Review Article Reprogramming with Small Molecules instead ...downloads.hindawi.com/journals/sci/2015/794632.pdf · Reprogramming with Small Molecules instead of Exogenous Transcription

Review ArticleReprogramming with Small Molecules instead ofExogenous Transcription Factors

Tongxiang Lin12 and Shouhai Wu1

1Guangzhou University of Chinese Medicine The Second Affiliated Hospital (Guangdong Provincial Hospital of Chinese Medicine)55 Neihuan W Road Higher Education Mega Center Guangzhou Guangdong 510006 China2Fujian Agriculture and Forestry University Stem Cell Research Center 15 Shangxiadian Road Cangshan DistrictFuzhou Fujian 350002 China

Correspondence should be addressed to Tongxiang Lin lintx69yahoocom

Received 29 November 2014 Revised 3 March 2015 Accepted 9 March 2015

Academic Editor Amanda C LaRue

Copyright copy 2015 T Lin and S Wu This is an open access article distributed under the Creative Commons Attribution Licensewhich permits unrestricted use distribution and reproduction in any medium provided the original work is properly cited

Induced pluripotent stem cells (iPSCs) could be employed in the creation of patient-specific stem cells which could subsequently beused in various basic and clinical applications However current iPSC methodologies present significant hidden risks with respectto genetic mutations and abnormal expression which are a barrier in realizing the full potential of iPSCs A chemical approach isthought to be a promising strategy for safety and efficiency of iPSC generation Many small molecules have been identified that canbe used in place of exogenous transcription factors and significantly improve iPSC reprogramming efficiency and quality Recentstudies have shown that the use of small molecules results in the generation of chemically induced pluripotent stem cells frommouse embryonic fibroblast cells These studies might lead to new areas of stem cell research and medical applications not onlyhuman iPSC by chemicals alone but also safe generation of somatic stem cells for cell based clinical trials and other researches Inthis paper we have reviewed the recent advances in small molecule approaches for the generation of iPSCs

1 Introduction

Human induced pluripotent stem cells (iPSCs) are simi-lar to human embryonic stem cells in that they have thepotential to differentiate into cells of all three germ layers[1ndash3] Elderly patients with injuries degenerative diseasesor cancers would benefit from stem cell-based regenerativemedical techniques The iPSC applications promise in celltransplantation and stimulate the regenerative medicine ofendogenous cells to rebuild tissues in vitro drug screeningand disease modeling

Initially adult cells were induced into iPSCs throughexogenous overexpression of the transcription factors Oct4(also known as Pou5f1) Sox2 cMyc and Klf4 Howeverefficiency of this technique is at very low level with around01 of mouse fibroblasts [4] and 001 of human fibroblastscell [2 5] The low efficiency and slow dynamics of thismethod posed serious potential problems for the genera-tion of iPSCs Besides low iPSC generation efficiency thereare some safety concerns regarding the overexpression of

the four aforementioned transcription factors involvinggenetic mutations gene insertions epigenetic changesincomplete reprogramming and immunogenicity [6ndash12]

To improve the efficiency and quality of iPSC inductionmuch effort has been applied in the development of newiPSC generation methods through the use of integrating andnonintegrating recombinant viruses [13ndash18] DNA expressionvectors [19] episomal vectors [20 21] minicircle vectors[22] and liposomalmagnetofection [23] Non-DNAmethodsinvolving proteins [24 25] mRNA molecules [26] andvarious chemical agents [27] have also been trialed and achemical method that produces chemically induced pluripo-tent stem cells (CiPSCs) appears to be the most promisingmethods [27]

Although the human iPSC by using chemicals only hasnot been developed yet human stem cells studied withsmall molecules are revealing further details about epigeneticremodeling Thus hopefully these researches might relieveconcerns about the specificity efficiency kinetics and safetyof generating human iPSCs and bring human iPSC closer

Hindawi Publishing CorporationStem Cells InternationalVolume 2015 Article ID 794632 11 pageshttpdxdoiorg1011552015794632

2 Stem Cells International

to effective clinical use [28ndash30] We here discuss the smallmolecules in iPSC generation including three types of com-pounds small molecules that may improve reprogrammingefficiency compounds that replace one or more reprogram-ming factors compound combination alone that is sufficientto induce mouse iPSC We also provide perspective views ofthe possibility of the iPSC generation from human somaticcells and its future applications

2 Compounds That May Improve theReprogramming Efficiency and Quality

For the first time Huangfu et al studied the compound appli-cation in iPSC generation they investigated the effects of thehistone deacetylase (HDAC) inhibitor valproic acid (VPA)and found that reprogramming efficiency was increased100-fold over that of the transcription factor method [31]Soon Ding group used BIX-01294 which inhibits histonemethyltransferase (HMT) by activating calcium channels inthe plasma membrane to improve reprogramming efficiency[32 33] To date the small molecules that have been used togenerate iPSCs can be categorized as epigenetic modifierswingless and integration site growth factor (WNT) signalmodulators moderators of cell senescence or modulatorsof metabolism [3 34 35] and the functions of the smallmolecules in iPSC generation are summarized in Figure 1Through these mechanisms the small molecules couldimprove iPSC generation efficiency andor could replacesome of Yamanaka factors The small molecules that mightenhance iPSC generation efficiency were collected in Table 1

21 Epigenetic Modification During programming cellsundergo changes at a global transcriptional level and alsoexperience epigenetic changes in their DNA along withhistone modifications [3 28 29 34 36 37] Small moleculessuch as Bix-01294 (Bix) target enzymes responsible for his-tone methylation and demethylation and increase expressionlevels of OCT4 and KLF4 during somatic cell reprogram-ming [32] Parnate a lysine-specific demethylase inhibitor(LSD1) mediates H3K4 demethylation enabling Oct4 andKLF4 induction of human keratinocytes into iPSCs whencombined with CHIR99021 a glycogen synthase kinase 3(GSK-3) inhibitor [31] DNA methyltransferase inhibitor5-azacytidine (5-Aza) or three other histone deacetylaseinhibitors (suberoylanilide hydroxamic acid trichostatin Aand valproic acid) can also improve reprogramming effi-ciency following the transduction of MEFs with four tran-scription factors [31 35 38 39]

To improve incomplete epigenetic reprogramming thesomatic memory can be erased and new epigenetics areestablished in iPSCs via treatment with trichostatin A [3839] Epigenetic modification using small molecules signifi-cantly increases iPSC reprogramming efficiency and reducesepigenetic memory retention [3 34 39]

22 WNT Signal Modulators The WNT pathway plays animportant role in self-renewal of embryonic stem cells (ESCs)[40] The CHIR99021 molecule an inhibitor of GSK-3120573

activates WNT signaling improving the efficiency of pro-gramming and eliminating the use of c-Myc [41]

When combined with other compounds they serve asreplacements for certain key transcription factors or theycan induce differentiation into specific cell types It hasbeen hypothesized that WNT pathway regulators couldplay a significant role in cellular reprogramming As anexample the GSK-3 beta inhibitor CHIR99021 can improvereprogramming efficacy by replacing c-Myc Another GSK3inhibitor kenpaullone can be used as a replacement forKLF4 however the three transcription factors Oct4 Sox2and c-Myc must still be used for successful reprogramming[42] Many similar small molecules have been found to beeffective in iPSC generation The screening of other WNTsignaling modulators has been discussed in detail by otherresearchers [34 39 40 43]

23 Moderators of Cell Senescence Cell senescence in repro-gramming is usually thought to contribute to slow dynamicsand low efficiency Stress response cell senescence and earlyprogramming characteristics of iPSCs involve expression ofp21cip1 and p16INK4ap19arf which are upregulated by p53Knocking out p53 improves iPSC generation efficiency andkinetics [44ndash51] However the p53 protein is a key tumorsuppressor and improving iPSC generation by blocking p53likely increases the risk of tumor formation [44ndash50 52] asshown in murine ESCs [51]

A common small molecule natural antioxidant vitaminC has been found to promote the formation of iPSCs frommouse and human somatic cells by indirectly lowering theexpression of p21 and p53 [53] Some small molecules havebeen found to be regulators of the pathways involved in cellsenescence with little risk of tumor formation [54] Theydownregulate the expression of various genes and result inimproved efficiency and dynamics during iPSC generation[55ndash57]

24 Modulators of Metabolism Growth of differentiatedadult somatic cells usually involves mitochondrial oxidativephosphorylation while pluripotent stem cells mainly employglycolysis metabolism [56ndash58] This metabolic programmingability is driven by hypoxia-inducible factor 1 alpha withc-Myc promoting glycolysis [58] Consistently PS48 anactivator of 31015840-phosphoinositide-dependent protein kinase1 in combination with other small molecules (a-83-01 andPD0325901) and sodium butyrate (a histone deacetylaseinhibitor) has been used to reprogram adult keratinocytesumbilical vein endothelial cells and amniotic fluid-derivedcells It has been shown that PS48 activates the phospho-inositide 3-kinaseAkt pathway upregulating gene expressionwhich promotes mitochondrial oxidative phosphorylationand glycolysis metabolism [59]

25 MET Mediated by Transforming Growth Factor- (TGF-)120573 Pathway Signaling Inhibitors Enhances ReprogrammingA mesenchymal-epithelial transition (MET) is a reversiblebiological process involving the transition from motile mul-tipolar or spindle-shaped mesenchymal cells to planar arrays

Stem Cells International 3

WNT Metabolism Epigeneticmodification MET Senescence

Differentiation

Pluripotentstem cell

Somaticcell

Dedifferentiation

Figure 1 Differentiation and reprogramming are influenced by various mechanisms Small molecules used to generate iPSCs can becategorized as epigenetic modifiers WNT signal modulators cell senescence moderators modulators of metabolism and regulators of METand cell deathsenescence pathways They can influence both differentiation and reprogramming (dedifferentiation)

of polarized epithelial cells Reprogramming of fibroblastcells or iPSCs inevitably involves MET with cells at variousstages of reprogramming undergoingmorphological changestowards an epithelial-like cell type [60 61]

Several researchers have shown that inhibition of theTGF-120573 signaling pathway enhances reprogramming throughderepressing the mesenchymal phenotype and inducingMET By combining two small molecules SB431542 (aninhibitor of the TGF-120573 receptor) and PD0325901 (a MEKinhibitor) we demonstrated a 100-fold improvement of effi-ciency of human iPSC generation [62]

Further features of MET were revealed in three otherstudies where small molecules were used to inhibit TGF-120573 signaling or E-cadherin upregulation Among the manyTGF-120573 inhibitors E-616452 (also known as Repsox) wasrecently found to be a functional substitute for SOX2 inmouse fibroblast reprogramming with OKM it also indi-rectly enhanced NANOG expression during the late stagesof reprogramming [63] The TGF-120573 receptor inhibitor a-83-01 combined with the protein arginine methyltransferaseinhibitor AMI-5 along with OCT4 has also been found topromote reprogramming [64] Small molecule modulatorsof signal pathways alone or in combination and sometimeswith epigenetic modifications induced by exogenous tran-scription factors affect reprogramming efficiency throughthe influence of an integrated cellular network

3 Compounds That Can ReplaceReprogramming Factors

To date many studies on small molecules that could be usedto replace reprogramming transcription factors have beenpublished Most of the small molecules might improve repro-gramming efficiency and also could replace some functionsof one or more Yamanaka factors Oct4 Sox2 Klf4 c-Myc ortheir combinations we summarize the representative studies(Table 2)

31 Compounds That Can Replace Oct4 in ReprogrammingOct4 is the master regulatory pluripotency gene and mayserve as a pluripotency determinant in reprogramming Sev-eral compounds have been claimed that might replace Oct4expression (Table 2) BIX01294 a G9a HMTase inhibitor wasfirst reported to induce miPSCs in place of Oct4 [32] RG108a DNMT inhibitor can replace Oct4 during mouse skeletalmuscle cell reprogramming into miPSCs where skeletalmuscle cells endogenously express Sox2Klf4 and c-Myc [33]

Cellular reprogramming involves profound alterations ingenome-wide gene expression that is precisely controlled bya hypothetical epigenetic code that can be created artificiallyby epigenetic modification in a sequence dependent mannerwith small molecules A recent report claimed that a specificDNA binding hairpin pyrrole-imidazole polyamides (PIPs)could be conjugated with chromatin modifying histonedeacetylase inhibitors like SAHA to epigenetically activatecertain pluripotent genes in mouse fibroblasts and identifieda novel compound termed SAHA-PIP delta [65] It coulddramatically induce the endogenous expression of Oct-34and Nanog and other pluripotency associated genes therebythe cells rapidly overcame the rate-limiting step of epithelialtransition in cellular reprogramming by switching ldquoONrdquo thecomplex transcriptional gene network [65]

However Oct4 could also be replaced by other com-pounds such as FSK under binodal ldquoSeesawrdquo model (Hou etal and Shu et al) This mechanism is mentioned in detail inthe section of the CiPSC Many of the small molecules usedto replace Oct4 might fall into this category

32 Small Molecules That Might Replace Sox2 The com-pounds 616452 (E-616452 Repsox) and SB431542 are trans-forming growth factor- (TGF-) beta inhibitors that couldreplace Sox2 during mouse and human iPSC generation[63] However 616452 does not actually act by inducingSox2 expression in the target cells rather it enables repro-gramming through the induction of Nanog transcription as


Table 1 Small molecules that enhance iPSC generation efficiency and quality

Target or signaling pathway Name Concentration Host cells Efficiency andnecessity Reference

HDAC inhibitor VPA 05ndash2mM Mousehuman gt100-fold Huangfu et al (2008)

[31]

HDAC inhibitor SAHA 5120583M Mouse 10-fold Huangfu et al (2008)[31]

HDAC inhibitor TSA 20 nM Mouse 10-fold Huangfu et al (2008)[31]

HDAC inhibitor Sodium butyrate 05ndash1mM Mousehuman 100-fold Mali et al (2010) [66]

DMNT inhibitor 5-aza-CR AZA 05mM Mouse 3-fold Mikkelsen et al(2008) [29]

DMNT inhibitor histone deacetylaseinhibitor RSC133 10 120583M Human 3-fold Lee et al (2012)

[10 67]Retinoic acid receptor agonist AM580 100 nM Mouse sim200-fold Wang et al (2011) [68]H3K4 demethylation inhibitor(epigenetic modulator)

Tranylcypromine(Parnate) 5ndash10 120583M Mouse 3-fold Li et al (2009) [35]

Epigenetic modulators DZNep 005ndash01 120583M Mouse 65-fold Hou et al (2013) [27]Retinoic acid receptor ligand TTNPB 1 120583M Mouse 15-fold Hou et al (2013) [27]

ALK4 ALK5 and ALK7 inhibitor SB431542 10 uM Human Thiazovivin andPD0325901 sim200 fold Lin et al (2009) [62]

Selective MEKERK inhibitor PD0325901 1 uM Human Thiazovivin andSB431542 sim200-fold Lin et al (2009) [62]

Rho-associated protein kinaseinhibitor Thiazovivin 1 uM Human PD0325901 and

SB431542 sim200-fold Lin et al (2009) [62]

Rho-associated protein kinaseinhibitor Y27632 10 uM Human Improve generation

and maintainingClaassen et al (2009)[69]

AKt-mediated inhibitor of GSK3-120573 Compound B6 1 120583M Mouse 3-fold Li et al (2009) [35]

GSK-3120573 inhibitor LSD1 inhibitor LiCl 5ndash10mM Mouse andhuman gt10-fold Wang et al (2011) [68]

TGF-120573 inhibitor A83-01 05120583M Mousehuman 7-fold Zhu et al (2010) [59]

Prolyl-4-hydroxylase inhibitor N-Oxalylglycine 1 120583M Human Zhu et al (2010) [59]

ALK4 inhibitor Compound B4(TGFb-RI) 1 120583M Mouse 4-fold Li and Rana (2012)

[70]mTOR inhibitor Rapamycin 03 nM Mouse 48-fold Chen et al (2011) [71]IP3K inhibitor Compound B8 1-2120583M Mouse 3-fold Li et al (2009) [35]

P38 kinase inhibitor Compound B10 1-2 120583M Mouse 3-fold Li and Rana (2012)[70]

cAMP agonist Prostaglandin E2 5 120583M Mouse Efficient in mixture Hou et al (2013) [27]cAMP agonist Rolipram 10 120583M Mouse Efficient in mixture Hou et al (2013) [27]cAMP-dependent protein kinaseactivator 8-Br-cAMP 01ndash05mM Human 65-fold Wang et al (2011) [68]

PDK1 activator

5-(4-Chloro-phenyl)-3-phenyl-pent-2-

enoic acid(PS48)

5120583M Human 15-fold Zhu et al (2010) [59]

HIF PHD1 and PHD2 inhibitor N-Oxalylglycine 1 uM Human 35-fold Zhu et al (2010) [59]

Phosphofructokinase 1 activator Fructose26-bisphosphate 10mM Human 2-fold Zhu et al (2010) [59]


Table 1 Continued


Hypoxia-inducible factor pathwayactivator Quercetin 1 120583m Human 3-fold Zhu et al (2010) [59]

Oxidative phosphorylation uncoupler DNP 1 120583M Human 2-fold Zhu et al (2010) [59]Note small molecules can improve reprogramming efficiency by epigenetic modifications or signaling pathway regulation Many of these small moleculesor compound combinations can also replace c-Myc or other transcription factors DNP 24-dinitrophenol DZNep 3-deazaneplanocin FSK forskolinHDAC histone deacetylase IP3K inositol triphosphate 3-kinase PDK1 phosphoinositide-dependent kinase 1 SAHA suberoylanilide hydroxamic acidTF transcription factor TSA trichostatin A VPA valproic acid 2-Me-5HT 2-methyl-5-hydroxytryptamine 5-aza-CR AZA 5-azacytidine 8-Br-cAMP 8-Bromoadenosine cyclic monophosphate

Table 2 Small molecule compounds that might replace Yamanaka factors

Replacementfor TF

Nameconcentration Concentration Host cell

species Function or target Reference

Oct4 Nanog SAHA-PIP delta 100 nM Mouse Histone deacetylaseinhibitor

Pandian et al (2014)[65]

Sox2 (with BIX) or Oct4 RG108 004ndash500120583M Mouse DMNT inhibitor Shi et al (2008)[32 33 72]

Oct4 BIX 05ndash2 120583M Mouse G9a HMTase inhibitor Shi et al (2008)[32 33 72]

Sox2 CHIR 3ndash10 120583M Mousehuman

GSK-3120573 inhibitor thatactivates Wnt signallingpathway

Li et al (2011) [73]

Klf4 Kenpaullone 5 120583M Mouse GSK-3CDKs inhibitor Lyssiotis et al (2009)[42]

Sox2 616452 (E-616452 Repsox) 1 120583M Mousehuman

TGF-120573 inhibitor (ALKinhibitor II)

Ichida et al (2009)[63]

Sox2 LY-364947 1 120583M Mouse TGF-120573 inhibitor Staerk et al (2011) [74]Sox2 Klf4 (withA-83-01) AMI-5 5120583M Mouse Protein arginine

methyltransferase inhibitor Yuan et al (2011) [64]

Sox2 Dasatinib 05120583M Mouse Src family tyrosine kinaseinhibitor Staerk et al (2011) [74]

Sox2 iPYrazine (iPY) 10 120583M Mouse Src family tyrosine kinaseinhibitor Staerk et al (2011) [74]

Sox2 PP1 10 120583M Mouse Src family tyrosine kinaseinhibitor Staerk et al (2011) [74]

Oct 4 with FSK and2-Me-5HT D4476 5120583M Mouse CK1 inhibitor Hou et al (2013) [27]

Sox2 BayK 2 120583M Mouse An L-channel calciumagonist

Shi et al (2008)[32 33 72]

Oct4 when used with2-Me-5HT and D4476 FSK 10ndash50 120583M Mouse cAMP agonist Hou et al (2013) [27]

Oct4 with FSK andD4476 2-Me-5HT 5 120583M Mouse 5-HT3 agonist Hou et al (2013) [27]

Sox2 Klf4 and C-Myc Oxysterol 05ndash1 120583M Mouse Sonic hedgehog signaling Moon et al (2011) [75]Sox2 Klf4 and C-Myc Purmorphamine 05ndash1 120583M Mouse Sonic hedgehog signaling Moon et al (2011) [75]Sox2 Klf4 and C-Myc Shh 500 ngmL Mouse Sonic hedgehog signaling Moon et al (2011) [75]Note small molecules can substitute for certain TFs andor improve reprogramming efficiency by epigenetic modifications or signaling pathway regulationBayK Bay K8644 BIX BIX-01294 CHIR CHIR99021 CK1 casein kinase 1 DNMT DNA methyltransferase DNP 24-dinitrophenol DZNep 3-deazaneplanocin FSK forskolin HDAC histone deacetylase G9a HMTase G9a histone ethyltransferase IP3K inositol triphosphate 3-kinase PDK1phosphoinositide dependent kinase 1 SAHA suberoylanilide hydroxamic acid TF transcription factor TSA trichostatin A VPA valproic acid 2-Me-5HT2-methyl-5-hydroxytryptamine 5-aza-CR AZA 5-azacytidine 8-Br-cAMP 8-bromoadenosine cyclic monophosphate


Table 3 Small molecules (VC6TFZ and 2i) that are used in mouse CiPSC

Target or signaling pathway Name and concentration TF to be replaced Efficiency ReferenceTGF beta pathwayALK5 inhibitor

Repsox (616452)5ndash10 uM Sox2 Myc Essential Hou et al (2013) [27]

Ichida et al (2009) [63]

PKA agonist Forskolin20ndash50 uM

Oct4 expression (with SKM)Klf2 klf4 expression Essential Hou et al (2013) [27]

WNT pathway regulator GSK3beta inhibitor

Chir9902110 uM Sox2 Myc Essential Hou et al (2013) [27]

Histone methylation modulatorlysine methyltransferase EZH2inhibitor

DENep50ndash100 nM Increase reprogramming Essential Hou et al (2013) [27]

Not specific TTNPB5 uM

Nuclease receptor signalingmodulator

Moreefficient Hou et al (2013) [27]

Not specific VPA05mM Histone deacetylase inhibitor More

efficient Hou et al (2013) [27]

PD0325901 selective MEKERKinhibitorChir99021WNT pathway regulator GSK3beta inhibitor

2iPD0325901

1 uMChir9902110 uM

Increase Oct4 Nanog Sox2expression

Moremature Hou et al (2013) [27]

Small molecules can substitute for all TFs through epigenetic modifications and signaling pathway regulations Hou et al [27] reported that CiPSC generationfrommEF was carried out in 3 steps of 16ndash20 days in VC6TF treatment and then 12ndash20 days in VC6TFZ and followed by 2i compounds regulations for 1 weekThe abbreviations of the small molecules are shown in Tables 1 and 2

reported Another TGF-b inhibitor LY-364947 can replaceSox2 in miPSC generation [74]

BayK8644 (BayK) an L-channel calcium agonist wasalso reported to be able to replace Sox2 in combination withBIX01294 during MEF reprogramming into miPSCs [32 3372] Shh purmorphamine and oxysterol the activator ofSonic hedgehog signaling have been reported to upregulateBmi1 Sox2 and N-Myc expression in mouse fibroblasts [75]

Staerk et al applied a cell-based high-throughput chem-ical screening method to identify small molecules that canreplace Sox2 duringmouse somatic cell reprogramming [74]From their Nanog reporter-based screening they discoveredthat the Pan-Src family kinase inhibitors iPYrazine dasatiniband PP1 could replace Sox2 in MEF reprogramming intomiPSCs [74]

33 Small Molecules That Might Replace Klf4 and c-MycWhile WNT signaling pathway regulators can improve iPSCgeneration efficiency they also could replace the function ofthe c-Myc Furthermore several smallmolecules can increasethe iPSC generation efficiency by replacing Klf4 and c-Mycduring somatic cell reprogramming into iPSCs Huangfuet al reprogrammed MEFs into miPSCs using VPA andtransducing three factors without introducing the oncogenec-My [31] Kenpaullone has been reported as a substitute forKlf4 in mouse cells although the underlying mechanism isunknown [42]

34 SmallMoleculesThatMight Replace Sox2 Klf4 and c-MycLi et al succeeded in generating miPSCs by transduction ofOct4 alone with the addition of VPA CHIR99021 616452and tranylcypromine (VC6T) in the culture medium [73]It is thought that the small-molecule combination including

VC6T facilitatedmiPSC generation by lowering severalmajorbarriers to the reprogramming process Really the first iPSCgeneration protocol by using complete chemical alone isbased on this protocol [27] Similarly many other smallmolecules combinations were also reported [29 64 66ndash6870ndash72 75ndash79]

35 Combination of 2i Provides Mouse ESC Ground Status forMature Mouse iPSC As reported before the combination ofchir99021 (GSK-3 beta inhibitor) and PD0325901 (mitogen-activated protein kinaseextracellular signal-regulated kinaseinhibitor) designated 2i is required to maintain the pluripo-tent ground state in mouse ES cells [80] Hou et al culturedthe partially reprogrammed cells in 2imediumwith leukemiainhibitory factor which induced the stable upregulation ofOct4 and Nanog transgene silencing and competence forsomatic and germline chimerism thereby the cells werecompletely reprogrammed into mouse CiPSCs [27]

4 Compound Cocktails Required toGenerate Chemical iPSCs (CiPSCs)

For the first time in history Hou et al obtained chemi-cally induced pluripotent stem cells from mouse embryonicfibroblast cells after extensive screening compounds andcompound cocktails (Table 3)

The CiPSC was induced through a very complicated pro-cedure with 3-step compound treatment shown in Figure 2To start they searched for small molecules that enabled mEFreprogramming in the absence of Oct4 usingmEF expressingOct4 promoter-driven GFP to identify small molecules [27]Three small molecules Forskolin (FSK or F) 2-methyl-5-hydroxytryptamine and D4476 were chemical substitutes


Differentiation

Pluripotentstem cell stem cell

Differentiation Differentiation

Somaticcell

mEFVC6FTVC6FTZ

Multipotent

Dedifferentiation Dedifferentiation Dedifferentiation

Figure 2 Small molecules based iPSC generation Small molecules can substitute for all TFs through epigenetic modifications and signalingpathway regulations Hou et al reported that CiPSC generation from mEF was carried out in 3 steps of 16ndash20 days in VC6TF treatment andthen 12ndash20 days in VC6TFZ and followed by 2i compounds regulations for 1 week The somatic cell mouse embryonic fibroblast (mEF) cellundergoes dedifferentiation and gains multipotent stem cell characteristics under the treatments of VC6TF and VC6TFZ steps and finallyobtains the full pluripotency in the medium containing 2i compounds (Chir99021 and PD0325901)

for Oct4 after screening 10 000 compounds FSK was thenchosen for subsequent studies Their early findings showedthat the compounds cocktail ldquoVC6Trdquo (VPA CHIR99021616452 and tranylcypromine) could inducemouse iPSCwitha single-gene transduction of Oct4 Thus they combinedthese two groups of small molecules into a cocktail VC6TFresulting in cells expressing E-cadherin [27] However Oct4and NANOG expression were not detectable in these cellssuggesting that reprogramming was incomplete [27]

They found one more compound 3-deazaneplanocin(DZNep or Z epigenetic modulator) whose chemicalinduced stem cell medium contained VC6TFZ [27] Toenhancemore pluripotent stem cell gene expression they fur-thermore treated the cell in mouse pluripotent ground statusconditions with ES medium containing LIF and PD0325901and CHIR99021 (2i medium)

They designed CiPSC protocol including 3 steps asfollows (a) the mEF cells were cultured in mESC mediumcontaining VC6FT for 16ndash20 days (b) the cells were culturedin the medium with VC6FTZ for 12ndash20 days (c) the cellswere cultured in mESC medium containing 2i (PD0325901and Chir99021) for 1 week The characteristics of CiPSCsresembled mESCs in terms of their gene expression pro-files epigenetic status and potential for differentiation andgermline transmissionTherefore they completed the CiPSCprepared from mEF solely using a combination of sevensmall-molecule compounds without using transduction ortransfection of TFs [27]

The group also found that among seven compoundsindividual small molecule of C 6 F Z was the most essentialingredient to CiPSC removal of any one of these compoundsfrom the cocktail might result in failure of CiPSC [27] Whilethese data are very interesting and clearly showed that CiPSCwas generated from mEF the principle of the CiPSC is notclear To explore the mechanism of the small moleculesbased iPSC the research group reported new findings onmechanisms of the pluripotent stem cell status [37]

For years it was generally believed that ESCs are main-tained by a shield of pluripotency factors which function inconcert with each other to prevent ESCs from differentiatinginto any lineage thus preserving ESCs at an undifferentiatedstate Noting that some small molecules could replace theYamanaka factors individually the combination of these

small molecules should be sufficient to replace the YamanakaFactors Unfortunately while some compounds could induceOct4 expression the key iPSC transcription factors and theother Yamanaka factors could not be replaced by a single setof small molecules Therefore Deng group performed manystudies to discover the chemical based iPSC generationmech-anism a binodal ldquoSeesawrdquo model for cell fate determinationthen was discovered [37]

Shu et al proposed a new model termed the ldquoSee-sawrdquo model in which the pluripotent state is a precariousbalancing equilibrium that results from continuous mutualcompetition between rival lineage specification forces Whilethe reprogramming factors that induce pluripotency havebeen identified primarily from embryonic stem cell- (ESC-)enriched pluripotency-associated factors pluripotency canbe induced with lineage specifiers that suppress ESC identityusing pluripotency rivals most of which are not enriched inESCs They found that OCT4 and SOX2 the core regulatorsof pluripotency can be replaced by lineage specifiers thatare involved in mesendodermal (ME) and ectodermal (ECT)specification respectively [37]

OCT4 and its substitutes attenuated the elevated expres-sion of a group of ECT genes whereas SOX2 and its substi-tutes curtailed a group of ME genes during reprogrammingSurprisingly the two counteracting lineage specifiers can syn-ergistically induce pluripotency in the absence of both OCT4and SOX2 [37] Therefore they concluded that the principleof the mouse CiPSC is that the groups of counteractinglineage specifier compounds VC6FTZ which belong to 2counteracting lineage specifiers were combined to induce theCiPSC [37]

After mouse CiPSC the next logical step is to try usingthe CiPSC approach to make human stem cells This binodalmodel shed light on fundamental questions regarding theestablishment of cellular identity during programming inmouse However there are a lot of differences betweenmouse pluripotent stem cells and human ones For exampleleukemia inhibitory factor LIF is a necessary factor formouse pluripotent stem cell whereas the bFGF is a keyessential supplement for human pluripotent stem cell cultureThe subtle ldquoSeesawrdquo balances of the pluripotent factors mightneed to be adjusted to specific conditions suitable for humanCiPSC


5 Conclusions and Perspectives

51 Is the Combination of Small Molecule CompoundsAlone Sufficient for Human iPSC Generation To date smallmolecules based iPSC have brought dramatically changesto iPSC research Some of these small molecules increasereprogramming efficiency and quality while others or com-binations of them replace iPSC factors Mouse CiPSCs wereobtained and thus the complete small molecule-based repro-gramming in a directed and deterministic manner has fun-damentally changed the reprogramming paradigm througha mechanism that involves the activation of endogenoustranscription factors by small molecules instead of exoge-nously expressed transcription factors Is the combination ofsmall molecule compounds alone sufficient for human iPSCgeneration

52 Extensively Screening for Novel Oct4 Substitutes andOther ESC-Related Transcription Factors To our knowledgehiPSC generation with small molecule cocktails has not yetbeen reported While most factors used as reprogrammingtransgenes can be replaced by other means Oct4 remainedessential until the development of human CiPSC technologyFurthermore the Oct4 expression level in the mouse CiPSCswas reported much lower than those in the mouse ESCs Inhuman iPSC generation activation of Oct4 expression andreaching a high enough level similar to that in human ESCare a key challenge New specific small molecule targeted toactivation of reprogramming factors such as Oct4 might beuseful strategy to solve this problem as recent report [65]Extensively screening for small molecules and compoundmodifications for novel Oct4 substitutes and other ESC-related transcription factors will cast light on the discoveryof molecular mechanisms for reprogramming and transdif-ferentiation which in turn facilitate the development of saferand more efficient stem cell resource for clinical applications

53 Mechanisms Governing Human CiPSC Generation MightNeed to Be Discovered Deng group found that the twocounteracting lineage specifiers can synergistically inducepluripotency in the absence of both OCT4 and SOX2 [37]While the Seesaw model might also work in human iPSCgeneration human pluripotent stem cell ground status condi-tions might be very different from mouse ones For exampleleukemia inhibitory factor LIF is necessary factor for mousepluripotent stem cell whereas the bFGF is a key essentialsupplement for human pluripotent stem cell culture As aresult similar ldquoseesawmodelrdquo of the pluripotent factors needsto be confirmed in human iPSC and different mechanismsthat specifically govern the human CiPSC generation mightneed to be discovered

In addition complete reprogramming in mouse CiPSCneed mouse ESC ground status compound combination ofchir99021 and PD0325901 designated 2i are required tomaintain the pluripotent ground state [80] and confirmedit is necessary for mouse CiPSC However 2i is useful butnot sufficient in maintaining the ground state for humanESCs while the compound Chir99021 is useful for iPSC

proliferation when it is used with three small moleculesduring two-step iPSC induction [81]

Furthermore the human CiPSC generation is so com-plicated that it needs a lot of cellular reorganization signal-ing pathways changes and extracellular matrix maintainingconditions to achieve final reprogrammed pluripotent stemcell status (Figure 3) The cells were induced by chemicalsthrough epigeneticmodifications that switch on pluripotencytranscription factors such as Oct4 Nang Sox2 Klf4 andc-Myc and were kept on through chromatin remodelingConversely genes responsible for differentiation must beturned off by the transcription machinery and kept silentthrough epigenetic mechanisms While critical chemicals forpluripotency were VC6TFZ (C6FZ were essential) in mouseCiPSC generation wemight have to regulatemore cell signal-ing pathways including TGF beta WNT ERK ROCK mito-chondria and other signaling pathways as found in humaniPSC generation through viral or nonviral transfection tillnow Therefore waiting to be discovered mechanisms mightalso be a lot more including cell signaling pathways as well asnuclear and mitochondria genomic activities (Figure 3)

54 Safety Issues Especially TumorConcernHave to Be Solvedbefore Clinical Applications Although human iPSCs providean alternative stem cell resource for regenerative medicinewe still hesitate before using them in clinic applications dueto safety concerns such as tumor risksThe iPSC generation isso complicated that cellular reorganizations signaling path-ways changes and extracellular matrix maintain conditionsto achieve final reprogramming goals Furthermore it isextremely demanding to obtain highly pure population ofthe target cells The small molecules based approaches mightprovide solutions for pure target adult stem cell withouttumor concerns

55 The Small Molecules Research Might Bring Close FutureApplications Not Only for iPSC Application but Also for AdultStem Cell Based Applications The small molecules in theiPSC generation might also provide useful information forthose studies in adult stem cell Direct reprogramming ofone type of adult stem cell to another one was suggestedas an attractive alternative for clinical applications Somestudies have developed small molecules that guide humantransdifferentiation of tissue-specific progenitor cells or stemcells Because adult stem cells such as bonemarrow stem cellsand hematopoietic stem cells are ready to be used in clinicalapplications the only obstacle is their limited proliferationThedevelopment of smallmolecules towork in adult stemcellself-renewals or to guide human somatic cells into progenitorcells will open avenues for the clinical application of thesetypes of progenitor cells and stem cells

Advances in the understanding of reprogrammingmech-anism and continuous development of small molecular toolsto enhance reprogramming will not only facilitate the possi-bility of generating safer and higher quality reprogrammedcells but also provide useful information for adult stem cellbased applications


Mitochondrion

ThiazovivinROCK

Myosin PPtase

PD0325901

Pl3kWNT

and otherpathways

p p

pERK

Oct4Sox2cMycKlf4Ac

AcAcAc

Ac

AcAc

Ac

NHNH

MTase

DNAMTase

SMsepigeneticregulators

S8431542ALKs inhibitor

ALKs

Cytokines

CH3 CH3 CH3

SMADs

MLCP

RasGTP

RasGTP

ActivinNodal

MWE12

Raf

RasGAP

McCP2 CH3

CH3

TGF-120573

TGF-120573 pathway

Integrin 120572 and 120573

p

CpG CpG CpGHDAC1HHHHH

Sin3A HDAC2

p53complex

RhoA

CpG

Histone

Figure 3 Human CiPSC perspective regulation pathways The human CiPSC generation is so complicated that needs a lot of cellularreorganization signaling pathways changes and extracellular matrixmaintaining conditions to achieve final reprogrammed pluripotent stemcell status The cells were induced by chemicals through epigenetic modifications that switch on pluripotency transcription factors such asOct4 Nang Sox2 Klf4 and c-Myc The cell signaling pathways including TGF beta WNT ERK ROCK mitochondria and other signalingpathways might need to be regulated in human CiPSC generation However waiting to be discovered mechanisms might also be some otherunknown cell signaling pathways as well as nuclear and mitochondria genomic activities

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

Acknowledgments

This work was supported by the State Key DevelopmentProgram of Basic Research of China (973 Program no2013CB966902 to Tongxiang Lin) and the National NaturalScience Foundation of China (Grant no 31271595 to Tongx-iang Lin) They like to give special thanks to Mr XiangyiLin San Diego California USA for his kind suggestion andrevision

References

[1] K Takahashi K Tanabe M Ohnuki et al ldquoInduction ofpluripotent stem cells from adult human fibroblasts by definedfactorsrdquo Cell vol 131 no 5 pp 861ndash872 2007

[2] J Yu M A Vodyanik K Smuga-Otto et al ldquoInduced pluripo-tent stem cell lines derived from human somatic cellsrdquo Sciencevol 318 pp 1917ndash1920 2007

[3] M H Chin M J Mason W Xie et al ldquoInduced pluripotentstem cells and embryonic stem cells are distinguished by geneexpression signaturesrdquo Cell Stem Cell vol 5 no 1 pp 111ndash1232009

[4] K Takahashi and S Yamanaka ldquoInduction of pluripotent stemcells from mouse embryonic and adult fibroblast cultures bydefined factorsrdquo Cell vol 126 no 4 pp 663ndash676 2006

[5] K Takahashi K Okita M Nakagawa and S Yamanaka ldquoIn-duction of pluripotent stem cells from fibroblast culturesrdquoNature Protocols vol 2 no 12 pp 3081ndash3089 2007

[6] K Okita T Ichisaka and S Yamanaka ldquoGeneration of germ-line-competent induced pluripotent stem cellsrdquo Nature vol448 no 7151 pp 313ndash317 2007

[7] Y Ohi H Qin C Hong et al ldquoIncomplete DNA methylationunderlies a transcriptional memory of somatic cells in humaniPS cellsrdquo Nature Cell Biology vol 13 no 5 pp 541ndash549 2011

[8] H Xu B A Yi H Wu et al ldquoHighly efficient derivation ofventricular cardiomyocytes from induced pluripotent stem cellswith a distinct epigenetic signaturerdquo Cell Research vol 22 no 1pp 142ndash154 2012

[9] O Bar-Nur H A Russ S Efrat and N Benvenisty ldquoEpigeneticmemory and preferential lineage-specific differentiation ininduced pluripotent stem cells derived from human pancreaticislet beta cellsrdquo Cell Stem Cell vol 9 no 1 pp 17ndash23 2011

[10] S B Lee D Seo D Choi et al ldquoContribution of hepatic lineagestage-specific donor memory to the differential potential ofinduced mouse pluripotent stem cellsrdquo Stem Cells vol 30 no5 pp 997ndash1007 2012

[11] R Lister M Pelizzola Y S Kida et al ldquoHotspots of aberrantepigenomic reprogramming in human induced pluripotentstem cellsrdquo Nature vol 471 no 7336 pp 68ndash73 2011


[12] T Zhao Z-N Zhang Z Rong and Y Xu ldquoImmunogenicity ofinduced pluripotent stem cellsrdquo Nature vol 474 no 7350 pp212ndash215 2011

[13] Y H Loh J C Yang A de los Angeles et al ldquoExcision of a viralreprogramming cassette by delivery of synthetic CremRNArdquo inCurrent Protocols in StemCell Biology chapter 4 unit 4A5 JohnWiley amp Sons Hoboken NJ USA 2012

[14] K Kaji K Norrby A Paca M Mileikovsky P Mohseni and KWoltjen ldquoVirus-free induction of pluripotency and subsequentexcision of reprogramming factorsrdquo Nature vol 458 no 7239pp 771ndash775 2009

[15] K Woltjen I P Michael P Mohseni et al ldquoPiggyBac transpo-sition reprograms fibroblasts to induced pluripotent stem cellsrdquoNature vol 458 no 7239 pp 766ndash770 2009

[16] M StadtfeldMNagaya J Utikal GWeir andKHochedlingerldquoInduced pluripotent stem cells generated without viral integra-tionrdquo Science vol 322 no 5903 pp 945ndash949 2008

[17] W Zhou and C R Freed ldquoAdenoviral gene delivery canreprogramhumanfibroblasts to induced pluripotent stem cellsrdquoStem Cells vol 27 no 11 pp 2667ndash2674 2009

[18] N Fusaki H Ban A Nishiyama K Saeki and M HasegawaldquoEfficient induction of transgene-free human pluripotent stemcells using a vector based on Sendai virus an RNA virus thatdoes not integrate into the host genomerdquo Proceedings of theJapan Academy Series B Physical and Biological Sciences vol 85no 8 pp 348ndash362 2009

[19] K Okita M Nakagawa H Hyenjong T Ichisaka and SYamanaka ldquoGeneration of mouse induced pluripotent stemcells without viral vectorsrdquo Science vol 322 no 5903 pp 949ndash953 2008

[20] J Yu K Hu K Smuga-Otto et al ldquoHuman induced pluripotentstem cells free of vector and transgene sequencesrdquo Science vol324 no 5928 pp 797ndash801 2009

[21] K Okita Y Matsumura Y Sato et al ldquoA more efficient methodto generate integration-free human iPS cellsrdquo Nature Methodsvol 8 pp 409ndash412 2011

[22] F Jia K D Wilson N Sun et al ldquoA nonviral minicircle vectorfor deriving human iPS cellsrdquo Nature Methods vol 7 pp 197ndash199 2010

[23] H Y Park E H Noh H-M Chung M-J Kang E Y Kimand S P Park ldquoEfficient generation of virus-free iPS cells usingliposomal magnetofectionrdquo PLoS ONE vol 7 no 9 Article IDe45812 2012

[24] H Zhou S Wu J Y Joo et al ldquoGeneration of inducedpluripotent stem cells using recombinant proteinsrdquo Cell StemCell vol 4 no 5 pp 381ndash384 2009

[25] D Kim C-H Kim J-I Moon et al ldquoGeneration of humaninduced pluripotent stem cells by direct delivery of reprogram-ming proteinsrdquo Cell Stem Cell vol 4 no 6 pp 472ndash476 2009

[26] L Warren P D Manos T Ahfeldt et al ldquoHighly efficientreprogramming to pluripotency and directed differentiation ofhuman cells with syntheticmodifiedmRNArdquoCell StemCell vol7 no 5 pp 618ndash630 2010

[27] P Hou Y Li X Zhang et al ldquoPluripotent stem cells inducedfrom mouse somatic cells by small-molecule compoundsrdquoScience vol 341 no 6146 pp 651ndash654 2013

[28] N Maherali R Sridharan W Xie et al ldquoDirectly repro-grammed fibroblasts show global epigenetic remodeling andwidespread tissue contributionrdquo Cell Stem Cell vol 1 no 1 pp55ndash70 2007

[29] T S Mikkelsen J Hanna X Zhang et al ldquoDissecting directreprogramming through integrative genomic analysisrdquo Naturevol 454 no 7200 pp 49ndash55 2008

[30] R Sridharan J Tchieu M J Mason et al ldquoRole of the murinereprogramming factors in the induction of pluripotencyrdquo Cellvol 136 no 2 pp 364ndash377 2009

[31] D Huangfu R Maehr W Guo et al ldquoInduction of pluripotentstem cells by defined factors is greatly improved by small-molecule compoundsrdquo Nature Biotechnology vol 26 no 7 pp795ndash797 2008

[32] Y Shi C Desponts J T Do H S Hahm H R Scholerand S Ding ldquoInduction of pluripotent stem cells from mouseembryonic fibroblasts by Oct4 and Klf4 with small-moleculecompoundsrdquo Cell Stem Cell vol 3 no 5 pp 568ndash574 2008

[33] Y Shi J T Do C Desponts H S Hahm H R Scholer andS Ding ldquoA combined chemical and genetic approach for thegeneration of induced pluripotent stem cellsrdquoCell StemCell vol2 pp 525ndash528 2008

[34] B Nie H Wang T Laurent and S Ding ldquoCellular reprogram-ming a small molecule perspectiverdquo Current Opinion in CellBiology vol 24 no 6 pp 784ndash792 2012

[35] W Li H Zhou R Abujarour et al ldquoGeneration of human-induced pluripotent stem cells in the absence of exogenousSox2rdquo Stem Cells vol 27 no 12 pp 2992ndash3000 2009

[36] R D Hawkins G C Hon L K Lee et al ldquoDistinct epigenomiclandscapes of pluripotent and lineage-committed human cellsrdquoCell Stem Cell vol 6 no 5 pp 479ndash491 2010

[37] J Shu C Wu Y Wu et al ldquoInduction of pluripotency in mousesomatic cells with lineage specifiersrdquo Cell vol 153 no 5 pp963ndash975 2013

[38] F Lluis L Ombrato E Pedone S Pepe B J Merrill and MP Cosma ldquoT-cell factor 3 (Tcf3) deletion increases somaticcell reprogramming by inducing epigenome modificationsrdquoProceedings of the National Academy of Sciences of the UnitedStates of America vol 108 no 29 pp 11912ndash11917 2011

[39] R Zhao and G Q Daley ldquoFrom fibroblasts to iPS cellsinduced pluripotency by defined factorsrdquo Journal of CellularBiochemistry vol 105 no 4 pp 949ndash955 2008

[40] H Niwa ldquoWnt whatrsquos Needed to maintain pluripotencyrdquoNature Cell Biology vol 13 no 9 pp 1024ndash1026 2011

[41] A Marson R Foreman B Chevalier et al ldquoWnt signalingpromotes reprogramming of somatic cells to pluripotencyrdquo CellStem Cell vol 3 no 2 pp 132ndash135 2008

[42] C A Lyssiotis R K Foreman J Staerk et al ldquoReprogrammingof murine fibroblasts to induced pluripotent stem cells withchemical complementation of Klf4rdquo Proceedings of the NationalAcademy of Sciences of the United States of America vol 106 no22 pp 8912ndash8917 2009

[43] J D Holland A Klaus A N Garratt andW Birchmeier ldquoWntsignaling in stem and cancer stem cellsrdquoCurrent Opinion in CellBiology vol 25 no 2 pp 254ndash264 2013

[44] Y Zhao X Yin H Qin et al ldquoTwo supporting factors greatlyimprove the efficiency of human iPSC generationrdquo Cell StemCell vol 3 no 5 pp 475ndash479 2008

[45] H Hong K Takahashi T Ichisaka et al ldquoSuppression ofinduced pluripotent stem cell generation by the p53-p21 path-wayrdquo Nature vol 460 no 7259 pp 1132ndash1135 2009

[46] T Kawamura J Suzuki Y V Wang et al ldquoLinking the p53tumour suppressor pathway to somatic cell reprogrammingrdquoNature vol 460 no 7259 pp 1140ndash1144 2009


[47] H Li M Collado A Villasante et al ldquoThe Ink4Arf locus is abarrier for iPS cell reprogrammingrdquo Nature vol 460 no 7259pp 1136ndash1139 2009

[48] J Utikal J M Polo M Stadtfeld et al ldquoImmortalizationeliminates a roadblock during cellular reprogramming into iPScellsrdquo Nature vol 460 no 7259 pp 1145ndash1148 2009

[49] R M Marion K Strati H Li et al ldquoA p53-mediated DNAdamage response limits reprogramming to ensure iPS cellgenomic integrityrdquo Nature vol 460 no 7259 pp 1149ndash11532009

[50] V Krizhanovsky and S W Lowe ldquoStem cells the promises andperils of p53rdquo Nature vol 460 no 7259 pp 1085ndash1086 2009

[51] T Lin C Chao S Saito et al ldquop53 induces differentiation ofmouse embryonic stem cells by suppressing Nanog expressionrdquoNature Cell Biology vol 7 no 2 pp 165ndash171 2005

[52] N Shevde ldquoStem cells flexible friendsrdquo Nature vol 483 no7387 pp S22ndashS26 2012

[53] M A Esteban T Wang B Qin et al ldquoVitamin C enhancesthe generation of mouse and human induced pluripotent stemcellsrdquo Cell Stem Cell vol 6 no 1 pp 71ndash79 2010

[54] D A Robinton and G Q Daley ldquoThe promise of inducedpluripotent stem cells in research and therapyrdquoNature vol 481no 7381 pp 295ndash305 2012

[55] A Banito S T Rashid J C Acosta et al ldquoSenescence impairssuccessful reprogramming to pluripotent stem cellsrdquo Genes ampDevelopment vol 23 no 18 pp 2134ndash2139 2009

[56] M G V Heiden L C Cantley and C B Thompson ldquoUnder-standing the warburg effect the metabolic requirements of cellproliferationrdquo Science vol 324 no 5930 pp 1029ndash1033 2009

[57] Y YoshidaK Takahashi KOkita T Ichisaka and S YamanakaldquoHypoxia enhances the generation of induced pluripotent stemcellsrdquo Cell Stem Cell vol 5 no 3 pp 237ndash241 2009

[58] A Prigione B Fauler R Lurz H Lehrach and J Adjaye ldquoThesenescence-related mitochondrialoxidative stress pathway isrepressed in human induced pluripotent stem cellsrdquo Stem Cellsvol 28 no 4 pp 721ndash733 2010

[59] S Zhu W Li H Zhou et al ldquoReprogramming of humanprimary somatic cells by OCT4 and chemical compoundsrdquo CellStem Cell vol 7 no 6 pp 651ndash655 2010

[60] R Li J Liang S Ni et al ldquoA mesenchymal-to-epithelial transi-tion initiates and is required for the nuclear reprogramming ofmouse fibroblastsrdquo Cell Stem Cell vol 7 no 1 pp 51ndash63 2010

[61] P Samavarchi-Tehrani A Golipour L David et al ldquoFunctionalgenomics reveals a BMP-driven mesenchymal-to-epithelialtransition in the initiation of somatic cell reprogrammingrdquo CellStem Cell vol 7 no 1 pp 64ndash77 2010

[62] T Lin R Ambasudhan X Yuan et al ldquoA chemical platform forimproved induction of human iPSCsrdquo Nature Methods vol 6no 11 pp 805ndash808 2009

[63] J K Ichida J Blanchard K Lam et al ldquoA small-moleculeinhibitor of tgf-120573 signaling replaces sox2 in reprogramming byinducing Nanogrdquo Cell Stem Cell vol 5 no 5 pp 491ndash503 2009

[64] X Yuan H Wan X Zhao S Zhu Q Zhou and S Ding ldquoBriefreport combined chemical treatment enables Oct4-inducedreprogramming frommouse embryonic fibroblastsrdquo StemCellsvol 29 no 3 pp 549ndash553 2011

[65] G N Pandian S Sato C Anandhakumar et al ldquoIdentificationof a small molecule that turns ON the pluripotency genecircuitry in human fibroblastsrdquo ACS Chemical Biology vol 9no 12 pp 2729ndash2736 2014

[66] P Mali B-K Chou J Yen et al ldquoButyrate greatly enhancesderivation of human induced pluripotent stem cells by promot-ing epigenetic remodeling and the expression of pluripotency-associated genesrdquo Stem Cells vol 28 no 4 pp 713ndash720 2010

[67] J Lee Y Xia M-Y Son et al ldquoA novel small molecule facilitatesthe reprogramming of human somatic cells into a pluripotentstate and supports the maintenance of an undifferentiatedstate of human pluripotent stem cellsrdquo Angewandte ChemieInternational Edition vol 51 no 50 pp 12509ndash12513 2012

[68] Q Wang X Xu J Li et al ldquoLithium an anti-psychotic druggreatly enhances the generation of induced pluripotent stemcellsrdquo Cell Research vol 21 no 10 pp 1424ndash1435 2011

[69] D A ClaassenMMDesler andA Rizzino ldquoROCK inhibitionenhances the recovery and growth of cryopreserved humanembryonic stem cells and human induced pluripotent stemcellsrdquo Molecular Reproduction and Development vol 76 no 8pp 722ndash732 2009

[70] Z Li and TM Rana ldquoA kinase inhibitor screen identifies small-molecule enhancers of reprogramming and iPS cell generationrdquoNature Communications vol 3 article 1085 2012

[71] T Chen L Shen J Yu et al ldquoRapamycin and other longevity-promoting compounds enhance the generation of mouseinduced pluripotent stem cellsrdquo Aging Cell vol 10 no 5 pp908ndash911 2011

[72] Y Shi M Zou E Y Baitei et al ldquoCannabinoid 2 receptorinduction by IL-12 and its potential as a therapeutic target forthe treatment of anaplastic thyroid carcinomardquo Cancer GeneTherapy vol 15 no 2 pp 101ndash107 2008

[73] Y Li Q Zhang X Yin et al ldquoGeneration of iPSCs from mousefibroblasts with a single gene Oct4 and small moleculesrdquo CellResearch vol 21 no 1 pp 196ndash204 2011

[74] J Staerk C A Lyssiotis L A Medeiro et al ldquoPan-src familykinase inhibitors replace Sox2 during the direct reprogrammingof somatic cellsrdquo Angewandte ChemiemdashInternational Editionvol 50 no 25 pp 5734ndash5736 2011

[75] J-H Moon J S Heo J S Kim et al ldquoReprogrammingfibroblasts into induced pluripotent stem cells with Bmi1rdquo CellResearch vol 21 no 9 pp 1305ndash1315 2011

[76] D-W Jung W-H Kim and D R Williams ldquoReprogramor reboot small molecule approaches for the production ofinduced pluripotent stem cells and direct cell reprogrammingrdquoACS Chemical Biology vol 9 no 1 pp 80ndash95 2014

[77] Z Pasha H K Haider and M Ashraf ldquoEfficient non-viralreprogramming of myoblasts to stemness with a single smallmolecule to generate cardiac progenitor cellsrdquo PLoS ONE vol6 no 8 Article ID e23667 2011

[78] S Durnaoglu S Genc and K Genc ldquoPatient-specific pluripo-tent stem cells in neurological diseasesrdquo Stem Cells Interna-tional vol 2011 Article ID 212487 17 pages 2011

[79] L HMak S N Georgiades E Rosivatz et al ldquoA smallmoleculemimicking a phosphatidylinositol (45)-bisphosphate bindingpleckstrin homology domainrdquoACS Chemical Biology vol 6 no12 pp 1382ndash1390 2011

[80] Q-L Ying J Wray J Nichols et al ldquoThe ground state ofembryonic stem cell self-renewalrdquoNature vol 453 no 7194 pp519ndash523 2008

[81] B Valamehr M Robinson R Abujarour et al ldquoPlatform forinduction and maintenance of transgene-free hiPSCs resem-bling ground state pluripotent stem cellsrdquo StemCell Reports vol2 no 3 pp 366ndash381 2014

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anatomy Research International

PeptidesInternational Journal of


Hindawi Publishing Corporation httpwwwhindawicom

International Journal of

Volume 2014

Zoology


Molecular Biology International

GenomicsInternational Journal of


The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014


BioinformaticsAdvances in

Marine BiologyJournal of



Signal TransductionJournal of


BioMed Research International

Evolutionary BiologyInternational Journal of



Biochemistry Research International

ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014


Genetics Research International


Advances in

Virolog y

Hindawi Publishing Corporationhttpwwwhindawicom

Nucleic AcidsJournal of

Volume 2014

Stem CellsInternational



Enzyme Research



Microbiology


to effective clinical use [28ndash30] We here discuss the smallmolecules in iPSC generation including three types of com-pounds small molecules that may improve reprogrammingefficiency compounds that replace one or more reprogram-ming factors compound combination alone that is sufficientto induce mouse iPSC We also provide perspective views ofthe possibility of the iPSC generation from human somaticcells and its future applications

2 Compounds That May Improve theReprogramming Efficiency and Quality

For the first time Huangfu et al studied the compound appli-cation in iPSC generation they investigated the effects of thehistone deacetylase (HDAC) inhibitor valproic acid (VPA)and found that reprogramming efficiency was increased100-fold over that of the transcription factor method [31]Soon Ding group used BIX-01294 which inhibits histonemethyltransferase (HMT) by activating calcium channels inthe plasma membrane to improve reprogramming efficiency[32 33] To date the small molecules that have been used togenerate iPSCs can be categorized as epigenetic modifierswingless and integration site growth factor (WNT) signalmodulators moderators of cell senescence or modulatorsof metabolism [3 34 35] and the functions of the smallmolecules in iPSC generation are summarized in Figure 1Through these mechanisms the small molecules couldimprove iPSC generation efficiency andor could replacesome of Yamanaka factors The small molecules that mightenhance iPSC generation efficiency were collected in Table 1

21 Epigenetic Modification During programming cellsundergo changes at a global transcriptional level and alsoexperience epigenetic changes in their DNA along withhistone modifications [3 28 29 34 36 37] Small moleculessuch as Bix-01294 (Bix) target enzymes responsible for his-tone methylation and demethylation and increase expressionlevels of OCT4 and KLF4 during somatic cell reprogram-ming [32] Parnate a lysine-specific demethylase inhibitor(LSD1) mediates H3K4 demethylation enabling Oct4 andKLF4 induction of human keratinocytes into iPSCs whencombined with CHIR99021 a glycogen synthase kinase 3(GSK-3) inhibitor [31] DNA methyltransferase inhibitor5-azacytidine (5-Aza) or three other histone deacetylaseinhibitors (suberoylanilide hydroxamic acid trichostatin Aand valproic acid) can also improve reprogramming effi-ciency following the transduction of MEFs with four tran-scription factors [31 35 38 39]

To improve incomplete epigenetic reprogramming thesomatic memory can be erased and new epigenetics areestablished in iPSCs via treatment with trichostatin A [3839] Epigenetic modification using small molecules signifi-cantly increases iPSC reprogramming efficiency and reducesepigenetic memory retention [3 34 39]

22 WNT Signal Modulators The WNT pathway plays animportant role in self-renewal of embryonic stem cells (ESCs)[40] The CHIR99021 molecule an inhibitor of GSK-3120573

activates WNT signaling improving the efficiency of pro-gramming and eliminating the use of c-Myc [41]

When combined with other compounds they serve asreplacements for certain key transcription factors or theycan induce differentiation into specific cell types It hasbeen hypothesized that WNT pathway regulators couldplay a significant role in cellular reprogramming As anexample the GSK-3 beta inhibitor CHIR99021 can improvereprogramming efficacy by replacing c-Myc Another GSK3inhibitor kenpaullone can be used as a replacement forKLF4 however the three transcription factors Oct4 Sox2and c-Myc must still be used for successful reprogramming[42] Many similar small molecules have been found to beeffective in iPSC generation The screening of other WNTsignaling modulators has been discussed in detail by otherresearchers [34 39 40 43]

23 Moderators of Cell Senescence Cell senescence in repro-gramming is usually thought to contribute to slow dynamicsand low efficiency Stress response cell senescence and earlyprogramming characteristics of iPSCs involve expression ofp21cip1 and p16INK4ap19arf which are upregulated by p53Knocking out p53 improves iPSC generation efficiency andkinetics [44ndash51] However the p53 protein is a key tumorsuppressor and improving iPSC generation by blocking p53likely increases the risk of tumor formation [44ndash50 52] asshown in murine ESCs [51]

A common small molecule natural antioxidant vitaminC has been found to promote the formation of iPSCs frommouse and human somatic cells by indirectly lowering theexpression of p21 and p53 [53] Some small molecules havebeen found to be regulators of the pathways involved in cellsenescence with little risk of tumor formation [54] Theydownregulate the expression of various genes and result inimproved efficiency and dynamics during iPSC generation[55ndash57]

24 Modulators of Metabolism Growth of differentiatedadult somatic cells usually involves mitochondrial oxidativephosphorylation while pluripotent stem cells mainly employglycolysis metabolism [56ndash58] This metabolic programmingability is driven by hypoxia-inducible factor 1 alpha withc-Myc promoting glycolysis [58] Consistently PS48 anactivator of 31015840-phosphoinositide-dependent protein kinase1 in combination with other small molecules (a-83-01 andPD0325901) and sodium butyrate (a histone deacetylaseinhibitor) has been used to reprogram adult keratinocytesumbilical vein endothelial cells and amniotic fluid-derivedcells It has been shown that PS48 activates the phospho-inositide 3-kinaseAkt pathway upregulating gene expressionwhich promotes mitochondrial oxidative phosphorylationand glycolysis metabolism [59]

25 MET Mediated by Transforming Growth Factor- (TGF-)120573 Pathway Signaling Inhibitors Enhances ReprogrammingA mesenchymal-epithelial transition (MET) is a reversiblebiological process involving the transition from motile mul-tipolar or spindle-shaped mesenchymal cells to planar arrays



Differentiation


Somaticcell

Dedifferentiation















[31]























PDK1 activator


enoic acid(PS48)





Table 1 Continued





Replacementfor TF









Li et al (2011) [73]












Shi et al (2008)[32 33 72]





















2iPD0325901

1 uMChir9902110 uM















Differentiation



Somaticcell

mEFVC6FTVC6FTZ

Multipotent
























Mitochondrion

ThiazovivinROCK

Myosin PPtase

PD0325901

Pl3kWNT

and otherpathways

p p

pERK

Oct4Sox2cMycKlf4Ac

AcAcAc

Ac

AcAc

Ac

NHNH

MTase

DNAMTase



ALKs

Cytokines

CH3 CH3 CH3

SMADs

MLCP

RasGTP

RasGTP

ActivinNodal

MWE12

Raf

RasGAP

McCP2 CH3

CH3

TGF-120573

TGF-120573 pathway


p


Sin3A HDAC2

p53complex

RhoA

CpG

Histone




Acknowledgments


References



























































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology



Differentiation


Somaticcell

Dedifferentiation















[31]























PDK1 activator


enoic acid(PS48)





Table 1 Continued





Replacementfor TF









Li et al (2011) [73]












Shi et al (2008)[32 33 72]





















2iPD0325901

1 uMChir9902110 uM















Differentiation



Somaticcell

mEFVC6FTVC6FTZ

Multipotent
























Mitochondrion

ThiazovivinROCK

Myosin PPtase

PD0325901

Pl3kWNT

and otherpathways

p p

pERK

Oct4Sox2cMycKlf4Ac

AcAcAc

Ac

AcAc

Ac

NHNH

MTase

DNAMTase



ALKs

Cytokines

CH3 CH3 CH3

SMADs

MLCP

RasGTP

RasGTP

ActivinNodal

MWE12

Raf

RasGAP

McCP2 CH3

CH3

TGF-120573

TGF-120573 pathway


p


Sin3A HDAC2

p53complex

RhoA

CpG

Histone




Acknowledgments


References



























































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology





[31]























PDK1 activator


enoic acid(PS48)





Table 1 Continued





Replacementfor TF









Li et al (2011) [73]












Shi et al (2008)[32 33 72]





















2iPD0325901

1 uMChir9902110 uM















Differentiation



Somaticcell

mEFVC6FTVC6FTZ

Multipotent
























Mitochondrion

ThiazovivinROCK

Myosin PPtase

PD0325901

Pl3kWNT

and otherpathways

p p

pERK

Oct4Sox2cMycKlf4Ac

AcAcAc

Ac

AcAc

Ac

NHNH

MTase

DNAMTase



ALKs

Cytokines

CH3 CH3 CH3

SMADs

MLCP

RasGTP

RasGTP

ActivinNodal

MWE12

Raf

RasGAP

McCP2 CH3

CH3

TGF-120573

TGF-120573 pathway


p


Sin3A HDAC2

p53complex

RhoA

CpG

Histone




Acknowledgments


References



























































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


Table 1 Continued





Replacementfor TF









Li et al (2011) [73]












Shi et al (2008)[32 33 72]





















2iPD0325901

1 uMChir9902110 uM















Differentiation



Somaticcell

mEFVC6FTVC6FTZ

Multipotent
























Mitochondrion

ThiazovivinROCK

Myosin PPtase

PD0325901

Pl3kWNT

and otherpathways

p p

pERK

Oct4Sox2cMycKlf4Ac

AcAcAc

Ac

AcAc

Ac

NHNH

MTase

DNAMTase



ALKs

Cytokines

CH3 CH3 CH3

SMADs

MLCP

RasGTP

RasGTP

ActivinNodal

MWE12

Raf

RasGAP

McCP2 CH3

CH3

TGF-120573

TGF-120573 pathway


p


Sin3A HDAC2

p53complex

RhoA

CpG

Histone




Acknowledgments


References



























































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


















2iPD0325901

1 uMChir9902110 uM















Differentiation



Somaticcell

mEFVC6FTVC6FTZ

Multipotent
























Mitochondrion

ThiazovivinROCK

Myosin PPtase

PD0325901

Pl3kWNT

and otherpathways

p p

pERK

Oct4Sox2cMycKlf4Ac

AcAcAc

Ac

AcAc

Ac

NHNH

MTase

DNAMTase



ALKs

Cytokines

CH3 CH3 CH3

SMADs

MLCP

RasGTP

RasGTP

ActivinNodal

MWE12

Raf

RasGAP

McCP2 CH3

CH3

TGF-120573

TGF-120573 pathway


p


Sin3A HDAC2

p53complex

RhoA

CpG

Histone




Acknowledgments


References



























































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


Differentiation



Somaticcell

mEFVC6FTVC6FTZ

Multipotent
























Mitochondrion

ThiazovivinROCK

Myosin PPtase

PD0325901

Pl3kWNT

and otherpathways

p p

pERK

Oct4Sox2cMycKlf4Ac

AcAcAc

Ac

AcAc

Ac

NHNH

MTase

DNAMTase



ALKs

Cytokines

CH3 CH3 CH3

SMADs

MLCP

RasGTP

RasGTP

ActivinNodal

MWE12

Raf

RasGAP

McCP2 CH3

CH3

TGF-120573

TGF-120573 pathway


p


Sin3A HDAC2

p53complex

RhoA

CpG

Histone




Acknowledgments


References



























































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology













Mitochondrion

ThiazovivinROCK

Myosin PPtase

PD0325901

Pl3kWNT

and otherpathways

p p

pERK

Oct4Sox2cMycKlf4Ac

AcAcAc

Ac

AcAc

Ac

NHNH

MTase

DNAMTase



ALKs

Cytokines

CH3 CH3 CH3

SMADs

MLCP

RasGTP

RasGTP

ActivinNodal

MWE12

Raf

RasGAP

McCP2 CH3

CH3

TGF-120573

TGF-120573 pathway


p


Sin3A HDAC2

p53complex

RhoA

CpG

Histone




Acknowledgments


References



























































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


Mitochondrion

ThiazovivinROCK

Myosin PPtase

PD0325901

Pl3kWNT

and otherpathways

p p

pERK

Oct4Sox2cMycKlf4Ac

AcAcAc

Ac

AcAc

Ac

NHNH

MTase

DNAMTase



ALKs

Cytokines

CH3 CH3 CH3

SMADs

MLCP

RasGTP

RasGTP

ActivinNodal

MWE12

Raf

RasGAP

McCP2 CH3

CH3

TGF-120573

TGF-120573 pathway


p


Sin3A HDAC2

p53complex

RhoA

CpG

Histone




Acknowledgments


References



























































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology
















































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology












































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology








Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology

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Review Article Reprogramming with Small Molecules instead ...downloads.hindawi.com/journals/sci/2015/794632.pdf · Reprogramming with Small Molecules instead of Exogenous Transcription