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1、组织干细胞生物学组织干细胞生物学 胡胡 以以 平平第二军医大学第二军医大学 交大交大20162016级研究生级研究生May 24,20161.All living organisms are composed of one or more cells.2.The cell is the most basic unit of life.Cell Theory(M.J.Schleiden,T.Schwann,1838)组织干细胞与组织器官的关系组织干细胞与组织器官的关系组织干细胞组织干细胞过渡细胞过渡细胞（前体细胞）（前体细胞）组织干细胞与医学科学发展组织干细胞与医学科学发展医学干预医学干预现代医
2、学现代医学未来医学未来医学过渡细胞过渡细胞（前体细胞）（前体细胞）特化细胞特化细胞（成熟细胞）（成熟细胞）干细胞干细胞（前体细胞）（前体细胞）特化细胞特化细胞特化功能特化功能无无无无有有分化程度分化程度（寿命短）（寿命短）（寿命长）（寿命长）过渡细胞过渡细胞组织干细胞的定义组织干细胞的定义Tissue-specific stem cellsAdult stem cellsSomatic stem cellstissue-specific stem cells are specialized cells found in tissues of adults,children and fetuse
3、s.These cells are committed to becoming a cell from their tissue of origin,but they still have the broad ability to become any one of these cells.Progenitor cellsProperties of Tissue-specific Stem CellsStem cells Early stem/progenitor cellsLate progenitor cells-Self-renewal(slowly cyling)-Multipoten
4、t(progeny in at least two lineages)-Long-term tissue reconstitution-Serially transplantable-Self-renewing?(more actitive dividing)-Multipotent(progeny in at least two lineages)-Long-term tissue reconstitution-Serially transplantable?-Non self-renewing(rapidly dividing)-Unipotent(progeny only one lin
5、eages)-Short-term tissue reconstitution-Not serially transplantable组织干细胞的增殖特征组织干细胞的增殖特征缓慢性缓慢性 增殖系统的自稳定性增殖系统的自稳定性 干细胞干细胞 过渡细胞过渡细胞 分化细胞分化细胞 （transit amplifying cell）小肠干细胞较其过渡细胞的分裂速度大约慢一倍；小肠干细胞较其过渡细胞的分裂速度大约慢一倍；70%70%的造血干细胞处于的造血干细胞处于G0期。期。缓慢性缓慢性A A：干细胞的对称分裂：干细胞的对称分裂 (symmetry division)B B：干细胞群体的不对称分裂：干细
6、胞群体的不对称分裂(populational asymmetry division)自稳性自稳性(self-maintenance)(self-maintenance)是指干是指干细胞可以在生物体生命期间中自我细胞可以在生物体生命期间中自我更新更新(self-renewing)(self-renewing)，并维持其自身，并维持其自身数目恒定的特性。数目恒定的特性。干细胞增殖系统的自稳性干细胞增殖系统的自稳性通过对称分裂和通过对称分裂和通过对称分裂和通过对称分裂和不对称分裂来实现不对称分裂来实现不对称分裂来实现不对称分裂来实现The Pins protein(in green)is asymm
7、etrically localized in the neuroblast revealing a polarity that is lacking from the overlying symmetrically dividing epithelial cells(where Pins,in green,is unlocalized).The outcome of this polarity in the neuroblast is the asymmetric localization of Miranda(in red),which will result in its segregat
8、ion to only one of the daughter cells and will make the daughter cells different from one another。组织干细胞的干性维持组织干细胞的干性维持组织干细胞的分化特性组织干细胞的分化特性组织干细胞组织干细胞 分化定向分化定向(神经干细胞、肝干细胞）神经干细胞、肝干细胞）前体细胞前体细胞 干细胞的可塑性干细胞的可塑性（Plasticity）转分化转分化(transdifferentiation)去分化去分化(dedifferentiation)转决定转决定(Transdetermination)直接诱导分化
9、直接诱导分化(Directly induced differentiation)A.Dedifferentiation B.Transdetermination C.Transdifferentiation D.Directly induced differentiation iPSiPS cellcellDE cellDE cellHepatocyteHepatocyteiHepSCiHepSCiHep CelliHep CellFibroblastFibroblast(Limited)(Limited)？Induction of functional hepatocyte-like cel
10、ls from mouse fibroblasts by defined factorsHuang P,et al:Nature 2011 May 11;475(7356):386-9Characterization of iHep cells invitroReprogramming Fibroblasts into Bipotential Hepatic Stem Cells by Defined FactorsYu B,et al:CellStemCell2013,13(3):328-40.iHepSCs differentiated into hepatocyte in vitroiH
11、epSCs differentiated into cholangiocytes invitroiHepSCs differentiated into hepatocyte invivoiHepSCs differentiated into cholangiocytes invivo造血干细胞的可塑性造血干细胞的可塑性Route Stem CellThe stem cell landscape depicted here illustrates the emerging characteristics of adult stem cells that include plasticity in
12、 cell fate,diversity of origin,and a multiplicity of tissue potentials.Stem cells(blue)are able to enter diverse tissue compartments from the blood stream(the stem cell highway)via on ramps and generate appropriate cell types in response to homing signals or growth factors depicted on billboards.In
13、theory,all choices are reversible.(Artwork by N.Gewertz and B.Colyear.)For over 1,500 years,alchemists struggled to convert base metals into gold.But their search for the legendary philosophers stone that could make such a transformation resulted in failure and the slide of this pseudoscience into d
14、isrepute.Not so in cell biology,however.In the December issue of NatureCellBiology,David Tosh and colleagues describe an equally startling transition the conversion of pancreatic cells into hepatocytes.AlisonMitchell 干细胞可塑性的思考干细胞可塑性的思考细胞分化相对性细胞分化相对性个体生长发育和结构与功能维持中的细胞行为个体生长发育和结构与功能维持中的细胞行为医学利用价值医学利用价
15、值(微环境的人为调整？微环境的人为调整？)（活化状态）（活化状态）胞外信号胞外信号分化启动分化启动 分化定向分化定向 （静息状态）（静息状态）组织再生、再生、细胞治胞治疗遗传调控、表观遗传调控遗传调控、表观遗传调控组织干干细胞含量极少胞含量极少组组织织干干细细胞胞人人为干干预如何识别？如何识别？如何标记？如何标记？如何示踪？如何示踪？现有技术能力可以对现有技术能力可以对单细胞进行遗传标记单细胞进行遗传标记分分 化化 调调 控控干细胞的微环境干细胞的微环境（干细胞巢，（干细胞巢，stem cell niche）外周的细胞、胞间基质、外周的细胞、胞间基质、分泌因子等分泌因子等Common feat
16、ures of adult stem cell niches干细胞巢（干细胞巢（Stem cell nicheStem cell niche）复杂性：空间位置、时间状态、复杂性：空间位置、时间状态、调控机制调控机制重要性：认识干细胞的生存特性重要性：认识干细胞的生存特性 建立干细胞生存的人工控制系统建立干细胞生存的人工控制系统Linheng Li and Ting Xie:Stem cell niche:Structure and function.Ann.Rev,Cell Dev.Biol.21:601-31,2005 Lander et al.What does the concept o
17、f the stem cell niche really mean today?BMC Biology 2012,10:19Nakada D,et al.Integrating Physiological Regulationwith Stem Cell and Tissue Homeostasis.Neuron 70,May 26,2011干干细胞与胞与组织器官衰老器官衰老干细胞干细胞过渡细胞过渡细胞 “实质细胞实质细胞”干细胞老化是组织器官（乃至个体）衰老的基础干细胞老化是组织器官（乃至个体）衰老的基础 如何干预干细胞的衰老如何干预干细胞的衰老？如何调整微环境如何调整微环境?抗衰老研究的新
18、思路癌干细胞癌干细胞（cancer stem cell）可能的定义可能的定义 The cells are defined functionally,specifically referring to tumor cells with unlimited renewal potential capable of forming tumors in immunodeficint mice.KPolyakandWCHahn.NatureMedicine12(3):296-300,2006癌干细胞不同于正常的干细胞癌干细胞不同于正常的干细胞 概念的产生概念的产生V Cohnheim(1875).Ste
19、m cells“misplaced”during embryonic development were the source of tumors that formed later in life.S Makino(1956),WR Bruce(1963),LJ Kleinsmith(1964),CH Park(1971),et al.来源于腹水、畸胎瘤、白血病的来源于腹水、畸胎瘤、白血病的单个单个肿瘤细肿瘤细胞可以产生一个新的肿瘤和胞可以产生一个新的肿瘤和异质性异质性的子代细胞的子代细胞。（肿瘤的克隆起源）（肿瘤的克隆起源）M Al-Hajj（2003）,et al.分离和鉴定了乳腺癌组织分
20、离和鉴定了乳腺癌组织中的癌干细胞中的癌干细胞以以Lin，ESA，CD44，CD24等为标志，等为标志，单独或联合分离，单独或联合分离，移植实验移植实验1)表面标志为表面标志为Lin-ESA+CD44+CD24-/LOW 的细胞（仅占的细胞（仅占2%）可以产生癌）可以产生癌2）在新产生的癌组织中仍有）在新产生的癌组织中仍有Lin-ESA+CD44+CD24-/LOW 细胞细胞组分的存在（表明可以更新）组分的存在（表明可以更新）肿瘤干细胞开始引起了广泛关注肿瘤干细胞开始引起了广泛关注“向肿瘤进军向肿瘤进军”4040多年，结果？多年，结果？早期诊断早期诊断转移转移复发复发问题问题原因：现有知识体系：
21、都是基于原因：现有知识体系：都是基于“肿瘤细胞肿瘤细胞”组织干细胞组织干细胞过渡细胞过渡细胞 “肿瘤细胞肿瘤细胞”肿瘤组织肿瘤组织（异质性）（异质性）Possible Mechanisms of Metastatic Relapse after Possible Mechanisms of Metastatic Relapse after Anticancer Therapy and Evaluation of Anti-CSC Treatments Anticancer Therapy and Evaluation of Anti-CSC Treatments VisvaderJE,etal
22、:CellStemCell10,June14,2012肿瘤干细胞肿瘤干细胞对传统治疗方法对传统治疗方法不敏感不敏感未来肿瘤防治研究的未来肿瘤防治研究的“根本根本”策略策略肿瘤干细胞肿瘤干细胞（特别是静息状态）（特别是静息状态）以以“根根”为为“靶靶”“肿瘤细胞肿瘤细胞”（“冠冠”）（“根根”）单细胞分析技术单细胞分析技术有效克服：有效克服：1 1）肿瘤干细胞数量极少（被掩盖）；）肿瘤干细胞数量极少（被掩盖）；2 2）肿瘤组织异质性（被干扰）。）肿瘤组织异质性（被干扰）。胡以平：关于肿瘤干细胞问题的一些思考（特邀述评）。中国肿瘤生物治疗杂志 15（3）：201-204，2008再生修复再生修复
23、涉及多种细胞类型、相互比例、结构组装等涉及多种细胞类型、相互比例、结构组装等一种分化细胞一种分化细胞 原位转分化原位转分化另一种分化细胞另一种分化细胞DeWitt ND,et al:TheEMBOJournal(2012)31,22442246 策略策略1 1（成纤维细胞）（成纤维细胞）（心肌细胞）（心肌细胞）（改变微环境）（改变微环境）策略策略2 2组织干细胞组织干细胞组织组织原位修复原位修复原位分化原位分化Watanabe,K.,etal:Nat.Biotechnol.25,681686.(2007)（改变微环境）（改变微环境）策略策略3 3基于脱细胞支架的组织工程基于脱细胞支架的组织工程
24、Endodermal Tissue Formation in 3D Stem Cell CultureDecelluarized&Recellularized LiverUnpublisheddata,HusLab肝干细胞研究的进展肝干细胞研究的进展 Cell TurnoverInjury Repair Replications of Hepatocyes&cholangiocytesYangerK.etal.CellStemCell.2014,15(3):340-9SchaubJR.etal.CellRep.2014,8(4):933-939MichalopoulosGK.etal.AJP.
25、2010,176(1):2-13GrompeM.CellStemCells.2014,15,257-258ImaiT.etal.PNAS.2001,98(8):4581-4586PonderKP.FASEBJ.1996,10(7):673-682 Replications of Hepatocyes&cholangiocytes Differentiation of Liver Stem CellsHuchM.etal.Nature.2013,494(7436):247-250FuruyamaK.etal.NatGenet.2011,43(1):34-41SemeraroR.etal.JHep
26、atol.2012,57(5):987-994 Differentiation of Liver Stem CellsEspaolR.etal.Gastroenterology.2012,(6):1564-75FuruyamaK.etal.NatGenet.2011,43(1):34-41ThompsonMD.etal.AJP.2010,177(4):1812-1822What is the Authentic Liver Stem Cell?Perplexity in the Liver Biomedical ResearchPerplexity in the Liver Biomedica
27、l ResearchLiver Stem CellsLiver Stem CellsLiver Parenchymal Cells Hepatocytes Hepatocytes Cholangiocytes Cholangiocytes Liver Progenitor CellsLiver Progenitor CellsHeterogeneity of Cell Compartment in the LiverHeterogeneity of Cell Compartment in the LiverWhat is the Lineage Specification of the Liv
28、er Stem Cell?Heterogegeity of Hepatocytes in the LiverHeterogegeity of Hepatocytes in the Liver雷章恒，等。雷章恒，等。第二军医大学学报第二军医大学学报 18(3):201-204,205-207,199718(3):201-204,205-207,1997 HBxHBsAgWild type liverHBcAgDifferences in the Differentiation State of Hepatocytes?Su J.,et al:Su J.,et al:Chinese Science
29、 BulletinChinese Science Bulletin,46(19):1625-25,2001,46(19):1625-25,2001Existence of Liver Primitive Cells肝脏强大的再生能力一直受到生物学家和医学家的普遍关注，加之肝脏肿瘤肝脏强大的再生能力一直受到生物学家和医学家的普遍关注，加之肝脏肿瘤发生学方面的大量实验资料，有人提出肝脏内有干细胞存在的假说。为了得发生学方面的大量实验资料，有人提出肝脏内有干细胞存在的假说。为了得到肝干细胞存在的直接证据，第二军医大学苏娟、胡以平等人对第到肝干细胞存在的直接证据，第二军医大学苏娟、胡以平等人对第131
30、3天小鼠天小鼠胚胎的肝脏组织进行体外培养，得到了具有干细胞特性的肝原始细胞系。进胚胎的肝脏组织进行体外培养，得到了具有干细胞特性的肝原始细胞系。进一步的胎肝细胞体内移植实验发现，此细胞系能在小鼠脾脏内形成具有肝细一步的胎肝细胞体内移植实验发现，此细胞系能在小鼠脾脏内形成具有肝细胞表型并呈克隆样生长的细胞集落，而且形成的脾结节数与移植的胎肝细胞胞表型并呈克隆样生长的细胞集落，而且形成的脾结节数与移植的胎肝细胞数之间呈正相关关系，从而证实了小鼠胎肝中肝干细胞的存在。这一研究为数之间呈正相关关系，从而证实了小鼠胎肝中肝干细胞的存在。这一研究为肝干细胞生物学特性的认识和利用奠定了基础。肝干细胞生物学特
31、性的认识和利用奠定了基础。(据据科学时报科学时报)据据科学时报科学时报20012001年年1111月月2121日日 Existence of Liver Progenitor CellsLi WL,et al.Li WL,et al.Stem CellsStem Cells 24(3):322332,200624(3):322332,2006Retrorsine +PHLi WL,et al.Li WL,et al.Stem CellsStem Cells 24(3):322332,200624(3):322332,2006Existence of Liver Progenitor Cells
32、CK19 Possible for the Tracing of Liver Stem Cells Development.2009;136(11):1951-60 Nat Genet.2011;43(1):34-41EpCAMSox9Lgr5Foxl1Foxl1Cre;Rosa26RlacZCK19:BrownLacZ:BlueHepatology.2009;49(3):920-9Nature.2013;494(7436):247-50Bi-potential:Bi-potential:Hepatocytes and Hepatocytes and CholangiocytesCholang
33、iocytesAuthenticAuthentic Liver stem Liver stem CellsCellsLineage Tracing of CK19-positive Cells in the LiverFACSSortingPick upSingle-CellsLiver Perfusion and Single-Cell AnalysisLiver Perfusion and Single-Cell Analysis IdentificationIdentificationof Cell Typesof Cell Types Construction of Construct
34、ion ofTranscriptome Library Transcriptome Library Deep SequencingDeep SequencingAnd TranscriptomeAnd Transcriptome Analyses Analyses Liver Stem CellsProgenitor CellsHepatocytesHepatocytesCholangiocytesLiver Perfusion and Single-Cell Analysis Liver Perfusion and Single-Cell Analysis Various Cell Type
35、s in CK19+Cell LineageBCharacteristics of Various Cell Lypes in CK19+Cell LineageACQuiescentStem CellsActivatedStem CellsCho precursorTACHepatocyteCholangiocyteHepatic precursorDifferentiation Lineage of Liver Stem CellsDifferentiation Lineage of Liver Stem CellsA BMolecular Signatures of Various Ce
36、ll Types in the Liver Stem Cell LineageMarkers Specific for Liver Stem CellsMarkers Specific for Liver Stem CellsActivation of Quiescent Liver Stem Cells Activation of Quiescent Liver Stem Cells in vivoin vivoQuiescentQuiescentStem CellsStem CellsActivatedActivatedStem CellsStem CellsImmunohistochem
37、istryAnalysisDDCActivation of Quiescent Liver Stem Cells Activation of Quiescent Liver Stem Cells by by Defined Factors Bi-potential Differentiation of Liver Stem Cells Bi-potential Differentiation of Liver Stem Cells in vitroin vitroBi-potential Differentiation of Liver Stem Cells Bi-potential Diff
38、erentiation of Liver Stem Cells in vitroin vitroContribution of the Liver Stem Cells to the Injured Liver Hepatocytes Hepatocytes Cholangiocytes Cholangiocytes Liver Liver Progenitor CellsProgenitor CellsLiver Liver Stem CellsStem CellsBiomedicineBiomedicine Issues Issues Targets for Liver Biomedicine Issues ResearchTargets for Liver Biomedicine Issues Research