Efficient conversion of ES cells into myogenic lineage using the gene-inducible system

Efficient conversion of ES cells into myogenic lineage usingthe gene-inducible systemShiro Ozasa a ,Shigemi Kimura a,*,Kaori Ito a,b ,Hiroe Ueno a ,Makoto Ikezawa a ,Makoto Matsukura a,b ,Kowashi Yoshioka a ,Kimi Araki c ,Ken-ich Yamamura c ,Kuniya Abe d ,Hitoshi Niwa e ,Teruhisa Miike aaDepartment of Child Development,Kumamoto University Graduate School,1-1-1Honjo,Kumamoto City,Kumamoto 862-8556,JapanbLaboratory of Clinical Pharmacology and Therapeutics,Sojo University,Kumamoto,Japan cDivision of Developmental Genetics,Kumamoto University Graduate School,Kumamoto,JapandResearch &Development Team for Mammalian Cellular Dynamics,RIKEN Tsukuba Institute,Tsukuba,JapaneLaboratory of Pluripotent Cell Studies,RIKEN Center for Developmental Biology,Kobe,JapanReceived 2April 2007Available online 17April 2007AbstractWe established genetically engineered ES (ZHTc6-MyoD)cells that harbor a tetracycline-regulated expression vector encoding myogenic transcriptional factor MyoD,for the therapy of muscle diseases,especially Duchenne muscular dystrophy (DMD).Almost all the ZHTc6-MyoD cells were induced into muscle lineage after removal of tetracycline.The undifferentiated ZHTc6-MyoD cells are Sca-1+and c-kit+,but CD34À,all well-known markers for mouse hematopoietic stem cells.In addition,they are able to maintain themselves in the undifferentiated state,even after one month of culture.Therefore,it is possible to obtain a large quantity of ZHTc6-MyoD cells in the undifferentiated state that maintain the potential to differentiate only into muscle lineage.Additionally,at two weeks post-injection of these cells into muscle of mdx ,a model mouse of DMD,clusters of dystrophin-positive myofibers were observed at the injection site.Therefore,ES cells have considerable therapeutic potential for treating muscle diseases.Ó2007Elsevier Inc.All rights reserved.Keywords:Embryonic stem cells;Tetracycline-regulatable system;Myogenic differentiation;Duchenne muscular dystrophyDuchenne muscular dystrophy (DMD)is a severe mus-cle degenerative disorder caused by mutations in the dys-trophin gene [1].Stem cell transplantation is considered to be one of the most promising therapies for the disease.Specifically,side population cells (SP cells)[2]derived from bone marrow have garnered considerable interest because of their ability to migrate into,and undergo myogenic spec-ification,in affected muscles [3].Similarly,SP cells derived from muscles have been shown to have similar characteris-tics as those from bone marrow.The migration of these cells into muscle after intravenous injection is insufficient for ameliorating the cumulative effect of muscle weaknesscaused by the disease [4,5]and a large dosage of cells may therefore need to be injected.However,given that less than 1%of these cells can be isolated from bone marrow,a limiting factor of this approach has been that these cells cannot be obtained in sufficiently large quantities.In addi-tion,these cells are difficult to culture in an undifferentiated condition for extended periods of time.By contrast,embryonic stem (ES)cells have consider-able advantages over SP cells:they can be cultured for extended periods in an undifferentiated and pluripotent condition.The possibility of invoking specific myogenic differentiation has been investigated in several studies.Thus far,however,these approaches have not succeeded in achieving the differentiation of ES cells only into muscle lineage.0006-291X/$-see front matter Ó2007Elsevier Inc.All rights reserved.doi:10.1016/j.bbrc.2007.04.032*Corresponding author.Fax:+81963735200.E-mail address:kimusige@kaiju.medic.kumamoto-u.ac.jp (S.Kimura)./locate/ybbrcBiochemical and Biophysical Research Communications 357(2007)957–963The tetracycline-regulatable(Tet-Off)gene expression system[6]has been shown to confer inducibility of trans-gene expression in several cell types in culture and in vivo.However,establishing the system in ES cells has proven problematic.This is probably due to the combina-tion of the relatively toxic effects of the tet repressor-VP16 fusion(tTA)and the tendency of ES cells to undergo trans-gene silencing.However,an ES cell line,ZHTc6,has been isolated,which is regulated by the Tet-Offsystem[7].Here, we propose a novel approach to controlling specific muscle lineage differentiation of ES cells by introducing MyoD cDNA into a tetracycline regulatory expression site in ZHTc6cells.Materials and methodsCell culture conditions and staining.ZHTc6are feeder-free ES cells derived from CGR8,which are129/Ola mouse-derived wild-type ES cells (Fig.1A)[7].ZHTc6cells carry a targeted integration of IRESzeo in one Oct3/4allele.They also carry an integrated gene trap construct, IRES hph:CAGtTA,that confers stable expression of the tetracycline-responsive tTA transactivator and a randomly integrated hCMV*-1-Oct3/ 4-IRES b geopA transgene.The hCMV*-1-Oct3/4-IRES b geopA transgene is comprised of the tetracycline-inducible promoter hCMV*-1(derived from pUHD10-3),the rabbit b-globin second intron,full-length Oct3/4 cDNA,and IRES b geopA unit[8].The ZHTc6ES cells were grown in maintenance medium,according to the protocols of Dr Niwa et al.[8,7].To induce myogenesis,maintenance medium was changed to differentiation medium[leukemia inhibitory fac-tor(LIF)(Invitrogen,CA,USA)was removed,and15%Knockout SR (Invitrogen,CA,USA)was changed to4%fetal bovine serum(FBS)].4%, 8%,and20%FBS were tested to see which concentration produced the best differentiation medium.The4%FBS was best,though the differen-tiation of ZHTc6-MyoD cells to myotubes also was observed with8%and 20%FBS(data not shown).X-gal staining was performed by standard protocol,and alkaline phosphatase activity was analyzed using an alkaline phosphatase staining kit(Sigma,MO,USA),Supertargeting.STV-MyoD was constructed for Supertargeting,as shown in Fig.1A[8].After the introduction of STV-MyoD to ZHTc6cells by electroporation,the cells were selected in the presence of200or300l g/ mL G418and100ng/mL doxycycline.Targeted clones were maintained in the presence of100ng/mL doxycycline and80l g/mL hygromycin (Invitrogen,CA,USA).Southern blotting.Genomic DNA from cells was digested with restriction enzyme Sac I(Takara,Mie,Japan),and Southern blot analysis was performed according to standard protocol.(Amersham,NJ,USA). An Eco RI–Sac I fragment of the LacZ gene as probe(Fig.1A)was labeled with[a-32P]dCTP(Amersham,NJ,USA)by using a Megaprime DNA labeling system(Amersham,NJ,USA).Western blotting.The cells were homogenized in100l l of a proteolytic solution(62.5l M Tris–HCl(pH6.8),20%Glycerol,2%SDS,0.05%b-mercaptoethanol,0.00125%bromophenol blue),boiled for10min,and then centrifuged.Two hundred micrograms of total proteins was taken from the supernatants and loaded on an SDS–polyacrylamide gel,withthe Fig.1.Generation of an inducible MyoD transgene integration by supertargeting.(A)Schematic of the supertargeting strategy for introduction of MyoD into a tetracycline-regulatable expression site.ZHTc6ES cells contain a tetracycline-regulated transgene comprising the hCMV*-1promoter,b-globin second intron,Oct-3/4open reading frame,and IRESgeopA selection marker.(B)Analysis of the supertargeting event in Gp ES cells.X-gal-positive clones(Gp)and X-gal-negative clones(Gn)were analyzed by Southern hybridization.C,control(the original ZHTc6cells before supertargeting);IRES, encephalomyocarditis internal ribosome entry site;hph,hygromycin phosphotransferase;CAG,CAG expression unit(cytomegalovirus enhancer,chicken b-actin promoter,and rabbit b-globin poly(A)signal);tTA,tetracycline-controlled transactivator;hCMV*-1,tTA-dependent promoter(tetR binding motif+human cytomegalovirus minimal promoter);b-geo,Escherichia coli b-galactosidase(LacZ)+neomycin phosphotransferase(neo r)fusion gene; loxP,site-specific recombination target sequence recognized by Cre recombinase;MC1tk,herpes simplex virus thymidine kinase(HSV tk)gene without pA under the control of MC1expression unit(PyF101mouse polyoma virus mutant enhancer+HSV tk minimal promoter).958S.Ozasa et al./Biochemical and Biophysical Research Communications357(2007)957–963use of a3%stacking gel and a4–12%gradient resolving gel.The frac-tionated proteins were transferred to nitrocellulose membranes(Bio-Rad, CA,USA)and then incubated with anti-MyoD1(Dako,Copenhagen, USA,Cat.No.M3512)at1:10,monoclonal anti-desmin(Sigma,MO, USA,Cat.No.D1033)at1:200,monoclonal anti-myogenin(Sigma,MO, USA,Cat.No.M5815)at1:20,monoclonal anti-dystrophin(Novocastra, Newcastle,USA,Cat.No.NCL-DYS2)at1:10dilutions.The resulting immune complexes were detected with affinity-purified anti-mouse IgG (H+L)(Vector Lab.Inc.,CA,USA)using ABC method(Vector Lab. Inc.,CA,USA).Flow cytometry.Undifferentiated and3-day-differentiated Gp1cells were stained withfluorescein isothiocyanate(FITC)-conjugated anti-mouse-CD34antibody(BD Bioscience,CA,USA,Cat.No.553733), phycoerythrin(PE)-conjugated anti-mouse-c-kit antibody(BD Bioscience, CA,USA,Cat.No.553355),and allophycocyanin(APC)-conjugated anti-mouse-Sca1antibody(BD Bioscience,CA,USA,Cat.No.17-5981-81). Approximately1·106cells were stained,and5000–10,000live cells were analyzed using a FACScalibur(BD Bioscience,CA,USA).Positively stained cells were quantitated using CellQuest software(BD Bioscience, CA,USA).Immunofluorescent microscopy of cells.Cells to be immunostained were grown on collagen type I-coated4-chamber slide glasses(IWAKI,Tokyo, Japan).After the cells werefixed,the immunostainings of cells were per-formed using the following antibodies:mouse monoclonal anti-Pax7 (R&D Systems,MN,USA,Cat.No.MAB1675)at1:20,the above anti-MyoD1at1:20,the above anti-desmin at1:100,mouse monoclonal anti-fast myosin heavy chain(fMHC)(Sigma,MO,USA,Cat.No.M4276)at 1:100,and rabbit polyclonal anti-dystrophin(Santa Cruz,CA,USA,Cat. No.SC-15376)at1:20dilutions.Secondary antibodies used were as fol-lows:rhodamine-conjugated goat anti-mouse IgG(Chemicon,CA,USA) at1:100for mouse antibodies,FITC-conjugated goat anti-rabbit IgG (Chemicon,CA,USA)at1:100,dilutions for rabbit antibody.After rinsing,thefluorescein was visualized using appropriatefilter sets.The percentages of positive cells and standard deviations for each specific antibody were calculated by counting the number of positive and negative cells based onfive high-powerfield digital images,paired with differential interference contrast microscopic images,taken randomly at200·.Intramuscular injection of Gp cells into mdx mice.Undifferentiated Gp1 cells were cultured for3days in the absence of doxycycline before injec-tion.After anesthesia,1·106Gp1cells were injected intramuscularly into the gastrocnemius muscles of8-week-old immunodeficient mdx-nude mice,and immunocompetent mdx mice.Two mdx-nude mice were sacri-ficed at7weeks post-injection for histochemistry of tissue sections.Four and two mdx mice were sacrificed at2and10weeks,respectively.Immunofluorescent microscopy of specimens.For histochemistry of tissue sections,samples were frozen in liquid nitrogen-cooled isopentane and cryostat-sectioned.The rabbit polyclonal anti-dystrophin(Santa Cruz,CA,USA,Cat.No.SC-15376)was used asfirst antibody,and FITC-conjugated anti-rabbit IgG antibody(Chemicon,CA,USA)was used as second antibody.For the quantification of dystrophin-positive myofibers,the gastrocnemius muscle sections with the highest number of dystrophin-positive myofibers were selected and the number of total dystrophin-positivefibers counted.C57BL/10wild-type mice(B10)were used for positive controls,and mdx mice were used for negative controls.Detection of injected cells by RT-PCR analysis.Total RNA from40to 50pieces of serial frozen sections with20l m thickness each,i.e.,the same segments of the injected mdx muscle used for immunohistochemistry,were isolated using TRIZOL Reagent(Invitrogen,CA,USA),according to the manufacturer’s instructions.After treatment with DNase I,the isolated RNA was reverse transcribed using Superscript II reverse transcriptase (Invitrogen,CA,USA)and random hexamer primers,according to the manufacturer’s instructions.RT-PCR analysis was performed in order to distinguish ES cells(wild-type)-differentiated myofibers from myofibers in mdx mouse,using the specific primers50-CTCTGCAAAGTTCTTTGA AAGAGTAA(forward)and50-GAAGTTTATTCATATGTTCTTCT AGC(reverse)[9].The PCR product includes exon23of the dystrophin gene,in which mdx mouse has the relevant point mutation.Additionally, the PCR product was digested by Mae III.ResultsIsolation of mouse-derived ES cell lines by tetracycline regulation(Tet-Off)Supertargeting is an effective strategy for producing Tet-OffSystem-regulated ES cells for controlling the MyoD gene[7,8](Fig.1A).G418-resistant clones were duplicated and screened for b-galactosidase expression in the presence of doxycycline by X-gal staining.b-Galactosidase-positive (Gp)and b-galactosidase-negative(Gn)clones were ana-lyzed by Southern hybridization(Fig.1B).A3.2-kb Sac I fragment was detected with a probe(Fig.1A)from the 50-end of LacZ gene in the Gp samples,which is indicative of the correct replacement of the Oct-3/4cDNA sequence with MyoD sequence.The Gn clone retained the4.8-kb fragment diagnostic for the original Oct-3/4transgene inte-gration in ZHTc6cells.Three clones,Gp1,Gp2and Gp3, were then analyzed further.They were named ZHTc6-MyoD cells.Induction of differentiation of Gp clonesThe Gp1,Gp2,and Gp3clones were observed to main-tain alkaline phosphatase activity even after one month of culture.After change to differentiation medium,the mor-phology of the cells was round initially,and then became long and spindly in appearance.By day7,almost all the cells began to fuse into myotubes,and occasional twitching of fused ES cells was observed,with some parts of the twitching myotubes becoming detached from the surface of the dish(Fig.2A and B)MyoD expression in the differ-entiated cells was confirmed by Western blot analysis (Fig.2C).Desmin expression in these differentiated cells was also detected from day4onwards after induction of differentiation,myogenin from day4onwards,and dystro-phin from day8onwards(Fig.2C).Pax7,MyoD,desmin,fMHC,and dystrophin expres-sion also were investigated by immunocytochemical analy-sis.Pax7expression was transiently observed only from day3to day5(Supplementary Figure1).The percentage of pax7-positive cells was highest on day3(68±11%) and declined to zero percent by day7.MyoD expression was observed from day3to day14and was localized in the nuclei(Supplementary Figure1);desmin expression was detected from day5to day14.Once MyoD-positive and desmin-positive cells appeared,the percentage of MyoD and desmin-positive cells observed reached nearly 100%,and these cells preserved their expression to the end of the experimental period.Fast myosin heavy chain and dystrophin expression were also detected,mainly on myotubes,from day10,and positive-percentages reached 66±18%and23±13%,respectively,by day14(Supple-mentary Figure1).By day14,more than half of the myo-tubes were detached from the dish,probably by contraction.However,the differentiated Gp1,Gp2,andS.Ozasa et al./Biochemical and Biophysical Research Communications357(2007)957–963959Gp3cells were unexpectedly negative for LacZ activity.The LacZ gene is driven by the MC1promoter,which is constitutive in itself.To further investigate the loss of LacZ expression upon differentiation,we performed RT-PCR analysis of Gp1cells.Transcripts of the LacZ and neo resistance genes were detected in the undifferentiatedGp1Fig.2.Phase contrast micrographs of Gp cells in the presence or absence of doxycycline and analysis of muscle-specific gene expressions after differentiation.(A)Gp1cells cultured in the presence of doxycycline formed colonies,indicative of undifferentiated state.(B)Gp1cells cultured for 10days in the absence of doxycycline morphed into long spindly cells.Bar,200l m.(C)Expression of dystrophin,desmin,MyoD,and myogenin detected by Western blot analysis during in vitro differentiation of Gp1cells investigated from day 0to 14.The MyoD expression in the differentiated cells was confirmed from day 2after the removal of doxycycline.Desmin was also detected from day 4,myogenin from day 4and dystrophin from day8.Fig.3.Flow cytometric analysis of undifferentiated and differentiated Gp cells.(A)Undifferentiated and (B)6-day-differentiated Gp1cells were stained for the expression of Sca1,c-kit,and CD34.In the undifferentiated cells,Sca1and c-kit were doubly expressed,whereas in the differentiated cells,only low expression of c-kit and Sca1was detected.CD34was rarely present in either undifferentiated or differentiated cells.960S.Ozasa et al./Biochemical and Biophysical Research Communications 357(2007)957–963cells but not in3-day-differentiated Gp1cells(data not shown).The cause of this problem may have been due to MC1promoter activity,which is suitable for gene expres-sion in immature cells,such as stem cells,but not in differ-entiated cells.Analysis of surface antigens of Gp clonesThe cell surface antigens,Sca1,c-kit,and CD34,are all well-established markers of mouse hematopoietic stem cells.The changes of these cell surface antigens in Gp1cells Fig.4.Detection of dystrophin expression of injected ZHTc6-MyoD cells in muscle of mdx-nude and mdx mouse.(A,B)A few dystrophin-positivefibers were detected in the tumor at7weeks post-injection into mdx-nude.(C)The clusters of dystrophin-positivefibers were observed at two weeks post-injection.(D)The serial section of(C).Arrows show dystrophin-negativefibers.(E)Positive control(B10mouse).(F)Negative control(mdx mouse).(A, C,E,and F)an immunohistochemical study with anti-dystrophin antibodies;(B,D)haematoxylin–eosin staining.Bar,50l m.(G)RT-PCR analysis for dystrophin expression of ES cell.Gp1-derived wild-type dystrophin expression is indicated by the25-bp ne1,DNA molecular weight marker XIII (50–750bp)(Roche,Germany);lane2,control B10muscle;lane3,control mdx muscle;lane4,injected mdx muscle.S.Ozasa et al./Biochemical and Biophysical Research Communications357(2007)957–963961pre-and post-differentiation were compared byflow cyto-metric analysis.The undifferentiated Gp1cells expressed Sca1and c-kit intensely(99.3%±0.6%and90.5%±0.8%,respectively), but expression levels decreased dramatically by day6 post-differentiation(13.3%±0.1%and 6.8%±0.3%, respectively;Fig.3).Conversely,little expression of CD34 was observed for both undifferentiated and differentiated Gp1cells(0.05%±0.02%and0.11%±0.08%,respec-tively;Fig.3).Transplantation of Gp cells into mdx miceTumor formation(20mm in diameter)was observed at the injected area of two mdx-nude mice at7weeks post-injec-tion.A few dystrophin-positivefibers at sarcolemma of myofibers,surrounded by non-myofiber cells that are thought to be non-differentiated injected ES cells,were detected at the injection areas(Fig.4A and B).However, the area did not include various organs like teratoma.In con-trast,tumors were not detected in the mdx mice,even at10 weeks post-injection,and clusters of many dystrophin-posi-tive myofibers in the injected area were observed(Fig.4C). Haematoxylin-eosin staining showed variation infiber size and centrally nucleated myofibers in these areas(Fig.4D). The average and standard deviation of the numbers of total dystrophin-positivefibers in the four injected mdx muscles was2727±330.The dystrophin-positive myofibers were negative for LacZ activity by X-gal assay(data not shown), which was consistent with the in vitro data shown above. Detection of dystrophin transcripts in mdx mice injected with Gp CellsThe normal dystrophin gene gave a150-bp band plus two25-bp fragments,whereas the mutated dystrophin gave a150-bp band plus a single50-bp fragment.Wild-type dys-trophin mRNAs were found in the muscles of injected mice (Fig.4G),suggesting that transplanted Gp1cells partici-pated in myogenesis.DiscussionWe established a mouse ES cell line(ZHTc6-MyoD), which not only proliferates indefinitely in an undifferenti-ated state in the presence of doxycycline,but also retains the potential to differentiate almost exclusively into myogenic lineage when exposed to a doxycycline-free environment.Morphologically,undifferentiated ZHTc6-MyoD cells are round,but fusing and twitching was observed at7days after differentiation.In addition,the myogenic conversion of the cells was confirmed by the expression of the myo-genic markers,desmin,myogenin(which is a skeletal mus-cle-specific transcriptional factor),and dystrophin[1]. These muscle-specific gene expressions were also confirmed by immunocytochemical analysis,using anti-Pax7,MyoD,desmin,fMHC,and dystrophin antibodies.In particular, Pax7is an important transcription factor for progenitor cells destined to differentiate into skeletal muscles[10]. The results,therefore,demonstrated that the differentiation of ZHTc6-MyoD cells into the skeletal muscle lineage was controlled by MyoD under a Tet-Offsystem.We demonstrated that the undifferentiated ZHTc6-MyoD cells were Sca-1+and c-kit+,but CD34À,a profile that is partly reminiscent of the expression pattern of bone marrow SP cells[3,11].However,this is not unique to ZHTc6-MyoD cells,because undifferentiated ZHTc6cells are similar to ZHTc6-MyoD(data not shown)regarding surface antigens[12–15].These results and alkaline phos-phatase staining show that our ZHTc6-MyoD cells,before removal of doxycycline,still retain an undifferentiated state.When ZHTc6-MyoD cells were differentiated into muscle lineage,a few cells still remained round in spite of the induction to differentiate.In order to confirm that they were still in the undifferentiated state,these round cells were extracted,returned to the growth condition and allowed to grow,and then again induced to differen-tiate into myotubes by removal of doxycycline.Interest-ingly,these round cells differentiated to myotubes(data not shown).In addition,the ZHTc6-MyoD cells died in the presence of gancyclovir,because the cells have a thymidine kinase gene(tk)(Fig.1A)(data not shown). These data showed that undifferentiated round cells were not contaminated with b-galactosidase-negative(Gn) clones,and they might maintain their undifferentiated state like muscle quiescent satellite cells and reserve cells [16].We then investigated whether ZHTc6-MyoD cells have the potential to differentiate into myofibers in vivo.At two weeks post-injection of ZHTc6-MyoD cells to muscles of mdx,clusters of dystrophin-positive myofibers were detected at the injection area.The average number of dys-trophin-positivefibers of the four injected mdx muscles was 2727±330.About1%of dystrophin-positivefibers,i.e., revertantfiber,are usually seen in the muscle section of mdx,but such a large number of positivefibers has never been observed before[17].We also confirmed dystrophin expression at the injection site with RT-PCR.These results revealed that our ZHTc6-MyoD cells also had capacity to differentiate into muscle lineage in vivo.CGR8(ES cell)cells and their derivatives were demon-strated to produce tumors approximately80%of the time when injected into immunocompetent syngenic mice[18]. In fact,after injection of our ZHTc6-MyoD cells into the muscle of mdx-nude mice,tumor formation was observed, but it was not teratoma.In the case of injection into immu-nocompetent mdx mice,tumor was not observed.Further experimental work is needed in order to determine the best way to avoid tumor formation.In summary,we successfully isolated ES cell lines that differentiate into the myogenic lineage in vitro by the regu-lation of the Tet-Offsystem.This system is excellent for962S.Ozasa et al./Biochemical and Biophysical Research Communications357(2007)957–963inducing ES cells into muscle cells more efficiently than any other in vitro methods.Additionally,our results showed that ES cells might have potential uses as in vivo therapeu-tic agents for muscle diseases such as DMD.AcknowledgmentsThis work was supported by a research grant for Ner-vous and Mental Disorders from the Ministry of Health, Labour,and Welfare,a grant for research in Brain Science from the Ministry of Health,Labour and Welfare,and a grant from the Ministry of Education,Science,Culture and Sports of Japan.Appendix A.Supplementary dataSupplementary data associated with this article can be found,in the online version,at doi:10.1016/j.bbrc.2007.04.032.References[1]M.Koenig,E.P.Hoffman,C.J.Bertelson,A.P.Monaco,C.Feener,L.M.Kunkel,Complete cloning of the Duchenne muscular dystrophy (DMD)cDNA and preliminary genomic organization of the DMD gene in normal and affected individuals,Cell50(1987)509–517. [2]M.A.Goodell,K.Brose,G.Paradis,A.S.Conner,R.C.Mulligan,Isolation and functional properties of murine hematopoietic stem cells that are replicating in vivo,J.Exp.Med.183(1996)1797–1806.[3]E.Gussoni,Y.Soneoka,C.D.Strickland,E.A.Buzney,M.K.Khan,A.F.Flint,L.M.Kunkel,R.C.Mulligan,Dystrophin expression inthe mdx mouse restored by stem cell transplantation,Nature401 (1999)390–394.[4]G.Ferrari,A.Stornaiuolo,F.Mavilio,Failure to correct murinemuscular dystrophy,Nature411(2001)1014–1015.[5]E.Gussoni,R.R.Bennett,K.R.Muskiewicz,T.Meyerrose,J.A.Nolta,I.Gilgoff,J.Stein,Y.M.Chan,H.G.Lidov,C.G.Bonnemann,A.Von Moers,G.E.Morris,J.T.Den Dunnen,J.S.Chamberlain,L.M.Kunkel,K.Weinberg,Long-term persistence of donor nuclei ina Duchenne muscular dystrophy patient receiving bone marrowtransplantation,J.Clin.Invest.110(2002)807–814.[6]M.Gossen,H.Bujard,Tight control of gene expression in mamma-lian cells by tetracycline-responsive promoters,Proc.Natl.Acad.Sci.USA89(1992)5547–5551.[7]H.Niwa,J.Miyazaki,A.G.Smith,Quantitative expression of Oct-3/4defines differentiation,dedifferentiation or self-renewal of ES cells, Nat.Genet.24(2000)372–376.[8]H.Niwa,T.Burdon,I.Chambers, A.Smith,Self-renewal ofpluripotent embryonic stem cells is mediated via activation of STAT3,Genes Dev.12(1998)2048–2060.[9]J.B.Shrager,A.Naji,A.M.Kelly,H.H.Stedman,A PCR-basedassay for the wild-type dystrophin gene transferred into the mdx mouse,Muscle Nerve15(1992)1133–1137.[10]P.Seale,L.A.Sabourin,A.Girgis-Gabardo,A.Mansouri,P.Gruss,M.A.Rudnicki,Pax7is required for the specification of myogenic satellite cells,Cell102(2000)777–786.[11]M.A.Goodell,M.Rosenzweig,H.Kim,D.F.Marks,M.DeMaria,G.Paradis,S.A.Grupp,C.A.Sieff,R.C.Mulligan,R.P.Johnson,Dye efflux studies suggest that hematopoietic stem cells expressing low or undetectable levels of CD34antigen exist in multiple species, Nat.Med.3(1997)1337–1345.[12]A.G.Elefanty,L.Robb,R.Birner,C.G.Begley,Hematopoietic-specific genes are not induced during in vitro differentiation of scl-null embryonic stem cells,Blood90(1997)1435–1447.[13]V.Ling,S.Neben,In vitro differentiation of embryonic stem cells:immunophenotypic analysis of cultured embryoid bodies,J.Cell Physiol.171(1997)104–115.[14]H.K.Mikkola,Y.Fujiwara,T.M.Schlaeger,D.Traver,S.H.Orkin,Expression of CD41marks the initiation of definitive hematopoiesis in the mouse embryo,Blood101(2003)508–516.[15]T.Miyagi,M.Takeno,H.Nagafuchi,M.Takahashi,N.Suzuki,Flk1+cells derived from mouse embryonic stem cells reconstitute hematopoiesis in vivo in SCID mice,Exp.Hematol.30(2002)1444–1453.[16]N.Yoshida,S.Yoshida,K.Koishi,K.Masuda,Y.Nabeshima,Cellheterogeneity upon myogenic differentiation:down-regulation of MyoD and Myf-5generates‘reserve cells’,J.Cell Sci.111(Pt6) (1998)769–779.[17]J.Zhao,K.Yoshioka,T.Miike,M.Miyatake,Developmental studiesof dystrophin-positivefibers in mdx,and DRP localization,J.Neurol.Sci.114(1993)104–108.[18]S.Gidekel,G.Pizov,Y.Bergman,E.Pikarsky,Oct-3/4is a dose-dependent oncogenic fate determinant,Cancer Cell4(2003)361–370.S.Ozasa et al./Biochemical and Biophysical Research Communications357(2007)957–963963。