Chemical transdifferentiation: closer to regenerative medicine · 2019. 9. 24. · epigenetic modiﬁcation. In this review, we brieﬂy examine the history of cell ... factors [42]

Chemical transdifferentiation: closer to regenerative medicine

Aining Xu, Lin Cheng (✉)

State Key Laboratory of Medical Genomics, Shanghai Institute of Hematology, Ruijin Hospital Affiliated to Shanghai Jiao Tong UniversitySchool of Medicine, Shanghai 200025, China

© Higher Education Press and Springer-Verlag Berlin Heidelberg 2016

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

Keywords cell therapy; cell transdifferentiation; chemical compounds; small molecules; tissue regeneration

Introduction

Cell differentiation, which produces functionally maturecells, is one of the most crucial events in the developmentof an organism. However, committed cells are not “frozen”as they develop. To generate desired cell types, the routinestability of the original cells could be disturbed and thecells that act like “marbles” in Waddington’s landscape rollacross the “valley” [1]. According to cross-antagonisms ina cascading landscape of unstable and stable cell states,transcription factors (TFs) play a crucial role in controllingcell fate. Over the past decades, novel technologies andscreening approaches have been applied to select appro-priate candidate TFs for lineage transdifferentiation [2].Although significant developments were achieved in thisfield, gene modification has been an obstacle in achievingthe goal of future regenerative medicine because of itssafety concerns.Small molecules are chemical compounds with low

molecular weight; these molecules exhibit crucial advan-tages and are a potential alternative in manipulating cellfate changes [3]. The biological effects of small molecules

are typically rapid. They do not bind covalently to theirtarget protein. Thus, they may be used reversibly. Smallmolecules are more stable and cost-effective than synthe-sized proteins and mRNAs, growth factors or cytokines.They are more easily synthesized, preserved, and standar-dized. Their effects could be improved by fine-tuning theirstructure, concentration, and combination. Small mole-cules could easily control signaling pathways andepigenetic modification.In this review, we briefly examine the history of cell

transdifferentiation and provide an overview on smallmolecule-induced transdifferentiation, which we refer to aschemical transdifferentiation. We also discuss its potentialclinical applications in regenerative medicine.

Nature of cell transdifferentiation

Cell transdifferentiation refers to the lineage switches fromone type of differentiated cells to another cell type.Evidences of transdifferentiation in nature have beendocumented. One of the well-known examples is the lensregeneration of adult newts [4], which was first observed asearly as 1895. Once the lens of adult newts are impaired,pigmented epithelial cells (PECs) from the dorsal irisinitiate transdifferentiation and reproduce the missing

REVIEW

Received January 26, 2016; accepted March 21, 2016

Correspondence: [email protected]

Front. Med. 2016, 10(2): 152–165DOI 10.1007/s11684-016-0445-z

tissue. This process begins with dedifferentiation, whichindicates that PECs lose their properties, such asmorphology and pigmentation. In this process, hyper-phosphorylated retinoblastoma protein loses its activityand dissociates from E2F, which allows the cells to re-enterthe cell cycle and proliferate to create a new lens vesicle.However, proliferation seems to be unnecessary becausetreatment with a cyclin-dependent kinase inhibitor does notcompletely stop the transdifferentiation [5]. The differ-entiation of the primary lens fiber manifests as thethickening of the internal layer of the lens vesicle andsynthesis of crystallins. Moreover, natural transdifferentia-tion also occurs in salamanders, Xenopus, jellyfishes [4],and worms [6]. Transdifferentiation is a rare event innature. Thus, the underlining mechanisms of this naturalprocess remain unknown. Multiple signaling pathways areinvolved in lens regeneration, including FGF, retinoic acid,TGFβ, Wnt, and hedgehog pathways (reviewed by Henry[7]). Interestingly, some members of the hedgehog path-way, such as Shh, Ihh, and Ptc2, which are expressed inembryonic lenses, can be detected again in the regeneratinglens [8]. Maki et al. demonstrated that epigeneticmodifiers, such as histone deacetylases (HDACs), arealso upregulated during this process [9].

Exogenous gene delivery carries forwardtransdifferentiation

The importance of TFs during lineage transdifferentiationwas demonstrated in late 1980s when the forced expressionof MyoD was determined to induce myotube formationfrom a fibroblast cell line [10,11]. However, this cell fateconversion is considered incomplete because the acquiredphenotype depends on the sustained overexpression ofMyoD. Over the next two decades, a few studiesdemonstrated that related lineages within the blood [12],endoderm [13], and nervous systems [14] can be convertedinto other cell types by overexpressing exogenous genes.In 2006, the advent of induced pluripotent stem cells(iPSCs) [15], which possibly transitioned between devel-opmentally distant cell types using a combination of fourpluripotent TFs, revitalized the field of lineage reprogram-ming and motivated researchers to determine sets of TFsthat may be crucial for cell transdifferentiation. To date,many studies have shown that in vitro transdifferentiationof cells in the same germ layers can be directed both inmice and humans by defined factors (reviewed by Deng[2]), which play key roles in maintaining and regulatingtarget cell functions. For the ectoderm, astrocytes can beconverted into GABAergic or glutamatergic neurons [16]and pericyte-derived cells of the adult human brain intoneurons [17]. For the mesoderm, fibroblasts were success-fully induced into functional adipocytes [18], cardiomyo-cytes [19,20], chondrocytes [21], endothelial cells [22],

hemogenic endothelial-like precursor cells [23], hemato-poietic progenitor cells [24,25], and macrophages [26].Proximal tubule cell line HK-2 is switched into a nephronprogenitor [27]. For the endoderm, pancreatic exocrinecells were directly changed into pancreatic β-like cells[28]. Further developments were achieved in remodelingcell types across distinct germ layers. Examples of thesecell types are neurons generated from fibroblasts [29–31]or hepatocytes [32], endothelial cells from amniotic cells[33], hepatocytes from fibroblasts [34,35], and monocytesfrom neural stem cells [36]. The transient introduction ofpluripotency factors plays an indirect role in transdiffer-entiation, including the generation of neural stem orprogenitor cells [37,38], cardiomyocytes [39], angioblast-like progenitor cells [40], endothelial cells [41], pancreaticlineages [42], and hepatocytes [43]. This predefined stepwas hypothesized to initiate the removal of the epigeneticmemory of the starting cells [44], which may requirefurther steps to improve the functional maturation ofconverted cells.Transdifferentiation in vivo, which maximizes the native

physiological niche, reduces the concerns related to in vitroculture and cell transplantation. Compared with cellconversion in vitro, higher conversion efficiency can beachieved and desirable cell types with better function canbe generated in vivo, which is probably attributed to theessential factors that the in situ niche provided. Forexample, in the induction of cardiomyocyte-like cells,murine cardiac fibroblasts could result in the robustgeneration of functional cardiomyocyte-like cells by theoverexpression of TFs (Gata4, Mef2c, and Tbx5) in vivo[45], whereas the induction in vitrowith the same TFs onlygenerated inefficient ones [46]. Several other in vivotransdifferentiation examples include pancreatic exocrinecells remodeled into β-cells [47], Sox9+ cells in liver intoinsulin-secreting ducts [48], non-myocytes into cardio-myocyte-like cells [49], astrocytes into neurons [50] orneuroblasts [51], embryonic and early postnatal callosalprojection neurons in layer II/III into corticofugal projec-tion neurons in layer V/VI [52], and pre/pro-B cells intohematopoietic stem or progenitor cells [53].

Current developments of chemicalcompounds promoting celltransdifferentiation

Small molecules, which reduce the safety concerns aboutgenetic manipulation for future clinical applications, hasgained increasing attention of researchers in regenerativemedicine. Small molecules can partially regulate geneexpression through four classes of mechanisms: signalingpathway modulators, which activate or repress componentsof signal transduction to regulate downstream transcriptionactivity; modulators of epigenetic proteins, which regulate

Aining Xu and Lin Cheng 153

the activity of epigenetic complexes; metabolic regulators,which adjust the cell state and shift the balance ofmetabolites that serve as ligands for proteins and cofactorsfor epigenetic proteins; and nuclear receptor agonists andantagonists, which directly modulate transcription byregulating the activity of nuclear receptors. To developsimpler and safer transdifferentiation methods for cell-based therapeutic applications, researchers aim to identifysmall molecules to replace exogenous genes, as previouslyreported.

Small molecules facilitating exogenousgene-driven transdifferentiation

Small molecules in chemical transdifferentiation starts as abooster of TF-based transdifferentiation. Increasing con-version efficiency is a pivotal issue in generating sufficientneurons because neurons are post-mitotic. Ladewig et al.established a minimalist approach to generate neuronsfrom fibroblasts by combining two neuronal specifiers(Ascl1 and Ngn2) with the small molecule-based inhibitionof glycogen synthase kinase-3β (GSK-3β) and SMADsignaling [54]. The results showed that the products oftransdifferentiation exceeded 200% (cell yield is calculatedas the percentage of ending cells in relation to the initialnumber of plated cells) and the final neuronal puritiesreached > 80%. Moreover, based on the previous studies,Liu et al. demonstrated that two other small molecules(forskolin and dorsomorphin) enable the TF Ngn2 toconvert human fetal lung fibroblasts into cholinergicneurons with significantly higher purity (>90%) andefficiency (up to 99% of Ngn2-expressing cells) [55]. Toobtain the functional neural crest from human postnatalfibroblasts, Kim et al. only used one single TF Sox10combined with environmental cues covering WNT acti-vator (CHIR99021 or BMP4) [56]. The generated inducedneural crest (iNC) can migrate into or be retained in theexpected regions in vivo. Approximately 40% of singleiNC clones could produce main downstream differentia-tion lineages (neurons, glia, smooth muscle cells, melano-cytes, chondrocytes, and adipocytes), which is comparableto 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 thereprogramming of adult human dermal fibroblasts trans-duced with OCT4 alone [57]. Three candidates wereidentified, including LPA (a phospholipid derivative),rolipram (a PDE4 inhibitor), and SP600125 (a JNKinhibitor). Furthermore, the researchers obtained expand-able human neural stem cells using a combination of Oct4overexpression and chemical cocktails. More recently, Leeet al. altered the fate of human blood progenitor into neural

stem cells with OCT4 alone [58]. The conversion wasfacilitated by inhibiting both the SMAD and GSK-3signaling pathways to overcome the restrictions on neuralfate conversion.In a similar study, mouse fibroblasts, which were

initially transduced with Oct4 alone, were exposed to acocktail of lineage-specific signals, including SB431542(ALK4/5/7 inhibitor), CHIR99021 (GSK3 inhibitor),parnate (LSD1/KDM1 inhibitor), and forskolin (adenylylcyclase activator), to achieve transdifferentiation intocardiac lineage [59]. Another study that was based on areporter system demonstrated that treatment withSB431542 in conjunction of inducible expression of theTFs enhances the conversion of mouse embryonicfibroblasts (MEFs) to induced cardiomyocytes [60]. Inthe pluripotency factor-based transdifferentiationdescribed above, adding JAK inhibitor to the late stageof inducting procession, which antagonizes an essentialpathway for embryonic stem cell maintenance (the LIF-STAT3 pathway), could suppress the establishment ofpluripotency while promoting the generation of epigeneti-cally 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 convertedinto proliferation competent definitive endoderm-like cells,pancreatic progenitor-like cells, and insulin-producing βand other pancreatic-like cells under a conditioned mediumwith diverse combinations of small molecules and solublefactors [42].

Chemical transdifferentiation withcomplete small molecules

Lineage reprogramming through the complete chemicalinduction medium without integrating exogenous genes isthe ultimate goal for generating target cells. Directtransdifferentiation with few chemicals in tumors wasachieved as early as 1982 where the demethylating agent5-Aza remodeled pre-B lymphoma into cells that expressmarkers of macrophages [61]. In the past five years,investigations about lineage transdifferentiation within thesame germ layer or across different germ layers with smallmolecules was carried out (Table 1).

Toward neural cells

Our previous study demonstrated that functional neuralprogenitor cells (NPCs) can be acquired from MEFs,mouse tail-tip fibroblasts, and human urinary cells usingchemical cocktails under hypoxia [62]. The chemicalcocktails were called VCR, including VPA, CHIR99021,and Repsox, which inhibit HDACs, GSK-3, and TGFβkinase pathways, respectively. NPCs were finally

154 Chemical transdifferentiation: closer to regenerative medicine

generated under lineage-specific culture conditions bypassing through intermediate compact cell colonies wherethe expression of Sox2 significantly increased. Thesechemically induced NPCs (ciNPCs) can proliferate, self-renew and exhibit similar transcription profiles as mousebrain-derived NPCs. ciNPCs can also differentiate intodifferent neural lineage cells both in vitro and in vivo underdefined culture mediums. Moreover, combining thealternative inhibitors of these signaling pathways alsofacilitates the transition from MEFs to NPCs. Withouthypoxia, Han et al. utilized more chemicals based on VCRto induce mouse fibroblasts into neural stem cells [63]. Thechemical cocktail includes A-83-01, CHIR99021, VPA,BIX01294, RG108, PD0325901, and vitamin C. Bahar-vand et al. utilized a suspension culture system in thepresence of 5-Aza instead of the traditional monolayerculture 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 coverthuman fibroblasts into neurons (hciNs) directly, thusbypassing the NPC state [65]. Importantly, hciNs thatwere derived from the familial fibroblasts of an Alzhei-mer’s disease patient by a chemical cocktail exhibitabnormal Aβ production. Neurons can also be inducedfrom human fibroblasts by an alternative chemical cocktailcontaining SB431542, CHIR99021, forskolin, pifithrin-α,LDN193189, and PD0325901, which accelerate mesench-ymal-to-epithelial transition, promote cell reprogramming,and facilitate neuronal conversion [66]. For starting cellsfrom mouse, Li et al. identified five chemical compoundsrobustly converting mouse fibroblasts into TUJ1-positiveneurons [67]. The chemical cocktail includes SB43152,CHIR99201, forskolin, I-BET151, and ISX9. A mechan-ical study demonstrates that I-BET151 disrupts thefibroblast-specific program and ISX9 activates neuron-specific genes. For more specific-type of neuron induction,

Table 1 Cell transdifferentiation and cell reprogramming enabled by complete chemical compounds

In vitro orin vivo

SpeciesCell types

Chemical compounds ReferencesStarting cells Ending cells

In vitro Mouse FibroblastsNeural stemcells

Intestinal epithelialcells

Pluropotent stem cells Repsox, CHIR99021, Forskolin, VPA, DZNep& PD0325901 with or w/o BrdU

[86–89]

In vitro MouseHuman

FibroblastsUrinal cells

Neural progenitor cells Repsox, CHIR99021 & VPA [62]

In vitro Human Fibroblasts Neural progenitor cells 5-Aza [64]

In vitro Mouse Fibroblasts Neural stem cells A-83-01, CHIR99021, VPA, BIX01294,RG108, PD0325901 & vitamin C

[63]

In vitro Mouse Fibroblasts Neurons SB431542, CHIR99021, Forskolin, ISX9& I-BET151

[67]

In vitro Mouse Fibroblasts Neurons SB431542 & ATRA [68]

In vitro Human Fibroblasts Neurons SB431542, CHIR99021, Forskolin,Pifithrin-α, LDN193189 & PD0325901

[66]

In vitro Human Fibroblasts Neurons Repsox, CHIR99021, Forskolin, VPA,SP600125, Go 6983 & Y-27632

[65]

In vitro Mouse Astrocytes Neurons Repsox & VPA [69]

In vivo Mouse Glial cells Neurons VPA [72]

In vitro Human Astrocytes Neurons SB431542, CHIR99021, VPA, LDN193189,DAPT, Tzv, TTNPB, SAG & Purmo

[70]

In vitro Human Fibroblasts Schwann cells SB431542, CP21, VPA & CB [73]

In vitro Mouse Fibroblasts Cardiomyocytes Repsox, CHIR99021, Forskolin, VPA, Parnate& TTNPB

[75]

In vitro Mouse Fibroblasts Cardiomyocytes A-83-01, CHIR99021, Forskolin, SC1 & (�)-BayK 8644

[74]

In vitro Human Fibroblasts Insulin-secreting cells 5-Aza [76]

In vitro Human Fibroblasts Insulin-secreting cells Nicotinamide [77]

In vitro Human Fibroblasts Endothelial cells Poly(I:C) [82]

In vitroIn vivo

Mouse Fibroblasts Endothelial cells RITA [81]

In vitro Human Granulosa cells Muscle cells 5-Aza [83]


GABAergic neurons were induced from mouse fibroblastscultured in conditioned medium from neurotrophin-3modified 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 ofthe delivery strategy and the retained epigenetic memory ofstarting fibroblasts. Thus, astrocytes are considered theideal starting candidate cell type for generating newneurons because of their proximity in lineage distance toneurons and their ability to proliferate after brain damage.Small molecules identified in the cell-converting assay invitro may be applied in vivo to enable the transdifferentia-tion of resident astrocytes into neurons. To this point, weidentified that the chemical cocktail composed of VPA andTranilast can convert mouse astrocytes into neurons invitro [69]. Human astroglial cells can also be repro-grammed efficiently into functional neurons under thetreatment of small molecules (SB431542, CHIR99021,VPA, LDN193189, DAPT, Tzv, TTNPB, SAG, andPurmo), which mediate epigenetic and transcriptionalregulation [70]. In both cases, the small molecules activatepro-neural TFs NueroD1 and NeuroG2. Moreover, onlyHDAC inhibitors (Trichostatin A or VPA) can directlyinduce malignant astrocytes into neuronal cells [71]. For invivo studies, VPA alone can convert glial cells into neuronsafter 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 peripheralnervous system, were also generated from humanfibroblasts with the stepwise induction by Noggin peptideswith small molecules, including SB431542, VPA, CP21,and compound B, which is the potent neural-inducingsmall molecule identified through high-throughput screen-ing [73].

Toward cardiomyocytes

To determine the combination of small molecules thatcould be used to induce pluripotent cells, You et al.selected 12 candidates that are functional substitutes forTFs or enhancers for iPSC induction [74]. The researchersfound that cTnT+ cells and not iPSCs were produced fromthe combination of small molecules. The gene expression,epigenetic status of cardiac-specific genes, and subcellularstructure of these induced cells were similar to that ofprimary cardiomyocytes. Five small molecules in thecocktail are abbreviated as “FASCB,” namely, forskolin,A-83-01, SC1, CHIR99021, and ( � )-Bay K 8644.Meanwhile, Xie’s group found that only small moleculesinducing chemically induced pluripotent cells (CiPSCs)and a specific culturing condition can induce MEFs andtail-tip fibroblasts into cardiomyocytes, which was con-firmed by lineage tracing using transgenic mouse [75].

Toward pancreatic cells

A total of 35% � 8.9% of adult human skin fibroblaststhat were exposed to 5-Aza for 18 h followed by a three-step protocol were successfully induced into endocrinepancreatic cells. These artificial cells shared manycharacteristics with primary pancreatic cells, such asepithelial morphology and the ability to secrete insulin inresponse to a physiological glucose challenge in vitro [76].Argibay’s group established a novel approach to transdif-ferentiate skin fibroblasts from type 1 diabetes patients intoinsulin-expressing clusters with a stepwise culture mediumincluding nicotinamide and exedin-4 [77]. Sasaki et al.turned a “straw” cell line of human hepatoma HepG2 cellsinto “gold” pancreatic-like cells using small moleculesCCl4 and D-galactosamine, which are severely hepatotoxicand ZnCl2, an agent that facilitates pancreatic developmentand function [78].

Toward adipocytes

The uncontrolled expansion of white adipocyte tissue canlead to obesity, whereas another type of fat brownadipocyte tissue serves as an opposite physiologicalfunction to dissipate energy. To increase the number oractivity of brown-like adipocytes in white adipose depots,two promising approaches were developed. One approachidentified one of the thyroid hormones triiodothyronine[79], and the other approach combined inhibitors of Januskinase (JAK) R406 and Tofacitinib [80] to promote awhite-to-brown metabolic conversion. These discoveriescould establish the foundation to further prevent diet-induced obesity and reduce the incidence and severity oftype 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 activatorRITA. 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 driveendothelial cell specification, such as exogenous endothe-lial cell growth factors, transdifferentiated human fibro-blasts into endothelial cells [82].The field of chemical transdifferentiation has gained

many achievements. However, the molecular mechanismof small molecules during transdifferentiation is yet to beexplained. Interestingly, small molecules targetingHDACs, GSK3, and TGFβ participate in mostly reportedchemical transdifferentiation (Table 2). Treatment with thesame small molecule could lead to extremely different celltypes. For example, 5-Aza that is treated with various


Table 2 Chemical compounds reported in chemical transdifferentiationCompound Alternative name Function Induced cells Structure

Repsox E616452 Potent and selective inhibitorof TGFβRI

Pluripotent stem cells, neuralprogenitor cells, neurons,cardiomyocytes

SB431542 301836-41-9 Potent, selective inhibitor ofTGFβRI, ALK4 and ALK7

Neurons, Schwann cells

A-83-01 909910-43-6 Selective inhibitor of TGFβRI,ALK4 and ALK7

Neural stem cells,cardiomyocytes

LDN193189 1062368-24-4 Highly selective antagonist ofBMP receptor isotypesALK2 and ALK3

Neurons

CHIR99021 CT99021 Highly selective GSK3inhibitor

Pluripotent stem cells, neuralprogenitor cells, neuralstem cells, neurons,cardiomyocytes

CP21 CP21R7 GSK3β inhibitor Schwann cells

Forskolin Colforsin Potent activator of theadenylate cyclase systemand the biosynthesis ofcyclic AMP

Pluripotent stem cells, neurons,cardiomyocytes

VPA Sodium valproate Histone deacetylase inhibitor Pluripotent stem cells, neuralprogenitor cells, neural stemcells, neurons, cardiomyocytes,Schwann cells


(Continued)Compound Alternative name Function Induced cells Structure

5-Aza 5-azacytidine DNA methyltransferaseinhibitor

Neural progenitor cells,insulin-secreting cells,muscle cells

RG108 N-phthalyl-L-tryptophan

DNA methyltransferaseinhibitor

Neural stem cells

BIX01294 935693-62-2 Histone lysinemethyltransferaseinhibitor

Neural stem cells

Parnate Tranylcypromine Inhibitor of lysine-specificdemethylase andmonoamine oxidase,also inhibits histonedemethylation

Pluripotent stem cells,cardiomyocytes

DZNep 3-deazaneplanocin S-adenosylhomocysteinehydrolase inhibitor andhistone methyltransferaseEZH2 inhibitor

Pluripotent stem cells

PD0325901 391210-10-9 Potent MEK1 and MEK2inhibitor

Pluripotent stem cells,neurons, neuron stem cells

Y-27632 N-(4-pyridyl)-4-(1-aminoethyl)cyclohexanecarb-oxamide

Selective p160ROCK inhibitor,also inhibits PRK2

Neurons

Tzv Thiazovivin Selective ROCK inhibitor Neurons

Pifithrin-α PFTα Inhibitor of p53 Neurons



RITA NSC 652287 MDM2-p53 interactioninhibitor

Endothelial cells

SAG Smoothenedagonist

Potent Smoothened receptoragonist; activates theHedgehog signalingpathway

Neurons

Purmo Purmorphamine Smoothened receptoragonist

Neurons

Poly(I:C) Polyinosinic-polycytidylicacid

Toll-like receptor 3 agonist Endothelial cells

SP600125 1,9-pyrazoloanthrone Selective Jun N-terminalkinase inhibitor

Neurons

ISX9 N-cyclopropyl-5-(2-thienyl)-3-isoxazolecar-boxamide

Neurogenic agent Neurons

I-BET151 GSK1210151A BET bromodomaininhibitor

Neurons

ATRA Retinoic acid Retinoic acid receptor agonist Neurons

Nicotinamide Vitamin B3 Inhibitor of poly(ADP-ribose)polymerase enzymes

NAD+precursor

Insulin-secretingcells



Go 6983 Goe 6983 Broad spectrum proteinkinase C inhibitor

Neurons

SC1 Pluripotin Dual inhibitor of extracellularsignal-regulated kinase 1and RasGAP

Cardiomyocytes

(�)-Bay K8644

71145-03-4 Ca2+ channel activator(L-type)

Cardiomyocytes

TTNPB Arotinoic acid Analog of retinoic acid Cardiomyocytes

BrdU 5-bromo-2-deoxyuridine

Synthetic thymidine analog Pluripotent stem cells

CB Compound-B Promote proliferation ofneural stem cells

Schwann cells

DAPT g-secretaseinhibitor IX

g-secretase inhibitor Neurons

Vitamin C L-ascorbic acid Antioxidant Neural stem cells


concentrations for a certain time can convert humanfibroblasts into functional NPCs or pancreatic cells ormuscle cells [83]. 5-Aza, a DNA methyltransferaseinhibitor, may cause chromatin de-condensation andinduce a short “dedifferentiation” state. Under definedculture conditions, the “dedifferentiated” intermediate cellsdifferentiate into mature cells. Another epigenetic reg-ulator, HDAC inhibitors, which change the chromatinstate, may play a similar role. TGFβ inhibitors mayundergo regulating transition between mesenchymal andepithelial state to promote cell reprogramming and helpconvert cell fate. However, the reason why GSK3 inhibitor,which probably activates WNT signaling, plays animportant role in these transitions need to be furtherinvestigated.

Perspectives

Although cell transdifferentiation is not an entirely newconcept, it has many potentials that have yet to bedetermined. In contrast to the differentiation fromembryonic stem cells or iPSCs, different functional celltypes could be more conveniently and more effectivelyderived from somatic cells, which are more easilyaccessible, in cell transdifferentiation. Moreover, directlineage transdifferentiation bypasses the potential tumor-igenicity, which may be caused by incomplete pluripotentcell differentiation. Through the overexpression of theexogenous genes, which is the most popular strategy in theearly stages, a large number of desired cell types weregenerated in both mouse and human cells as mentionedabove. To address gene manipulation and safety issues,researchers applied modified RNAs, as well as hormonemixtures, as an alternative strategy to induce lineageconversion [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 fromMEFs, mouse neonatal fibroblasts, mouse adult fibroblasts,adipose-derived stem cells, neural stem cells, and intestinalepithelial cells using a cocktail of small moleculesVC6TFZ (VPA, CHIR99021, 616452, tranylcypromine,forskolin, and DZNep) [86–88]. Xie et al. determined thatthe full chemical-induced cell reprogramming could besignificantly enhanced by BrdU [89]. Considering thesignificant roles of small molecules in maintaining anddifferentiating stem cells [90] and reprogramming cells[91], more studies sought to harness the potential of smallmolecules on cell transdifferentiation. At present, smallmolecules are known not only as facilitating factors forexogenous genes but also as master factors of lineagetransdifferentiation. Nevertheless, high-throughput andhigh-definition screening technology must be explored todetermine small-molecule candidates to replace exogenousgenes and achieve in complete chemical transdifferentia-tion. In addition, directly targeting certain TFs by smallmolecule compounds is difficult. Previous reports pointedout that E-cadherin [92] and orphan nuclear receptor Esrrb[93] can replace TFs in somatic cell reprogramming. Thesefindings demonstrate the potential of small molecules intargeting nuclear receptors to achieve lineage conversion.To some extent, stem or progenitor cells that can be

grafted are more desirable. Thus, improving the generationor enhancing their functions using small molecule-basedapproaches must be thoroughly examined in the future.Additionally, by maximizing in vivo niche, chemicaltransdifferentiation in vivo may provide broader prospectsfor clinical applications [94]. Small molecules havelimitations, such as unexpected side effects, which shouldbe considered in clinical applications.

Fig. 1 Chemical transdifferentiation leads to tissue regeneration. Transplantation of chemically induced cells from easily accessible cells by smallmolecules in vitro may help tissue regeneration after injury, which is closer to clinical translation for cell-based therapy. Administration of drugsidentified in in vitro assay may directly convert resident cells around locally damaged sites into desirable cells in vivo and improve functional recoveryof injured tissue or organs, which is one of the terminal goals for regenerative medicine.


The field of regenerative medicine has undergonebreakthroughs and significant growth in recent years.Although chemical transdifferentiation still needs to bethoroughly analyzed, this approach is certainly a potentialmethod in cell-based regeneration (Fig.1).

Acknowledgements

This work was supported by the National Basic Research Program ofChina (No. 31301129), Shanghai Rising-Star Program (No.16QA1402800), and the Ministry of Science and Technology (No.

2013CB966801).

Compliance with ethics guidelines

Aining Xu and Lin Cheng declare that they have no financialconflicts of interest. This manuscript is a review article and does notinvolve a research protocol requiring approval by a relevant

institutional review board or ethics committee.

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