivo activity127. This drug discovery approach includes a patient-derived iPSC model as one of the validation steps by taking advantage of the patient-specific and disease-relevant properties of motor neurons derived from patient iPSCs.

Overall, iPSC-based drug screening has been used to evaluate more than 1,000 compounds for several diseases ()121,125,128,129, and several clinical candidates have been identified ()126,127,130. However, these studies require considerable time (several weeks or more) to differentiate iPSCs into disease-relevant cell types. Although this may not seem long for phenotypic screening, a shorter differentiation period is preferable to avoid variation in cell quality. Therefore, faster and more stable differentiation methods that result in higher maturity and purity are being sought. An alternative approach is to perform drug screen using cells derived from direct conversion131,132. Direct conversion forces the target somatic cells (e.g. fibroblasts) to express cell-specific transcription factors and reprogram one somatic cell state to another somatic cell state without passing through the iPSC state49,132. Direct conversion has been used to reprogram myocardial cells, liver cells, neural cells, or other type of somatic cells from a different type of somatic cells, such as fibroblasts. As an advantage of direct conversion, authentic human neurons that reflect important aspects of cellular aging can be generated50. However, the non-renewable source of cells provided by this approach may not be applicable for large-scale drug screening. The forced expression of transcription factors also offers the potential to differentiate patient iPSCs much more rapidly. In a recent study, forced expression of MYOD1 (myogenic differentiation gene), a master regulator of skeletal muscle differentiation, was used to produce new cellular models of intractable muscle disease pathologies such as Miyoshi myopathy133 and Duchenne-type muscular dystrophy134.

Large scale drug screening using human iPSCs

Drug from iPSC research in clinical trials

An important point in pathology research using iPSCs is the nature of the control group. For genetic disorders, a control group can be created by conducting gene correction of the mutant allele in patient iPSCs. Comparisons between various groups of iPSCs (healthy, patient, and gene-corrected patient iPSCs) can be conducted to validate the results of drug screening119. iPSCs also make invaluable models in the case of sporadic diseases. In these cases, because no causal mutation is known, the nature of a control group is difficult to establish, but disease-relevant single-nucleotide polymorphisms (SNPs) can be considered instead65. As described in the Perspective section below, for future drug screening, sporadic disease iPSCs should allow for investigation of whether the disease is caused by genetic factors such as SNPs, somatic mutation/mosaicism, or epigenetic factors. These developments could further open the door to personalized drug screening using iPSCs135.

Another application is drug repositioning using disease-specific iPSCs. In drug repositioning, existing drugs already approved for specific diseases are tested to find new applications for other diseases. For example, a human iPSC model derived from achondroplasia patients with fibroblast growth factor receptor 3 (FGFR3) mutations showed that patient iPSCs did not differentiate well into cartilage tissue136. Using this model, a screen for molecules that rescue chondrogenically differentiated iPSCs from the defective cartilage phenotype identified several statins, which are approved drugs for cardiovascular disease. The same study found that statins could promote the growth of shortened limbs in a mouse model of FGFR3-linked disease. These results indicate that statins may be repositioned as candidate drugs for achondroplasia136. As another example of drug repositioning, the anti-epileptic drug ezogabine was found to be effective in an iPSC model of the motor neuron disease amyotrophic lateral sclerosis (ALS) and is now undergoing clinical trial137. In this study, the authors showed the effect of ezogabine on an iPSC model derived from not only ALS patients with mutations in the superoxide dismutase 1 (SOD1) gene, but also ALS patients with mutations in other genes linked to ALS, such as C9orf72 and FUS. It has also been demonstrated that ALS patient-derived iPSC motor neurons initially exhibit a hyper-excitable state, followed by a decrease in excitability138, suggesting that early intervention with ezogabine treatment may be required for the treatment of ALS patients. The observation of similar drug response in different patient groups allowed generalizing the drug responsiveness across ALS patient types. Drug discovery using patient iPSCs derived from multiple genetic forms is of great value, because it allows testing the drug responsiveness in a broad patient population. In contrast, it is hard to analyze the effect of a drug on multiple mouse models simultaneously.

The development of new drugs is enormously costly, mostly because of failures, particularly those in late-stage clinical trials, which are in part due to unanticipated side effects139,140. Many unpredicted adverse effects of new candidate drugs can occur, with cardiac and liver toxicity being of special concern. Consequently, there is considerable interest in approaches that could more effectively predict the likelihood of candidate drugs to cause serious side effects, thereby enabling the selection of candidates that are less likely to fail due to toxicity in late-stage trials.

Lethal arrhythmias with a QT prolongation account for 21% of total cardiac toxicities141. QT prolongation is an adverse effect related to human Ether-a-go-go Related Gene (hERG) channels. Cardiac safety testing has been mainly dependent on the hERG assay, because blocking the hERG current is considered to be associated with the deadly ventricular arrhythmia named torsades de pointes (or TdP). It has been discovered that 4060% of drugs that inhibit hERG channel current do not cause QT prolongation142,143. These false positive results from the hERG assay have hindered the development of promising drugs. Preclinical strategies have been proposed to detect drug-induced electrophysiological cardiotoxicity using in vitro human ion channel assays, human-based in silico reconstructions, and human stem cell-derived cardiomyocytes144. Recent efforts have shown that multi-electrode arrays (MEA) assays using human iPSC-derived cardiomyocytes may offer a reliable, cost-effective surrogate for preclinical in vitro testing145 that could be used to assess pro-arrhythmic risk146.

For hepatotoxicity, hepatocyte cell lines or human primary hepatocytes are widely used. However, there are limitations to these models too, including cell resources, loss of function due to freezing-thawing, and lot-to-lot variation. Recently, human ESC/iPSC-derived hepatic cells were generated that express functional molecules such as CYP3A4 and uptake Indocyanine Green147 responding to known hepatotoxic drugs148. Functional 3D liver organ buds have also been reported, which may result in better drug screening99.

Finally, regarding the nervous system, a platform that assesses adverse drug effects using pluripotent stem cells is now being developed. To conduct such an assessment, the analysis of alterations in the gene expression of cells in the nervous system, such as neuronal cells, mesenchymal stem cells, and vascular endothelial cells derived from human ESCs in a culture dish has been proposed149.

However, there are several obstacles associated with iPSC-based therapy that will need to be addressed before routine clinical applications can begin153. One concern is the risk of tumorigenicity from ESCs and iPSCs154. Because pluripotent cells are maintained in culture for prolonged periods of time, they can accumulate karyotypic abnormalities, copy number variants, and loss of heterozygosity155. Hence prior to clinical use, iPSC-derived products need to be carefully screened for the lack of potentially risky genetic alterations155 and rigorously tested to ensure their purity, quality, and sterility. Increased knowledge on the basic biology of pluripotency induction and maintenance will also help us to reduce the risk of mutation development and genetic instability associated with human iPSC derivation and maintenance.

Although the products differentiated from iPSCs have not been shown to generate teratomas, it is critical to ensure the final product does not contain undifferentiated cells that have the potential to generate teratomas. Accordingly, improved protocols for differentiating human iPSCs into desired cell types with precise identity and cellular functions are needed. To this end, small molecule inhibitors that have been shown to induce selective and complete cell death of undifferentiated human pluripotent stem cells without affecting their differentiated derivatives have been identified156,157. Treatment of the iPSC-derived cellular product with these inhibitors may reduce the potential tumorigenicity. Another potential solution is to sort the iPSC-derived cells before transplantation through positive selection for desired cell types and negative selection against human ESC markers using fluorescence-activated cell sorting (FACS). Lastly, the risk of tumorigenecity can be tested in animal models prior to transplant. However, this approach may not be applicable to patients with rapid disease progression due to the long period of time associated with animal tests.

Compliance with good manufacturing practice (GMP) is mandatory before human transplantation of cell therapies. Once cells are safely delivered, ideally patients should be monitored for the development of potential tumors and activation of the immune system158. One approach for tumor monitoring may be to assess the enhanced angiogenesis that often accompanies teratoma formation, which can be detected using 64Cu-labeled cyclic arginine-glycine-aspartic acid tetramer (64Cu-DOTA-RGD4) radiotracer with positron emission tomography (PET) imaging159. Another approach may be to use a combination of serum biomarkers (e.g., carcinoembryonic antigen, -fetoprotein, or human chorionic gonadotropin) and magnetic resonance imaging (MRI) screening as described recently160. However, it is worth noting that these approaches would be mostly useful at the preclinical stage, especially if they are already part of the imaging procedure required for evaluating an endpoint. Their feasibility and necessity for future human trials remain to be determined.

The lack of an effective method of inducing immune tolerance is a major roadblock for human ESC-based therapies. ESCs were once considered immune-privileged due to the low expression of major histocompatibility complex (MHC) class I, MHC II, and costimulatory molecules161. Although undifferentiated ESCs might be immune-privileged, their differentiated derivatives can trigger cellular and humoral immune responses162. By contrast, autologous iPSCs may avoid the high cost and serious side effects associated with lifelong immunosuppression required for allogeneic cell transplantation163. Despite some controversy over the immunogenicity of undifferentiated iPSCs164, recent studies demonstrate that differentiation of iPSCs could result in loss of immunogenicity165167.

The application of cells derived from individual patients own iPSCs or iPSCs from matched donors may become a cornerstone of precision medicine, and has the important advantage that there should be no need for long-term immune suppression to preserve the transplante cells. Indeed, the first iPSC clinical trial used RPEs from autologous iPSCs derived from the patient. Using autologous iPSC products for personalized cell therapy seems ideal for orphan diseases, as massive cell banking is not required. However, for more common diseases, especially acute common diseases such as cerebrovascular accident (CVA) or myocardial infarction (MI), autologous iPSC therapy may not be practical for large number of patients given the high cost and lengthy period of time needed for careful validation of each cell line. For these reasons, the second phase of the iPSC-RPE trial in Japan will be employing allogeneic products168.

The allogeneic iPSC approach could also bring down the cost for iPSC-based cell therapy. Excluding high startup cost, each iPSC line costs ~$10,00020,000 to produce169. Meeting cGMP requirements increases this cost substantially170. Costs are even higher, by approximately $800,000169, to generate an iPSC-derived tissue product suitable for clinical use (e.g., differentiation of iPSC-neuronal cells for CVA, iPSC-cardiomyocytes for MI, or iPSC-RPE cells for macular degeneration). Banking iPSCs for allogeneic transplant has the potential to reduce cost because one production may be used for multiple patients. To facilitate allogeneic transplant, the effectiveness of conventional immunosuppressive protocols and newer regimen of co-stimulatory blockers for inducing immunotolerance will need to be improved in preclinical and clinical settings171,172. Moreover, understanding how pluripotent stem cells interact with the immune system and why they may be more tolerance-inducing than other transplanted cells may lead to the identification of new immunosuppressive mechanisms and strategies163. Furthermore, transplantation to immune-privileged sites may serve as a possible strategy to overcome immune rejection. Incorporating recent advances in genome editing strategies to create universally accepted donor cells could be another alternative approach173.

The combination of the human iPSC platform with the recently developed gene editing and 3D organoid technologies could make human iPSCs an even more powerful cellular resource for stem cell-based cell therapy development. As a proof-of-principle, mouse iPSCs corrected through gene editing have been used to generate hematopoietic progenitors for successful treatment of sickle cell anemia in a mouse model174. Furthermore, the integration of genetically corrected human iPSCs with 3D organoids could allow tissues to be generated as sources for organ replacement therapies97. Indeed, human iPSC-derived liver organoids have been shown to successfully generate functional human liver-like tissues in transplanted mice in a proof-of-principle study99. However, there are still challenges to overcome for such approaches to become applicable in human cell therapy. For example, the potential off-target effects associated with gene editing need to be addressed, as do the limitations of organoids, as described in the section of iPSC-based disease modeling.

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Induced pluripotent stem cell technology: a decade of progress