Chemical transdifferentiation: closer to regenerative medicine

Abstract

Cell transdifferentiation, which directly switches one type of differentiated cells into another cell type, is more advantageous than cell reprogramming to generate pluripotent cells and differentiate them into functional cells. This process is crucial in regenerative medicine. However, the cell-converting strategies, which mainly depend on the virus-mediated expression of exogenous genes, have clinical safety concerns. Small molecules with compelling advantages are a potential alternative in manipulating cell fate conversion. In this review, we briefly retrospect the nature of cell transdifferentiation and summarize the current developments in the research of small molecules in promoting cell conversion. Particularly, we focus on the complete chemical compound-induced cell transdifferentiation, which is closer to the clinical translation in cell therapy. Despite these achievements, the mechanisms underpinning chemical transdifferentiation remain largely unknown. More importantly, identifying drugs that induce resident cell conversion in vivo to repair damaged tissue remains to be the end-goal in current regenerative medicine.

Keywords

cell therapy / cell transdifferentiation / chemical compounds / small molecules / tissue regeneration

Introduction

Cell differentiation, which produces functionally mature cells, is one of the most crucial events in the development of an organism. However, committed cells are not “frozen” as they develop. To generate desired cell types, the routine stability of the original cells could be disturbed and the cells that act like “marbles” in Waddington’s landscape roll across the “valley” [ 1]. According to cross-antagonisms in a cascading landscape of unstable and stable cell states, transcription factors (TFs) play a crucial role in controlling cell fate. Over the past decades, novel technologies and screening approaches have been applied to select appropriate candidate TFs for lineage transdifferentiation [ 2]. Although significant developments were achieved in this field, gene modification has been an obstacle in achieving the goal of future regenerative medicine because of its safety concerns.

Small molecules are chemical compounds with low molecular weight; these molecules exhibit crucial advantages and are a potential alternative in manipulating cell fate changes [ 3]. The biological effects of small molecules are typically rapid. They do not bind covalently to their target protein. Thus, they may be used reversibly. Small molecules are more stable and cost-effective than synthesized proteins and mRNAs, growth factors or cytokines. They are more easily synthesized, preserved, and standardized. Their effects could be improved by fine-tuning their structure, concentration, and combination. Small molecules could easily control signaling pathways and epigenetic modification.

In this review, we briefly examine the history of cell transdifferentiation and provide an overview on small molecule-induced transdifferentiation, which we refer to as chemical transdifferentiation. We also discuss its potential clinical applications in regenerative medicine.

Nature of cell transdifferentiation

Cell transdifferentiation refers to the lineage switches from one type of differentiated cells to another cell type. Evidences of transdifferentiation in nature have been documented. One of the well-known examples is the lens regeneration of adult newts [ 4], which was first observed as early as 1895. Once the lens of adult newts are impaired, pigmented epithelial cells (PECs) from the dorsal iris initiate transdifferentiation and reproduce the missing tissue. This process begins with dedifferentiation, which indicates that PECs lose their properties, such as morphology and pigmentation. In this process, hyper-phosphorylated retinoblastoma protein loses its activity and dissociates from E2F, which allows the cells to re-enter the cell cycle and proliferate to create a new lens vesicle. However, proliferation seems to be unnecessary because treatment with a cyclin-dependent kinase inhibitor does not completely stop the transdifferentiation [ 5]. The differentiation of the primary lens fiber manifests as the thickening of the internal layer of the lens vesicle and synthesis of crystallins. Moreover, natural transdifferentiation also occurs in salamanders, Xenopus, jellyfishes [ 4], and worms [ 6]. Transdifferentiation is a rare event in nature. Thus, the underlining mechanisms of this natural process remain unknown. Multiple signaling pathways are involved in lens regeneration, including FGF, retinoic acid, TGFβ, Wnt, and hedgehog pathways (reviewed by Henry [ 7]). Interestingly, some members of the hedgehog pathway, such as Shh, Ihh, and Ptc2, which are expressed in embryonic lenses, can be detected again in the regenerating lens [ 8]. Maki et al. demonstrated that epigenetic modifiers, such as histone deacetylases (HDACs), are also upregulated during this process [ 9].

Exogenous gene delivery carries forward transdifferentiation

The importance of TFs during lineage transdifferentiation was demonstrated in late 1980s when the forced expression of MyoD was determined to induce myotube formation from a fibroblast cell line [ 10, 11]. However, this cell fate conversion is considered incomplete because the acquired phenotype depends on the sustained overexpression of MyoD. Over the next two decades, a few studies demonstrated that related lineages within the blood [ 12], endoderm [ 13], and nervous systems [ 14] can be converted into other cell types by overexpressing exogenous genes. In 2006, the advent of induced pluripotent stem cells (iPSCs) [ 15], which possibly transitioned between developmentally distant cell types using a combination of four pluripotent TFs, revitalized the field of lineage reprogramming and motivated researchers to determine sets of TFs that may be crucial for cell transdifferentiation. To date, many studies have shown that in vitro transdifferentiation of cells in the same germ layers can be directed both in mice and humans by defined factors (reviewed by Deng [ 2]), which play key roles in maintaining and regulating target cell functions. For the ectoderm, astrocytes can be converted into GABAergic or glutamatergic neurons [ 16] and pericyte-derived cells of the adult human brain into neurons [ 17]. For the mesoderm, fibroblasts were successfully induced into functional adipocytes [ 18], cardiomyocytes [ 19, 20], chondrocytes [ 21], endothelial cells [ 22], hemogenic endothelial-like precursor cells [ 23], hematopoietic progenitor cells [ 24, 25], and macrophages [ 26]. Proximal tubule cell line HK-2 is switched into a nephron progenitor [ 27]. For the endoderm, pancreatic exocrine cells were directly changed into pancreatic b-like cells [ 28]. Further developments were achieved in remodeling cell types across distinct germ layers. Examples of these cell types are neurons generated from fibroblasts [ 29– 31] or hepatocytes [ 32], endothelial cells from amniotic cells [ 33], hepatocytes from fibroblasts [ 34, 35], and monocytes from neural stem cells [ 36]. The transient introduction of pluripotency factors plays an indirect role in transdifferentiation, including the generation of neural stem or progenitor cells [ 37, 38], cardiomyocytes [ 39], angioblast-like progenitor cells [ 40], endothelial cells [ 41], pancreatic lineages [ 42], and hepatocytes [ 43]. This predefined step was hypothesized to initiate the removal of the epigenetic memory of the starting cells [ 44], which may require further steps to improve the functional maturation of converted cells.

Transdifferentiation in vivo, which maximizes the native physiological niche, reduces the concerns related to in vitro culture and cell transplantation. Compared with cell conversion in vitro, higher conversion efficiency can be achieved and desirable cell types with better function can be generated in vivo, which is probably attributed to the essential factors that the in situ niche provided. For example, in the induction of cardiomyocyte-like cells, murine cardiac fibroblasts could result in the robust generation of functional cardiomyocyte-like cells by the overexpression of TFs (Gata4, Mef2c, and Tbx5) in vivo [ 45], whereas the induction in vitro with the same TFs only generated inefficient ones [ 46]. Several other in vivo transdifferentiation examples include pancreatic exocrine cells remodeled into b-cells [ 47], Sox9⁺ cells in liver into insulin-secreting ducts [ 48], non-myocytes into cardiomyocyte-like cells [ 49], astrocytes into neurons [ 50] or neuroblasts [ 51], embryonic and early postnatal callosal projection neurons in layer II/III into corticofugal projection neurons in layer V/VI [ 52], and pre/pro-B cells into hematopoietic stem or progenitor cells [ 53].

Current developments of chemical compounds promoting cell transdifferentiation

Small molecules, which reduce the safety concerns about genetic manipulation for future clinical applications, has gained increasing attention of researchers in regenerative medicine. Small molecules can partially regulate gene expression through four classes of mechanisms: signaling pathway modulators, which activate or repress components of signal transduction to regulate downstream transcription activity; modulators of epigenetic proteins, which regulate the activity of epigenetic complexes; metabolic regulators, which adjust the cell state and shift the balance of metabolites that serve as ligands for proteins and cofactors for epigenetic proteins; and nuclear receptor agonists and antagonists, which directly modulate transcription by regulating the activity of nuclear receptors. To develop simpler and safer transdifferentiation methods for cell-based therapeutic applications, researchers aim to identify small molecules to replace exogenous genes, as previously reported.

Small molecules facilitating exogenous gene-driven transdifferentiation

Small molecules in chemical transdifferentiation starts as a booster of TF-based transdifferentiation. Increasing conversion efficiency is a pivotal issue in generating sufficient neurons because neurons are post-mitotic. Ladewig et al. established a minimalist approach to generate neurons from fibroblasts by combining two neuronal specifiers (Ascl1 and Ngn2) with the small molecule-based inhibition of glycogen synthase kinase-3b (GSK-3b) and SMAD signaling [ 54]. The results showed that the products of transdifferentiation exceeded 200% (cell yield is calculated as the percentage of ending cells in relation to the initial number of plated cells) and the final neuronal purities reached>80%. Moreover, based on the previous studies, Liu et al. demonstrated that two other small molecules (forskolin and dorsomorphin) enable the TF Ngn2 to convert human fetal lung fibroblasts into cholinergic neurons with significantly higher purity (>90%) and efficiency (up to 99% of Ngn2-expressing cells) [ 55]. To obtain the functional neural crest from human postnatal fibroblasts, Kim et al. only used one single TF Sox10 combined with environmental cues covering WNT activator (CHIR99021 or BMP4) [ 56]. The generated induced neural crest (iNC) can migrate into or be retained in the expected regions in vivo. Approximately 40% of single iNC clones could produce main downstream differentiation lineages (neurons, glia, smooth muscle cells, melanocytes, chondrocytes, and adipocytes), which is comparable to the neural crest derived from embryonic stem cells. Under basal culture conditions containing three chemicals, namely, A-83-01, CHIR99021, and sodium butyrate, which could enhance the formation of neural stem cells, Zhu et al. screened potential chemicals to facilitate the reprogramming of adult human dermal fibroblasts transduced with OCT4 alone [ 57]. Three candidates were identified, including LPA (a phospholipid derivative), rolipram (a PDE4 inhibitor), and SP600125 (a JNK inhibitor). Furthermore, the researchers obtained expandable human neural stem cells using a combination of Oct4 overexpression and chemical cocktails. More recently, Lee et al. altered the fate of human blood progenitor into neural stem cells with OCT4 alone [ 58]. The conversion was facilitated by inhibiting both the SMAD and GSK-3 signaling pathways to overcome the restrictions on neural fate conversion.

In a similar study, mouse fibroblasts, which were initially transduced with Oct4 alone, were exposed to a cocktail of lineage-specific signals, including SB431542 (ALK4/5/7 inhibitor), CHIR99021 (GSK3 inhibitor), parnate (LSD1/KDM1 inhibitor), and forskolin (adenylyl cyclase activator), to achieve transdifferentiation into cardiac lineage [ 59]. Another study that was based on a reporter system demonstrated that treatment with SB431542 in conjunction of inducible expression of the TFs enhances the conversion of mouse embryonic fibroblasts (MEFs) to induced cardiomyocytes [ 60]. In the pluripotency factor-based transdifferentiation described above, adding JAK inhibitor to the late stage of inducting procession, which antagonizes an essential pathway for embryonic stem cell maintenance (the LIF-STAT3 pathway), could suppress the establishment of pluripotency while promoting the generation of epigenetically plastic intermediate cells [ 39].

Li et al. also demonstrated that engineered MEFs expressing inducible Yamanaka factors (Oct4, Sox2, Klf4, and c-Myc) at an early stage can be further converted into proliferation competent definitive endoderm-like cells, pancreatic progenitor-like cells, and insulin-producing b and other pancreatic-like cells under a conditioned medium with diverse combinations of small molecules and soluble factors [ 42].

Chemical transdifferentiation with complete small molecules

Lineage reprogramming through the complete chemical induction medium without integrating exogenous genes is the ultimate goal for generating target cells. Direct transdifferentiation with few chemicals in tumors was achieved as early as 1982 where the demethylating agent 5-Aza remodeled pre-B lymphoma into cells that express markers of macrophages [ 61]. In the past five years, investigations about lineage transdifferentiation within the same germ layer or across different germ layers with small molecules was carried out (Table 1).

Toward neural cells

Our previous study demonstrated that functional neural progenitor cells (NPCs) can be acquired from MEFs, mouse tail-tip fibroblasts, and human urinary cells using chemical cocktails under hypoxia [ 62]. The chemical cocktails were called VCR, including VPA, CHIR99021, and Repsox, which inhibit HDACs, GSK-3, and TGFb kinase pathways, respectively. NPCs were finally generated under lineage-specific culture conditions by passing through intermediate compact cell colonies where the expression of Sox2 significantly increased. These chemically induced NPCs (ciNPCs) can proliferate, self-renew and exhibit similar transcription profiles as mouse brain-derived NPCs. ciNPCs can also differentiate into different neural lineage cells both in vitro and in vivo under defined culture mediums. Moreover, combining the alternative inhibitors of these signaling pathways also facilitates the transition from MEFs to NPCs. Without hypoxia, Han et al. utilized more chemicals based on VCR to induce mouse fibroblasts into neural stem cells [ 63]. The chemical cocktail includes A-83-01, CHIR99021, VPA, BIX01294, RG108, PD0325901, and vitamin C. Baharvand et al. utilized a suspension culture system in the presence of 5-Aza instead of the traditional monolayer culture to induce human fibroblasts into NPCs [ 64].

Based on our NPC induction protocol, Hu et al. added small molecules that are known to promote neural differentiation of NPCs, which include forskolin, SP600125, GO6983, and Y-27632, with VCR, to covert human fibroblasts into neurons (hciNs) directly, thus bypassing the NPC state [ 65]. Importantly, hciNs that were derived from the familial fibroblasts of an Alzheimer’s disease patient by a chemical cocktail exhibit abnormal Ab production. Neurons can also be induced from human fibroblasts by an alternative chemical cocktail containing SB431542, CHIR99021, forskolin, pifithrin-a, LDN193189, and PD0325901, which accelerate mesenchymal-to-epithelial transition, promote cell reprogramming, and facilitate neuronal conversion [ 66]. For starting cells from mouse, Li et al. identified five chemical compounds robustly converting mouse fibroblasts into TUJ1-positive neurons [ 67]. The chemical cocktail includes SB43152, CHIR99201, forskolin, I-BET151, and ISX9. A mechanical study demonstrates that I-BET151 disrupts the fibroblast-specific program and ISX9 activates neuron-specific genes. For more specific-type of neuron induction, GABAergic neurons were induced from mouse fibroblasts cultured in conditioned medium from neurotrophin-3 modified olfactory ensheathing cells plus SB431542, retinoic acid, and GDNF for three weeks [ 68].

Treating neurological disorders by transplanting induced neuronal cells from fibroblasts is still limited because of the delivery strategy and the retained epigenetic memory of starting fibroblasts. Thus, astrocytes are considered the ideal starting candidate cell type for generating new neurons because of their proximity in lineage distance to neurons and their ability to proliferate after brain damage. Small molecules identified in the cell-converting assay in vitro may be applied in vivo to enable the transdifferentiation of resident astrocytes into neurons. To this point, we identified that the chemical cocktail composed of VPA and Tranilast can convert mouse astrocytes into neurons in vitro [ 69]. Human astroglial cells can also be reprogrammed efficiently into functional neurons under the treatment of small molecules (SB431542, CHIR99021, VPA, LDN193189, DAPT, Tzv, TTNPB, SAG, and Purmo), which mediate epigenetic and transcriptional regulation [ 70]. In both cases, the small molecules activate pro-neural TFs NueroD1 and NeuroG2. Moreover, only HDAC inhibitors (Trichostatin A or VPA) can directly induce malignant astrocytes into neuronal cells [ 71]. For in vivo studies, VPA alone can convert glial cells into neurons after a stabbing injury with low efficiency [ 72].

Except for neural stem or progenitor cells and neurons, Schwann cells, the major glial cell type of the peripheral nervous system, were also generated from human fibroblasts with the stepwise induction by Noggin peptides with small molecules, including SB431542, VPA, CP21, and compound B, which is the potent neural-inducing small molecule identified through high-throughput screening [ 73].

Toward cardiomyocytes

To determine the combination of small molecules that could be used to induce pluripotent cells, You et al. selected 12 candidates that are functional substitutes for TFs or enhancers for iPSC induction [ 74]. The researchers found that cTnT⁺ cells and not iPSCs were produced from the combination of small molecules. The gene expression, epigenetic status of cardiac-specific genes, and subcellular structure of these induced cells were similar to that of primary cardiomyocytes. Five small molecules in the cocktail are abbreviated as “FASCB,” namely, forskolin, A–83-01, SC1, CHIR99021, and (±)-Bay K 8644. Meanwhile, Xie’s group found that only small molecules inducing chemically induced pluripotent cells (CiPSCs) and a specific culturing condition can induce MEFs and tail-tip fibroblasts into cardiomyocytes, which was confirmed by lineage tracing using transgenic mouse [ 75].

Toward pancreatic cells

A total of 35%±8.9% of adult human skin fibroblasts that were exposed to 5-Aza for 18 h followed by a three-step protocol were successfully induced into endocrine pancreatic cells. These artificial cells shared many characteristics with primary pancreatic cells, such as epithelial morphology and the ability to secrete insulin in response to a physiological glucose challenge in vitro [ 76]. Argibay’s group established a novel approach to transdifferentiate skin fibroblasts from type 1 diabetes patients into insulin-expressing clusters with a stepwise culture medium including nicotinamide and exedin-4 [ 77]. Sasaki et al. turned a “straw” cell line of human hepatoma HepG2 cells into “gold” pancreatic-like cells using small molecules CCl₄ and D-galactosamine, which are severely hepatotoxic and ZnCl₂, an agent that facilitates pancreatic development and function [ 78].

Toward adipocytes

The uncontrolled expansion of white adipocyte tissue can lead to obesity, whereas another type of fat brown adipocyte tissue serves as an opposite physiological function to dissipate energy. To increase the number or activity of brown-like adipocytes in white adipose depots, two promising approaches were developed. One approach identified one of the thyroid hormones triiodothyronine [ 79], and the other approach combined inhibitors of Janus kinase (JAK) R406 and Tofacitinib [ 80] to promote a white-to-brown metabolic conversion. These discoveries could establish the foundation to further prevent diet-induced obesity and reduce the incidence and severity of type 2 diabetes by changing cell fate.

Toward endothelial cells

Cardiac fibroblasts can transdifferentiate into endothelial-like cells after cardiac injury in response to a p53 activator RITA. This transition contributed to neovascularization, which could enhance overall cardiac function [ 81]. Another example is that toll-like receptor 3 agonist Poly (I:C), together with microenvironmental cues that drive endothelial cell specification, such as exogenous endothelial cell growth factors, transdifferentiated human fibroblasts into endothelial cells [ 82].

The field of chemical transdifferentiation has gained many achievements. However, the molecular mechanism of small molecules during transdifferentiation is yet to be explained. Interestingly, small molecules targeting HDACs, GSK3, and TGFb participate in mostly reported chemical transdifferentiation (Table 2). Treatment with the same small molecule could lead to extremely different cell types. For example, 5-Aza that is treated with various concentrations for a certain time can convert human fibroblasts into functional NPCs or pancreatic cells or muscle cells [ 83]. 5-Aza, a DNA methyltransferase inhibitor, may cause chromatin de-condensation and induce a short “dedifferentiation” state. Under defined culture conditions, the “dedifferentiated” intermediate cells differentiate into mature cells. Another epigenetic regulator, HDAC inhibitors, which change the chromatin state, may play a similar role. TGFb inhibitors may undergo regulating transition between mesenchymal and epithelial state to promote cell reprogramming and help convert cell fate. However, the reason why GSK3 inhibitor, which probably activates WNT signaling, plays an important role in these transitions need to be further investigated.

Perspectives

Although cell transdifferentiation is not an entirely new concept, it has many potentials that have yet to be determined. In contrast to the differentiation from embryonic stem cells or iPSCs, different functional cell types could be more conveniently and more effectively derived from somatic cells, which are more easily accessible, in cell transdifferentiation. Moreover, direct lineage transdifferentiation bypasses the potential tumorigenicity, which may be caused by incomplete pluripotent cell differentiation. Through the overexpression of the exogenous genes, which is the most popular strategy in the early stages, a large number of desired cell types were generated in both mouse and human cells as mentioned above. To address gene manipulation and safety issues, researchers applied modified RNAs, as well as hormone mixtures, as an alternative strategy to induce lineage conversion [ 84, 85]. However, the instability and cost are the limitations of this approach.

Small molecules, which are stable and cost-effective, are becoming a popular alternative to control cell outcomes. Deng’s group first successfully generated CiPSCs from MEFs, mouse neonatal fibroblasts, mouse adult fibroblasts, adipose-derived stem cells, neural stem cells, and intestinal epithelial cells using a cocktail of small molecules VC6TFZ (VPA, CHIR99021, 616452, tranylcypromine, forskolin, and DZNep) [ 86– 88]. Xie et al. determined that the full chemical-induced cell reprogramming could be significantly enhanced by BrdU [ 89]. Considering the significant roles of small molecules in maintaining and differentiating stem cells [ 90] and reprogramming cells [ 91], more studies sought to harness the potential of small molecules on cell transdifferentiation. At present, small molecules are known not only as facilitating factors for exogenous genes but also as master factors of lineage transdifferentiation. Nevertheless, high-throughput and high-definition screening technology must be explored to determine small-molecule candidates to replace exogenous genes and achieve in complete chemical transdifferentiation. In addition, directly targeting certain TFs by small molecule compounds is difficult. Previous reports pointed out that E-cadherin [ 92] and orphan nuclear receptor Esrrb [ 93] can replace TFs in somatic cell reprogramming. These findings demonstrate the potential of small molecules in targeting nuclear receptors to achieve lineage conversion.

To some extent, stem or progenitor cells that can be grafted are more desirable. Thus, improving the generation or enhancing their functions using small molecule-based approaches must be thoroughly examined in the future. Additionally, by maximizing in vivo niche, chemical transdifferentiation in vivo may provide broader prospects for clinical applications [ 94]. Small molecules have limitations, such as unexpected side effects, which should be considered in clinical applications.

The field of regenerative medicine has undergone breakthroughs and significant growth in recent years. Although chemical transdifferentiation still needs to be thoroughly analyzed, this approach is certainly a potential method in cell-based regeneration (Fig.1).

References

[1] Waddington CH. The Strategy of the Genes: a Discussion of Some Aspects of Theoretical Biology. London: Allen & Unwin, 1957

[2] Xu J, Du Y, Deng H. Direct lineage reprogramming: strategies, mechanisms, and applications. Cell Stem Cell 2015; 16(2): 119–134

[3] Xu Y, Shi Y, Ding S. A chemical approach to stem-cell biology and regenerative medicine. Nature 2008; 453(7193): 338–344

[4] Tsonis PA, Madhavan M, Tancous EE, Del Rio-Tsonis K. A newt’s eye view of lens regeneration. Int J Dev Biol 2004; 48(8-9): 975–980

[5] Tsonis PA, Madhavan M, Call M K, Gainer S, Rice A, Del Rio-Tsonis K. Effects of a CDK inhibitor on lens regeneration. Wound Repair Regen 2004;12:24–29

[6] Hajduskova M, Ahier A, Daniele T, Jarriault S. Cell plasticity in Caenorhabditis elegans: from induced to natural cell reprogramming. Genesis 2012; 50(1): 1–17

[7] Henry JJ, Thomas AG, Hamilton PW, Moore L, Perry KJ. Cell signaling pathways in vertebrate lens regeneration. Curr Top Microbiol Immunol 2013; 367: 75–98

[8] Tsonis PA, Vergara MN, Spence JR, Madhavan M, Kramer EL, Call MK, Santiago WG, Vallance JE, Robbins DJ, Del Rio-Tsonis K. A novel role of the hedgehog pathway in lens regeneration. Dev Biol 2004; 267(2): 450–461

[9] Maki N, Martinson J, Nishimura O, Tarui H, Meller J, Tsonis PA, Agata K. Expression profiles during dedifferentiation in newt lens regeneration revealed by expressed sequence tags. Mol Vis 2010; 16: 72–78

[10] Davis RL, Weintraub H, Lassar AB. Expression of a single transfected cDNA converts fibroblasts to myoblasts. Cell 1987; 51(6): 987–1000

[11] Tapscott SJ, Davis RL, Thayer MJ, Cheng PF, Weintraub H, Lassar AB. MyoD1: a nuclear phosphoprotein requiring a Myc homology region to convert fibroblasts to myoblasts. Science 1988; 242(4877): 405–411

[12] Kulessa H, Frampton J, Graf T. GATA-1 reprograms avian myelomonocytic cell lines into eosinophils, thromboblasts, and erythroblasts. Genes Dev 1995; 9(10): 1250–1262

[13] Shen CN, Slack JM, Tosh D. Molecular basis of transdifferentiation of pancreas to liver. Nat Cell Biol 2000; 2(12): 879–887

[14] Heins N, Malatesta P, Cecconi F, Nakafuku M, Tucker KL, Hack MA, Chapouton P, Barde YA, Götz M. Glial cells generate neurons: the role of the transcription factor Pax6. Nat Neurosci 2002; 5(4): 308–315

[15] Yamanaka S, Takahashi K.Induction of pluripotent stem cells from mouse fibroblast cultures. Protein, Nucleic acid, Enzyme (Tanpakushitsu Kakusan Koso) 2006; 51: 2346–2351 (in Japanese)

[16] Heinrich C, Blum R, Gascón S, Masserdotti G, Tripathi P, Sánchez R, Tiedt S, Schroeder T, Götz M, Berninger B. Directing astroglia from the cerebral cortex into subtype specific functional neurons. PLoS Biol 2010; 8(5): e1000373

[17] Karow M, Sánchez R, Schichor C, Masserdotti G, Ortega F, Heinrich C, Gascón S, Khan MA, Lie DC, Dellavalle A, Cossu G, Goldbrunner R, Götz M, Berninger B. Reprogramming of pericyte-derived cells of the adult human brain into induced neuronal cells. Cell Stem Cell 2012; 11(4): 471–476

[18] Kajimura S, Seale P, Kubota K, Lunsford E, Frangioni JV, Gygi SP, Spiegelman BM. Initiation of myoblast to brown fat switch by a PRDM16-C/EBP-beta transcriptional complex. Nature 2009; 460(7259): 1154–1158

[19] Nam YJ, Song K, Luo X, Daniel E, Lambeth K, West K, Hill JA, DiMaio JM, Baker LA, Bassel-Duby R, Olson EN. Reprogramming of human fibroblasts toward a cardiac fate. Proc Natl Acad Sci USA 2013; 110(14): 5588–5593

[20] Wada R, Muraoka N, Inagawa K, Yamakawa H, Miyamoto K, Sadahiro T, Umei T, Kaneda R, Suzuki T, Kamiya K, Tohyama S, Yuasa S, Kokaji K, Aeba R, Yozu R, Yamagishi H, Kitamura T, Fukuda K, Ieda M. Induction of human cardiomyocyte-like cells from fibroblasts by defined factors. Proc Natl Acad Sci USA 2013; 110(31): 12667–12672

[21] Hiramatsu K, Sasagawa S, Outani H, Nakagawa K, Yoshikawa H, Tsumaki N. Generation of hyaline cartilaginous tissue from mouse adult dermal fibroblast culture by defined factors. J Clin Invest 2011; 121(2): 640–657

[22] Han JK, Chang SH, Cho HJ, Choi SB, Ahn HS, Lee J, Jeong H, Youn SW, Lee HJ, Kwon YW, Cho HJ, Oh BH, Oettgen P, Park YB, Kim HS. Direct conversion of adult skin fibroblasts to endothelial cells by defined factors. Circulation 2014; 130(14): 1168–1178

[23] Pereira CF, Chang B, Qiu J, Niu X, Papatsenko D, Hendry CE, Clark NR, Nomura-Kitabayashi A, Kovacic JC, Ma’ayan A, Schaniel C, Lemischka IR, Moore K. Induction of a hemogenic program in mouse fibroblasts. Cell Stem Cell 2013; 13(2): 205–218

[24] Batta K, Florkowska M, Kouskoff V, Lacaud G. Direct reprogramming of murine fibroblasts to hematopoietic progenitor cells. Cell Reports 2014; 9(5): 1871–1884

[25] Szabo E, Rampalli S, Risueño RM, Schnerch A, Mitchell R, Fiebig-Comyn A, Levadoux-Martin M, Bhatia M. Direct conversion of human fibroblasts to multilineage blood progenitors. Nature 2010; 468(7323): 521–526

[26] Feng R, Desbordes SC, Xie H, Tillo ES, Pixley F, Stanley ER, Graf T. PU.1 and C/EBPα/β convert fibroblasts into macrophage-like cells. Proc Natl Acad Sci USA 2008; 105(16): 6057–6062

[27] Hendry CE, Vanslambrouck JM, Ineson J, Suhaimi N, Takasato M, Rae F, Little MH. Direct transcriptional reprogramming of adult cells to embryonic nephron progenitors. J Am Soc Nephrol 2013; 24(9): 1424–1434

[28] Lemper M, Leuckx G, Heremans Y, German MS, Heimberg H, Bouwens L, Baeyens L. Reprogramming of human pancreatic exocrine cells to b-like cells. Cell Death Differ 2015; 22(7): 1117–1130

[29] Chanda S, Ang CE, Davila J, Pak C, Mall M, Lee QY, Ahlenius H, Jung SW, Südhof TC, Wernig M. Generation of induced neuronal cells by the single reprogramming factor ASCL1. Stem Cell Rep 2014; 3(2): 282–296

[30] Vierbuchen T, Ostermeier A, Pang ZP, Kokubu Y, Südhof TC, Wernig M. Direct conversion of fibroblasts to functional neurons by defined factors. Nature 2010; 463(7284): 1035–1041

[31] Sheng C, Zheng Q, Wu J, Xu Z, Wang L, Li W, Zhang H, Zhao XY, Liu L, Wang Z, Guo C, Wu HJ, Liu Z, Wang L, He S, Wang XJ, Chen Z, Zhou Q. Direct reprogramming of Sertoli cells into multipotent neural stem cells by defined factors. Cell Res 2012; 22(1): 208–218

[32] Marro S, Pang ZP, Yang N, Tsai MC, Qu K, Chang HY, Südhof TC, Wernig M. Direct lineage conversion of terminally differentiated hepatocytes to functional neurons. Cell Stem Cell 2011; 9(4): 374–382

[33] Ginsberg M, James D, Ding BS, Nolan D, Geng F, Butler JM, Schachterle W, Pulijaal VR, Mathew S, Chasen ST, Xiang J, Rosenwaks Z, Shido K, Elemento O, Rabbany SY, Rafii S. Efficient direct reprogramming of mature amniotic cells into endothelial cells by ETS factors and TGFb suppression. Cell 2012; 151(3): 559–575

[34] Huang P, He Z, Ji S, Sun H, Xiang D, Liu C, Hu Y, Wang X, Hui L. Induction of functional hepatocyte-like cells from mouse fibroblasts by defined factors. Nature 2011; 475(7356): 386–389

[35] Sekiya S, Suzuki A. Direct conversion of mouse fibroblasts to hepatocyte-like cells by defined factors. Nature 2011; 475(7356): 390–393

[36] Forsberg M, Carlén M, Meletis K, Yeung MS, Barnabé-Heider F, Persson MA, Aarum J, Frisén J. Efficient reprogramming of adult neural stem cells to monocytes by ectopic expression of a single gene. Proc Natl Acad Sci USA 2010; 107(33): 14657–14661

[37] Corti S, Nizzardo M, Simone C, Falcone M, Donadoni C, Salani S, Rizzo F, Nardini M, Riboldi G, Magri F, Zanetta C, Faravelli I, Bresolin N, Comi GP. Direct reprogramming of human astrocytes into neural stem cells and neurons. Exp Cell Res 2012; 318(13): 1528–1541

[38] Wang L, Wang L, Huang W, Su H, Xue Y, Su Z, Liao B, Wang H, Bao X, Qin D, He J, Wu W, So KF, Pan G, Pei D. Generation of integration-free neural progenitor cells from cells in human urine. Nat Methods 2013; 10(1): 84–89

[39] Efe JA, Hilcove S, Kim J, Zhou H, Ouyang K, Wang G, Chen J, Ding S. Conversion of mouse fibroblasts into cardiomyocytes using a direct reprogramming strategy. Nat Cell Biol 2011; 13(3): 215–222

[40] Kurian L, Sancho-Martinez I, Nivet E, Aguirre A, Moon K, Pendaries C, Volle-Challier C, Bono F, Herbert JM, Pulecio J, Xia Y, Li M, Montserrat N, Ruiz S, Dubova I, Rodriguez C, Denli AM, Boscolo FS, Thiagarajan RD, Gage FH, Loring JF, Laurent LC, Izpisua Belmonte JC. Conversion of human fibroblasts to angioblast-like progenitor cells. Nat Methods 2013; 10(1): 77–83

[41] Li J, Huang NF, Zou J, Laurent TJ, Lee JC, Okogbaa J, Cooke JP, Ding S. Conversion of human fibroblasts to functional endothelial cells by defined factors. Arterioscler Thromb Vasc Biol 2013; 33(6): 1366–1375

[42] Li K, Zhu S, Russ HA, 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(2): 228–236

[43] Zhu S, Rezvani M, Harbell J, Mattis AN, Wolfe AR, Benet LZ, Willenbring H, Ding S. Mouse liver repopulation with hepatocytes generated from human fibroblasts. Nature 2014; 508(7494): 93–97

[44] Lumelsky N. Small molecules convert fibroblasts into islet-like cells avoiding pluripotent state. Cell Metab 2014; 19(4): 551–552

[45] Qian L, Huang Y, Spencer CI, Foley A, Vedantham V, Liu L, Conway SJ, Fu JD, Srivastava D. In vivo reprogramming of murine cardiac fibroblasts into induced cardiomyocytes. Nature 2012; 485(7400): 593–598

[46] Ieda M, Fu JD, Delgado-Olguin P, Vedantham V, Hayashi Y, Bruneau BG, Srivastava D. Direct reprogramming of fibroblasts into functional cardiomyocytes by defined factors. Cell 2010; 142(3): 375–386

[47] Zhou Q, Brown J, Kanarek A, Rajagopal J, Melton DA. In vivo reprogramming of adult pancreatic exocrine cells to β-cells. Nature 2008; 455(7213): 627–632

[48] Banga A, Akinci E, Greder LV, Dutton JR, Slack JM.In vivo reprogramming of Sox9⁺ cells in the liver to insulin-secreting ducts. Proc Natl Acad Sci USA 2012; 109(38): 15336–15341

[49] Song K, Nam YJ, Luo X, Qi X, Tan W, Huang GN, Acharya A, Smith CL, Tallquist MD, Neilson EG, Hill JA, Bassel-Duby R, Olson EN. Heart repair by reprogramming non-myocytes with cardiac transcription factors. Nature 2012; 485(7400): 599–604

[50] Torper O, Pfisterer U, Wolf DA, Pereira M, Lau S, Jakobsson J, Björklund A, Grealish S, Parmar M. Generation of induced neurons via direct conversion in vivo. Proc Natl Acad Sci USA 2013; 110(17): 7038–7043

[51] Niu W, Zang T, Zou Y, Fang S, Smith DK, Bachoo R, Zhang CL. In vivo reprogramming of astrocytes to neuroblasts in the adult brain. Nat Cell Biol 2013; 15(10): 1164–1175

[52] Rouaux C, Arlotta P. Direct lineage reprogramming of post-mitotic callosal neurons into corticofugal neurons in vivo. Nat Cell Biol 2013; 15(2): 214–221

[53] Riddell J, Gazit R, Garrison BS, Guo G, Saadatpour A, Mandal PK, Ebina W, Volchkov P, Yuan GC, Orkin SH, Rossi DJ. Reprogramming committed murine blood cells to induced hematopoietic stem cells with defined factors. Cell 2014; 157(3): 549–564

[54] Ladewig J, Mertens J, Kesavan J, Doerr J, Poppe D, Glaue F, Herms S, Wernet P, Kögler G, Müller FJ, Koch P, Brüstle O. Small molecules enable highly efficient neuronal conversion of human fibroblasts. Nat Methods 2012; 9(6): 575–578

[55] Liu ML, Zang T, Zou Y, Chang JC, Gibson JR, Huber KM, Zhang CL. Small molecules enable neurogenin 2 to efficiently convert human fibroblasts into cholinergic neurons. Nat Commun 2013; 4: 2183

[56] Kim YJ, Lim H, Li Z, Oh Y, Kovlyagina I, Choi IY, Dong X, Lee G. Generation of multipotent induced neural crest by direct reprogramming of human postnatal fibroblasts with a single transcription factor. Cell Stem Cell 2014; 15(4): 497–506

[57] Zhu S, Ambasudhan R, Sun W, Kim HJ, Talantova M, Wang X, Zhang M, Zhang Y, Laurent T, Parker J, Kim HS, Zaremba JD, Saleem S, Sanz-Blasco S, Masliah E, McKercher SR, Cho YS, Lipton SA, Kim J, Ding S. Small molecules enable OCT4-mediated direct reprogramming into expandable human neural stem cells. Cell Res 2014; 24(1): 126–129

[58] Lee JH, Mitchell RR, McNicol JD, Shapovalova Z, Laronde S, Tanasijevic B, Milsom C, Casado F, Fiebig-Comyn A, Collins TJ, Singh KK, Bhatia M. Single transcription factor conversion of human blood fate to NPCs with CNS and PNS developmental capacity. Cell Reports 2015; 11(9): 1367–1376

[59] Wang H, Cao N, Spencer CI, 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 Reports 2014; 6(5): 951–960

[60] Ifkovits JL, Addis RC, Epstein JA, Gearhart JD. Inhibition of TGFb signaling increases direct conversion of fibroblasts to induced cardiomyocytes. PLoS ONE 2014; 9(2): e89678

[61] Boyd AW, Schrader JW. Derivation of macrophage-like lines from the pre-B lymphoma ABLS 8.1 using 5-azacytidine. Nature 1982; 297(5868): 691–693

[62] Cheng L, Hu W, Qiu B, Zhao J, Yu Y, Guan W, Wang M, Yang W, Pei G. Generation of neural progenitor cells by chemical cocktails and hypoxia. Cell Res 2014; 24(6): 665–679

[63] Han YC, Lim Y, Duffieldl MD, Li H, Liu J, Abdul Manaph NP, Yang M, Keating DJ, Zhou XF. Direct reprogramming of mouse fibroblasts to neural stem cells by small molecules. Stem Cells Int 2016; 2016: 4304916

[64] Mirakhori F, Zeynali B, Kiani S, Baharvand H. Brief azacytidine step allows the conversion of suspension human fibroblasts into neural progenitor-like cells. Cell J 2015; 17(1): 153–158

[65] 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(2): 204–212

[66] Dai P, Harada Y, Takamatsu T. Highly efficient direct conversion of human fibroblasts to neuronal cells by chemical compounds. J Clin Biochem Nutr 2015; 56(3): 166–170

[67] 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(2): 195–203

[68] Xu H, Wang Y, He Z, Yang H, Gao WQ. Direct conversion of mouse fibroblasts to GABAergic neurons with combined medium without the introduction of transcription factors or miRNAs. Cell Cycle 2015; 14(15): 2451–2460

[69] Cheng L, Gao L, Guan W, Mao J, Hu W, Qiu B, Zhao J, Yu Y, Pei G. Direct conversion of astrocytes into neuronal cells by drug cocktail. Cell Res 2015; 25(11): 1269–1272

[70] Zhang L, Yin JC, Yeh H, Ma NX, Lee G, Chen XA, Wang Y, Lin L, Chen L, Jin P, Wu GY, Chen G. Small molecules efficiently reprogram human astroglial cells into functional neurons. Cell Stem Cell 2015; 17(6): 735–747

[71] Zhang L, Li P, Hsu T, Aguilar HR, Frantz DE, Schneider JW, Bachoo RM, Hsieh J. Small-molecule blocks malignant astrocyte proliferation and induces neuronal gene expression. Differentiation 2011; 81(4): 233–242

[72] Ghasemi-Kasman M, Hajikaram M, Baharvand H, Javan M. MicroRNA-mediated in vitro and in vivo direct conversion of astrocytes to neuroblasts. PLoS ONE 2015; 10(6): e0127878

[73] Thoma EC, Merkl C, Heckel T, Haab R, Knoflach F, Nowaczyk C, Flint N, Jagasia R, Jensen Zoffmann S, Truong HH, Petitjean P, Jessberger S, Graf M, Iacone R. Chemical conversion of human fibroblasts into functional Schwann cells. Stem Cell Rep 2014; 3(4): 539–547

[74] Park G, Yoon BS, Kim YS, Choi SC, Moon JH, Kwon S, Hwang J, Yun W, Kim JH, Park CY, Lim DS, Kim YI, Oh CH, You S. Conversion of mouse fibroblasts into cardiomyocyte-like cells using small molecule treatments. Biomaterials 2015; 54: 201–212

[75] Fu Y, Huang C, Xu X, Gu H, Ye Y, Jiang C, Qiu Z, Xie X. Direct reprogramming of mouse fibroblasts into cardiomyocytes with chemical cocktails. Cell Res 2015; 25(9): 1013–1024

[76] Pennarossa G, Maffei S, Campagnol M, Tarantini L, Gandolfi F, Brevini TA. Brief demethylation step allows the conversion of adult human skin fibroblasts into insulin-secreting cells. Proc Natl Acad Sci USA 2013; 110(22): 8948–8953

[77] Pereyra-Bonnet F, Gimeno ML, Argumedo NR, Ielpi M, Cardozo JA, Giménez CA, Hyon SH, Balzaretti M, Loresi M, Fainstein-Day P, Litwak LE, Argibay PF. Skin fibroblasts from patients with type 1 diabetes (T1D) can be chemically transdifferentiated into insulin-expressing clusters: a transgene-free approach. PLoS ONE 2014; 9(6): e100369

[78] Kanoh Y, Tomotsune D, Shirasawa S, Yoshie S, Ichikawa H, Yokoyama T, Mae S, Ito J, Mizuguchi M, Matsumoto K, Yue F, Sasaki K. In vitro transdifferentiation of HepG2 cells to pancreatic-like cells by CCl₄, D-galactosamine, and ZnCl₂. Pancreas 2011; 40(8): 1245–1252

[79] Korac A, Cakic-Milosevic M, Ukropina M, Grubic M, Micunovic K, Petrovic V, Buzadzic B, Jankovic A, Vasilijevic A, Korac B. White adipocytes transdifferentiation into brown adipocytes induced by triiodothyronine. In: EMC 2008 14th European Microscopy Congress 1–5 September 2008, Aachen, Germany. Springer-Verlag Berlin Heidelberg, 2008:123–124

[80] Moisan A, Lee YK, Zhang JD, Hudak CS, Meyer CA, Prummer M, Zoffmann S, Truong HH, Ebeling M, Kiialainen A, Gérard R, Xia F, Schinzel RT, Amrein KE, Cowan CA. White-to-brown metabolic conversion of human adipocytes by JAK inhibition. Nat Cell Biol 2015; 17(1): 57–67

[81] Ubil E, Duan J, Pillai IC, Rosa-Garrido M, Wu Y, Bargiacchi F, Lu Y, Stanbouly S, Huang J, Rojas M, Vondriska TM, Stefani E, Deb A. Mesenchymal-endothelial transition contributes to cardiac neovascularization. Nature 2014; 514(7524): 585–590

[82] Sayed N, Wong WT, Ospino F, Meng S, Lee J, Jha A, Dexheimer P, Aronow BJ, Cooke JP. Transdifferentiation of human fibroblasts to endothelial cells: role of innate immunity. Circulation 2015; 131(3): 300–309

[83] Brevini TA, Pennarossa G, Rahman MM, Paffoni A, Antonini S, Ragni G, deEguileor M, Tettamanti G, Gandolfi F. Morphological and molecular changes of human granulosa cells exposed to 5-azacytidine and addressed toward muscular differentiation. Stem Cell Rev 2014; 10(5): 633–642

[84] Nie T, Hui X, Gao X, Nie B, Mao L, Tang X, Yuan R, Li K, Li P, Xu A, Liu P, Ding S, Han W, Cooper GJ, Wu D. Conversion of non-adipogenic fibroblasts into adipocytes by a defined hormone mixture. Biochem J 2015; 467(3): 487–494

[85] Warren L, Manos PD, Ahfeldt T, Loh YH, Li H, Lau F, Ebina W, Mandal PK, Smith ZD, Meissner A, Daley GQ, Brack AS, Collins JJ, Cowan C, Schlaeger TM, Rossi DJ. Highly efficient reprogramming to pluripotency and directed differentiation of human cells with synthetic modified mRNA. Cell Stem Cell 2010; 7(5): 618–630

[86] 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 small-molecule compounds. Science 2013; 341(6146): 651–654

[87] Zhao Y, Zhao T, Guan J, Zhang X, Fu Y, Ye J, Zhu J, Meng G, Ge J, Yang S, Cheng L, Du Y, Zhao C, Wang T, Su L, Yang W, Deng H. A XEN-like state bridges somatic cells to pluripotency during chemical reprogramming. Cell 2015; 163(7): 1678–1691

[88] Ye J, Ge J, Zhang X, Cheng L, Zhang Z, He S, Wang Y, Lin H, Yang W, Liu J, Zhao Y, Deng H. Pluripotent stem cells induced from mouse neural stem cells and small intestinal epithelial cells by small molecule compounds. Cell Res 2016; 26(1): 34–45

[89] Long Y, Wang M, Gu H, Xie X. Bromodeoxyuridine promotes full-chemical induction of mouse pluripotent stem cells. Cell Res 2015; 25(10): 1171–1174

[90] Li W, Li K, Wei W, Ding S. Chemical approaches to stem cell biology and therapeutics. Cell Stem Cell 2013; 13(3): 270–283

[91] Lin T, Wu S. Reprogramming with small molecules instead of exogenous transcription factors. Stem Cells Int 2015; 2015: 794632

[92] Chen T, Yuan D, Wei B, Jiang J, Kang J, Ling K, Gu Y, Li J, Xiao L, Pei G. E-cadherin-mediated cell-cell contact is critical for induced pluripotent stem cell generation. Stem Cells 2010; 28(8): 1315–1325

[93] Feng B, Jiang J, Kraus P, Ng JH, Heng JC, Chan YS, Yaw LP, Zhang W, Loh YH, Han J, Vega VB, Cacheux-Rataboul V, Lim B, Lufkin T, Ng HH. Reprogramming of fibroblasts into induced pluripotent stem cells with orphan nuclear receptor Esrrb. Nat Cell Biol 2009; 11(2): 197–203

[94] Cheng L. Novel strategy for treating neural disease. Sci China Life Sci 2014; 57(9): 947–948

RIGHTS & PERMISSIONS

Higher Education Press and Springer-Verlag Berlin Heidelberg