review article cellular reprogramming using defined...

Review ArticleCellular Reprogramming Using Defined Factors and MicroRNAs

Takanori Eguchi123 and Takuo Kuboki4

1Division of Molecular and Cell Biology Department of Radiation Oncology Beth Israel Deaconess Medical CenterHarvard Medical School 3 Blackfan Circle Center for Life Science 6 Boston MA 02115 USA2Department of Dental Pharmacology Okayama University Graduate School of Medicine Dentistry and Pharmaceutical Sciences2-5-1 Shikata-cho Okayama 700-8525 Japan3Advanced Research Center for Oral and Craniofacial Sciences Okayama University Dental SchoolGraduate School of MedicineDentistry and Pharmaceutical Sciences Okayama 700-8525 Japan4Department of Oral Rehabilitation and Regenerative Medicine Okayama University Graduate School of MedicineDentistry and Pharmaceutical Sciences 2-5-1 Shikata-cho Okayama 700-8525 Japan

Correspondence should be addressed to Takanori Eguchi eguchiokayama-uacjp

Received 11 December 2015 Revised 8 March 2016 Accepted 10 April 2016

Academic Editor Huricha Baigude

Copyright copy 2016 T Eguchi and T Kuboki This is an open access article distributed under the Creative Commons AttributionLicense which permits unrestricted use distribution and reproduction in any medium provided the original work is properlycited

Development of human bodies organs and tissues contains numerous steps of cellular differentiation including an initial zygoteembryonic stem (ES) cells three germ layers and multiple expertized lineages of cells Induced pluripotent stem (iPS) cellshave been recently developed using defined reprogramming factors such as Nanog Klf5 Oct34 (Pou5f1) Sox2 and Myc Thisoutstanding innovation is largely changing life science and medicine Methods of direct reprogramming of cells into myocytesneurons chondrocytes and osteoblasts have been further developed using modified combination of factors such as N-myc L-mycSox9 andmicroRNAs in defined celltissue culture conditionsMesenchymal stem cells (MSCs) and dental pulp stem cells (DPSCs)are also emergingmultipotent stem cells with particular microRNA expression signatures It was shown that miRNA-720 had a rolein cellular reprogramming through targeting the pluripotency factor Nanog and induction of DNA methyltransferases (DNMTs)This review reports histories topics and idea of cellular reprogramming

1 Introduction

A term of cellular ldquoreprogrammingrdquo has been major afterthe development of induced pluripotent stem (iPS) cells [1]For the development of iPSCs Dr Shinya Yamanaka wasawardedNobel prize in physiology andmedicine in 2012TheiPS cells are embryonic stem (ES) cells-like pluripotent cellsinduced using defined factors The definition of ldquoreprogram-mingrdquo in the narrow sense is like artificial dedifferentiation(reprogram) of cells such as skin cells into ES cells-likepluripotent stem cells Mesenchymal stem cells (MSCs)haematopoietic stem cells (HSCs) or neuronal stem cells(NSCs) are also multipotent stem cells which are inter-mediate cells between more matured cells and pluripotentstem cells These intermediate stem cells have been alsoinvestigated in reprogramming studies More recently a newconcept termed ldquodirect reprogrammingrdquo has been developed

Direct reprogramming is reprogramming of cells such asskin cells into another type of differentiated cells in anotherlineage

2 Stem Cells Germ Layers andTissue Development

In order to understand cellular reprogramming we needsome basic knowledge regarding tissue development Anembryo is a multicellular diploid eukaryote in its earlieststage of development from the time of fertilization throughsexual reproduction until birth hatch or germination EScells are pluripotent stem cells derived from the inner cellmass of a blastocyst an early-stage preimplantation embryoIn a beginning step of embryonic development from ES cellsand the blastocyst three germ layers are generated ectodermmesoderm and endoderm

Hindawi Publishing CorporationStem Cells InternationalVolume 2016 Article ID 7530942 12 pageshttpdxdoiorg10115520167530942

2 Stem Cells International

21 Ectoderm Ectoderm emerges and originates from theouter layer of germ cells The word ectoderm comes fromthe Greek ektos meaning outside and derma meaning skinThe ectoderm differentiates to form the nervous system(spine peripheral nerves and brain) and tooth enamel viaameloblasts and epidermis (the outer part of integument)Ectoderm also forms the lining of the mouth (oral mucosa)anus nostrils sweat glands hair and nails In vertebratesthe ectoderm has three parts external ectoderm also knownas surface ectoderm the neural crest and neural tube Thelatter two are known as neuroectoderm as described belowEstablished ectodermal markers are 120573-III-tubulin and Otx2Sasai et al reported that ectodermal factor XFDL156 (Zfp12Zfp74) restricts mesodermal differentiation by inhibiting p53that is required for mesodermal differentiation [2]

22 Neuroectoderm Neurulation and Neural Crest Forma-tion process of the neural tube neural crest cells and the epi-dermis is called neurulationThe neural tube cells give rise tothe central nervous system (CNS) Neural crest cells give riseto the peripheral and enteric nervous system melanocytesfacial cartilage and the dentin of teeth The epidermal cellregion gives rise to the epidermis hair nails sebaceousglands olfactory andmouth epithelium as well as eyes All ofthe organs that arise from the ectoderm originate from twoadjacent tissue layers the epithelium and the mesenchymeOrganogenesis of the ectoderm is mediated by signals suchas FGF TGF120573 Wnt and the hedgehog family FGF-9 whichis expressed in epidermis but not in the mesenchyme is a keyfactor in the initiation of tooth germ development FGF-10helps to stimulate epithelial cell (ameloblast) proliferation inorder to make larger tooth germs Mammalian teeth developfrom oral ectoderm and neural crest ectoderm derived frommesenchyme There are over 170 subtypes of ectodermaldysplasia

23 Odontogenesis (Tooth Development) Tooth germ is anaggregation of cells that eventually forms a tooth These cellsare derived from the ectoderm of the first pharyngeal archand the ectomesenchyme of the neural crest [3 4] The toothgerm is organized into three parts the enamel organ thedental papilla and the dental sac (or dental follicle) The cellsin the enamel organ give rise to ameloblasts which produceenamel The location where the outer enamel epithelium andinner enamel epithelium join is called the cervical loop Thegrowth of cervical loop cells into the deeper tissues formsHertwig Epithelial Root Sheath which determines the rootshape of the tooth During the development there are strongsimilarities between keratinization and amelogenesis [5 6]Keratin is also present in epithelial cells of tooth germ A thinfilm of keratin is present on erupted tooth so calledNasmythrsquosmembrane or enamel cuticle The dental papilla containscells that develop into odontoblasts which are dentin-forming cells Mesenchymal cells within the dental papillaare responsible for the formation of tooth pulp The dentalsac (or dental follicle) gives rise to cementoblasts osteoblastsand fibroblasts Cementoblasts form the cementum of atooth Osteoblasts give rise to the alveolar bone surroundingthe roots of teeth Fibroblasts are involved in developing

periodontal ligaments which connect tooth cementum to thealveolar bone

Tooth have a bone-like structure and nature whoseoutside is armored by cortical calcified enamel layer Interme-diate ivory-like dentin layer of tooth contains dendrite-likeprocess of odontoblasts Inside of tooth is called dental pulpwhich has bone-marrow-like space and contains nerves vas-culatures haematopoietic cells and matrix-producing cellsodontoblasts The odontoblasts are cells lining on the surfaceof dentinmatrix from the pulp inside and extending dendrite-like process through the dentinal tube These structuresand functions of odontoblasts are similar to osteoblastswhich are lining on the surface of bone matrix from thebone marrow As odontoblasts are extending dendrite-likeprocess osteocytes are extending dendrites through the bonecanaliculi Both odontoblasts and osteocytes locate close tonerves and are the sensors as well Such bone-marrow-likefeature of dental pulp ldquotooth marrowrdquo has given us an ideaof dental pulp stem cells (DPSCs) or tooth marrow stromalcells (TMSCs) that may contain multiple stem cells includingMSCs HSCs and NSCs [7ndash11] Stem cells from humanexfoliated deciduous teeth (SHEDs) have been shown to beuseful as therapeutic tools [12]

24 Mesoderm Mesoderm has been known as a resource ofmuscle (including smooth cardiac skeletal tongue masti-cation and facial expressions) bone cartilage blood vesselsblood cells urogenital structures such as kidney gonads andtheir associated ducts A group of cells in ectoderm trans-forms via epithelial-mesenchymal transition (EMT)migratesbetween ectoderm and endoderm and then formsmesodermMesoderm has the capacity to induce the growth of otherstructures such as the neural plate the precursor to thenervous system Mesoderm is formed through a processcalled gastrulation There are three components the paraxialmesoderm the intermediate mesoderm and the lateral platemesodermTheparaxialmesoderm gives rise tomesenchymeof the head and organizes into somites that give rise tomuscletissue cartilage and bone and subcutaneous tissue of skinsSignals for somite differentiation are derived from structuressurrounding mesoderm such as notochord neural tube andepidermis Established markers of mesoderm are 120572-smoothmuscle actin (120572-SMA) and brachyury

Muscle satellite cells (myosatellite cells) have been shownas precursors of mature skeletal muscle cells [13] The musclesatellite cells have a potential to provide additional myonucleito their parental muscle fiber or return to a quiescentstate [14] Tendon and ligament have been known to bemesenchymal lineage as well

25 Bone Marrow Stromal Cells and Mesenchymal Stem CellsBone marrow stromal cells (BMSCs) have been shown tocontain mesenchymal stem cells (MSCs) that have abilitiesto differentiate to multiple lineages such as osteoblastschondrocytes and adipocytes Osteoblasts are lining on thesurface of and producing bone matrix while osteocytes thefurther differentiated cells of osteoblasts are embedded in thelacuna in the calcifiedmatrix extending communicative den-drites into canaliculi for mechanotransduction [15] Defined

Stem Cells International 3

transcription factors can induce differentiation of MSCs intoexpertized lineages for example Sox9 in initial chondroge-nesis [16] Runx2 in osteoblast precursor and chondrocytehypertrophy [17] OsterixSp7 in later osteogenesis [18 19]PPAR120574 in adipogenesis [20] and MyoD in myogenesis [21]

26 Endothelial Cells Haematopoietic Stem Cells and BloodCells Haematopoietic stem cells (HSCs) and cardiovascularsystemhave been known to be differentiated frommesodermWhether blood cells arise from mesodermal cells mes-enchymal progenitors bipotent endothelial-haematopoieticprecursors or haemogenic endothelial cells had remainedcontroversial but haemangioblasts have been known todifferentiate to endothelial cells as well as to blood cellsLancrin et al showed that the haemangioblast generateshaematopoietic cells through a haemogenic endotheliumstage [22] Eilken et al showed that using new imaging andcell-tracking methods embryonic endothelial cells could behaemogenic [23] Boisset et al showed that using in vivoimaging the dynamic de novo emergence of phenotypicallydefined HSCs which were Sca1(+) c-kit(+) and CD41(+)directly from ventral aortic haemogenic endothelial cells[24] Bertrand et al (2010) showed that HSCs derive directlyfrom aortic endothelium during development [25] Chenet al showed that Runx1 is required for the endothelialto haematopoietic cell transition but not thereafter [26]Kissa and Herbomel showed that blood stem cells emergefrom aortic endothelium by an endothelial-haematopoietictransition [27]

Blood haematopoietic cells have been known to bederived frommesoderm and located in the red bonemarrowHSCs are the stem cells that give rise to all the other bloodcells through the process of haematopoiesis [28] The HSCsgive rise to the myeloid and lymphoid lineages of blood cellsMyeloid cells includemonocytes macrophages (M1 andM2)neutrophils basophils eosinophils erythrocytes dendriticcells (DCs) megakaryocytes or its products platelets Bone-resolving multinucleated osteoclasts are one of the maturecells derived from monocytes that differentiate to mononu-cleic osteoclast precursor leading to osteoclastic cell fusionLymphoid cells include T cells (T helper 1 cells T helper 2cells and T helper 17 cells also known as Th1 Th2 andTh17cells etc) B cells and natural killer (NK) cells HSCs consti-tute 1 10000 of cells in the myeloid tissue in bone marrowArtery vein nerves and lymphatic vessels are entering andextending in bone As part of the lymphatic system lymphvessels are complementary to the cardiovascular system

27 Endoderm Including Definitive Endoderm and Mesoen-doderm Endoderm is an origin of multiple organs suchas pancreas (Pdx1 Ngn3 Ins are the markers) liver gutintestine lung thyroid gland and thymus urinary systemsuch as urinary bladder and urethra The endoderm consistsof flattened cells which subsequently become columnar Itforms the epithelial lining ofmultiple systemsThe embryonicendoderm develops into the interior lining of the digestivetube and respiratory tube

Endoderm gives rise to definitive endoderm (DE)and visceral endoderm Interestingly a part of DE forms

mesendoderm that is originated from common precursorcells derived from mesoderm and endoderm The DE givesrise to the gastrointestinal organs such as stomach pancreasliver and intestine Recent studies have identified severalgerm layer-specific markers of the early DE Sox17 is a DE-specific marker [29] CXCR4 (C-X-C chemokine receptortype 4) which is expressed in the mesoderm as well asin the DE and is used in combination with E-cadherinwhich is also expressed in ES cells and ectoderm for theprospective isolation of embryonic or ES cell-derivedDE cells[30] Dr Kumersquos group identified DAF1 (decay acceleratingfactor)CD55 and Cerberus1 (Cer1) as novel DE markers[31 32] Shiraki et al reported differentiation and character-ization of ES cells into three germ layers using mesoderm-derived feeder M15 cells [33 34] In this study stimulationwith a combination of activin and bFGF induced ES celldifferentiation to mesendoderm endoderm and pancreaticprecursor cells stimulation with Bmp7 induced differenti-ation to mesoderm the addition of p38 MAPK inhibitorSB203580 induced differentiation to neuroectoderm Furtherlong-term culture enabled neuroectodermal differentiationto neuron astrocytes and oligodendrocytes andmesodermaldifferentiation to osteoblasts and adipocytes It was alsoshown that stimulation of endoderm with a combination ofHGF and dexamethasone induced differentiation to hepato-cytes

Characterization of stem cells in the development ofbodies organs and tissues has been our scientific interestswhile the multipotency of differentiation proliferation andself-renewal have been emerging in clinical applications

3 Induced Pluripotent Stem Cells

In 2006 Takahashi and Yamanaka reported induction ofpluripotent stem (iPS) cells from mouse adult fibroblast cul-tures by defined factors [1] Before this study numerous tran-scription factors (TFs) had been shown to be critical to induceor maintain certain cellular differentiation stages includingES cells Dr Yamanakarsquos group aimed to know which factorswere required for induction of ES cells-like pluripotencyMore than 20 transcription factors and their combinationswere examined using retrovirus-mediated transduction inskin fibroblasts Nanog is one of the TFs that are expressedin ES cells and Nanog promoter-driven green fluorescenceprotein (GFP) reporter was used as a marker for the screen-ing of the TFs Another marker is the colony formationability of pluripotent stem cells Utilizing these significantfeatures Dr Yamanakarsquos group finally discovered that EScells-like colonies were induced from skin fibroblasts usingcombination of four defined factors Klf4 Oct-34 (Pou5f1)Sox2 and c-Myc The ES cells had been known to havean ability to form teratoma upon injection to experimentalanimals Both ES and iPS cells generated from skin fibroblastsindeed formed teratoma upon subcutaneous injection tomice Finally the ESC-like iPSCs were shown to be able togive rise to embryosThese results were enough to say that thecells were pluripotentThe iPS cells were next generated fromhuman dermal fibroblasts [35] So far established markers ofES and iPS cells are SSEA-3 SSEA-4 TRA-1-60 TRA-1-81


TSmesodermal

p53

miR-34miR-720

Nanog N-myc Oct4

Mdm2 E3 ligase

HDACSirT1

Sox2CTGF

miR-200family

miR-302367cluster

EMT

Tob2

Dazap2

Slain1

Lif

BMP

anta

goni

st

Jak

Stat Bmp Cdh1E-cadherin

Klf24

Intercellularadhesion

Zeb1

Zeb2

Mesenchymaldifferentiation

Tgfbr2

Snai2

Fn1

RhoC

Reprogramming pluripotency

G1S transition

G1 S

Rbl2(p130)Rbl1

(p107)Rb1

(pRb)

Smad3

Apoptosis

Bcl2

TS

Cdk46

CcnD

Cdk2CcnE Survival

p21-Cdkn1aG1S arrest

p16-Cdkn2a(Ink4aArt)

Figure 1 Summary of published intermolecular interactions featuring pluripotency-associated miRNAs and their targets Factors positiveto induction of pluripotency was shown in red Inhibitory factors to induction of pluripotency was shown in blue Epithelial-mesenchymaltransition (EMT) cell cycle and cell death and survival were featured Molecularly targeted drugs are shown with drug marks TS tumorsuppressor

Oct-4 (Pou5f1) Nanog Sox2 Klf4 MycN Lin28 CriptoFbx15 Dnmt3b Fgf4 Gdf3 Rex1 miR-200c miR-302 familymiR-369-3p and miR-369-5p

31 Improvement of Methods in Generation of iPS Cells Afterthis breakthrough of iPS cells researchers have made theeffort to reduce risks of oncogenesis which could be inducedduring the reprogramming The risk included the usage ofviral vectors that could be integrated into genomic DNAand the usage of an oncogene c-myc Replacement of theviral vectors to plasmid vectors enabled to generate iPScells while induction efficiency was reduced [36] One cluewas that the oncogenic c-myc is a member of Myc familythat includes other two members N-myc and L-myc DrYamanakarsquos group replaced the c-myc to N-myc and L-myc[37] Indeed iPS cells were generated using N-myc instead ofc-myc

It had been known that p53ndashp21 pathway served as abarrier in tumorigenicity (Figure 1) Dr Yamanakarsquos groupdemonstrated that the p53ndashp21 pathway was also a barrier inthe generation of iPS cells [38]The discovery of iPS cells andthe recovery from the tumorigenic risk have made the cellsmore feasible toward clinical applications

32 Clinical Application of Pluripotent Stem Cells The appli-cation of ES and iPS cells has been thought for regenerativemedicine The cells or tissues generated by the reprogram-mingmight be useful for transplantation therapies Hotta andYamanaka reviewed clinical applications of pluripotent stemcells in their recent publication entitled ldquoFrom Genomics toGeneTherapy Induced Pluripotent StemCellsMeet GenomeEditingrdquo [39] The worldrsquos first phase I clinical trial using hEScells was aimed to treat patients with spinal cord injuriesIn October 2010 oligodendrocyte progenitor cells derivedfrom H1 human ES cells were injected into the site of spinalcord damage Unfortunately the trial was terminated in 2013after the biotech company Geron decided to withdraw fromthe stem cell business The second clinical trial using hEScells investigated Stargardtrsquos macular dystrophy (SMD) andadvanced dry age-related macular degeneration (Dry AMD)by using retinal pigment epithelium (RPE) cells derived fromahumanES cell lineMA09Thefirst patients for each trial wastreated in 2011 As a phase I study to test feasibility and safetythere were no signs of tumorigenicity of apparent rejection[40] The third example of an ES cell-related clinical trial isfor type I diabetes Pancreatic precursor cells (called PEC-01cells) from CyT203 human ES cells [41] were encapsulated


into a device Encaptra and transplanted into a patient in2014 as a phase III clinical trial In all trials transplantationof human ES cells-derived products was allogenic and hasbeen conducted without matching HLA types Therefore thecurrent applications are mainly limited to immune privilegedtissues such as eyes and spinal cord iPS cells have beeninvestigated in an autologous transplantation setting InSeptember 2014 the first transplantation of RPE cells derivedfrom iPS cells [42] for Wet AMD was conducted by DrTakahashirsquos group at RIKEN CDB in Japan

A potential application of iPS cells has been advocatedthat this concept and methods can be useful for generationof disease model in which cells can be reprogrammed frompatient cells Such patient-derived iPS cells can be reasonablyused for drug screening Yahata et al (2011) reported anti-amyloid 120573 (A120573) drug screening platform using human-iPScells-derived neurons for the treatment of Alzheimerrsquos disease[43] Dr Takahashirsquos group (2014) reported integration-freeiPS cells derived from retinitis pigmentosa patient for diseasemodeling [44] Suzuki et al reported that pluripotent cellmodels of Fanconi anemia identify the early pathologicaldefect in human haemoangiogenic progenitors [45]

Genetic linkage analysis and recent genome-wide associ-ation studies (GWAS) meta-analysis have identified humangenetic variationmutations associated with diverse dis-eases and physical characteristics [46] Genetic variationsmutations with diseases could be corrected to normal orlower risk variation with genome editing technologies usingCRISPRCas9 that requires noncoding guide RNA (gRNA)[47] Indeed Kazuki et al reported complete genetic correc-tion of iPS cells from Duchenne muscular dystrophy (DMD)[48] Morishima et al reported that genetic correction ofHAX1 in iPS cells from a patient with severe congenitalneutropenia improves defective granulopoiesis [49] Furtherapplication of iPS cells in combination with genome editingtherapy and for generation of disease model in terms of drugdiscovery is ongoing

4 MicroRNAs Involving PluripotentStem Cells

MicroRNAs (miRNAs) are short noncoding RNAs that targetlong RNAs leading to inhibition of translation andor pro-motion of mRNA degradation miRNAs are generated frommiRNA cluster or intron of coding RNA These resources ofmiRNA are transcripts of genomic DNA miRNA also havebeen thought to be useful as biomarkers tools for cellularreprogramming and therapeutic targetsOnemiRNA speciestargets multiple long RNA leading to translational inhibitionof multiple proteins Nevertheless several challenges tocontrol cellular stemness and differentiation by miRNA havebeen done

41 MicroRNA-302367 Cluster Targets Multiple FactorsInvolving Epithelial-Mesenchymal and G1-S TransitionsmiRNA-induced pluripotent stem cells (mirPSC) werefirstly reported using human skin cancer cells Lin et alreported that miR-302 reprogrammed human skin cancercells into a pluripotent ESC-like state [50] This group firstly

reported that the miR-302 family members (miR-302s) wereexpressed most abundantly in slowly growing human EScells and ldquoquicklyrdquo decreased after cell differentiation andproliferation Therefore they hypothesized that miR-302scould be one of the key factors essential for maintenanceof ESC-renewal and pluripotency The miR-302-transfectedcells expressed key ES cell markers such as Oct34 SSEA-34Sox2 and Nanog but also had a highly demethylated genomesimilar to a reprogrammed zygotic genome Microarrayanalysis further revealed that genome-wide gene expressionpatterns between the mirPSCs and human ES cells H1 andH9 shared over 86 similarity Using molecular guidancein vitro this mirPSC could differentiate into distinct tissuecell types such as neurons chondrocytes fibroblasts andspermatogonia-like cells Based on these findings this groupconcluded that miR-302s function to reprogram cancer cellsinto an ES-like pluripotent state but also to maintain thestem cell state under a feeder-free cultural condition

Thereafter Barroso-del Jesus et al (2009) released per-spectives regarding the miR-302367 cluster as a potentialstemness regulator in ES cells [51] Later miRNA profilingstudies reproduced that miR-302 was essentially expressedspecifically in ES and iPS cells and lost upon differentiationand proliferation One such profiling was carried out byWilson et al who reported microRNA profiling of human-iPS cells termed microRNAomes [52] This group confirmedthat the presence of a signature group of miRNAs that isupregulated in both iPS and hES cells such as the miR-302367 and miR-1792 clusters Another miRNA profilingwas carried out by Stadler et al who reported the charac-terization of microRNAs involved in a state of ES cells [53]This group showed that defined hES cells-enriched miRNAgroups (miR-302 miR-17 miR-515 families and the miR-371-373 cluster) were downregulated ldquorapidlyrdquo in response todifferentiation

One arising question was what factors were targetedby miR-302 The first report of targets of miR-302 in ESand iPS cells was regarding cell cycle regulators Card et alshowed that Oct4Sox2-regulated miR-302 targeted mRNAencoding cyclin D1 in hES cells [54] (Figure 1) Lin etal reported that maintaining slowly proliferating adhesivestem cells will require inhibition of cell cycle by miR-302367 that targets cyclin D1 and cyclin-dependent kinases246 (Cdk2 Cdk4 and Cdk6) [55] Nevertheless cell cyclemust be rotated for mitosis of iPS and ES cells A tumorsuppressor p53 had been shown to upregulate p21sdotCdkn1a(Cdk inhibitor 1a) p21Cdkn1 had been known to inhibitCdk2cyclin E (CcnE) complex leading to G1S arrest Wanget al reported that ES cell-specific microRNAs regulated G1-S transition and promote rapid proliferation [56] In thisstudy G1-S arrest factors including p21Cdkn1a p130Rbl2and Lats2 were shown to be targeted by 51015840-UAAAGUGC -containing or AAAGUGCU -containing microRNAs suchas miR-291 miR-294 miR-295 and miR-302d This workexplained how the miR-302 overcame the G1-S arrest atthe G1-S transition Lin et al (2011) reported that miR-302targeted cosuppression of four ldquoepigeneticrdquo regulators lysinedemethylase KDM1 (also known as LSD1AOF2) AOF1p66MECP1 and MECP2 [57]


Targeting TGF120573 signaling by miR-302 may reprogramcells toward generation of iPS and mirPS cells throughinduction of mesenchymal-epithelial transition (MET) theacquisition of intercellular adhesion Pluripotent stem cellshave characters to form colonies along with acquirementof intercellular adhesion Intercellular adhesion is knownlargely to be lost during EMT in tissue development Themost significant inducer of EMT is TGF120573 and loss of TGF120573signaling can induce epithelial phenotypes with intercellularadhesion Thus the generation of iPS cells may require METalong with the acquisition of intercellular adhesion

Sequencing of RNA transcripts revealed that a pre-miRNA cluster encoded five miRNAs including miR-302a-302b -302c -302d (miR-302s) and miR-367 termed miR-302367 cluster Liao et al reported that the miR-302367cluster enhanced somatic cell reprogramming (SCR) byaccelerating anMET through targeting TGF120573 type II receptor(TGFbR2) and increased E-cadherin expression [58] BMPsignaling had been known as being required for maintenanceof ES cells Lipchina et al reported that miR-302367 clusterpromotes BMP signaling by targeting BMP inhibitors TOB2DAZAP2 and SLAIN1 [59] (Figure 1) Li et al reported thatnot only miR-302 but also miR-93 targets mRNA encodingTGFbR2 to enhance generation of iPS cells [60] Anokye-Danso et al reported miRNA-302367-mediated reprogram-ming of mouse and human somatic cells to pluripotency[61] This work showed an extremely higher efficiency of EScell-like colony formation with ES cell-like morphology andexpression of markers using miR-302367 cluster comparedto OSKM-iPS In this study the number of colonies with EScell-like morphology per 100000 cells was 10396 cells usingmiR-302367 and only 3 with OSKM in this work Howeverone says that for years this work has not been reproducedat all in any other groups or extensively used (given thatthe efficiency in the paper is strikingly high) Poleganovet al reported that human fibroblasts and ldquoblood-derivedendothelial progenitorrdquo cells were efficiently reprogrammedby transduction of nonmodifiedmiR-302367 cluster in a sin-gle vector leading to immune evasion [62]These works usingmiR-302367 showed that blocking mesenchymal TGF120573 sig-naling and maintenance of BMP signaling were requiredfor generation and maintenance of iPS cells Switching fromBMP to TGF120573 signaling may enable cells to differentiate tomesenchymal lineagephenotype

Faherty et al reported that CCN2CTGF increased miR-302s expression level [63] (Figure 1) CCN2 has been knownto associate with diverse extracellular proteins includinggrowth factors cell surface molecules such as receptors andintegrin and extracellular matrix proteins leading to modu-lation of these partners [64 65] Thus CCN2CTGF may beuseful as a growth factor supporting cellular reprogrammingthrough induction of miR-302

42 Combination of miR-200c Plus miR-302 s and miR-369s Family miR-200 family has been shown to repress EMTthrough targeting Zeb1Tcf8deltaEF1 a master transcriptionfactor that represses E-cadherin gene (CDH1) Miyoshi etal reported ldquoreprogramming of mouse and human cells topluripotency using mature microRNAsrdquo [66 67] This group

used a combination ofmiR-200c plusmiR-302 s andmiR-369s family of miRNAs without using OSKM factors Miyoshiet al showed similarity and difference between mirPS andES cells in gene expression levels of markers such as NanogOct4 Cripto Dppa5 Eras Fbx15 and miRNAs on whichthey focused Leonardo et al featured these successfulmaturemiRNA-driven reprogramming in a review entitled ldquothefunctions of microRNAs in pluripotency and reprogram-mingrdquo [68]

43 miR-34a Induced by Tumor Suppressor p53 Targets Multi-ple Pluripotency Factors Cell Cycle Genes and AntiapoptoticBcl2 The p53-p21 pathway had been known as a tumorsuppressor to G1-S arrest axis (Figure 1) Hong et al showedsuppression of iPS cell generation by the p53ndashp21 path-way [38] In this study iPS cell generation efficiency waslargely increased by knockdown and knockout of p53 Inother words loss of the tumor suppressor p53 may inducepluripotency in carcinogenesis In the p53-null backgroundiPS cells were generated from terminally differentiated Tlymphocytes

Interestingly in 2007 four groups reported thatmicroRNA-34 family members were a novel transcriptionaltarget of tumor suppressor p53 in human colon cancercells or in mouse embryonic fibroblasts (MEFs) [69ndash74]In these works it was shown that miR-34 family miRNAsmediated cellular senescence (growth arrest) in humancolon cancer cells or MEFs and targeted a program of genespromoting cell cycle progression such as E2F Cdk4 CcnE2and Met (Figure 1) Referring to these works mentionedabove Choi et al showed that miR-34 provided a barrierfor somatic cell reprogramming [75] This work showedOSMK-triggered p53-dependent induction of miR-34 s inMEFs This work also showed that miR-34a deficiency inmice significantly increased reprogramming efficiency andkinetics while miR-34a and p21 the downstream factors ofp53 were cooperatively barrier of somatic reprogrammingIt was shown that suppression of reprogramming bymiR-34a was due to repression of pluripotency genesNanog Sox2 and N-myc Cole et al showed miR-34aas a candidate tumor suppressor gene in neuroblastomathrough targeting antiapoptotic Bcl2 and pluripotency factorN-myc [76] (Figure 1) Removing such barrier factors area reasonable strategy for the establishment of barrier-freereprogramming in terms of efficient and reproduciblereprogramming

5 Direct Reprogramming

The reprogramming of cells to iPS cells gives the cellspossessing abilities of pluripotency and self-renewal Anotheridea has been challenged that cells in a mature lineage couldbe directly transdifferentiated into the other lineages usingdefined factors termed as ldquodirect reprogrammingrdquo SinceEMT and MET have been shown during the developmentof body and cancer the induced transition from one lineageto the other is realistic Moreover the short cut direct repro-gramming without iPSC generationmay be more reasonableefficient and beneficial for clinical application


51 Induced Chondrocyte-Like Cells (iChon) Dr NobuyukiTsumakirsquos group have reported the direct reprogrammingof human dermal fibroblasts to chondrocytes using definedfactors [77ndash84] A key idea was replacing Sox2 one of theindispensable factors for the iPSC generation to its brotherSox9 that is a master transcription factor for chondrogenesisThis group has established a model of type II collagenopathyskeletal dysplasia using direct conversion and iPS cells [8586]

52 Induced Neuronal Stem Cells (iNSC) and Induced Neurons(iN) The combination of miR-124 and two TFs MYT1L andBRN2 was sufficient for direct reprogramming of postnataland adult human primary dermal fibroblasts to functionalneurons [87] Han et al reported direct reprogramming offibroblasts into NSCs using defined factors including SMOKfactor plus TCF3E47 [88] The requirement of 120573-cateninsignaling for induced NSC (iNSC) was shown in this studybecause the TCF3 is a recipient TF of the Wnt120573-cateninsignaling

Not addition but subtraction of defined factors haschallenged for the iNSC generation Ring et al reporteddirect reprogramming of mouse and human fibroblasts intomultipotent NSCs with only a single factor Sox2 in an ldquoNSCpermissive culture conditionrdquo [89] Wapinski et al reported ahierarchicalmechanism in the direct conversion of fibroblastsinto induced neurons (iN) cells mediated by Ascl1 Brn2 andMyt1L transcription factors [90]

Guo et al reported in vivo direct reprogramming ofreactive glial cells into functional neurons after brain injuryand in an Alzheimerrsquos disease model [91] In the studyretroviral expression of a single neural TF NeuroD1 was usedfor direct reprogramming of reactive glial cells into functionalneurons in vivo in the cortex of stab-injured or Alzheimerrsquosdisease model mice

53 Induced Cardiomyocyte-Like Cells (iCM) Fu et alreported direct reprogramming of human fibroblasts towardcardiomyocyte-like state [92] This group first reported thatthree TFs (GATA4MEF2C andTBX5) termedGMTdirectlyreprogrammed nonmyocyte mouse heart cells into inducedcardiomyocyte-like cells (iCMs) Then this group showedthat GMT plus ESRRG and MESP1 induced global cardiacgene expression and phenotypic shifts in human fibrob-lasts In addition myocardin ZFPM2 and TGF120573 signalingwere shown to be important for the iCM reprogrammingJayawardena et al reported that a combination of miR-1-133 -208 and -499 was capable of inducing direct cellularreprogramming of fibroblasts into cardiomyocyte-like cells invitro [93]

54 Induced Vascular Endothelial Cells (iVEC) Vascularendothelial cells were generated from fibroblasts via partial-iPSC reprogramming with four TFs required for iPSC gen-eration and subsequent transduction of SETSIP with VEGF[94] This group showed the SETSIP translocated to nucleiupon stimulation with VEGF and bound to the VE-cadherinpromoter and increased VE-cadherin expression levels andEC differentiation

55 Induced Osteoblast-Like Cells (iOB) Direct conversionof human fibroblasts into functional osteoblasts by definedfactors was recently reported Yamamoto et al reported thatosteogenic transcription factors Runx2 and OsterixSp7in combination with Oct4 and L-myc drastically inducedfibroblasts to produce calcified bone matrix and osteoblas-tic markers [95] This group reported that the inducedosteoblasts (iOBs) into bone defects contributed to bonerepair in mice In addition iOBs did not require continuousexpression of the exogenous genes to maintain their pheno-types Mizoshiri et al reported that L-myc in combinationwith either Oct34 Oct6 or Oct9 enabled the conversionof fibroblasts to osteoblasts [96] Alternatively Oct9 plus N-myc had the strongest capability of inducing osteoblasticphenotype Prior to these iOBs studies Song et al reportedthat loss of Wnt120573-catenin signaling caused cell fate shift ofpreosteoblasts to adipocytes [97]

MicroRNAs have been used for generation of iPSC anddirect reprogramming as described above We have had anidea that miRNA produced during osteogenic differentiationfrom stem cells (termed OstemiR) may be biomarkers toolsand therapeutic targets which are beneficial for patientssuffering fromosteoporosis bone fracture genetic osteogenicdisorders arthritis and aging We screened such OstemiR[98] (a partial list was shown in Table 1) The OstemiRdatabase provides gene expression signature of miRNA andmRNA that were altered in osteoblasticosteocytic differenti-ation of MSCs The combination of some OstemiR can be atool for direct reprogramming to osteogenic lineage

6 miR-720 Promotes Dental Pulp (Stem)Cell Differentiation via TargetingPluripotent Stem Cell Factor Nanog

We reported that microRNA-720 (miR-720) controlled stemcell phenotype proliferation and differentiation of humandental pulp cells [99] Dental pulp includes mesenchymal(stem) cells peripheral nerves blood vessels and bloodThe mesenchymal stem cells have been known to have anability to differentiate to mature odontoblasts that produceextracellular matrix proteins We challenged a hypothe-sis that dental pulp could include multipotent stem cellsSide population (SP) cells which had been known to beenriched with stem cells having pluripotency in dental pulpcells were separated from main population (MP) cells withfluorescence-activated cell sorting (FACS) usingHoechst blueand Hoechst red ABCG2 Nanog and Oct-4 were expressedat higher levels while a level ofmiR-720 expression was lowerin SP cells compared to MP cells miR-720 was predicted totarget Nanog and this targeting was experimentally proven(Figure 1) Neutralization of miR-720 using anti-miR-720 wasshown to repress odontoblastic differentiation

7 miR-720 Promotes Dental Pulp (Stem)Cell Differentiation through Induction ofDNA Methyltransferases

We showed that miR-720 positively regulated expressionof DNA methyltransferases Dnmt3a and Dnmt3b as well


Table1Listof

Oste

miR

andtheirp

otenttargets

PTMposttransla

tionalm

odificatio

nfactor

Growth

factor

Receptor

Sign

alP

TMTranscrip

tionfactor

Epigeneticregu

lator

Cellcycleregu

lator

Others

miR-30a

miR-30d

miR-30e

NovIgfB

dnf

Lrp6

LifR

NotchIgf2R

Igf1R

Integrin

a5a4

Integrin

b3Senp

5Sox9R

unx2Smad12

FoxO3

Klf9

11Z

eb2Sn

ai1NcoA1

HoxB8

Lin28b

Tet1

Tet3SirT

1Hdac5E

edM

bd6

CyclinE2

T2K

Cdk

6HspA5

Grp78

miR-30b

miR-30c

Spp1O

pnC

cn2

Lrp6

LifR

Sox9R

unx2Lin28ahn

RnpA

3Pcgf5Zb

tb4144

SirT1Eed

HspA5

Grp78

miR-21

TgfB1Fgf1

Lrp6

Tgfb

R2B

mpR

2Ac

vR1cLifR

ItgB8

SkiSm

ad7

Sox25K

lf3512

Msx1

Tet1

ReckT

imp3

miR-16miR-503

Wnt3a4Fgf7

IhhVe

gfAIgf1

BmpR

1aA

cvR2

bInsR

Igf1R

Smad7Sm

urf1

Smurf2

Sox5M

ybM

ybL1FosL1

Runx

1T1Cy

clin

E1D

1D2D3T2

M2

Cdk

5r16

Hspg2R

eck

miR-155

Gdf6Fgf7

TgfbR2

AcvR2

bLrp1b

Tcf4Smad12

Klf3

FosSp13

SirT1

CyclinD1

SmarcA

D1

claud

in1

miR-541

Wnt11

Integrin1205723

Baz1bSp1

Ago1

Adam

ts7

Timp2


In addition overexpression of Dnmt3a and Dnmt3b pro-moted odontoblastic differentiation of dental pulp cells andrepressed Nanog expression Thus it was suggested thatodontoblastic differentiation of dental pulp cells requiredmiR-720 that repressed Nanog and induces Dnmt3a andDnmt3b

Prior to our study roles for Dnmt3ab in promotion ofHSC differentiation had been shown Okano et al showedthat Dnmt3a and Dnmt3b were essential for de novo methy-lation and mammalian development using Dnmt3a andDnmt3b deficient mice [100] Mutations in human DNMT3Bwere found in immunodeficiency centromeric instabilityand facial anomalies syndrome (ICF syndrome) in this studyChallen et al (2012) showed that Dnmt3a was essentialfor HSC differentiation [101] In this study loss of Dnmt3ain HSCs led to higher expression of HSC multipotencygenes Runx1 Gata3 and Nr4a2 Thus it was suggested thatHSCs could differentiate into B cells via Dnmt3b-dependenthypermethylation of Runx1 Gata3 Nr4a2 and Vasn genesChallen et al (2014) reported that Dnmt3a and Dnmt3bmight promote DNA methylation in Ctnnb1 which encodes120573-catenin stem cell self-renewal factor CcnD1 a G1-phasecyclin gene PparG an adipogenic transcription factor andVegfA and Jag1 genes [102] The roles for Dnmt in HSCdifferentiation may be similar with that in DPSCs Thustargeting 120573-catenin PAPR120574 and CcnD1 by Dnmt3a3b isthought to promote differentiation of cells in odontoblasticand osteoblastic lineage

These works suggested that direct reprogramming tomature odontoblasts as well as osteoblasts could be chal-lenged using a combination of miR-720 and Dnmt3a3boverexpression Another idea is that dental pulp (stem) cellsare a good resource of cells having potential for generationof iPS cells from dental pulp (stem) cells Oda et al reportedhighly efficient generation of iPS cells from human thirdmolar mesenchymal stromal cells [103]

8 Conclusion

Improvement of concept materials and methods in cellularreprogramming has given huge progress in life science andmedicine Firstly growth factors and transcription factorswere defined useful for generation of iPS cells Later studieshave shown microRNAs are also useful for the generationof iPS cells Defined factors including growth factors TFsand miRNAs have been shown to be useful for cellular repro-gramming The combination of the cellular reprogrammingwith genome editing is ongoing in life science and medicinetoward benefits for patients in human society

Competing Interests

The authors declare no competing financial interests

Acknowledgments

The authors thank Drs Ken Watanabe Emilio Satoshi HaraMistuaki Ono Yuta Mishima Ken-ichi Kozaki Satoshi Kub-otaMasaharuTakigawa and Stuart K Calderwood for useful

discussion This work was supported by JSPS KAKENHIGrant no JP16K11722 (to Takanori Eguchi) JP25670818 andJP15K15708 (to Takuo Kuboki) and by a Joint Center forRadiation Therapy grant at Harvard Medical School 2015 (toTakanori Eguchi)

References

[1] 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

[2] N Sasai R YakuraDKamiya YNakazawa andY Sasai ldquoEcto-dermal factor restricts mesoderm differentiation by inhibitingp53rdquo Cell vol 133 no 5 pp 878ndash890 2008

[3] I Thesleff A Vaahtokari and A-M Partanen ldquoRegulationof organogenesis Common molecular mechanisms regulatingthe development of teeth and other organsrdquo The InternationalJournal of Developmental Biology vol 39 no 1 pp 35ndash50 1995

[4] IThesleff A Vaahtokari P Kettunen and T Aberg ldquoEpithelial-mesenchymal signaling during tooth developmentrdquo ConnectiveTissue Research vol 32 no 1ndash4 pp 9ndash15 1995

[5] P D Toto J J OrsquoMalley and E R Grandel ldquoSimilarities ofkeratinization and amelogenesisrdquo Journal of Dental Researchvol 46 no 3 pp 602ndash607 1967

[6] G Gustafson and B Sundstrom ldquoEnamel morphologicalconsiderationsrdquo Journal of Dental Research vol 54 no 2supplement pp B114ndashB120 1975

[7] M G Domingues M M M Jaeger V C Araujo and N SAraujo ldquoExpression of cytokeratins in human enamel organrdquoEuropean Journal ofOral Sciences vol 108 no 1 pp 43ndash47 2000

[8] F H Gage ldquoMammalian neural stem cellsrdquo Science vol 287 no5457 pp 1433ndash1438 2000

[9] S Gronthos M Mankani J Brahim P G Robey and S ShildquoPostnatal human dental pulp stem cells (DPSCs) in vitro andin vivordquo Proceedings of the National Academy of Sciences of theUnited States of America vol 97 no 25 pp 13625ndash13630 2000

[10] S Gronthos J Brahim W Li et al ldquoStem cell properties ofhuman dental pulp stem cellsrdquo Journal of Dental Research vol81 no 8 pp 531ndash535 2002

[11] K Sakai A Yamamoto K Matsubara et al ldquoHuman dentalpulp-derived stem cells promote locomotor recovery aftercomplete transection of the rat spinal cord by multiple neuro-regenerative mechanismsrdquoThe Journal of Clinical Investigationvol 122 no 1 pp 80ndash90 2012

[12] K Akiyama C Chen S Gronthos and S Shi ldquoLineagedifferentiation of mesenchymal stem cells from dental pulpapical papilla and periodontal ligamentrdquo in Odontogenesis pp111ndash121 Humana Press 2012

[13] Y X Wang and M A Rudnicki ldquoSatellite cells the engines ofmuscle repairrdquo Nature Reviews Molecular Cell Biology vol 13no 2 pp 127ndash133 2012

[14] A Asakura M A Rudnicki and M Komaki ldquoMuscle satel-lite cells are multipotential stem cells that exhibit myogenicosteogenic and adipogenic differentiationrdquoDifferentiation vol68 no 4-5 pp 245ndash253 2001

[15] L F Bonewald ldquoThe amazing osteocyterdquo Journal of Bone andMineral Research vol 26 no 2 pp 229ndash238 2011

[16] V Lefebvre P Li and B de Crombrugghe ldquoA new longform of Sox5 (LminusSox5) Sox6 and Sox9 are coexpressed inchondrogenesis and cooperatively activate the type II collagengenerdquoThe EMBO Journal vol 17 no 19 pp 5718ndash5733 1998


[17] C A Yoshida T Furuichi T Fujita et al ldquoCore-binding factor120573interacts with Runx2 and is required for skeletal developmentrdquoNature Genetics vol 32 no 4 pp 633ndash638 2002

[18] K Nakashima X Zhou G Kunkel et al ldquoThe novel zinc finger-containing transcription factor osterix is required for osteoblastdifferentiation and bone formationrdquo Cell vol 108 no 1 pp 17ndash29 2002

[19] T Koga Y Matsui M Asagiri et al ldquoNFAT and Osterixcooperatively regulate bone formationrdquo Nature Medicine vol11 no 8 pp 880ndash885 2005

[20] B B Lowell ldquoPPAR120574 an essential regulator of adipogenesis andmodulator of fat cell functionrdquo Cell vol 99 no 3 pp 239ndash2421999

[21] MA Rudnicki P N J Schnegelsberg RH Stead T BraunH-H Arnold and R Jaenisch ldquoMyoD or Myf-5 is required for theformation of skeletal musclerdquo Cell vol 75 no 7 pp 1351ndash13591993

[22] C Lancrin P Sroczynska C Stephenson T Allen V Kouskoffand G Lacaud ldquoThe haemangioblast generates haematopoieticcells through a haemogenic endothelium stagerdquoNature vol 457no 7231 pp 892ndash895 2009

[23] H M Eilken S-I Nishikawa and T Schroeder ldquoContinuoussingle-cell imaging of blood generation from haemogenicendotheliumrdquo Nature vol 457 no 7231 pp 896ndash900 2009

[24] J-C Boisset W van Cappellen C Andrieu-Soler N GaljartE Dzierzak and C Robin ldquoIn vivo imaging of haematopoieticcells emerging from themouse aortic endotheliumrdquoNature vol464 no 7285 pp 116ndash120 2010

[25] J Y Bertrand N C Chi B Santoso S Teng D Y R Stainierand D Traver ldquoHaematopoietic stem cells derive directly fromaortic endothelium during developmentrdquo Nature vol 464 no7285 pp 108ndash111 2010

[26] M J Chen T Yokomizo B M Zeigler E Dzierzak and N ASpeck ldquoRunx1 is required for the endothelial to haematopoieticcell transition but not thereafterrdquo Nature vol 457 no 7231 pp887ndash891 2009

[27] K Kissa and P Herbomel ldquoBlood stem cells emerge from aorticendothelium by a novel type of cell transitionrdquoNature vol 464no 7285 pp 112ndash115 2010

[28] S J Morrison and D T Scadden ldquoThe bone marrow niche forhaematopoietic stem cellsrdquo Nature vol 505 no 7483 pp 327ndash334 2014

[29] MKanai-Azuma Y Kanai JMGad et al ldquoDepletion of defini-tive gut endoderm in Sox17-null mutant micerdquo Developmentvol 129 no 10 pp 2367ndash2379 2002

[30] M Yasunaga S Tada S Torikai-Nishikawa et al ldquoInductionand monitoring of definitive and visceral endoderm differenti-ation of mouse ES cellsrdquo Nature Biotechnology vol 23 no 12pp 1542ndash1550 2005

[31] N Shiraki S Harada S Ogaki K Kume and S KumeldquoIdentification of DAF1CD55 a novel definitive endodermmarkerrdquo Cell Structure and Function vol 35 no 2 pp 73ndash802010

[32] H Iwashita N Shiraki D Sakano et al ldquoSecreted cerberus1 as amarker for quantification of definitive endodermdifferentiationof the pluripotent stem cellsrdquo PLoS ONE vol 8 no 5 Article IDe64291 2013

[33] N Shiraki Y Higuchi S Harada et al ldquoDifferentiation andcharacterization of embryonic stem cells into three germ layersrdquoBiochemical and Biophysical Research Communications vol 381no 4 pp 694ndash699 2009

[34] N Shiraki S Ogaki and S Kume ldquoProfiling of embryonic stemcell differentiationrdquoThe Review of Diabetic Studies vol 11 no 1pp 102ndash114 2014

[35] 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

[36] 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

[37] M Nakagawa M Koyanagi K Tanabe et al ldquoGeneration ofinduced pluripotent stem cells without Myc from mouse andhuman fibroblastsrdquoNature Biotechnology vol 26 no 1 pp 101ndash106 2008

[38] H Hong K Takahashi T Ichisaka et al ldquoSuppression ofinduced pluripotent stem cell generation by the p53ndashp21 path-wayrdquo Nature vol 460 no 7259 pp 1132ndash1135 2009

[39] A Hotta and S Yamanaka ldquoFrom genomics to gene therapyinduced pluripotent stem cells meet genome editingrdquo AnnualReview of Genetics vol 49 pp 47ndash70 2015

[40] S D Schwartz J-P Hubschman G Heilwell et al ldquoEmbryonicstem cell trials for macular degeneration a preliminary reportrdquoThe Lancet vol 379 no 9817 pp 713ndash720 2012

[41] E Kroon L A Martinson K Kadoya et al ldquoPancreaticendoderm derived from human embryonic stem cells gener-ates glucose-responsive insulin-secreting cells in vivordquo NatureBiotechnology vol 26 no 4 pp 443ndash452 2008

[42] H Kamao M Mandai S Okamoto et al ldquoCharacterization ofhuman induced pluripotent stem cell-derived retinal pigmentepithelium cell sheets aiming for clinical applicationrdquo Stem CellReports vol 2 no 2 pp 205ndash218 2014

[43] N Yahata M Asai S Kitaoka et al ldquoAnti-A120573 drug screeningplatform using human iPS cell-derived neurons for the treat-ment of Alzheimerrsquos diseaserdquo PLoS ONE vol 6 no 9 ArticleID e25788 2011

[44] Z-B Jin S Okamoto P Xiang andM Takahashi ldquoIntegration-free induced pluripotent stem cells derived from retinitis pig-mentosa patient for disease modelingrdquo Stem Cells TranslationalMedicine vol 1 no 6 pp 503ndash509 2012

[45] N M Suzuki A Niwa M Yabe et al ldquoPluripotent cell modelsof fanconi anemia identify the early pathological defect inhuman hemoangiogenic progenitorsrdquo Stem Cells TranslationalMedicine vol 4 no 4 pp 333ndash338 2015

[46] P M Visscher M A Brown M I McCarthy and J Yang ldquoFiveyears of GWAS discoveryrdquo The American Journal of HumanGenetics vol 90 no 1 pp 7ndash24 2012

[47] P D Hsu E S Lander and F Zhang ldquoDevelopment andapplications ofCRISPR-Cas9 for genome engineeringrdquoCell vol157 no 6 pp 1262ndash1278 2014

[48] Y Kazuki M Hiratsuka M Takiguchi et al ldquoComplete geneticcorrection of iPS cells from Duchenne muscular dystrophyrdquoMolecular Therapy vol 18 no 2 pp 386ndash393 2010

[49] T Morishima K-I Watanabe A Niwa et al ldquoGenetic cor-rection of HAX1 in induced pluripotent stem cells from apatient with severe congenital neutropenia improves defectivegranulopoiesisrdquo Haematologica vol 99 no 1 pp 19ndash27 2014

[50] S-L Lin D C Chang S Chang-Lin et al ldquoMir-302 reprogramshuman skin cancer cells into a pluripotent ES-cell-like staterdquoRNA vol 14 no 10 pp 2115ndash2124 2008

[51] A Barroso-del Jesus G Lucena-Aguilar and PMenendez ldquoThemiR-302-367 cluster as a potential stemness regulator in ESCsrdquoCell Cycle vol 8 no 3 pp 394ndash398 2009


[52] K D Wilson S Venkatasubrahmanyam F Jia N Sun A JButte and J C Wu ldquoMicroRNA profiling of human-inducedpluripotent stem cellsrdquo Stem Cells and Development vol 18 no5 pp 749ndash757 2009

[53] B Stadler I Ivanovska K Mehta et al ldquoCharacterization ofmicroRNAs involved in embryonic stem cell statesrdquo Stem Cellsand Development vol 19 no 7 pp 935ndash950 2010

[54] D A G Card P B Hebbar L Li et al ldquoOct4Sox2-regulatedmiR-302 targets cyclin D1 in human embryonic stem cellsrdquoMolecular and Cellular Biology vol 28 no 20 pp 6426ndash64382008

[55] S-L Lin D C Chang S-Y Ying D Leu and D T S WuldquoMicroRNA miR-302 inhibits the tumorigenecity of humanpluripotent stem cells by coordinate suppression of the CDK2and CDK46 cell cycle pathwaysrdquo Cancer Research vol 70 no22 pp 9473ndash9482 2010

[56] YWang S Baskerville A Shenoy J E Babiarz L Baehner andR Blelloch ldquoEmbryonic stem cell-specific microRNAs regulatethe G1-S transition and promote rapid proliferationrdquo NatureGenetics vol 40 no 12 pp 1478ndash1483 2008

[57] S-L Lin D C Chang C-H Lin S-Y Ying D Leu and DT S Wu ldquoRegulation of somatic cell reprogramming throughinducible mir-302 expressionrdquo Nucleic Acids Research vol 39no 3 pp 1054ndash1065 2011

[58] B Liao X Bao L Liu et al ldquoMicroRNA cluster 302ndash367 enhances somatic cell reprogramming by accelerating amesenchymal-to-epithelial transitionrdquoThe Journal of BiologicalChemistry vol 286 no 19 pp 17359ndash17364 2011

[59] I Lipchina Y Elkabetz M Hafner et al ldquoGenome-wideidentification of microRNA targets in human ES cells revealsa role for miR-302 in modulating BMP responserdquo Genes ampDevelopment vol 25 no 20 pp 2173ndash2186 2011

[60] Z Li C-S Yang K Nakashima and T M Rana ldquoSmall RNA-mediated regulation of iPS cell generationrdquoThe EMBO Journalvol 30 no 5 pp 823ndash834 2011

[61] F Anokye-Danso C M Trivedi D Juhr et al ldquoHighly effi-cient miRNA-mediated reprogramming of mouse and humansomatic cells to pluripotencyrdquo Cell Stem Cell vol 8 no 4 pp376ndash388 2011

[62] M A Poleganov S Eminli T Beissert et al ldquoEfficient repro-gramming of human fibroblasts and blood-derived endothelialprogenitor cells using nonmodified RNA for reprogrammingand immune evasionrdquoHuman GeneTherapy vol 26 no 11 pp751ndash766 2015

[63] N Faherty S P Curran H OrsquoDonovan et al ldquoCCN2CTGFincreases expression of miR-302 micrornas which target theTGF120573 type II receptor with implications for nephropathic cellphenotypesrdquo Journal of Cell Science vol 125 no 23 pp 5621ndash5629 2012

[64] S Kubota and M Takigawa ldquoCellular and molecular actions ofCCN2CTGF and its role under physiological and pathologicalconditionsrdquo Clinical Science vol 128 no 3 pp 181ndash196 2015

[65] S Kubota and M Takigawa ldquoThe CCN family acting through-out the body recent research developmentsrdquo BiomolecularConcepts vol 4 no 5 pp 477ndash494 2013

[66] N Miyoshi H Ishii H Nagano et al ldquoReprogramming ofmouse and human cells to pluripotency using mature microR-NAsrdquo Cell Stem Cell vol 8 no 6 pp 633ndash638 2011

[67] S Miyazaki H Yamamoto N Miyoshi et al ldquoEmergingmethods for preparing iPS cellsrdquo Japanese Journal of ClinicalOncology vol 42 no 9 pp 773ndash779 2012

[68] T R Leonardo H L Schultheisz J F Loring and L CLaurent ldquoThe functions of microRNAs in pluripotency andreprogrammingrdquo Nature Cell Biology vol 14 no 11 pp 1114ndash1121 2012

[69] H Tazawa N Tsuchiya M Izumiya and H NakagamaldquoTumor-suppressive miR-34a induces senescence-like growtharrest through modulation of the E2F pathway in human coloncancer cellsrdquo Proceedings of the National Academy of Sciences ofthe United States of America vol 104 no 39 pp 15472ndash154772007

[70] V Tarasov P Jung B Verdoodt et al ldquoDifferential regulation ofmicroRNAs by p53 revealed by massively parallel sequencingmiR-34a is a p53 target that induces apoptosis and G1-arrestrdquoCell Cycle vol 6 no 13 pp 1586ndash1593 2007

[71] T-C Chang E A Wentzel O A Kent et al ldquoTransactivationof miR-34a by p53 broadly influences gene expression andpromotes apoptosisrdquoMolecular Cell vol 26 no 5 pp 745ndash7522007

[72] L He X He L P Lim et al ldquoA microRNA component of thep53 tumour suppressor networkrdquo Nature vol 447 no 7148 pp1130ndash1134 2007

[73] X He L He and G J Hannon ldquoThe guardianrsquos little helpermicroRNAs in the p53 tumor suppressor networkrdquo CancerResearch vol 67 no 23 pp 11099ndash11101 2007

[74] H Hermeking ldquop53 Enters the MicroRNAWorldrdquo Cancer Cellvol 12 no 5 pp 414ndash418 2007

[75] Y J Choi C-P Lin J J Ho et al ldquoMiR-34 miRNAs providea barrier for somatic cell reprogrammingrdquo Nature Cell Biologyvol 13 no 11 pp 1353ndash1360 2011

[76] K A Cole E F Attiyeh Y P Mosse et al ldquoA functionalscreen identifies miR-34a as a candidate neuroblastoma tumorsuppressor generdquo Molecular Cancer Research vol 6 no 5 pp735ndash742 2008

[77] K Hiramatsu S Sasagawa H Outani K Nakagawa HYoshikawa and N Tsumaki ldquoGeneration of hyaline cartilagi-nous tissue from mouse adult dermal fibroblast culture bydefined factorsrdquoThe Journal of Clinical Investigation vol 121 no2 pp 640ndash657 2011

[78] N Tsumaki ldquoDirected induction of chondrogenic cells frommouse dermal fibroblast culturerdquo Arthritis Research ampTherapyvol 14 supplement 1 article O27 2012

[79] N Tsumaki ldquoDirect cell reprogramming to chondrogenic cellsfrom dermal fibroblast culturerdquo Clinical Calcium vol 22 no 5pp 659ndash667 2012

[80] H Outani M Okada A Yamashita K Nakagawa HYoshikawa andN Tsumaki ldquoDirect induction of chondrogeniccells from human dermal fibroblast culture by defined factorsrdquoPLoS ONE vol 8 no 10 Article ID e77365 2013

[81] Y Minegishi K Hosokawa and N Tsumaki ldquoTime-lapseobservation of the dedifferentiation process in mouse chondro-cytes using chondrocyte-specific reportersrdquo Osteoarthritis andCartilage vol 21 no 12 pp 1968ndash1975 2013

[82] N TsumakiMOkada andA Yamashita ldquoiPS cell technologiesand cartilage regenerationrdquo Bone vol 70 pp 48ndash54 2015

[83] N Tsumaki ldquoCartilage regeneration using induced pluripotentstem cell technologiesrdquo in A Tissue Regeneration Approach toBone and Cartilage Repair pp 85ndash98 Springer 2015

[84] A Yamashita M Morioka Y Yahara et al ldquoGeneration ofscaffoldless hyaline cartilaginous tissue from human iPSCsrdquoStem Cell Reports vol 4 no 3 pp 404ndash418 2015


[85] N Tsumaki ldquoAnalysis of musculoskeletal systems and theirdiseases Use of iPS cells in regenerative therapy and drugdiscovery for cartilage diseasesrdquo Clinical Calcium vol 25 no8 pp 1162ndash1168 2014

[86] M Okada S Ikegawa M Morioka et al ldquoModeling type IIcollagenopathy skeletal dysplasia by directed conversion andinduced pluripotent stem cellsrdquoHumanMolecular Genetics vol24 no 2 Article ID ddu444 pp 299ndash313 2015

[87] R AmbasudhanM Talantova R Coleman et al ldquoDirect repro-gramming of adult human fibroblasts to functional neuronsunder defined conditionsrdquo Cell Stem Cell vol 9 no 2 pp 113ndash118 2011

[88] DWHan N Tapia A Hermann et al ldquoDirect reprogrammingof fibroblasts into neural stem cells by defined factorsrdquoCell StemCell vol 10 no 4 pp 465ndash472 2012

[89] K L Ring L M Tong M E Balestra et al ldquoDirect reprogram-ming of mouse and human fibroblasts into multipotent neuralstem cells with a single factorrdquo Cell Stem Cell vol 11 no 1 pp100ndash109 2012

[90] O LWapinski T Vierbuchen K Qu et al ldquoHierarchical mech-anisms for direct reprogramming of fibroblasts to neuronsrdquoCell vol 155 no 3 pp 621ndash635 2013

[91] Z Guo L Zhang Z Wu Y Chen F Wang and G Chen ldquoInvivo direct reprogramming of reactive glial cells into functionalneurons after brain injury and in anAlzheimerrsquos diseasemodelrdquoCell Stem Cell vol 14 no 2 pp 188ndash202 2014

[92] J-D Fu N R Stone L Liu et al ldquoDirect reprogramming ofhuman fibroblasts toward a cardiomyocyte-like staterdquo Stem CellReports vol 1 no 3 pp 235ndash247 2013

[93] T M Jayawardena B Egemnazarov E A Finch et alldquoMicroRNA-mediated in vitro and in vivo direct reprogram-ming of cardiac fibroblasts to cardiomyocytesrdquo CirculationResearch vol 110 no 11 pp 1465ndash1473 2012

[94] A Margariti B Winkler E Karamariti et al ldquoDirect repro-gramming of fibroblasts into endothelial cells capable of angio-genesis and reendothelialization in tissue-engineered vesselsrdquoProceedings of the National Academy of Sciences of the UnitedStates of America vol 109 no 34 pp 13793ndash13798 2012

[95] K Yamamoto T Kishida Y Sato et al ldquoDirect conversionof human fibroblasts into functional osteoblasts by definedfactorsrdquo Proceedings of the National Academy of Sciences of theUnited States of America vol 112 no 19 pp 6152ndash6157 2015

[96] N Mizoshiri T Kishida K Yamamoto et al ldquoTransduction ofOct6 or Oct9 gene concomitant with Myc family gene inducedosteoblast-like phenotypic conversion in normal human fibrob-lastsrdquo Biochemical and Biophysical Research Communicationsvol 467 no 4 pp 1110ndash1116 2015

[97] L Song M Liu N Ono F R Bringhurst H M Kronenbergand J Guo ldquoLoss of wnt120573-catenin signaling causes cell fateshift of preosteoblasts fromosteoblasts to adipocytesrdquo Journal ofBone and Mineral Research vol 27 no 11 pp 2344ndash2358 2012

[98] T Eguchi KWatanabe E S HaraM Ono T Kuboki and S KCalderwood ldquoOstemiR a novel panel ofmicroRNAbiomarkersin osteoblastic and osteocytic differentiation from mesencymalstem cellsrdquo PLoS ONE vol 8 no 3 Article ID e58796 2013

[99] E S Hara M Ono T Eguchi et al ldquomiRNA-720 controlsstem cell phenotype proliferation and differentiation of humandental pulp cellsrdquo PLoS ONE vol 8 no 12 Article ID e835452013

[100] M Okano D W Bell D A Haber and E Li ldquoDNA methyl-transferases Dnmt3a and Dnmt3b are essential for de novo

methylation and mammalian developmentrdquo Cell vol 99 no 3pp 247ndash257 1999

[101] G A Challen D Sun M Jeong et al ldquoDnmt3a is essential forhematopoietic stem cell differentiationrdquo Nature Genetics vol44 no 1 pp 23ndash31 2012

[102] G A Challen D Sun A Mayle et al ldquoDnmt3a and Dnmt3bhave overlapping and distinct functions in hematopoietic stemcellsrdquo Cell Stem Cell vol 15 no 3 pp 350ndash364 2014

[103] Y Oda Y Yoshimura H Ohnishi et al ldquoInduction of pluripo-tent stem cells from human third molar mesenchymal stromalcellsrdquo The Journal of Biological Chemistry vol 285 no 38 pp29270ndash29278 2010

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anatomy Research International

PeptidesInternational Journal of


Hindawi Publishing Corporation httpwwwhindawicom

International Journal of

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Molecular Biology International

GenomicsInternational Journal of


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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


21 Ectoderm Ectoderm emerges and originates from theouter layer of germ cells The word ectoderm comes fromthe Greek ektos meaning outside and derma meaning skinThe ectoderm differentiates to form the nervous system(spine peripheral nerves and brain) and tooth enamel viaameloblasts and epidermis (the outer part of integument)Ectoderm also forms the lining of the mouth (oral mucosa)anus nostrils sweat glands hair and nails In vertebratesthe ectoderm has three parts external ectoderm also knownas surface ectoderm the neural crest and neural tube Thelatter two are known as neuroectoderm as described belowEstablished ectodermal markers are 120573-III-tubulin and Otx2Sasai et al reported that ectodermal factor XFDL156 (Zfp12Zfp74) restricts mesodermal differentiation by inhibiting p53that is required for mesodermal differentiation [2]

22 Neuroectoderm Neurulation and Neural Crest Forma-tion process of the neural tube neural crest cells and the epi-dermis is called neurulationThe neural tube cells give rise tothe central nervous system (CNS) Neural crest cells give riseto the peripheral and enteric nervous system melanocytesfacial cartilage and the dentin of teeth The epidermal cellregion gives rise to the epidermis hair nails sebaceousglands olfactory andmouth epithelium as well as eyes All ofthe organs that arise from the ectoderm originate from twoadjacent tissue layers the epithelium and the mesenchymeOrganogenesis of the ectoderm is mediated by signals suchas FGF TGF120573 Wnt and the hedgehog family FGF-9 whichis expressed in epidermis but not in the mesenchyme is a keyfactor in the initiation of tooth germ development FGF-10helps to stimulate epithelial cell (ameloblast) proliferation inorder to make larger tooth germs Mammalian teeth developfrom oral ectoderm and neural crest ectoderm derived frommesenchyme There are over 170 subtypes of ectodermaldysplasia

23 Odontogenesis (Tooth Development) Tooth germ is anaggregation of cells that eventually forms a tooth These cellsare derived from the ectoderm of the first pharyngeal archand the ectomesenchyme of the neural crest [3 4] The toothgerm is organized into three parts the enamel organ thedental papilla and the dental sac (or dental follicle) The cellsin the enamel organ give rise to ameloblasts which produceenamel The location where the outer enamel epithelium andinner enamel epithelium join is called the cervical loop Thegrowth of cervical loop cells into the deeper tissues formsHertwig Epithelial Root Sheath which determines the rootshape of the tooth During the development there are strongsimilarities between keratinization and amelogenesis [5 6]Keratin is also present in epithelial cells of tooth germ A thinfilm of keratin is present on erupted tooth so calledNasmythrsquosmembrane or enamel cuticle The dental papilla containscells that develop into odontoblasts which are dentin-forming cells Mesenchymal cells within the dental papillaare responsible for the formation of tooth pulp The dentalsac (or dental follicle) gives rise to cementoblasts osteoblastsand fibroblasts Cementoblasts form the cementum of atooth Osteoblasts give rise to the alveolar bone surroundingthe roots of teeth Fibroblasts are involved in developing

periodontal ligaments which connect tooth cementum to thealveolar bone

Tooth have a bone-like structure and nature whoseoutside is armored by cortical calcified enamel layer Interme-diate ivory-like dentin layer of tooth contains dendrite-likeprocess of odontoblasts Inside of tooth is called dental pulpwhich has bone-marrow-like space and contains nerves vas-culatures haematopoietic cells and matrix-producing cellsodontoblasts The odontoblasts are cells lining on the surfaceof dentinmatrix from the pulp inside and extending dendrite-like process through the dentinal tube These structuresand functions of odontoblasts are similar to osteoblastswhich are lining on the surface of bone matrix from thebone marrow As odontoblasts are extending dendrite-likeprocess osteocytes are extending dendrites through the bonecanaliculi Both odontoblasts and osteocytes locate close tonerves and are the sensors as well Such bone-marrow-likefeature of dental pulp ldquotooth marrowrdquo has given us an ideaof dental pulp stem cells (DPSCs) or tooth marrow stromalcells (TMSCs) that may contain multiple stem cells includingMSCs HSCs and NSCs [7ndash11] Stem cells from humanexfoliated deciduous teeth (SHEDs) have been shown to beuseful as therapeutic tools [12]

24 Mesoderm Mesoderm has been known as a resource ofmuscle (including smooth cardiac skeletal tongue masti-cation and facial expressions) bone cartilage blood vesselsblood cells urogenital structures such as kidney gonads andtheir associated ducts A group of cells in ectoderm trans-forms via epithelial-mesenchymal transition (EMT)migratesbetween ectoderm and endoderm and then formsmesodermMesoderm has the capacity to induce the growth of otherstructures such as the neural plate the precursor to thenervous system Mesoderm is formed through a processcalled gastrulation There are three components the paraxialmesoderm the intermediate mesoderm and the lateral platemesodermTheparaxialmesoderm gives rise tomesenchymeof the head and organizes into somites that give rise tomuscletissue cartilage and bone and subcutaneous tissue of skinsSignals for somite differentiation are derived from structuressurrounding mesoderm such as notochord neural tube andepidermis Established markers of mesoderm are 120572-smoothmuscle actin (120572-SMA) and brachyury

Muscle satellite cells (myosatellite cells) have been shownas precursors of mature skeletal muscle cells [13] The musclesatellite cells have a potential to provide additional myonucleito their parental muscle fiber or return to a quiescentstate [14] Tendon and ligament have been known to bemesenchymal lineage as well

25 Bone Marrow Stromal Cells and Mesenchymal Stem CellsBone marrow stromal cells (BMSCs) have been shown tocontain mesenchymal stem cells (MSCs) that have abilitiesto differentiate to multiple lineages such as osteoblastschondrocytes and adipocytes Osteoblasts are lining on thesurface of and producing bone matrix while osteocytes thefurther differentiated cells of osteoblasts are embedded in thelacuna in the calcifiedmatrix extending communicative den-drites into canaliculi for mechanotransduction [15] Defined












TSmesodermal

p53

miR-34miR-720

Nanog N-myc Oct4

Mdm2 E3 ligase

HDACSirT1

Sox2CTGF

miR-200family

miR-302367cluster

EMT

Tob2

Dazap2

Slain1

Lif

BMP

anta

goni

st

Jak


Klf24


Zeb1

Zeb2


Tgfbr2

Snai2

Fn1

RhoC


G1S transition

G1 S

Rbl2(p130)Rbl1

(p107)Rb1

(pRb)

Smad3

Apoptosis

Bcl2

TS

Cdk46

CcnD

Cdk2CcnE Survival










































Table1Listof

Oste

miR

andtheirp

otenttargets

PTMposttransla

tionalm

odificatio

nfactor

Growth

factor

Receptor

Sign

alP

TMTranscrip

tionfactor

Epigeneticregu

lator

Cellcycleregu

lator

Others

miR-30a

miR-30d

miR-30e

NovIgfB

dnf

Lrp6

LifR

NotchIgf2R

Igf1R

Integrin

a5a4

Integrin

b3Senp

5Sox9R

unx2Smad12

FoxO3

Klf9

11Z

eb2Sn

ai1NcoA1

HoxB8

Lin28b

Tet1

Tet3SirT

1Hdac5E

edM

bd6

CyclinE2

T2K

Cdk

6HspA5

Grp78

miR-30b

miR-30c

Spp1O

pnC

cn2

Lrp6

LifR

Sox9R

unx2Lin28ahn

RnpA

3Pcgf5Zb

tb4144

SirT1Eed

HspA5

Grp78

miR-21

TgfB1Fgf1

Lrp6

Tgfb

R2B

mpR

2Ac

vR1cLifR

ItgB8

SkiSm

ad7

Sox25K

lf3512

Msx1

Tet1

ReckT

imp3

miR-16miR-503

Wnt3a4Fgf7

IhhVe

gfAIgf1

BmpR

1aA

cvR2

bInsR

Igf1R

Smad7Sm

urf1

Smurf2

Sox5M

ybM

ybL1FosL1

Runx

1T1Cy

clin

E1D

1D2D3T2

M2

Cdk

5r16

Hspg2R

eck

miR-155

Gdf6Fgf7

TgfbR2

AcvR2

bLrp1b

Tcf4Smad12

Klf3

FosSp13

SirT1

CyclinD1

SmarcA

D1

claud

in1

miR-541

Wnt11

Integrin1205723

Baz1bSp1

Ago1

Adam

ts7

Timp2





8 Conclusion


Competing Interests


Acknowledgments



References



















































































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology












TSmesodermal

p53

miR-34miR-720

Nanog N-myc Oct4

Mdm2 E3 ligase

HDACSirT1

Sox2CTGF

miR-200family

miR-302367cluster

EMT

Tob2

Dazap2

Slain1

Lif

BMP

anta

goni

st

Jak


Klf24


Zeb1

Zeb2


Tgfbr2

Snai2

Fn1

RhoC


G1S transition

G1 S

Rbl2(p130)Rbl1

(p107)Rb1

(pRb)

Smad3

Apoptosis

Bcl2

TS

Cdk46

CcnD

Cdk2CcnE Survival










































Table1Listof

Oste

miR

andtheirp

otenttargets

PTMposttransla

tionalm

odificatio

nfactor

Growth

factor

Receptor

Sign

alP

TMTranscrip

tionfactor

Epigeneticregu

lator

Cellcycleregu

lator

Others

miR-30a

miR-30d

miR-30e

NovIgfB

dnf

Lrp6

LifR

NotchIgf2R

Igf1R

Integrin

a5a4

Integrin

b3Senp

5Sox9R

unx2Smad12

FoxO3

Klf9

11Z

eb2Sn

ai1NcoA1

HoxB8

Lin28b

Tet1

Tet3SirT

1Hdac5E

edM

bd6

CyclinE2

T2K

Cdk

6HspA5

Grp78

miR-30b

miR-30c

Spp1O

pnC

cn2

Lrp6

LifR

Sox9R

unx2Lin28ahn

RnpA

3Pcgf5Zb

tb4144

SirT1Eed

HspA5

Grp78

miR-21

TgfB1Fgf1

Lrp6

Tgfb

R2B

mpR

2Ac

vR1cLifR

ItgB8

SkiSm

ad7

Sox25K

lf3512

Msx1

Tet1

ReckT

imp3

miR-16miR-503

Wnt3a4Fgf7

IhhVe

gfAIgf1

BmpR

1aA

cvR2

bInsR

Igf1R

Smad7Sm

urf1

Smurf2

Sox5M

ybM

ybL1FosL1

Runx

1T1Cy

clin

E1D

1D2D3T2

M2

Cdk

5r16

Hspg2R

eck

miR-155

Gdf6Fgf7

TgfbR2

AcvR2

bLrp1b

Tcf4Smad12

Klf3

FosSp13

SirT1

CyclinD1

SmarcA

D1

claud

in1

miR-541

Wnt11

Integrin1205723

Baz1bSp1

Ago1

Adam

ts7

Timp2





8 Conclusion


Competing Interests


Acknowledgments



References



















































































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology



































Table1Listof

Oste

miR

andtheirp

otenttargets

PTMposttransla

tionalm

odificatio

nfactor

Growth

factor

Receptor

Sign

alP

TMTranscrip

tionfactor

Epigeneticregu

lator

Cellcycleregu

lator

Others

miR-30a

miR-30d

miR-30e

NovIgfB

dnf

Lrp6

LifR

NotchIgf2R

Igf1R

Integrin

a5a4

Integrin

b3Senp

5Sox9R

unx2Smad12

FoxO3

Klf9

11Z

eb2Sn

ai1NcoA1

HoxB8

Lin28b

Tet1

Tet3SirT

1Hdac5E

edM

bd6

CyclinE2

T2K

Cdk

6HspA5

Grp78

miR-30b

miR-30c

Spp1O

pnC

cn2

Lrp6

LifR

Sox9R

unx2Lin28ahn

RnpA

3Pcgf5Zb

tb4144

SirT1Eed

HspA5

Grp78

miR-21

TgfB1Fgf1

Lrp6

Tgfb

R2B

mpR

2Ac

vR1cLifR

ItgB8

SkiSm

ad7

Sox25K

lf3512

Msx1

Tet1

ReckT

imp3

miR-16miR-503

Wnt3a4Fgf7

IhhVe

gfAIgf1

BmpR

1aA

cvR2

bInsR

Igf1R

Smad7Sm

urf1

Smurf2

Sox5M

ybM

ybL1FosL1

Runx

1T1Cy

clin

E1D

1D2D3T2

M2

Cdk

5r16

Hspg2R

eck

miR-155

Gdf6Fgf7

TgfbR2

AcvR2

bLrp1b

Tcf4Smad12

Klf3

FosSp13

SirT1

CyclinD1

SmarcA

D1

claud

in1

miR-541

Wnt11

Integrin1205723

Baz1bSp1

Ago1

Adam

ts7

Timp2





8 Conclusion


Competing Interests


Acknowledgments



References



















































































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology





8 Conclusion


Competing Interests


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

review article cellular reprogramming using defined...

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