sibs干细胞诱导分化技术part12htyang

1、杨杨 黄黄 恬恬2012学年学年SIBS 研究生第一学期实验生物学课程研究生第一学期实验生物学课程 20121225中国科学院上海生命科学研究院中国科学院上海生命科学研究院上海交通大学医学院上海交通大学医学院干细胞分化诱导技术干细胞分化诱导技术健康科学研究所健康科学研究所分子心脏学课题组分子心脏学课题组中科院干细胞生物学重点实验室中科院干细胞生物学重点实验室lGeneral backgroundlMechanisms of cardiac differentiationlStrategies for in vitro differentiation of pluripotent stem ce

2、lls (PSCs) to cardiomyocyteslEnrichment and purification of cardiomyocytes derived from PSCslPerspectivesWhat we are going to discusslGeneral backgroundlMechanisms of cardiac differentiationlStrategies for in vitro differentiation of pluripotent stem cells (PSCs) to cardiomyocyteslEnrichment and pur

3、ification of cardiomyocytes derived from PSCslPerspectivesWhat we are going to discussES cells iPS cells tissue stem cells核移植ES细胞体细胞核移植胚胎干细胞胚胎干细胞 诱导性多能干细胞诱导性多能干细胞 组织干细胞组织干细胞Self-renewalDifferentiation potential Embryonic stem cellsEmbryonic stem (ES) cells 胚胎干细胞胚胎干细胞Embryonic carcinoma (EC) cells胚胎癌

4、细胞胚胎癌细胞Embryonic germ (EG) cells胚胎生殖细胞胚胎生殖细胞Embryonic stem (ES) cells 胚胎干细胞胚胎干细胞Embryonic Stem Cells1954 mEG cells Chiquoine AD 1981 mES cells Evans MJ & Kaufmann MH1981 mEC cells Martin G1980 hEC cells Andrews PW et al.1989 hEC cells Pera MF et al. (multipotent)1998 hES cells Thomson JA 1998 hE

5、G cells Shamblott MJ, Gearheart JD2006 miPS cells Takahashi K & Yamanaka S2007 hiPS cells Yamanaka S & George Q. DaleyHistory: pluripotent stem cellsESC lines of other species l Sheep ES cells. Handyside A, Hooper ML, Kaufman MH, Wilmut I. Rouxs Arch Dev Biol 196:18590, 1987l Rabbit ES cells

6、. Graves KH, Moreadith RW. Mol Reprod Dev 36:42433, 1993l Swine ES cells. MB Wheeler. Reprod Fertil Dev 6:5638, 1994l Bovine ES cells. Cherny RA, Stokes TM, Merei J, Lom L, Brandon MR, Williams L. Reprod Fertil Dev 6:56975, 1994l ES-like cells from early zebrafish embryos. Sun L, Bradford CS, Ghosh

7、C, Collidi P, Barnes DW. Mol Mar Biol Biotechnol 4:1939, 1995l Primate ES cells. Thomson JA, Kalishman J, Golos TG, Durning M, Harris CP, Becker RA, et al. Proc Natl Acad Sci USA 92:78448, 1995l Avian ES cells. Pain B, Clark ME, Shen M, Nakazawa H, Sakurai M, Samarut J, et al. Development 122:233948

8、, 1996l Rat ES cells. Buehr M, Smith A. Cell. 26;135(7):1287-98, 2008 Dec; Li P, Ying QL. Cell. 26;135(7):1299-310, 2008 Dec .1937, Verona, ItalyUniversity of Utah, Howard HughesMedical Institute1941, United KingdomCardiff University, Cardiff, United Kingdom1925, Halifax,United KingdomUniversity of

9、North Carolina at Chapel Hill, Chapel Hill, NC, USAOliver SmithiesGene KO in mice Using Mouse ESCs forGene Targeting & Gene TrapGene TargetingPrize motivation: “for their discoveries of principles for introducing specific gene modifications in mice by the use of embryonic stem cells.”The Nobel P

10、rize in Physiology or Medicine 2007Mario R. CapecchiSir Martin J. EvansInduction of pluripotent stem cells from mouse embryonic and adult fibroblast cultures by defined factors通过向胚胎和成体成纤维细胞中转入特定基因使通过向胚胎和成体成纤维细胞中转入特定基因使之逆分化为多潜能干细胞之逆分化为多潜能干细胞Takahashi K, Yamanaka SCell. 2006 Aug 25; 126(4):663-76. Dep

11、t of Stem Cell BiologyInstitute for Frontier Medical SciencesKyoto University Gladstone Institute of Cardiovascular Disease, San Francisco, 93-96 96-99, Osake City Uni; 99-05 Nara Ins of Science TechQ1：How to select candidate genes？Oct-4NanogSox2 etcES cellsC-MycSTAT3Klf4 etcTumor cellsQ2: how to id

12、entification?All 24 factorsYes24-1 factorsYesNoIdentification 10 indispensable genesAll 10 factorsYes,more clones10-1 factorsYesNo4-1 factorsNo clonesOct 4,Sox2, c-myc, and Klf4Takahashi K,. Yamanaka S. Cell. 2006 Aug 25;126(4):663-76Mouse fibroblasts to undifferentiated, pluripotentstem cells (indu

13、ced pluripotent stem (iPS) cells)Induction of Pluripotent Stem Cells from Adult Human Fibroblasts by Defined FactorsOCT4, SOX2, KLF4, c-MYCKazutoshi Takahashi, Shinya YamanakaCell 131, 112, November 30, 2007Teratoma Formation from Human iPS CellsRetroviraltransductionReseedingon feederColonyPicking

14、up(A) Time schedule of iPS cell generation.(B) Morphology of HDF.(D) Typical image of hES cell-like colony.(E) Morphology of established iPS cell line at passage number 6 (clone 201B7).(IN) Immunocytochemistry for SSEA-3 (I), SSEA-4 (J), TRA-1-60 (K), TRA-1-81 (L), TRA-2-49/6E (M), and Nanog (N). Nu

15、clei were stained with Hoechst 33342 (blue). Bars = 200 mm (BE), and 100 mm (IN). James A. Thomson: (OCT4, SOX2, NANOG, and LIN28)John B. GurdonShinya Yamanaka(山中伸弥山中伸弥) 1933, United KingdomGurdon Institute, Cambridge, United Kingdom1962, Osaka, Kyoto University, Kyoto, Japan & Gladstone Institu

16、tes, San Francisco, CA,USA细胞核移植细胞核移植与克隆与克隆iPSCsPrize motivation: for the discovery that mature cells can be reprogrammed to become pluripotent”The Nobel Prize in Physiology or Medicine 2012 分分 化化 多能性多能性自我更新自我更新多能干细胞的特征多能干细胞的特征Early mouse embryogenesisembryonic body progressionES cellearly erimitive

17、ectodermlate primitive ectodermprimitivevisceral endodermparietal endodermgastrulationmesodermendodermectodermembryonic and adult cell types4.5 d.p.c.5.5 d.p.c.6.0 d.p.c.7.0 d.p.c.2 3 d3 4 d4 5d5 8 dIcm/ESPrimitive ectodermMesodermPrimitive endodermVisceral endodermParietal endodermTrophectodermExtr

18、aembryonic ectodermModified from Reprod Fertil. Dev 1998;10:31-47 Charles E. Murry and Gordon KellerCell 132:661-680, 2008(原肠胚形成原肠胚形成)再现了胚胎的再现了胚胎的发育过程发育过程Major sources of starting cells for generating cardiomyocytes Myocardial Infarction (50% of cardiovascular diseases) Myocardial infarction remains

19、 the leading cause of death from CVDl Human pluripotent stem cells (hPSCs: hESCs & hiPSCs)l Adult heart-derived cardiac progenitor cells (CPCs)l Reprogrammed fibroblastshESC-derived cardiomyocytes electrically couple and suppress arrhythmias in injured heartsYuji Shiba, , Charles E. Murry &

20、Michael A. Laflamme. Nature, 2012; 000:1-4Consistent 1:1 hostgraft couplingCharles E. MurryProfessorCenter for Cardiovascular BiologyUniversity of WashingtonMichael A. LaflammeAssociate ProfessorMechanical function improved Ventricular tachycardia reduced ESCs iPSCsDrug discoveryDisease modelsEarly

21、developmentCell transplantationRegulationMaturation?Efficiency?Purification?What can pluripotent stem cells contribute to?Direct differentiationFrom laboratory to bedside:myocardial differentiationSwijnenburg RJ, Wu JC. Curr Opin Biotechnol. 2007, 18:3845Clinical hurdles for the transplantation of p

22、luripotent stem cellsDrug screenTGlGeneral backgroundlMechanisms of cardiac differentiationlStrategies for in vitro differentiation of pluripotent stem cells (PSCs) to cardiomyocyteslEnrichment and purification of cardiomyocytes derived from PSCslPerspectivesWhat we are going to discussWu & Mumm

23、ery et al. Cell 2008,132,537 CVP, cardiovascular progenitors EC, endothelial cells SMC: smooth muscle cells CP, cardiomyocyte progenitors MC, mature cardiomyocytesCardiac differentiation from ESCs/cardiac progenitor cellsOrigins of Cardiac Progenitor Cells in the Developing HeartSchematic of current

24、 knowledge of factors involved in hPSC Cardiac Differentiationelectrical maturationCM differentiationcardiac specificationmesoderm speciationmesoderm differentiationepithelial tomesenchymal transitionPaul W. Burridge, Gordon Keller, Joseph D. Gold, and Joseph C Wu Cell Stem Cell 2012 JanModified fro

25、m Noseda, et al. Circ. Res. 2011PluripotentMesodermCardiac MesodermCardiacProgenitorsCardiomyocytesendodermSignals for Cardiac Specification during Embryogenesis and PSC DifferentiationmicroRNAs: a class of cell lineage determinantsmicroRNAsA class of 21-25 nucleotide non-coding RNAs that regulate t

26、he expression of target genes through binding to the 3-untranslated region (3UTR) of mRNAs, mediating de-stabilitzation or suppressed translation of the mRNA targets A single miRNA can regulate the expression of numerous genes;Spatial and temporal expression of miRNAs Ivey et al., Cell Stem Cells 7:

27、36-41, 2010Example 1MicroRNAs regulate the switch between self-renewal and differentiationNa Xu et al., Cell 137: 647-658, 2009Pluripotent Cellsself-renewaldifferentiationl Endogenous miR-145 represses the 3 UTR of OCT4, SOX2, and KLF4; l Increased miR-145 expression inhibits hESC self renewal, repr

28、esses expression of pluripotency genes, and induces lineage-restricted differentiation.Kathryn N. Ivey et al., Cell Stem Cell 2, 219-229, 2008MicroRNAs regulate cell lineage specificationDll-1: Notch ligand Delta-like 1Relative expression0.00.40.80370370.40.00.8Relative expressionDifferentiation tim

29、e (days)03704812370410121680Differentiation time (days)1.21.2Expression patterns of microRNAs during differentiation miR-AmiR-BmiR-CmiR-DCardiacmyocytes mESCsCVS & cardiac progenitorsmesodermday 0day 7day 3day 3.5-5.5miR-28Let-7cmiR-375Day 3_1Day 3_2Day 3_3Day 3_4Day 7_1Day 7_2Day 7_3Day 7_4ESC_

30、1ESC_2ESC_3AmiR-424miR-489miR-335miR-130amiR-361miR-302amiR-125bmiR-132miR-130bmiR-195miR-199bmiR-100Let-7dmiR-21Let-7bmiR-296miR-615miR-181a王嘉王嘉1.00.02.03.03.5Relative expression of miR-125b0135710Differentiation time (days)0.51.52.5UCCCUGAGACCCUAACUUGUGA UCCCUGAGACCCUAACUUGUGA H.sapiensR.norvegicu

31、sM.musculusUCCCUGAGACCUCAAGUGUGA C.elegans UCCCUGAGACCCUAACUUGUGA miR-125bmiR-125bmiR-125blin-4 (first discovered in 1984)Expression pattern of miR-125b during ESCs differentiation lBoth miR-125a and miR-125b are highly expressed in mouse spinal cord, brain and liver lO n l y m i R - 1 2 5 b i s det

32、ectable in several other tissues, such as the heart and lung, and skeletal musclemiR-125b is dispensable in maintaining self-renewal of mESCsRelative expression of miR-125b0.00.40.81.21.6LNA-Scr125b-Inhibitor*Oct4Nanogm28sRex1LNA-Scr125b-InhibitorLNA-Scr: scramble LNACLNA-Scr125b-InhibitorDRelative

33、expression0.00.20.60.8Rex1Oct4Nanog1.20.41.0125b-inhibitor LNA-Scr E125b-inhibitor LNA-Scr SSEA-1+82.2%SSEA-1+83.9%ABMiR-125b is dispensable in maintaining the characteristics of mESCscon125b-4125b-13wtABRelative expression0.00.30.60.9Rex1Oct4Nanog125b-13 wt con125b-4 1.21.5Cwtcon125b-4125b-13SSEA-1

34、+93.0%SSEA-1+91.1%SSEA-1+91.0%SSEA-1+90.9%NanogOct4Rex1m28scontrol125b-4wt10243Relative expression of miR-125b0135710Differentiation time (days)56125b-13 wt control125b-4 H1 neomiR-125bDEDownregulation of miR-125b is required for the initiation of ESC differentiationABCMiR-125b inhibits mesoderm and

35、 cardiomyocytes differentiation of mESCsBeating EBs (%)Differentiation time (days)78910121440608010020LNA-Scr 125b-InhibitorADBwt con 125b-4125b-13 CmiR-125b represses adipogenic and chondrogenic differentiation of ESCsAOil Red O wtcon125b-4125b-13Alizarin Red S adipogenic cellscartilage cellsBPparg

36、Col2a1m28sSox9Adipoqm28swtcon125b-4125b-13wtcon125b-4125b-13Col2a1Sox9Relative expression0.00.20.40.6125b-13 wt con125b-4 0.81.01.2PpargAdipoqRelative expression0.00.20.40.60.81.0*miR-125b represses endoderm differentiation of ESCswtcontrol125b-4125b-13AfpGata6Gata4m28sDab2Foxa2 1 11 10 00 00 01 11

37、13 33 33 33 35 55 55 57 77 77 71010101010105 57 710100 0Differentiation time (days)Tmprss2125b-13AFPDab2b-b-actinactinwtcontrol125b-4013 5 7 100246Gata60 13 57 1001234Dab2*580 13 57 10Relative expression0246Afp*8wt con 125b-4125b-13 Differentiation time (days)LMC unpublished dataTuj1MAP2NucleiNuclei

38、control125b-4125b-13wtmiR-125b does not affect ectoderm differentiation of ESCsDifferentiation time (days)wtcon125b-4125b-13m28s1 11 10 00 00 01 11 13 33 33 33 35 55 55 57 77 77 71010101010105 57 710100 0Fgf5NestinSox1LMC unpublished dataDifferentiation time (days)Fgf50123013 5 7 10Nestin01234013 57

39、 104Relative expressionWhats the potential molecular target of miR-125b in cardiac differentiation Mouse LIN283 UTR0.01.02.03.00135710Differentiation time (days)Relative expression of Lin282.51.50.51.00.02.03.03.5Relative expression of miR-125b0135710Differentiation time (days)0.51.52.5 Lin28 is the

40、 target of miR-125b in ESCs5AGGGACUCUAACGAGUACAUG 3miR-125bwt-Lin28 3UTRmutant-Lin28 3UTRLin28-3UTR5 UCCCUGAGACCCUAACUUGUGA 35AGGACGGCUAACGAGUACAUG 3wt-Lin28 3UTR0.00.40.81.21.6Relative luc expression*2.0mutant-Lin28 3UTRmiR-24 vector miR-125bmiR-24Lin28wtcontrol125b-4125b-13miR-24Lin28wtcontrol125b

41、-4125b-13m28sb-actinb-actinUndifferentiated ES cellsActinLin28controlmiR-125b-4miR-125b-131 10 00 01 11 13 33 33 35 55 55 57 77 77 7 1010101010100 0Differentiation time (days)Differentiation time (days)ImmunoblotLin28/Actin ratio*con 125b-4 125b-1301357100.01.20.81.60.42.0Lin28 is negatively regulat

42、ed by miR-125b in early differentiating ESCsmiR-125b?Lin28 cardiogenesis Lin28ActinncwtsiLin28-#1 wt nc 0.00.51.01.52.0ImmunoblotLin28/Actin ratio*2.5siLin28-#2 siLin28-#1 siLin28-#2 Lin28 is dispensable in maintaining self-renewal of mESCswtncsiLin28-1#siLin28-2#AALP activity siLin28-#1 wt nc siLin

43、28-#2 NanogOct4Rex1m28ssiLin28-#1 wt nc siLin28-#2 SSEA-1+ cells (%)060801002040120wtmiR-125b4miR125b13miR125b4-Lin28miR125b13-Lin28Lin28Lin28ncwtsiLin28-#1 siLin28-#2 b-actinb-actinb-actinb-actin020406080100Beating EBs (%)120Differentiation time (days)789101214wt nc siLin28-#1siLin28-#2 02040607891

44、01214Differentiation time (days)80miR-125b4 miR-125b13miR-125b4-Lin28miR-125b13-Lin28 Phenotypes of miR-125b overexpressing cells are mimicked by down-regulation of Lin28 and rescued by re-introduction of Lin28 in mESCsLet-7gRelative expression0.00.10.20.3*Let-7aRelative expression0.00.20.40.6*wtcon

45、125b-4125b-134-Lin2813-Lin28Let-7dRelative expression0.00.10.20.30.4*Igf2Hmga2wtcontrol125b-4125b-134-Lin2813-Lin28m28swtcon125b-4125b-134-Lin2813-Lin28wtcon125b-4125b-134-Lin2813-Lin28MiR-125b regulates the expression of let-7 and igf2Lin28Igf2Let-7Hmga2Mesoderm/cardiogenesiskoshiro M, et al., natu

46、re cell biology (10): 567-574, 2008Olivier G, et al., development biology (227): 135-145, 2000Lin28 pathway is involved in mesoderm formationmiR-125b-Lin28 act as a switch controlling the correct lineage commitmentSelf-renewalDifferentiationHigh miR-125bLin28let-7 familymesoderm/cardiogenesisHmga2lo

47、w miR-125bLin28Igf2let-7Pluripotency targetsendodermStem Cells Dev. 2012;21(9):1524-37 Cardiac transcriptional cascadesPuceat, M., and Jaconi, M. Ca2+ signalling in cardiogenesis. Cell Calcium (2005).Example 2Ca2+-signaling pathways in differentiating cardioblastsPuceat, M., and Jaconi, M. Ca2+ sign

48、alling in cardiogenesis. Cell Calcium (2005). ES cellsEBsCa2+ image in embryoid bodies (EBs)Sauer H., et al. Experimental Cell Res. 238: 1322, 1998 Ca2+ influx- +- + intracellular Ca2+ releaseLMC unpublished data key pattern forming events during early vertebrate development Coordinating cell moveme

49、nts;cell fate specification and morphogenesis; establishment of the basic embryonic axes;Slusarshi et al., Develop Bio 307:1-13, 2007PLCPKCCa2+ ATPaseIP3RRyR内质网内质网L型钙通道型钙通道酪氨酸激酶酪氨酸激酶受体受体FGF ATPG蛋白耦联蛋白耦联受体受体PIP2DAGIP3细胞浆细胞浆质膜质膜钙钙ATP酶酶RyR: 雷诺丁受体雷诺丁受体IP3R: 三磷酸肌醇受体三磷酸肌醇受体calcineurin NFATGATAMEF2CNFATpNF

50、ATCaM CamKII细胞核细胞核PIP2：二磷酸磷脂酰肌醇：二磷酸磷脂酰肌醇DAG: 二脂酰甘油二脂酰甘油IP3： 三磷酸肌醇三磷酸肌醇 PKC：蛋白激酶：蛋白激酶CCaM: 钙调蛋白钙调蛋白NFAT: T细胞激活的核因子细胞激活的核因子 Regulation of cytosolic Ca2+ signalingRole of IP3R3 during ESC differentiation胚胎干细胞胚胎干细胞分化的终末细胞分化的终末细胞凋亡凋亡自我更新自我更新Ca2+信号信号?IP3R3tubulin0 1 2 3 4 5ES cell differentiation daysC#4

51、WT C #2 #4 M28sOct4NanogRex-1020406080100WTC#2 #4SSEA-1 positive cells (%)梁冀梁冀WTC#2#4Undifferentiation status maintains unchanged in IP3R3 KD ESCsC, siEGFP ESCs#2,#4, two ESC clones of siIP3R3Alkaline phosphatase activityBrdU HochestOverlapwtsiEGFP#2#4day0day3day5020406080100wtsiEGFP #2#4Percentages

52、 (%) of S-G2-M cellswtsiEGFP#2#4M28sMef2cNkx2.5aMHCaMHCMlc-2vhochesta-actinina-actininhochestMLC-2vwtsiEGFP#2#4wtsiEGFP#2#4IP3R3 KD impairs differentiationof cardiomyocytes789 10 12 14 16beating EBs (%)020406080100120wtsiEGFP#2#4Differentiation time (days)Does IP3R3 control specific lineage differen

53、tiation?IP3R3 KD also inhibited chondrogenic and adipogenic differentiationwtsiEGFP#2#4aM28sPPARgsox9Col2a1adiponectinsiEGFP#2#4wtbsiEGFP#4wtM28sAlican blue Oil red O WTEGFP#2#4d10 EBsAFPDab2IM-positive cell (%)020406080wtEGFP #2#4AFPDab2IP3R3 KD impairs differentiation of endodermendodermal markers

54、:hepatic cell markers CK8, cytokertin 8; HNF3b, hepatocyte nuclear factor 3 bAFP: a-fetoprotein; AIBI, albumin 1visceral endodermal markersSdc4, Syndecan-4; tmpress2; Dab-2, disabled-2M28sDab2CK8HNF3bAlB1AFPsdc4tmpress2siEGFP#2#4wtMap210 20 10 EGFP#2#4wtTuj/hochestNeuron differentiation day 13NF-Mma

55、p2Tuj1M28sEGFP#2#4wtNeuronal differentiation remains Unchanged in IP3R3 KD ESCsMap2/hochestneuronal markers: Map2: microtubule-associated protein 2Tuj1: III b-tubulin NF-M: neurofilament genes Differentiation days2345678013573456789891011121314681012145678910111278910111214891011121301357wtsiEGFP#2#489101112131401357468101214166789101112131415Ct ValueCt ValueCt ValueGata6Gata4BrachyuryRex-1Oct-4Fgf5GscNestinCt Value*Hand1*Differentiation daysDifferentiation da