HHS Public Access Author manuscript Author Manuscript
Drug Target Rev. Author manuscript; available in PMC 2016 October 21. Published in final edited form as: Drug Target Rev. 2016 ; 3(2): 34–38.
Translating Stem Cell Biology Into Drug Discovery Ilyas Singeç and Anton Simeonov National Institutes of Health (NIH). National Center for Advancing Translational Sciences (NCATS). Division of Pre-Clinical Innovation (DPI)
Abstract Author Manuscript
Pluripotent stem cell research has made extraordinary progress over the last decade. The robustness of nuclear reprogramming of somatic cells into induced pluripotent stem cells (iPSCs) has created entirely novel opportunities for drug discovery and personalized regenerative medicine. Patient- and disease-specific iPSCs can be expanded indefinitely and differentiated into relevant cell types of different organ systems. As the utilization of iPSCs is becoming a key enabling technology across various scientific disciplines, there are still important challenges that need to be addressed. Here we review the current state and reflect on the issues that the stem cell and translational communities are facing in bringing iPSCs closer to clinical application.
Quality Assurance of iPSCs
Author Manuscript Author Manuscript
Pluripotent stem cells are characterized by extensive self-renewal under appropriate cell culture conditions and differentiation into all cell types of the human body. The groundbreaking discovery by Shinya Yamanaka and colleagues1,2 over a decade ago that somatic cells can be reprogrammed into embryonic-like cells by defined factors has reinvigorated biomedical research in general and human biology-oriented drug discovery in particular. After quickly establishing that somatic cells - independent of tissue of origin, age, and reprogramming technique - can be robustly reverted back and stably maintained at a pluripotent state3,4, the field has moved on to generating new iPSC lines from patients with monogenic and complex genetic diseases as well as those of unknown etiology. Studying relevant cell types in a dish in order to better understand the molecular underpinnings of intractable human diseases holds a great promise to develop novel mechanism-based concepts in drug screening. To enable such disease modeling, predictive toxicology, and regenerative cell therapy applications, major international efforts and public-private partnerships are underway to generate large iPSC banks considering ethnic and diseasespecific backgrounds5. While many new iPSC lines are being generated, important questions remain to be answered in order to ensure acceptable standardization, comprehensive characterization, rigorous quality control, and safety. Significant challenges remain to be addressed in order to overcome lack of experimental reproducibility, uncontrolled differentiation protocols, genome instability of reprogrammed and differentiated cells, and transplantation of unwanted cells with potential teratoma formation risk. Recent work has demonstrated the equivalence of genetically matched iPSCs and embryonic stem cell lines (ESCs)6. This is an important comparison, since ESCs are still considered the gold standard for bona fide pluripotency. In addition, this study highlighted that genetic
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background can be a major confounding factor for comparative studies that rely solely on genetic and epigenetic signatures, and also underscore the importance of being careful when drawing conclusions based on comparing only a few iPSC lines, which is currently the main approach in the field. Moreover, further systematic studies are necessary to establish criteria that can reliably distinguish “high-quality” iPSCs from “poor-quality” iPSCs. At present, the most widely used parameters to characterize iPSCs include morphology, karyotype analysis, immunophenotyping (e.g. surface makers, transcription factors), multilineage differentiation (i.e. ectoderm, mesoderm, endoderm) by generating embryoid bodies, and in vivo teratoma formation. Other approaches suggested as a measure of pluripotency include bioinformatics analysis based on gene expression7,8. However, it still debated if these approaches are acceptable to completely replace teratoma formation. Working with iPSCs is labor-intensive and costly and identifying “high-quality” iPSCs is a critical goal, which should be prioritized as early as possible in the drug discovery process. Nevertheless, it remains unclear if the currently available assays and protocols are sufficient and rigorous enough for clinical and translational purposes.
Reproducible Differentiation Protocols
Author Manuscript Author Manuscript
The remarkable biological versatility of pluripotent stem cells is based on their developmental potential to give rise to all somatic cell types, while not undergoing cellular senescence in the pluripotent state. Attempts to harness this potential of unlimited cell growth and differentiation started after the successful derivation of the first human ESC lines9. Several approaches have been used over the years to differentiate pluripotent cells into desired lineages and cell types, including overgrowing cells and culturing them as neurospheres10, forming free-floating embryoid bodies and picking neural rosettes11, coculture with stromal cells12, using recombinant proteins (e.g. Noggin)13, small molecule inhibitors such as SB431542 alone14 or in combination with recombinant Noggin15. While over the years there has been a trend to move from empirical methods to directed differentiation strategies by targeting specific cell signaling pathways, there are still major knowledge gaps and biological unknowns that hinder the formulation of highly reproducible differentiation protocols to yield large numbers of mature cell types at high purity (Figure 1). How can the stem cell field overcome the challenge of heterogeneous cultures with immature cell types generated over extended periods of time (weeks to months) and exposed to a flurry of small molecules and recombinant proteins, often used at supraphysiological concentrations? How can we comprehensively characterize cell type identities across different developmental stages? How can we identify and manipulate relevant cell signaling pathways and new small molecules that target these pathways? Bringing solutions to these questions is central to firmly establishing the iPSC technology in drug discovery and clinical applications. To tackle these widely accepted challenges, the National Institutes of Health (NIH), as part of its Regenerative Medicine Program (RMP), has recently launched the Stem Cell Translation Laboratory (SCTL). The SCTL is located within the National Center for Advancing Translational Sciences (NCATS) and is dedicated to bring the iPSC technology closer to drug discovery and regenerative medicine applications. (https:// commonfund.nih.gov/stemcells/index).
Drug Target Rev. Author manuscript; available in PMC 2016 October 21.
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Automation and Industrialized Scale-Up of iPSC Cultures Precisely formulating robust differentiation protocols requires deeper and actionable insights into the basic biology of pluripotent cells. The resulting information and reference datasets should then allow to better control cell fate choice and terminal differentiation. To this end, it is critical to perform dose-response experiments, elucidate signal strengths, and carefully document the net effect of combinatorial manipulations of relevant pathways. The importance of combining genomic and proteomic methods is underscored by the fact that mRNA abundance often does not predict protein expression levels, particularly during dynamic transitions16. Integration and analysis of such complex orthogonal datasets by systems biology methods should enable establishing a roadmap that is practical and costefficient enough to be used across many different laboratories and translational research settings.
Author Manuscript Author Manuscript
Routine manual cell culture work is inherently influenced by the physico-chemical environment as well as human bias. Typically, scientists working in cell culture have their own style and preference in performing daily tasks. Variations in carrying out specific tasks such as adding fresh medium at varying time points, cell passaging at varying cell densities, and other more or less significant or subtle effects can all have a strong impact on the experimental outcome and the quality of the end product. Standardizing routine cell culture and the manufacturing process in cell engineering are critical steps, with automated robotic cell culture systems providing unique opportunities to establish standard operating procedures (SOPs), reduce human bias, and control cell culture conditions more precisely. Using the robotic CompacT SelecT system17 combined with chemically defined medium18, we have started to establish protocols that can maintain and expand pluripotent stem cells under highly controlled feeder-free conditions (Figure 2). The fully automated workflow allows controlling each step via computer-aided commands. This real-time system ensures that the scientist can conveniently control and monitor cell culture around-the-clock. For instance, daily media change can be performed consistently at exact times. Any possible technical error is immediately transmitted to the scientist in charge. Integrated cell counter, viability assessment, and image-based passaging are key features for standardization and documentation. In summary, automation provides the following advantages, which will help to implement standardized utilization of iPSCs for translational research:
Author Manuscript
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Expansion and maintenance of multiple cell lines
•
Cell counting and viability measurement
•
Sub-culturing and cell passaging
•
Cell seeding
•
Transient transfection
•
Harvesting at exact time points
•
Reducing the risk of cell culture contamination
•
Incubation of up to 182 T-flasks and up to 420 plates in current configuration
Drug Target Rev. Author manuscript; available in PMC 2016 October 21.
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Plating into 96, 384 and 1536-well plates suitable for high-throughput and high-content screening
Industrialized scale-up and access to large numbers of properly differentiated cell types will impact future health care by leveraging both personalized medicine and treatment of large patient cohorts.
Transforming Drug Discovery by Human Biology
Author Manuscript
The traditional drug discovery process is painstakingly slow and inefficient19–21. The high attrition rate and failure of potential drugs due to safety issues and lack of efficacy in patients detected only after many years of investment suggest to critically re-visit the usefulness of pre-clinical models that have been relied upon in the past. Indeed, overdependence on animal models and immortalized cell lines in the pre-clinical stage is very risky and often times of limited predictive value for the outcome in the clinical phase22–25. More than 90% of drugs developed based on animal models fail in clinical trials23. To address at least some of the obstacles, drug repurposing has emerged as a valuable strategy. The underlying concept, which is to find new indications for existing drugs, can avoid redundant and costly pre-clinical studies and Phase 1 safety trials21. Along these lines, the National Center for Advancing Translational Sciences (NCATS) has compiled the largest public repository of approved and clinical phase drugs, the NCATS Pharmaceutical Collection (NPC), aimed at broadly sharing this resource for drug repositioning, toxicology studies, and chemical genomic profiling26,27..
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The two main strategies of small molecule drug discovery can be classified as either targetbased or phenotypic28. While target-based approaches aim to modulate a specific molecular target of interest, the phenotypic approach is intended to modify a disease-associated phenotype in cells or whole organisms. Comparing the success rate of both approaches has led to a renaissance of phenotypic screens28. However, setting up informative phenotypic screens requires a renewable cell source and validation of cellular assays that faithfully recapitulate human physiology. Patient- and disease-specific iPSCs are uniquely suited for developing novel human biology-focused paradigms for drug discovery and first-in-class therapies. Once the challenges discussed above are sufficiently addressed (i.e. quality assurance, reproducible and scalable differentiation), iPSC-based models will be broadly implemented in all stages of pre-clinical drug development. For instance, high-throughput and high-content screening, hit validation, structure-activity relationship studies, and toxicology assays will directly benefit from the consistency of utilizing the same cell types(s) derived from genotyped patients with known clinical history. A few studies started to employ pluripotent cells for drug screening29–32. As we learn to robustly differentiate and scale up iPSC-derived cells, it is apparent that more systematic and extensive screening studies using much larger compound libraries will follow in the near future. Beyond using human cells in screening, careful analysis of iPSC-based cellular assays will also provide insights into the complexity of human disease and leverage the understanding of genotype-phenotype relations and pinpoint novel cellular and molecular disease mechanisms33–36. Elucidating genetic diseases, particularly those with underlying strong
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cell-autonomous effects, may indeed enable robust and streamlined identification and validation of new ‘druggable’ targets, signaling pathways, and disease phenotypes. In a different approach, the wealth of genetic data obtained by genome-wide association studies (GWAS) can be interrogated by probing disease-relevant cell types. Follow-up experiments and prioritization of hundreds of potential disease targets based on GWAS data is a significant problem. This is further complicated by the fact that the majority of hits are found in regulatory elements of unknown significance and not in protein-coding regions37. It is again desirable that these targets be directly investigated in relevant human cells from affected patients under defined laboratory conditions. The versatility of the iPSC technology (Figure 3) can be further enhanced by the power of functional genomics (e.g. RNAi) and genome editing such as the CRISPR/Cas9 system, which allows precise site-specific genetic manipulations. The interrogation of iPSC-based micro-physiological systems (‘tissue-chips’) and 3D model systems38 will bring exciting novel opportunities, too. Together, these cell and gene engineering technologies will transform modern day drug discovery and shed also new light on designing successful clinical trials.
Conclusion
Author Manuscript
The discovery of iPSCs and the work of the last decade have moved this remarkable technology closer to drug screening and clinical applications. Improving the robustness of differentiation protocols and implementing SOPs for scale-up and standardized assays will firmly establish the iPSC technology across many different translational communities. Such an integrated precision medicine strategy that is genetics-based, cell type-specific, and clinical data-informed, will enable to interrogate and treat human diseases following the principles of evidence-based medicine. Novel therapeutics based on truly novel disease mechanisms should eventually return on investment and significantly increase the efficiency of drug discovery for common and rare diseases.
Supplementary Material Refer to Web version on PubMed Central for supplementary material.
Acknowledgments We are grateful to Pınar Ormanoğlu and Steven Titus for excellent ongoing support. We thank all our colleagues at NCATS and the NIH Common Fund (Regenerative Medicine Program) for their collaboration and commitment to help patients.
References Author Manuscript
1. Takahashi K, Yamanaka S. Induction of pluripotent stem cells from mouse embryonic and adult fibroblast cultures by defined factors. Cell. 2006; 126(4):663–76. [PubMed: 16904174] 2. Takahashi K, Tanabe K, Ohnuki M, Narita M, Ichisaka T, Tomoda K, Yamanaka S. Induction of pluripotent stem cells from adult human fibroblasts by defined factors. Cell. 2007; 131(5):861–72. [PubMed: 18035408] 3. Aoi T, Yae K, Nakagawa M, Ichisaka T, Okita K, Takahashi K, Chiba T, Yamanaka S. Generation of pluripotent stem cell from adult mouse liver and stomach cells. Science. 2008; 321(5889):699–702. [PubMed: 18276851]
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4. Okita K, Nakagawa M, Hyenjong H, Ichisaka T, Yamanaka S. Generation of mouse induced pluripotent stem cells without viral vectors. Science. 2008; 322(5903):949–53. [PubMed: 18845712] 5. Wilmut I, Leslie S, Martin NG, Peschanski M, Rao M, et al. Development of a global network of induced pluripotent stem cell haplobanks. Regen Med. 2015; 10(3):235–8. [PubMed: 25933231] 6. Choi J, Lee S, Mallard W, Clement K, Tagliazucchi GM, Lim H, Choi IY, Ferrari F, Tsankov AM, Pop R, Lee G, Rinn JL, Meissner A, Park PJ, Hochedlinger. A comparision of genetically matched cell lines reveals the equivalence of human iPSCs and ESCs. Nat Biotechnol. 2015; 33(11):1173– 81. [PubMed: 26501951] 7. Müller FJ, Laurent LC, Kostka D, Ulitsky I, Williams R, Lu C, Park IH, Rao MS, Shamir R, Schwartz PH, Schmidt NO, Loring JF. Regulatory networks define phenotypic classes for human stem cell lines. Nature. 2008; 455(7211):401–5. [PubMed: 18724358] 8. Tsankov AM, Akopian V, Pop R, Chetty S, Gifford CA, Daheron L, Tsankova NM, Meissner A. A qPCR ScoreCard quantifies the differentiation potential of human pluripotent stem cells. Nat Biotechnol. 2015; 33(11):1182–92. [PubMed: 26501952] 9. Thomson JA, Itskovitz-Eldor J, Shapiro SS, Waknitz MA, Swiergiel JJ, Marshall VS, Jones JM. Science. 1998; 282(5391):1145–7. [PubMed: 9804556] 10. Reubinoff BE, Itsykson P, Turestsky T, Pera MF, Reinhartz E, Itzik A, Ben-Hur T. Neural progenitors from human embryonic stem cells. Nat Biotechnol. 2001; 19(2):1134–40. [PubMed: 11731782] 11. Zhang SC, Wernig M, Duncan ID, Brüstle O, Thomson JA. In vitro differentiation of transplantable neural precursors from human embryonic stem cells. Nat Biotechnol. 2001; 19(12):1129–33. [PubMed: 11731781] 12. Perrier AL, Tabar V, Barberi T, Rubio ME, Bruses J, Topf N, Harrison NL, Studer L. Derivation of midbrain dopamine neurons from human embryonic stem cells. Proc Natl Acad Sci USA. 2004; 101(34):12543–8. [PubMed: 15310843] 13. Pera MF, Andrade J, Houssami S, Reubinoff B, Trounson A, Stanley EG, Ward-van Oostwaard D, Mummery C. Regulation of human embryonic stem cell differentiation by BMP-2 and its antagonist noggin. J Cell Sci. 2004; 117:1269–80. [PubMed: 14996946] 14. Smith JR, Vallier L, Lupo G, Alexander M, Harris WA, Pedersen RA. Inhibition of Activin/Nodal signaling promotes specification of human embryonic stem cells into neuroectoderm. Dev Biol. 2008; 313(1):107–17. [PubMed: 18022151] 15. Chambers SM, Fasano CA, Papapetrou EP, Tomishima M, Sadelain M, Studer L. Highly efficient neural conversion of human ES and iPS cells by dual inhibition of SMAD signaling. Nat Biotechnol. 2009; 27(3):275–80. [PubMed: 19252484] 16. Liu Y, Beyer A, Aebersold R. On the dependency of cellular protein levels on mRNA abundance. Cell. 2016; 165(3):534–50. 17. Thomas RJ, Anderson D, Chandra A, Smith NM, Young LE, Williams D, Denning C. Automated, scalable culture of human embryonic stem cells in feeder-free conditions. Biotechnol Bioeng. 102(6):1636–44. 18. Chen G, Gulbranson DR, Hou Z, Bolin JM, Ruotti V, Probasco MD, Smuga-Otto K, Howden SE, Diol NR, Propson NE, Wagner R, Lee GO, Antosiewicz-Bourget J, Teng JM, Thomson JA. Chemically defined conditions for human iPSC derivation and culture. Nat Methods. 2011; 8(5): 424–9. [PubMed: 21478862] 19. Pangalos MN, Schechter LE, Hurko O. Drug development for CNS disorders: strategies for balancing risk and reducing attrition. Nat Rev Drug Discov. 2007; 6(7):521–32. [PubMed: 17599084] 20. Scannell JW, Blanckley A, Boldon H, Warrington B. Diagnosing the decline in pharmaceutical R&D efficiency. Nat Rev Drug Discov. 2012; 11(3):191–200. [PubMed: 22378269] 21. Tobinick EL. The value of drug repositioning in the current pharmaceutical market. Drug News Perspect. 2009; 22(2):119–25. [PubMed: 19330170] 22. Nestler EJ, Hyman SE. Animal models of neuropsychiatric disorders. Nat Neurosci. 2010; 13(10): 1161–9. [PubMed: 20877280]
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23. van der Worp HB, Howells DW, Sena ES, Porritt MJ, Rewell S, O’Collins V, Macleod MR. Can animal models of disease reliably inform human studies? PLoS Med. 2010; 7(3):e1000245. [PubMed: 20361020] 24. Seok J, Warren HS, Cuenca AG, Mindrinos MN, Baker HV, et al. Genomic responses in mouse models poorly mimic human inflammatory diseases. Proc Natl Acad Sci USA. 2013; 110(9):3507– 12. [PubMed: 23401516] 25. O’Connor MD. The 3R principle: advancing clinical application of human pluripotent stem cells. Stem Cell Res Ther. 2013; 4(2):21. [PubMed: 23510719] 26. Huang R, Southall N, Wang Y, Yasgar A, Shinn P, Jadhav A, Nguyen DT, Austin CP. The NCGC pharmaceutical collection: a comprehensive resource of clinically approved drugs enabling repurposing and chemical genomics. Sci Transl Med. 2011; 3(80):80ps16. 27. Lee JA, Shinn P, Jaken S, Oliver S, Willard FS, et al. Novel Phenotypic outcomes identified for a public collection of approved drugs from a publicly accessible panel of assays. PLoS One. 2015; 10(7):e)130796. 28. Swinney DC, Anthony J. How were new medicines discovered? Nat. Rev Drug Discov. 2011; 10(7):507–19. 29. Makhortova NR, Hayhurst M, Cerqueira A, Sinor-Anderson AD, Zhao WN, et al. A screen for regulators of survival of motor neuron protein levels. Nat Chem Biol. 2011; 7(8):544–52. [PubMed: 21685895] 30. Lee G, Ramirez CN, Kim H, Zeltner N, Liu B, et al. Large-scale screening using familial dysautonomia induced pluripotent stem cells identifies compounds that rescue IKBKAP expression. Nat Biotechnol. 2012; 30(12):1244–8. [PubMed: 23159879] 31. Choi SM, Kim Y, Shim JS, Park JT, Wang RH, et al. Efficient drug screening and gene correction for treating liver disease using patient-specific stem cells. Hepatology. 2013; 57(6):2458–68. [PubMed: 23325555] 32. Charbord J, Poydenot P, Bonnefond C, Feyeux M, Casagrande F, et al. High throughput screening for inhibitors of REST in neural derivatives of human embryonic stem cells reveals a chemical compound that promotes expression of neuronal genes. Stem Cells. 2013; 31(9):1816–28. [PubMed: 23712629] 33. Grskovic M, Javaherian A, Strulovici B, Daley GQ. Induced pluripotent stem cells—opportunties for disease modeling and drug discovery. Nat Rev Drug Discov. 2011; 10(12):915–29. [PubMed: 22076509] 34. Matsa E, Burridge PW, Wu JC. Human stem cells for modeling heart disease and for drug discovery. Sci Transl Med. 2014; 6(239):239ps6. 35. Kimbrel EA, Lanza R. Current status of pluripotent stem cells: moving the first therapies to the clinic. Nat Rev Drug Discov. 2015; 14(10):681–92. [PubMed: 26391880] 36. Plenge RM, Scolnick EM, Altshuler D. Validating therapeutic targets through human genetics. Nat Rev Drug Discov. 2013; 12(8):581–94. [PubMed: 23868113] 37. Schizophrenia Working Group of the Psychiatric Genomics Consortium. Biological insights from 108 schizophrenia-associated genetic loci. Nature. 2014; 511(7510):421–7. [PubMed: 25056061] 38. Schwartz MP, Hou Z, Propson NE, Zhang J, Engstrom CJ, et al. Human pluripotent stem cellderived neural constructs for predicting neural toxicity. Proc Natl Acad Sci USA. 2015; 112(40): 12516–21. [PubMed: 26392547]
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Figure 1.
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Reprogramming of somatic cells into iPSCs by transient expression of defined transcription factors (OCT4, SOX2, KLF4, C-MYC) is a robust and highly reproducible procedure. However, efficient and controlled multistep differentiation of iPSCs into transient phenotypes and mature functional cells and precise characterization of these cell type identities across developmental states are currently among the greatest challenges in stem cell biology.
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Figure 2.
The CompacT SelecT automated cell culture platform allows standardization and scale-up of iPSCs and their differentiated progeny. Some key features and components of the system are highlighted with arrows. HTS, High-throughput screening.
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Author Manuscript Author Manuscript Figure 3.
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Summary of the many biomedical opportunities enabled by the iPSC technology (“patient in a dish”). For instance, after generating specific neuronal subtypes representative of various neurotransmitter systems (e.g. glutamatergic, GABAergic, dopaminergic), these cells can be investigated as pure and mixed cultures and subjected to various manipulations (e.g. polypharmacology) for new target identification (ID), predictive toxicology, and other purposes.
Author Manuscript Drug Target Rev. Author manuscript; available in PMC 2016 October 21.
Drug Target Rev. Author manuscript; available in PMC 2016 October 21. Published in final edited form as: Drug Target Rev. 2016 ; 3(2): 34–38.
Translating Stem Cell Biology Into Drug Discovery Ilyas Singeç and Anton Simeonov National Institutes of Health (NIH). National Center for Advancing Translational Sciences (NCATS). Division of Pre-Clinical Innovation (DPI)
Abstract Author Manuscript
Pluripotent stem cell research has made extraordinary progress over the last decade. The robustness of nuclear reprogramming of somatic cells into induced pluripotent stem cells (iPSCs) has created entirely novel opportunities for drug discovery and personalized regenerative medicine. Patient- and disease-specific iPSCs can be expanded indefinitely and differentiated into relevant cell types of different organ systems. As the utilization of iPSCs is becoming a key enabling technology across various scientific disciplines, there are still important challenges that need to be addressed. Here we review the current state and reflect on the issues that the stem cell and translational communities are facing in bringing iPSCs closer to clinical application.
Quality Assurance of iPSCs
Author Manuscript Author Manuscript
Pluripotent stem cells are characterized by extensive self-renewal under appropriate cell culture conditions and differentiation into all cell types of the human body. The groundbreaking discovery by Shinya Yamanaka and colleagues1,2 over a decade ago that somatic cells can be reprogrammed into embryonic-like cells by defined factors has reinvigorated biomedical research in general and human biology-oriented drug discovery in particular. After quickly establishing that somatic cells - independent of tissue of origin, age, and reprogramming technique - can be robustly reverted back and stably maintained at a pluripotent state3,4, the field has moved on to generating new iPSC lines from patients with monogenic and complex genetic diseases as well as those of unknown etiology. Studying relevant cell types in a dish in order to better understand the molecular underpinnings of intractable human diseases holds a great promise to develop novel mechanism-based concepts in drug screening. To enable such disease modeling, predictive toxicology, and regenerative cell therapy applications, major international efforts and public-private partnerships are underway to generate large iPSC banks considering ethnic and diseasespecific backgrounds5. While many new iPSC lines are being generated, important questions remain to be answered in order to ensure acceptable standardization, comprehensive characterization, rigorous quality control, and safety. Significant challenges remain to be addressed in order to overcome lack of experimental reproducibility, uncontrolled differentiation protocols, genome instability of reprogrammed and differentiated cells, and transplantation of unwanted cells with potential teratoma formation risk. Recent work has demonstrated the equivalence of genetically matched iPSCs and embryonic stem cell lines (ESCs)6. This is an important comparison, since ESCs are still considered the gold standard for bona fide pluripotency. In addition, this study highlighted that genetic
Singeç and Simeonov
Page 2
Author Manuscript Author Manuscript
background can be a major confounding factor for comparative studies that rely solely on genetic and epigenetic signatures, and also underscore the importance of being careful when drawing conclusions based on comparing only a few iPSC lines, which is currently the main approach in the field. Moreover, further systematic studies are necessary to establish criteria that can reliably distinguish “high-quality” iPSCs from “poor-quality” iPSCs. At present, the most widely used parameters to characterize iPSCs include morphology, karyotype analysis, immunophenotyping (e.g. surface makers, transcription factors), multilineage differentiation (i.e. ectoderm, mesoderm, endoderm) by generating embryoid bodies, and in vivo teratoma formation. Other approaches suggested as a measure of pluripotency include bioinformatics analysis based on gene expression7,8. However, it still debated if these approaches are acceptable to completely replace teratoma formation. Working with iPSCs is labor-intensive and costly and identifying “high-quality” iPSCs is a critical goal, which should be prioritized as early as possible in the drug discovery process. Nevertheless, it remains unclear if the currently available assays and protocols are sufficient and rigorous enough for clinical and translational purposes.
Reproducible Differentiation Protocols
Author Manuscript Author Manuscript
The remarkable biological versatility of pluripotent stem cells is based on their developmental potential to give rise to all somatic cell types, while not undergoing cellular senescence in the pluripotent state. Attempts to harness this potential of unlimited cell growth and differentiation started after the successful derivation of the first human ESC lines9. Several approaches have been used over the years to differentiate pluripotent cells into desired lineages and cell types, including overgrowing cells and culturing them as neurospheres10, forming free-floating embryoid bodies and picking neural rosettes11, coculture with stromal cells12, using recombinant proteins (e.g. Noggin)13, small molecule inhibitors such as SB431542 alone14 or in combination with recombinant Noggin15. While over the years there has been a trend to move from empirical methods to directed differentiation strategies by targeting specific cell signaling pathways, there are still major knowledge gaps and biological unknowns that hinder the formulation of highly reproducible differentiation protocols to yield large numbers of mature cell types at high purity (Figure 1). How can the stem cell field overcome the challenge of heterogeneous cultures with immature cell types generated over extended periods of time (weeks to months) and exposed to a flurry of small molecules and recombinant proteins, often used at supraphysiological concentrations? How can we comprehensively characterize cell type identities across different developmental stages? How can we identify and manipulate relevant cell signaling pathways and new small molecules that target these pathways? Bringing solutions to these questions is central to firmly establishing the iPSC technology in drug discovery and clinical applications. To tackle these widely accepted challenges, the National Institutes of Health (NIH), as part of its Regenerative Medicine Program (RMP), has recently launched the Stem Cell Translation Laboratory (SCTL). The SCTL is located within the National Center for Advancing Translational Sciences (NCATS) and is dedicated to bring the iPSC technology closer to drug discovery and regenerative medicine applications. (https:// commonfund.nih.gov/stemcells/index).
Drug Target Rev. Author manuscript; available in PMC 2016 October 21.
Singeç and Simeonov
Page 3
Author Manuscript
Automation and Industrialized Scale-Up of iPSC Cultures Precisely formulating robust differentiation protocols requires deeper and actionable insights into the basic biology of pluripotent cells. The resulting information and reference datasets should then allow to better control cell fate choice and terminal differentiation. To this end, it is critical to perform dose-response experiments, elucidate signal strengths, and carefully document the net effect of combinatorial manipulations of relevant pathways. The importance of combining genomic and proteomic methods is underscored by the fact that mRNA abundance often does not predict protein expression levels, particularly during dynamic transitions16. Integration and analysis of such complex orthogonal datasets by systems biology methods should enable establishing a roadmap that is practical and costefficient enough to be used across many different laboratories and translational research settings.
Author Manuscript Author Manuscript
Routine manual cell culture work is inherently influenced by the physico-chemical environment as well as human bias. Typically, scientists working in cell culture have their own style and preference in performing daily tasks. Variations in carrying out specific tasks such as adding fresh medium at varying time points, cell passaging at varying cell densities, and other more or less significant or subtle effects can all have a strong impact on the experimental outcome and the quality of the end product. Standardizing routine cell culture and the manufacturing process in cell engineering are critical steps, with automated robotic cell culture systems providing unique opportunities to establish standard operating procedures (SOPs), reduce human bias, and control cell culture conditions more precisely. Using the robotic CompacT SelecT system17 combined with chemically defined medium18, we have started to establish protocols that can maintain and expand pluripotent stem cells under highly controlled feeder-free conditions (Figure 2). The fully automated workflow allows controlling each step via computer-aided commands. This real-time system ensures that the scientist can conveniently control and monitor cell culture around-the-clock. For instance, daily media change can be performed consistently at exact times. Any possible technical error is immediately transmitted to the scientist in charge. Integrated cell counter, viability assessment, and image-based passaging are key features for standardization and documentation. In summary, automation provides the following advantages, which will help to implement standardized utilization of iPSCs for translational research:
Author Manuscript
•
Expansion and maintenance of multiple cell lines
•
Cell counting and viability measurement
•
Sub-culturing and cell passaging
•
Cell seeding
•
Transient transfection
•
Harvesting at exact time points
•
Reducing the risk of cell culture contamination
•
Incubation of up to 182 T-flasks and up to 420 plates in current configuration
Drug Target Rev. Author manuscript; available in PMC 2016 October 21.
Singeç and Simeonov
Page 4
•
Author Manuscript
Plating into 96, 384 and 1536-well plates suitable for high-throughput and high-content screening
Industrialized scale-up and access to large numbers of properly differentiated cell types will impact future health care by leveraging both personalized medicine and treatment of large patient cohorts.
Transforming Drug Discovery by Human Biology
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The traditional drug discovery process is painstakingly slow and inefficient19–21. The high attrition rate and failure of potential drugs due to safety issues and lack of efficacy in patients detected only after many years of investment suggest to critically re-visit the usefulness of pre-clinical models that have been relied upon in the past. Indeed, overdependence on animal models and immortalized cell lines in the pre-clinical stage is very risky and often times of limited predictive value for the outcome in the clinical phase22–25. More than 90% of drugs developed based on animal models fail in clinical trials23. To address at least some of the obstacles, drug repurposing has emerged as a valuable strategy. The underlying concept, which is to find new indications for existing drugs, can avoid redundant and costly pre-clinical studies and Phase 1 safety trials21. Along these lines, the National Center for Advancing Translational Sciences (NCATS) has compiled the largest public repository of approved and clinical phase drugs, the NCATS Pharmaceutical Collection (NPC), aimed at broadly sharing this resource for drug repositioning, toxicology studies, and chemical genomic profiling26,27..
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The two main strategies of small molecule drug discovery can be classified as either targetbased or phenotypic28. While target-based approaches aim to modulate a specific molecular target of interest, the phenotypic approach is intended to modify a disease-associated phenotype in cells or whole organisms. Comparing the success rate of both approaches has led to a renaissance of phenotypic screens28. However, setting up informative phenotypic screens requires a renewable cell source and validation of cellular assays that faithfully recapitulate human physiology. Patient- and disease-specific iPSCs are uniquely suited for developing novel human biology-focused paradigms for drug discovery and first-in-class therapies. Once the challenges discussed above are sufficiently addressed (i.e. quality assurance, reproducible and scalable differentiation), iPSC-based models will be broadly implemented in all stages of pre-clinical drug development. For instance, high-throughput and high-content screening, hit validation, structure-activity relationship studies, and toxicology assays will directly benefit from the consistency of utilizing the same cell types(s) derived from genotyped patients with known clinical history. A few studies started to employ pluripotent cells for drug screening29–32. As we learn to robustly differentiate and scale up iPSC-derived cells, it is apparent that more systematic and extensive screening studies using much larger compound libraries will follow in the near future. Beyond using human cells in screening, careful analysis of iPSC-based cellular assays will also provide insights into the complexity of human disease and leverage the understanding of genotype-phenotype relations and pinpoint novel cellular and molecular disease mechanisms33–36. Elucidating genetic diseases, particularly those with underlying strong
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cell-autonomous effects, may indeed enable robust and streamlined identification and validation of new ‘druggable’ targets, signaling pathways, and disease phenotypes. In a different approach, the wealth of genetic data obtained by genome-wide association studies (GWAS) can be interrogated by probing disease-relevant cell types. Follow-up experiments and prioritization of hundreds of potential disease targets based on GWAS data is a significant problem. This is further complicated by the fact that the majority of hits are found in regulatory elements of unknown significance and not in protein-coding regions37. It is again desirable that these targets be directly investigated in relevant human cells from affected patients under defined laboratory conditions. The versatility of the iPSC technology (Figure 3) can be further enhanced by the power of functional genomics (e.g. RNAi) and genome editing such as the CRISPR/Cas9 system, which allows precise site-specific genetic manipulations. The interrogation of iPSC-based micro-physiological systems (‘tissue-chips’) and 3D model systems38 will bring exciting novel opportunities, too. Together, these cell and gene engineering technologies will transform modern day drug discovery and shed also new light on designing successful clinical trials.
Conclusion
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The discovery of iPSCs and the work of the last decade have moved this remarkable technology closer to drug screening and clinical applications. Improving the robustness of differentiation protocols and implementing SOPs for scale-up and standardized assays will firmly establish the iPSC technology across many different translational communities. Such an integrated precision medicine strategy that is genetics-based, cell type-specific, and clinical data-informed, will enable to interrogate and treat human diseases following the principles of evidence-based medicine. Novel therapeutics based on truly novel disease mechanisms should eventually return on investment and significantly increase the efficiency of drug discovery for common and rare diseases.
Supplementary Material Refer to Web version on PubMed Central for supplementary material.
Acknowledgments We are grateful to Pınar Ormanoğlu and Steven Titus for excellent ongoing support. We thank all our colleagues at NCATS and the NIH Common Fund (Regenerative Medicine Program) for their collaboration and commitment to help patients.
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Figure 1.
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Reprogramming of somatic cells into iPSCs by transient expression of defined transcription factors (OCT4, SOX2, KLF4, C-MYC) is a robust and highly reproducible procedure. However, efficient and controlled multistep differentiation of iPSCs into transient phenotypes and mature functional cells and precise characterization of these cell type identities across developmental states are currently among the greatest challenges in stem cell biology.
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Figure 2.
The CompacT SelecT automated cell culture platform allows standardization and scale-up of iPSCs and their differentiated progeny. Some key features and components of the system are highlighted with arrows. HTS, High-throughput screening.
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Summary of the many biomedical opportunities enabled by the iPSC technology (“patient in a dish”). For instance, after generating specific neuronal subtypes representative of various neurotransmitter systems (e.g. glutamatergic, GABAergic, dopaminergic), these cells can be investigated as pure and mixed cultures and subjected to various manipulations (e.g. polypharmacology) for new target identification (ID), predictive toxicology, and other purposes.
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