Pharmacological Reprogramming of Somatic Cells for Regenerative

Jan 8, 2017 - pluripotent stem cells (iPSCs), which can replicate indefinitely and give rise to any somatic cell type. This reprogramming can be achie...
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Pharmacological Reprogramming of Somatic Cells for Regenerative Medicine Min Xie,† Shibing Tang,† Ke Li, and Sheng Ding* Gladstone Institutes, 1650 Owens Street, San Francisco, California 94158, United States CONSPECTUS: Lost or damaged cells in tissues and organs can be replaced by transplanting therapeutically competent cells. This approach requires methods that effectively manipulate cellular identities and properties to generate sufficient numbers of desired cell types for transplantation. These cells can be generated by reprogramming readily available somatic cells, such as fibroblasts, into induced pluripotent stem cells (iPSCs), which can replicate indefinitely and give rise to any somatic cell type. This reprogramming can be achieved with genetic methods, such as forced expression of pluripotency-inducing transcription factors (TFs), which can be further improved, or even avoided, with pharmacological agents. We screened chemical libraries for such agents and identified small molecules that enhance TF-mediated pluripotency induction in somatic cells. We also developed cocktails of small molecules that can functionally replace combinations of TFs required to induce pluripotency in mouse and human somatic cells. Importantly, we devised and established a general strategy to develop effective pharmacological cocktails for specific cellular reprogramming processes. In the search for useful small molecules, we also discovered and characterized previously unknown mechanisms pertinent to cellular reprogramming. A more direct method to access scarce cells for cell transplantation is transdifferentiation, which uses combinations of cell-type specific TFs to reprogram fibroblasts into the target somatic cell types across lineage boundaries. We created an alternative strategy for cellular transdifferentiation that epigenetically activates somatic cells by pairing temporal treatment with reprogramming molecules and tissue-specific signaling molecules to generate cells of multiple lineages. Using this cell-activation and signaling-directed (CASD) transdifferentiation paradigm, we converted fibroblasts into a variety of somatic cells found in major organs, such as the heart, brain, pancreas, and liver. Specifically, we induced, isolated, and expanded (long-term) lineagespecific progenitor cells that can give rise to a defined range of cell types relevant to specific tissues or organs. Transplanting these progenitor cells or their progeny was therapeutically beneficial in animal models of diseases and organ damage. Importantly, we developed chemically defined conditions, without any genetic factors, that convert fibroblasts into cells of the cardiac and neural lineages, further extending the realm of pharmacological reprogramming of cells. Continuously advancing technologies in pharmacological reprogramming of cells may benefit and advance regenerative medicine. The established pharmacological tools have already been applied to enhance the processes of cellular reprogramming and improve the quality of cells for their clinical applications. The rapidly increasing number of readily available bioactive chemical tools will fuel our efforts to reprogram cells for transplantation therapies.

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they can replicate indefinitely without losing their pluripotency.6 Interestingly, fibroblasts and other somatic cells can be reprogrammed into induced PSCs (iPSCs) (Figure 1).7 Under certain conditions, iPSCs can be guided to become desired somatic cell types through directed differentiation (Figure 1), so they could produce unlimited quantities of therapeutic cells for cell transplantation.8 Additionally, with transdifferentiation, one type of somatic cell can be directly reprogrammed into another type without first going through a pluripotent state (Figure 1).9

REGENERATIVE MEDICINE AND CELLULAR REPROGRAMMING When relevant cells in tissues or organs are severely lost or defective, body parts fail to work properly. With regenerative medicine, such as cell transplantation,1 we might replenish these lost cells, grow new tissues, and repair damaged organs.2 These therapies often require engineering cellular functions and identities. Unfortunately, committed somatic cells in mammals cannot change their acquired identities, and they are restricted to a particular cell fate.3 Nonetheless, through reprogramming,4 we can overcome the barriers between cellular hierarchies and lineage boundaries to precisely manipulate somatic cell fates for regenerative medicine (Figure 1). While specialized somatic cells, such as fibroblasts, can only maintain their state and have limited propagation potential, stem cells can extensively self-renew and naturally develop into other cell types. Pluripotent stem cells (PSCs), such as those derived from early embryos,5 can develop into any somatic cell type, and © 2017 American Chemical Society

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CELLULAR REPROGRAMMING MEDIATED BY TRANSCRIPTION FACTORS Cellular reprogramming involves profound changes in cellular fate via gene transcription, a process that produces all RNAs. In Received: January 8, 2017 Published: April 28, 2017 1202

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advantages and wide applicability of pharmacological agents in the manipulation of cell fates.20 In particular, the pharmacological approach for cellular reprogramming can complement, reduce, or even avoid the use of pluripotency-inducing TFs.21 In the following sections, we will highlight how we applied this approach to reprogram cells for therapeutic transplantation. Pharmacological agents can modulate gene expression through innate cellular mechanisms.20 For example, gene transcription is regulated by epigenetic mechanisms that involve chemical modifications of chromatin, such as DNA methylation, histone methylation, and acetylation.22 Small molecules that modulate the activities of enzymes23 responsible for these modifications can help to remodel chromatin structure and poise it for the transcription of genes needed for cellular reprogramming. Interestingly, small-molecule epigenetic modulators can promote reprogramming of somatic cells toward pluripotency. For example, BIX-01294, an inhibitor of histone methyltransferase G9a, can induce the generation of iPSCs from mouse neural progenitor cells ectopically expressing the three TFs Sox2, Klf4, and c-Myc.24 Combining RG108 (Table 1), an inhibitor of DNA methyltransferase, with BIX-01294 (Table 1) can induce pluripotency in mouse embryonic fibroblasts (MEFs) ectopically expressing only the two TFs Oct4 and Klf4.25 Pharmacological agents can also modulate the expression of genes through cell signaling pathways. Signaling proteins, such as growth factors, can interact with receptor proteins on the cell surface to trigger cascades of biochemical events inside the cell, transducing external signals into the cell to change gene expression.26 Small molecules can also interact with the biochemical components of signaling pathways to either enhance or suppress signaling events and consequently affect gene expression.20 With small-molecule modulators, we can alter signaling pathways to facilitate cellular reprogramming toward pluripotency. For example, dual inhibition of the TGFβ (by SB431542, Table 1) and MAPK/ERK (by PD0325901, Table 1) pathways increased the efficiency of pluripotency induction by promoting mesenchymal-to-epithelial transition.27 Additionally, CHIR99021 (Table 1), an inhibitor of glycogen synthase kinase 3 (GSK3), enhanced Wnt signaling and induced pluripotency in MEFs transduced with Oct4 and Klf4.28 We also used small molecules to combinatorially modulate epigenetic and signaling mechanisms to compensate for the absence of most TFs during pluripotency induction. We combined A83-01 and AMI-5 (Table 1), inhibitors of the TGFβ pathway and protein arginine methylation and histone lysine methylation, respectively, to enable the Oct4-mediated generation of iPSCs from MEFs.29 We also refined a chemical cocktail that contains functionally diverse small molecules that induce pluripotency in human somatic cells.30 In this study, dual inhibition of TGFβ (by A83-01) and MAPK/ERK (by PD0325901) induced pluripotency mediated by Oct4 and Klf4 in neonatal human epidermal keratinocytes, which was further improved by adding PS48 (Table 1) and sodium butyrate (NaB)31 (an inhibitor of histone deacetylase, Table 1) to enable the Oct4-mediated pluripotency induction in these human somatic cells. Adding CHIR99021 and Parnate28 [an inhibitor of lysine-specific demethylase 1 (LSD1); Table 1] to this cocktail further improved its ability to reprogram adult human epidermal keratinocytes, which are difficult to reprogram. An optimized protocol using these six small molecules effectively generated iPSCs from adult human epidermal keratinocytes with forced expression of only Oct4 (Scheme 1).

Figure 1. Cellular reprogramming for cell transplantation in regenerative medicine.

cells, gene transcription is primarily regulated by transcription factors (TFs) that target genes by recognizing and binding to particular regions of DNA.10 These TFs interact with DNA, RNA, and proteins to modify chromatin structure, recruit and organize components of the transcriptional machinery, and either activate or suppress the transcription of their target genes. Endogenous TFs cooperatively maintain gene regulatory networks (GRNs)11 that confer identities onto cells by activating and suppressing the transcription of genes crucial to distinct cell types.12 Cell type-specific TFs can be delivered into target cells and forcefully expressed13 to rewire GRNs in cells, reprogram cell fates, and switch cellular identities. This overwhelming power of exogenous TFs on cellular identities was first demonstrated in a study that transdifferentiated fibroblasts into skeletal muscle cells, in which the fibroblasts were transfected with the complementary DNA of Myod, a myogenic TF.14 In 2006, the Yamanaka lab generated iPSCs by virally transducing four pluripotency-inducing TFs, Oct4, Sox2, Klf4, and c-Myc (OSKM), into mouse fibroblasts.15 This groundbreaking work demonstrated the feasibility of manipulating cell fates with exogenously delivered TFs. Similar methods were soon developed to supply less controversial, autologous human iPSCs16,17 for clinical development,18 disease modeling, and drug discovery.19

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PHARMACOLOGICAL REPROGRAMMING OF SOMATIC CELLS TOWARD PLURIPOTENCY Despite the initial success of TF-based reprogramming methods, the process of pluripotency induction is slow and the efficiency of iPSC generation is generally low. Many efforts have worked to enhance these processes, including the use of pharmacological agents, such as small molecules and growth factors. These agents can be conveniently incorporated into culture conditions for cells, and their effects can be temporally and dose-dependently controlled. Also, diverse pharmacological agents with defined effects are readily available and serve as a large pool of candidates for selection and tests. Extensive studies have demonstrated the 1203

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Accounts of Chemical Research Table 1. Representative Chemicals and Pharmacological Agents Used in Cellular Reprogramming (Alphabetically)

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Accounts of Chemical Research Table 1. continued

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Scheme 1. Oct4-Mediated, Small Molecule-Assisted Pluripotency Induction in Adult Human Epidermal Keratinocytes

Figure 2. A general strategy to screen pharmacological agents and optimize a pharmacological cocktail for cellular reprogramming.

Figure 3. Cell-activation and signaling-directed (CASD) transdifferention vs pluripotency induction and directed differentiation.

the pharmacological cocktail to improve it synergistically. The resulting cocktail can be optimized iteratively to achieve the desired outcome of cellular reprogramming. In this study, we also used PS48 to uncover a fundamental mechanism underlying reprogramming: PS48 facilitates a metabolic switch from mitochondrial oxidation in somatic cells to glycolysis in PSCs.30 With subsequent chemical screening, we identified autophagy-inducing small molecules (such as SMER28, Table 1) that increase the efficiency of pluripotency induction. We also characterized an ATG5-independent, but Ulk1/Rab9-dependent, noncanonical autophagy pathway that mediates mitochondrial clearance and metabolic reprogramming

This study illustrates a general strategy that we have adopted to develop effective pharmacological cocktails for specific reprogramming processes. Such a strategy involves constructing an initial pharmacological cocktail, screening new candidate agents, and incorporating the beneficial agents into the cocktail (Figure 2). We normally construct the initial cocktail from pharmacological agents known to facilitate the desired cellular reprogramming. We then employ this cocktail to the reprogramming process (either TF-driven or TF-free) that needs to be enhanced. In the screening of new candidate agents, we add individual agent and observe for improvement of the reprogramming process. Having identified the beneficial agents, we incorporate them into 1206

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Scheme 2. Generation of Therapeutic Cardiac Progenitor Cells via Cell-Activation and Signaling-Directed Transdifferentiation, Their Expansion, and Cardiac-Specific Differentiation

to glycolysis.32 These studies demonstrated that screening of small molecules can help to reveal new tool compounds and previously unknown mechanisms.

progenitor cells through CASD transdifferentiation, but also to maintain, specify, and mature them. Zhang et al. developed conditions to generate cardiac progenitor cells (CPCs).40 These CPCs could expand in vitro and provide larger numbers of cells to heal an infarcted heart, likely due to their ability to give rise to cardiomyocytes, endothelial cells (ECs), and smooth muscle cells (SMCs) in vivo. To harness the therapeutic potential of CPCs, we developed a proper pharmacological condition to capture and maintain this cell population (Scheme 2). The established transdifferentiation process involves brief overexpression of OSKM, followed by 2 days of treatment with the Wnt signaling enhancer CHIR99021 to direct MEFs toward the cardiac lineage (Scheme 2). We also added JAK Inhibitor I (JI1) (Table 1) to this process to guard against the generation of iPSCs (Scheme 2).37 To further drive cardiac transdifferentiation and capture the putative CPCs, we tested and optimized combinations of molecules that modulate signaling pathways pertinent to the cardiac differentiation of PSCs.41 We found that CPCs were effectively induced and enriched by treatment with JI1 and a cocktail containing two growth factors [BMP4 and activin A (ActA)] and two smallmolecule signaling modulators (CHIR99021 and SU5402; Table 1) (Scheme 2) for 2 weeks. This cocktail also repressed the spontaneous differentiation of CPCs and stabilized them for long-term self-renewal. When cultured in medium supplemented with this cocktail, the number of CPCs increased over ten-billionfold through repeated passaging (Scheme 2). These CPCs could differentiate into cardiomyocytes, ECs, and SMCs in vitro, suggesting their proper developmental potential. When transplanted into infarcted hearts of mice, they spontaneously differentiated into the three cardiovascular cell types in vivo, contributed to the growth of new muscle tissues and blood vessels in the damaged regions, and improved heart function. Another group also reprogrammed mouse fibroblasts into expandable CPCs using cardiac lineage-specific genes combined with signaling molecules.42 Cells of the endodermal lineage, such as those found in the pancreas and liver, are highly valuable for transplantation therapy. With pharmacological agents, we generated multiple endodermal cell types through CASD transdifferentiation and differentiated them into therapeutically useful cells. Li et al. efficiently converted MEFs into definitive endodermal cells by transiently expressing OSKM and simultaneously treating with ActA (a growth factor that induces endodermal differentiation of PSCs),43 BIX-01294, lithium chloride (LiCl), and 2-phospho-L-

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CELLULAR TRANSDIFFERENTIATION MEDIATED BY TRANSCRIPTION FACTORS Tissue-specific TFs33 and microRNAs34 have been used to induce transdifferentiation of somatic cells, such as converting human fibroblasts into neurons.35 Alternatively, a common set of reprogramming TFs can induce partial reprogramming and, in conjunction with tissue-specific signaling, can selectively induce multiple cell lineages. Inducing pluripotency requires prolonged, continuous forced expression of OSKM (Figure 3), which provides the opportunity to intercept the process at its early stage.36 Indeed, exogenous OSKM may remodel the epigenome of somatic cells to allow expression of previously suppressed genes required for cell-fate conversion.36 We hypothesized that briefly expressing exogenous OSKM, while insufficient to establish pluripotency in committed somatic cells, could activate cells for an identity switch by remodeling their epigenome. These activated, plastic cells can then develop into specific cell lineages under the direction of signals sent by growth factors and smallmolecule modulators of signaling pathways, processes that partially resemble the directed differentiation of iPSCs (Figure 3). On the basis of this hypothesis, we devised a strategy of cellactivation and signaling-directed (CASD) transdifferentiation that transforms fibroblasts into alternative cell lineages through transient, ectopic expression of OSKM and exposure to growth factors or small-molecule signaling modulators or both (Figure 3). We first demonstrated the versatility of this CASD strategy in transdifferentiating MEFs into cells of the cardiac37 and neural lineages.38

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PROGENITOR CELLS GENERATED THROUGH TRANSDIFFERENTIATION Further characterizations revealed that proliferative, lineagespecific progenitor cells are generated during CASD transdifferentiation. Relying on established findings in the directed differentiation of stem cells and progenitor cells,39 lineagespecific progenitor cells can be either kept undifferentiated and expanded into large quantities or controllably differentiated into specialized cell types for various applications. Pharmacological agents have been used to not only create multiple lineage-specific 1207

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Scheme 3. Generation of Therapeutic Pancreatic Progenitor Cells via Cell-Activation and Signaling-Directed Transdifferentiation and Their Differentiation

Scheme 4. Oct4-Mediated, Small Molecule-Assisted Cell Activation and Signaling-Directed Transdifferentiation of Mouse Embryonic Fibroblasts into Cardiomyocytes

Scheme 5. Pharmacological Generation of Neural Stem Cells and Their Neural-Specific Differentiation

example, human fibroblasts transduced with Oct4 and Klf4 and then cultured in media containing various combinations of growth factors and small molecules converted to endothelial cells.49 Additionally, ectopically expressing Oct4 in human fibroblasts and simultaneously treating them with six small molecules transformed them into neural stem cells (NSCs).50

ascorbic acid trisodium salt (pVc) (Table 1) (Scheme 3).44 We then identified a cocktail containing retinoic acid (RA), A83-01, LDE225 (Table 1), and pVc that can effectively specify these definitive endodermal cells into pancreatic progenitor cells (Scheme 3). When transplanted into type 1 diabetic mice lacking functional pancreatic β cells, these pancreatic progenitor cells gave rise to glucose-responsive, insulin-secreting pancreatic β cells in vivo and ameliorated hyperglycemia. Using optimized combinations of growth factors, small molecules, and TFs, we also induced the CASD transdifferentiation of human fibroblasts into definitive endodermal cells and specified them into functional pancreatic β cells.45 Expandable, endodermal progenitor cells were also generated through CASD transdifferentiation to subsequently afford abundant hepatocytes that can repopulate injured livers in mice and improve their survival.46

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PHARMACOLOGICALLY DRIVEN CELLULAR TRANSDIFFERENTIATION To avoid completely the use of pluripotency-inducing TFs, we further developed CASD transdifferentiation solely driven by pharmacological agents. We used pharmacological agents to successfully transdifferentiate MEFs into expandable NSCs that can give rise to multiple neural cell types (Scheme 5).51 To find combinations of pharmacological agents for effective neural transdifferentiation, we performed iterative screening. We devised a basal medium containing LDN-193189 (Table 1), A83-01, CHIR99021, and the growth factor bFGF, all of which help to induce pluripotency in somatic cells or differentiate PSCs into neural cells. To evaluate the outcome of neural transdifferentiation, we treated MEFs with this basal medium and an additional individual compound for 10 days and then measured the number of cells expressing Sox and Nestin (characteristic of NSCs). Using this screening system, we identified small molecules that promote the generation of target NSCs. We then added these molecules to the original basal medium to improve it. With this improved medium, we then searched for additional agents that promote neural transdifferentiation. We thus identified an effective, neural lineage-inducing cocktail containing nine components (LDN-193189, A83-01,

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PHARMACOLOGICALLY ASSISTED CELLULAR TRANSDIFFERENTIATION Using small molecules that enhance cellular reprogramming and functionally replace TFs in these processes, we eliminated most of the pluripotency-inducing TFs in CASD-cardiac transdifferentiation. Wang et al. found that a brief ectopic expression of a single TF Oct4 followed by treatment with a cocktail containing Parnate, forskolin (Table 1),47 SB431542, and CHIR99021, converted MEFs into the cardiac lineage (Scheme 4).48 These cells further matured into cardiomyocytes with BMP4 (Scheme 4). This process generated cardiomyocytes through cardiac precursor cells instead of iPSCs. We also developed conditions to replace some of the pluripotency-inducing TFs in CASD transdifferentiation. For 1208

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Accounts of Chemical Research Scheme 6. Pharmacological Cardiac Transdifferentiation of Human Foreskin Fibroblasts

CHIR99021, bFGF, Hh-Ag1.5, RA, RG108, Parnate, and SMER28) (Table 1). When cultured in a medium containing these components for 10 days, approximately 30% of MEFs were converted into NSCs, which could be serially expanded and differentiated into three neural cell types, neurons, oligodendrocytes, and astrocytes, both in vitro and in vivo (Scheme 5). Other laboratories also reported pharmacological conditions that induce transdifferentiation of mouse and human fibroblasts into neurons.52,53 We recently developed a chemical cocktail that pharmacologically drives CASD transdifferentiation of human fibroblasts into cardiomyocytes.54 Here, we screened for optimal combinations of small molecules that activate human somatic cells for cardiac transdifferentiation. We sought to improve the basal cocktail of four small molecules (Parnate, forskolin, SB431542, and CHIR99021) that enabled Oct4-mediated cardiac transdifferentiation.48 We treated human foreskin fibroblasts with this basal cocktail and an additional individual compound for 6 days. Then, we cultured the cells in a cardiac induction medium containing CHIR99021, ActA, BMP4, and VEGF for another 5 days, at which point we quantified the expression of cardiac genes in the resulting cells. Compounds that promoted the expression of cardiac genes were incorporated into the cocktail for further screening of other candidates, and nonessential compounds were subsequently removed from the cocktail. Through this process, we identified an optimal combination of nine compounds (9C), A83-01, AS8351, BIX01294, CHIR99021, JNJ10198409, OAC2, SC1, SU16F, and Y27632 (Table 1), that enabled the cardiac transdifferentiation of fibroblasts. Treatment with 9C followed by culturing in the cardiac induction medium robustly converted human fibroblasts into cardiac mesoderm cells, which can further differentiate into cardiomyocytes in vitro (Scheme 6). When transplanted into infarcted hearts of mice, these cardiac mesoderm cells gave rise to cardiomyocytes in vivo and partially repaired damaged cardiac muscle. Our study demonstrated the feasibility of pharmacologically driven cardiac transdifferentiation and revealed mechanisms underlying this process. In the nuclei of fibroblasts treated with 9C, we observed a more open chromatin landscape, especially for cardiogenic genes at a later reprogramming time point. These observations indicate that 9C remodels the epigenome of the starting fibroblasts and renders the cells susceptible to cardiogenic signals. These findings suggest that we could develop a common set of small molecules that act in the early step of CASD transdifferentiation to convert fibroblasts into multiple cell lineages.

The expanding pharmacological toolbox will continue to improve cellular reprogramming for clinical applications. For example, pharmacological agents have already been widely employed in chemically defined culture media for the derivation and maintenance of human iPSCs to ensure their high quality.36 Cellular transdifferentiation, on the other hand, may offer unique therapeutic advantages by avoiding the involvement of tumorigenic PSCs.57 With the transgenic and pharmacological tools that generate and direct the differentiation of iPSCs, we used CASD transdifferentiation to generate a wide variety of therapeutically valuable cells, a paradigm that will likely continue to inspire creative approaches to cellular transdifferentiation. Small molecules that facilitate cellular reprogramming may translate into pharmaceutical drugs that regenerate cells, tissues, or organs in situ.58 This approach will benefit from targeted delivery and controlled release of drugs that rely heavily on chemical approaches.59 Thus, with new discoveries by the chemistry community, we expect that the field of cellular reprogramming will rapidly advance and benefit efforts that support regenerative medicine.

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AUTHOR INFORMATION

Corresponding Author

*Phone: (415) 734-2717. Fax: (415) 355-0141. E-mail: sheng. [email protected]. ORCID

Sheng Ding: 0000-0002-3354-1263 Author Contributions †

M.X. and S.T. contributed equally to this Account.

Notes

The authors declare no competing financial interest. Biographies Min Xie received a B.S. in Chemistry from Peking University in 2003 and a Ph.D. in Organic Chemistry from the University of Illinois at Urbana−Champaign in 2010. He is now a postdoctoral fellow at the Gladstone Institutes. Xie is participating in the discovery and preclinical development of small-molecule drugs for the treatment of cancers, neurodegenerative diseases, and metabolic diseases. Shibing Tang received a B.S. in Chemistry from Jilin University in 2006 and a Ph.D. in Organic Chemistry from Lanzhou University in 2011. Tang is now a postdoctoral fellow at the Gladstone Institutes under the supervision of Sheng Ding. His research focuses on discovering small molecules that control cell fate and function, elucidating their molecular targets, and examining their therapeutic potential in disease models.

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OUTLOOK Cellular reprogramming creates new opportunities in regenerative medicine, but the method must be significantly improved to fulfill its therapeutic potential.55 Excitingly, technologies in cellular reprogramming have tremendously advanced. Cell transplantation therapies using human iPSC-derived progenies under study in clinical trials are already showing great promise.56

Ke Li received a B.S. from Shandong University in 2004. In 2009, she earned a Ph. D. degree in biochemistry and cell biology from the Chinese Academy of Science, where she also worked as an assistant professor. In 2010, she joined Sheng Ding’s lab as a postdoctoral fellow. Her research focuses on transdifferentiation of somatic cells. 1209

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(17) Yu, J.; Vodyanik, M. A.; Smuga-Otto, K.; Antosiewicz-Bourget, J.; Frane, J. L.; Tian, S.; Nie, J.; Jonsdottir, G. A.; Ruotti, V.; Stewart, R.; Slukvin, I. I.; Thomson, J. A. Induced pluripotent stem cell lines derived from human somatic cells. Science 2007, 318, 1917−1920. (18) Tani, K. Towards the safer clinical translation of human induced pluripotent stem cell-derived cells to regenerative medicine. Mol. Ther.– Methods Clin. Dev. 2015, 2, 15032. (19) Tang, S.; Xie, M.; Cao, N.; Ding, S. Patient-Specific Induced Pluripotent Stem Cells for Disease Modeling and Phenotypic Drug Discovery. J. Med. Chem. 2016, 59, 2−15. (20) Liu, K.; Yu, C.; Xie, M.; Li, K.; Ding, S. Chemical modulation of cell fate in stem cell therapeutics and regenerative medicine. Cell Chem. Biol. 2016, 23, 893−916. (21) Xie, M.; Cao, N.; Ding, S. Small molecules for cell reprogramming and heart repair: progress and perspective. ACS Chem. Biol. 2014, 9, 34− 44. (22) Quina, A. S.; Buschbeck, M.; Di Croce, L. Chromatin structure and epigenetics. Biochem. Pharmacol. 2006, 72, 1563−1569. (23) Finley, A.; Copeland, R. A. Small molecule control of chromatin remodeling. Chem. Biol. 2014, 21, 1196−1210. (24) Shi, Y.; Do, J. T.; Desponts, C.; Hahm, H. S.; Schöler, H. R.; Ding, S. A combined chemical and genetic approach for the generation of induced pluripotent stem cells. Cell Stem Cell 2008, 2, 525−528. (25) Shi, Y.; Desponts, C.; Do, J. T.; Hahm, H. S.; Schöler, H. R.; Ding, S. Induction of pluripotent stem cells from mouse embryonic fibroblasts by Oct4 and Klf4 with small-molecule compounds. Cell Stem Cell 2008, 3, 568−574. (26) Barolo, S.; Posakony, J. W. Three habits of highly effective signaling pathways: principles of transcriptional control by developmental cell signaling. Genes Dev. 2002, 16, 1167−1181. (27) Lin, T.; Ambasudhan, R.; Yuan, X.; Li, W.; Hilcove, S.; Abujarour, R.; Lin, X.; Hahm, H. S.; Hao, E.; Hayek, A.; Ding, S. A chemical platform for improved induction of human iPSCs. Nat. Methods 2009, 6, 805−808. (28) Li, W.; Zhou, H.; Abujarour, R.; Zhu, S.; Joo, Y. J.; Lin, T.; Hao, E.; Schöler, H. R.; Hayek, A.; Ding, S. Generation of human-induced pluripotent stem cells in the absence of exogenous Sox2. Stem Cells 2009, 27, 2992−3000. (29) Yuan, X.; Wan, H.; Zhao, X.; Zhu, S.; Zhou, Q.; Ding, S. Brief report: combined chemical treatment enables Oct4-induced reprogramming from mouse embryonic fibroblasts. Stem Cells 2011, 29, 549−553. (30) Zhu, S.; Li, W.; Zhou, H.; Wei, W.; Ambasudhan, R.; Lin, T.; Kim, J.; Zhang, K.; Ding, S. Reprogramming of human primary somatic cells by OCT4 and chemical compounds. Cell Stem Cell 2010, 7, 651−655. (31) Mali, P.; Chou, B. K.; Yen, J.; Ye, Z.; Zou, J.; Dowey, S.; Brodsky, R. A.; Ohm, J. E.; Yu, W.; Baylin, S. B.; Yusa, K.; Bradley, A.; Meyers, D. J.; Mukherjee, C.; Cole, P. A.; Cheng, L. Butyrate greatly enhances derivation of human induced pluripotent stem cells by promoting epigenetic remodeling and the expression of pluripotency-associated genes. Stem Cells 2010, 28, 713−720. (32) Ma, T.; Li, J.; Xu, Y.; Yu, C.; Xu, T.; Wang, H.; Liu, K.; Cao, N.; Nie, B.; Zhu, S.; Xu, S.; Li, K.; Wei, W.; Wu, Y.; Guan, K.; Ding, S. Atg5independent autophagy regulates mitochondrial clearance and is essential for iPSC reprogramming. Nat. Cell Biol. 2015, 17, 1379−1387. (33) Xu, J.; Du, Y.; Deng, H. Direct lineage reprogramming: strategies, mechanisms, and applications. Cell Stem Cell 2015, 16, 119−134. (34) Moradi, S.; Asgari, S.; Baharvand, H. Concise review: harmonies played by microRNAs in cell fate reprogramming. Stem Cells 2014, 32, 3−15. (35) Ambasudhan, R.; Talantova, M.; Coleman, R.; Yuan, X.; Zhu, S.; Lipton, S. A.; Ding, S. Direct reprogramming of adult human fibroblasts to functional neurons under defined conditions. Cell Stem Cell 2011, 9, 113−118. (36) Takahashi, K.; Yamanaka, S. A decade of transcription factormediated reprogramming to pluripotency. Nat. Rev. Mol. Cell Biol. 2016, 17, 183−193. (37) Efe, J. A.; Hilcove, S.; Kim, J.; Zhou, H.; Ouyang, K.; Wang, G.; Chen, J.; Ding, S. Conversion of mouse fibroblasts into cardiomyocytes

Sheng Ding is the William K. Bowes, Jr., Distinguished Investigator at the Gladstone Institutes and a professor in the Department of Pharmaceutical Chemistry at the University of California, San Francisco. He obtained a B.S. in chemistry from Caltech in 1999 and a Ph.D. in chemistry from The Scripps Research Institute in 2003. Before moving to Gladstone in 2011, Ding was an assistant professor and then an associate professor of chemistry at The Scripps Research Institute from 2003 to 2011. Ding has developed and applied innovative chemical approaches to stem cell biology and regeneration, with a focus on discovering and characterizing small molecules that control cell fate/ function, including stem cell maintenance, differentiation, and reprogramming.

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ACKNOWLEDGMENTS We thank many collaborators and members of the Ding lab who contributed to our scientific endeavors. We thank Dr. Crystal Herron from the Gladstone Institutes for critically reading and editing this manuscript. We apologize to all scientists whose research could not be properly discussed and cited in this Account owing to space limitations.

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REFERENCES

(1) Sánchez, A.; Schimmang, T.; García-Sancho, J. Cell and tissue therapy in regenerative medicine. Adv. Exp. Med. Biol. 2012, 741, 89− 102. (2) Mao, A. S.; Mooney, D. J. Regenerative medicine: Current therapies and future directions. Proc. Natl. Acad. Sci. U. S. A. 2015, 112, 14452−14459. (3) Ferrell, J. E., Jr. Bistability, bifurcations, and Waddington’s epigenetic landscape. Curr. Biol. 2012, 22, R458−466. (4) Ladewig, J.; Koch, P.; Brüstle, O. Leveling Waddington: the emergence of direct programming and the loss of cell fate hierarchies. Nat. Rev. Mol. Cell Biol. 2013, 14, 225−236. (5) Wobus, A. M.; Boheler, K. R. Embryonic stem cells: prospects for developmental biology and cell therapy. Physiol. Rev. 2005, 85, 635−78. (6) Chen, K. G.; Mallon, B. S.; McKay, R. D.; Robey, P. G. Human pluripotent stem cell culture: considerations for maintenance, expansion, and therapeutics. Cell Stem Cell 2014, 14, 13−26. (7) Stadtfeld, M.; Hochedlinger, K. Induced pluripotency: history, mechanisms, and applications. Genes Dev. 2010, 24, 2239−2263. (8) Fox, I. J.; Daley, G. Q.; Goldman, S. A.; Huard, J.; Kamp, T. J.; Trucco, M. Stem cell therapy. Use of differentiated pluripotent stem cells as replacement therapy for treating disease. Science 2014, 345, 1247391. (9) Graf, T. Historical origins of transdifferentiation and reprogramming. Cell Stem Cell 2011, 9, 504−516. (10) Todeschini, A. L.; Georges, A.; Veitia, R. A. Transcription factors: specific DNA binding and specific gene regulation. Trends Genet. 2014, 30, 211−219. (11) Macneil, L. T.; Walhout, A. J. Gene regulatory networks and the role of robustness and stochasticity in the control of gene expression. Genome Res. 2011, 21, 645−657. (12) Holmberg, J.; Perlmann, T. Maintaining differentiated cellular identity. Nat. Rev. Genet. 2012, 13, 429−439. (13) Nayerossadat, N.; Maedeh, T.; Ali, P. A. Viral and nonviral delivery systems for gene delivery. Adv. Biomed. Res. 2012, 1, 27. (14) Davis, R. L.; Weintraub, H.; Lassar, A. B. Expression of a single transfected cDNA converts fibroblasts to myoblasts. Cell 1987, 51, 987− 1000. (15) Takahashi, K.; Yamanaka, S. Induction of pluripotent stem cells from mouse embryonic and adult fibroblast cultures by defined factors. Cell 2006, 126, 663−676. (16) 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, 861−872. 1210

DOI: 10.1021/acs.accounts.7b00020 Acc. Chem. Res. 2017, 50, 1202−1211

Article

Accounts of Chemical Research using a direct reprogramming strategy. Nat. Cell Biol. 2011, 13, 215− 222. (38) Kim, J.; Efe, J. A.; Zhu, S.; Talantova, M.; Yuan, X.; Wang, S.; Lipton, S. A.; Zhang, K.; Ding, S. Direct reprogramming of mouse fibroblasts to neural progenitors. Proc. Natl. Acad. Sci. U. S. A. 2011, 108, 7838−7843. (39) Williams, L. A.; Davis-Dusenbery, B. N.; Eggan, K. C. SnapShot: directed differentiation of pluripotent stem cells. Cell 2012, 149, 1174. (40) Zhang, Y.; Cao, N.; Huang, Y.; Spencer, C. I.; Fu, J. D.; Yu, C.; Liu, K.; Nie, B.; Xu, T.; Li, K.; Xu, S.; Bruneau, B. G.; Srivastava, D.; Ding, S. Expandable Cardiovascular Progenitor Cells Reprogrammed from Fibroblasts. Cell Stem Cell 2016, 18, 368−381. (41) Burridge, P. W.; Keller, G.; Gold, J. D.; Wu, J. C. Production of de novo cardiomyocytes: human pluripotent stem cell differentiation and direct reprogramming. Cell Stem Cell 2012, 10, 16−28. (42) Lalit, P. A.; Salick, M. R.; Nelson, D. O.; Squirrell, J. M.; Shafer, C. M.; Patel, N. G.; Saeed, I.; Schmuck, E. G.; Markandeya, Y. S.; Wong, R.; Lea, M. R.; Eliceiri, K. W.; Hacker, T. A.; Crone, W. C.; Kyba, M.; Garry, D. J.; Stewart, R.; Thomson, J. A.; Downs, K. M.; Lyons, G. E.; Kamp, T. J. Lineage Reprogramming of Fibroblasts into Proliferative Induced Cardiac Progenitor Cells by Defined Factors. Cell Stem Cell 2016, 18, 354−367. (43) D’Amour, K. A.; Agulnick, A. D.; Eliazer, S.; Kelly, O. G.; Kroon, E.; Baetge, E. E. Efficient differentiation of human embryonic stem cells to definitive endoderm. Nat. Biotechnol. 2005, 23, 1534−1541. (44) Li, K.; Zhu, S.; Russ, H. A.; Xu, S.; Xu, T.; Zhang, Y.; Ma, T.; Hebrok, M.; Ding, S. Small molecules facilitate the reprogramming of mouse fibroblasts into pancreatic lineages. Cell Stem Cell 2014, 14, 228− 236. (45) Zhu, S.; Russ, H. A.; Wang, X.; Zhang, M.; Ma, T.; Xu, T.; Tang, S.; Hebrok, M.; Ding, S. Human pancreatic beta-like cells converted from fibroblasts. Nat. Commun. 2016, 7, 10080. (46) Zhu, S.; Rezvani, M.; Harbell, J.; Mattis, A. N.; Wolfe, A. R.; Benet, L. Z.; Willenbring, H.; Ding, S. Mouse liver repopulation with hepatocytes generated from human fibroblasts. Nature 2014, 508, 93−97. (47) Hou, P.; Li, Y.; Zhang, X.; Liu, C.; Guan, J.; Li, H.; Zhao, T.; Ye, J.; Yang, W.; Liu, K.; Ge, J.; Xu, J.; Zhang, Q.; Zhao, Y.; Deng, H. Pluripotent stem cells induced from mouse somatic cells by smallmolecule compounds. Science 2013, 341, 651−654. (48) Wang, H.; Cao, N.; Spencer, C. I.; Nie, B.; Ma, T.; Xu, T.; Zhang, Y.; Wang, X.; Srivastava, D.; Ding, S. Small molecules enable cardiac reprogramming of mouse fibroblasts with a single factor, Oct4. Cell Rep. 2014, 6, 951−960. (49) Li, J.; Huang, N. F.; Zou, J.; Laurent, T. J.; Lee, J. C.; Okogbaa, J.; Cooke, J. P.; Ding, S. Conversion of human fibroblasts to functional endothelial cells by defined factors. Arterioscler., Thromb., Vasc. Biol. 2013, 33, 1366−1375. (50) Zhu, S.; Ambasudhan, R.; Sun, W.; Kim, H. J.; Talantova, M.; Wang, X.; Zhang, M.; Zhang, Y.; Laurent, T.; Parker, J.; Kim, H. S.; Zaremba, J. D.; Saleem, S.; Sanz-Blasco, S.; Masliah, E.; McKercher, S. R.; Cho, Y. S.; Lipton, S. A.; Kim, J.; Ding, S. Small molecules enable OCT4-mediated direct reprogramming into expandable human neural stem cells. Cell Res. 2014, 24, 126−129. (51) Zhang, M.; Lin, Y.-H.; Sun, Y.-J.; Zhu, S.; Zheng, J.; Liu, K.; Cao, N.; Li, K.; Huang, Y.; Ding, S. Pharmacological Reprogramming of Fibroblasts into Neural Stem Cells by Signaling-Directed Transcriptional Activation. Cell Stem Cell 2016, 18, 653−667. (52) Hu, W.; Qiu, B.; Guan, W.; Wang, Q.; Wang, M.; Li, W.; Gao, L.; Shen, L.; Huang, Y.; Xie, G.; Zhao, H.; Jin, Y.; Tang, B.; Yu, Y.; Zhao, J.; Pei, G. Direct Conversion of Normal and Alzheimer’s Disease Human Fibroblasts into Neuronal Cells by Small Molecules. Cell Stem Cell 2015, 17, 204−212. (53) Li, X.; Zuo, X.; Jing, J.; Ma, Y.; Wang, J.; Liu, D.; Zhu, J.; Du, X.; Xiong, L.; Du, Y.; Xu, J.; Xiao, X.; Wang, J.; Chai, Z.; Zhao, Y.; Deng, H. Small-Molecule-Driven Direct Reprogramming of Mouse Fibroblasts into Functional Neurons. Cell Stem Cell 2015, 17, 195−203. (54) Cao, N.; Huang, Y.; Zheng, J.; Spencer, C. I.; Zhang, Y.; Fu, J. D.; Nie, B.; Xie, M.; Zhang, M.; Wang, H.; Ma, T.; Xu, T.; Shi, G.; Srivastava,

D.; Ding, S. Conversion of human fibroblasts into functional cardiomyocytes by small molecules. Science 2016, 352, 1216−1220. (55) Dimmeler, S.; Ding, S.; Rando, T. A.; Trounson, A. Translational strategies and challenges in regenerative medicine. Nat. Med. 2014, 20, 814−821. (56) Ilic, D.; Devito, L.; Miere, C.; Codognotto, S. Human embryonic and induced pluripotent stem cells in clinical trials. Br. Med. Bull. 2015, 116, 19−27. (57) Lee, A. S.; Tang, C.; Rao, M. S.; Weissman, I. L.; Wu, J. C. Tumorigenicity as a clinical hurdle for pluripotent stem cell therapies. Nat. Med. 2013, 19, 998−1004. (58) Mohamed, T. M.; Stone, N. R.; Berry, E. C.; Radzinsky, E.; Huang, Y.; Pratt, K.; Ang, Y. S.; Yu, P.; Wang, H.; Tang, S.; Magnitsky, S.; Ding, S.; Ivey, K. N.; Srivastava, D. Chemical Enhancement of In Vitro and In Vivo Direct Cardiac Reprogramming. Circulation 2017, 135, 978−995. (59) Tibbitt, M. W.; Dahlman, J. E.; Langer, R. Emerging Frontiers in Drug Delivery. J. Am. Chem. Soc. 2016, 138, 704−717.

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DOI: 10.1021/acs.accounts.7b00020 Acc. Chem. Res. 2017, 50, 1202−1211