MSCsandcancerrevised

间充质干细胞和肿瘤,Wen GuanmeiDept. of Pathophysiology, GZMC,内容提要,MSC: 恶性转化的靶细胞 。MSC ：肿瘤的发生过程中的“双刃剑”。问题和展望。,MSC: Mesenchymal stem cells,Martin Evans,Mario R. Capecchi,Oliver Smithies,The Nobel Prize in Physiology or Medicine 2007,“利用胚胎干细胞在老鼠身上引入特定基因修饰 ,胚胎干细胞 (Embryonic Stem Cells, ESCs),优势,障碍,解决办法,治疗性克隆Therapeutical clone,2002年3月，美国怀特黑德生物医学研究所借助克隆技术成功对实验鼠进行了胚胎干细胞治疗，首次在动物身上证实“治疗性克隆”技术是可行的。,干细胞库Stem cell database (SCDB),2004年5月，世界首个国家胚胎干细胞库在英国伦敦建立，它可为糖尿病、癌症、帕金森氏病和阿尔茨海默氏症等疾病的研究和治疗储存、提供干细胞 。,成体干细胞 (Adult Stem Cells, ASCs ),优势,不足,间充质干细胞 （MSCs）,An adult tissue-derived undifferentiated cell with extended proliferative capability and multilineage differentiation potential .,A.J. Friedenstein (1924-1998),来源,生物学特性,临床应用,MSCs,（中胚层） Mesodermal 骨髓、肌肉、脂肪、扁桃体,（内胚层）Endodermal 胸腺,（外胚层）Ectodermal 皮肤、毛囊、硬脊膜和牙髓,产前或围产期组织脐带或脐带血 胎盘,Bone marrow MSC, BMMSCs,HSCs,MSCs,来源,生物学特性,临床应用,MSCs,MSCs 的鉴定标准,贴壁生长表达特定的表面分子多向分化潜能 -国际细胞治疗协会(ISCT),生物学特性,分离, 培养 和 扩增。形态学特点。 细胞周期。,形态学特点,倒置相差显微镜激光共聚焦 电子显微镜,对MSCs细胞鉴定标准的完善：95 MSC ：CD73 (ecto-5-nucleotidase), CD90 (Thy-1), and CD105 (endoglin), 和 2% 表达 CD34, CD45, CD11b or CD14, CD19,or CD79 (B-cell marker), and HLA-DR (marker of stimulated MSCs),细胞周期,1,2,CTX,60Co,NOD/SCID,3,分化潜能,体外分化,MSCs: CD90 (red),激光共聚焦： GFP+MSCs,Adipocytes:Nile Red,体外分化,显微注射,来源,生物学特性,临床应用,MSCs,可塑性,细胞治疗组织工程基因治疗再生医学,应用潜能,MSCs从实验室到临床，我们应该考虑的问题是什么?生物安全性?恶性转化的靶细胞?MSCs & 肿瘤的发生,MSC: 恶性转化的靶细胞,MSC: 恶性转化的靶细胞,体外: 恶性转化实验性诱导自发转化体内: MSC-来源的肿瘤,实验诱导,hMSC-hTERT.长期培养的人MSC 端粒酶过表达.EWS-FLI-1癌基因转染对小鼠MSCs .,hTERT: human telomerase reverse transcriptase,hTERT gene,hMSCs,hMSCs-TERT2, 4, 20,恶性转化 In vitro In vivo,自发转化,Spontaneous Transformation of Cultured Mouse Bone MarrowDerived Stromal Cells. Cancer Res. 2006; 66: 22.,Loss of contact inhibition,Development of malignant sarcomas following injection of B6.6. (C57BL/6) P6 MSCs into nude mice.,Features of malignancy and differentiation in sarcomas following injection of B6.8.P6 MSCs into SCID mice.,Karyotype analysis of mouse MSCs.,Karyotype analysis of mouse MSCs.,Summary,mMSCs (P3) :非整倍体.染色体不稳定. 肌肉注射：形成侵袭性的骨肉瘤。静脉注射：形成肺结节病灶.保留了干细胞的特性.,可能机制,端粒酶和 c-myc 过表达.细胞融合.染色体复制过程中没有发生细胞分离.在随后的有丝分裂中发生转位和融合.,Chromosome translocation,50% 长期培养(4-5 months)的脂肪源性的 人 MSCs 发生自发转化.,huMSCs,Transformation,In vitro: long-term,In vivo: NOD/SCID mice,solid tumors,Long-term culture,Morphology,FACS,Elongated spindle shape a small compact morphology,46, XY,47, XY,TMC: Transformed mesenchymal stem cells,Genes differentially expressed in MSC versus TMC,Lane1-3: presenescence MSC,Lane2: post-senescence MSC,Lane4: senescence MSC,Lane5-7: TMC,A:,Telomerase activity,Sample1: presenescence MSC,Sample 2: post-senescence MSC,Sample 3-5: TMC,B:,In vivo analysis of TMC,Organs extracted from mice inoculated with EGFP-TMC show fluorescent tumor nodules in (A) lung, (B) kidney, (C) liver, (D) brain, (E) heart, (F) striated muscle, (G) ovary, and (H) suprarenal gland. I -J (H&E staining ) K-L, peroxidase-conjugated anti-human mitochondrial mAb staining of lung infiltrated with TMC,恶性转化的靶细胞,Tumor,Cancer stem cells,MSC,体外: 恶性转化.体内: MSC来源的肿瘤.,Gastric cancer originating from bone marrow-derived cells,Helicobacter induced gastric cancer is derived from BMDCs,8 wk infectionX-gal staining,20-30 wk infectionX-gal stainingdysplasia,-Galactosidase (IHC)GIN (gastrointestinal intraepithelial neoplasia),X-gal staining,Brdu incorporation,WT+mock,-galactosidase staining (blue),-galactosidase staining (blue)TFF2 (red),WT+H.feils,ROSA 26+H.feils,ROSA 26+H.feils,-galactosidase expression of stomachs from C57BL/6 mice transplanted with ROSA26 marrow,A ,C: Mock-infected miceB , D: H. felis infected mice,E: Glands within GIN from an infected mouse transplanted with WT marrowF: Glands within GIN from an infected mouse transplanted with ROSA26 marrow(red), colocalized with cytokeratin (green),B: GFP+CD45+ (P4) /CK- ; C: XY,Cytokeratin,FISH,D: GFP+CD45- (P5) /CK+ ; E: XY,F: GFP-CD45- (P6) /CK+ ; G: XX,X chromosome (red)Y chromosome (green),Female to female,Male to female,IHC,FISH,B,C: IHC for GFP (brown) in tumor cellsE,F: FISH for Y chromosome (green), cytokeratin( red), nuclei (black),Multiple Tumor Types May Originate from Bone MarrowDerived Cells (2006) . Sarcoma derived from MSCs (2007).,A,B,C,D,E,F,G,H,I,转化的MSC的特性,可能机制:染色体不稳定性.异常端粒酶活性.基因突变.促癌蛋白.Nucleostemin (核干细胞因子, 2002)Bmi-1自我更新功能障碍.微环境: 肿瘤 “龛”.,MSCs & 肿瘤的发生,肿瘤中MSC的异常.在肿瘤的发生发展中的作用.MSC向肿瘤病灶迁移和聚集.MSC 影响肿瘤的生长. 抗肿瘤作用.促进肿瘤细胞生长.,Aberrant MSC in tumors,在肿瘤的发生发展中的作用,MSC诱导人乳腺癌细胞株（MCF-7）发生以下改变：抑制细胞聚集.提高增殖能力.下调E-cadherin 的表达.,MCF-7,+MSCs,+MSCs-CM,+MSCsCytokeratin,cytokeratin peptide 18,Con.,+IL-6,E-cadherin,E-cadherin expression in MCF-7.,+MSC,control,+MSC-CM,+IL-6,MSC向肿瘤病灶迁移和聚集,MSCs home to tumor sites.Primary tumors can trigger systemic cues that could potentially activated MSCs to migrate to the tumor via blood stream.The tumor microenvironment is a complex ecology of numerous cell types, growth factors, and ECM molecules.,“双刃剑”,抗肿瘤促进肿瘤生长,Immunosuppressive effect of mesenchymal stem cells favors tumor growth in allogeneic animals.,Murine B16 melanoma cell line,Allogenenic bone marrow MSCs,促进肿瘤生长,1. Gelatin pieces (arrow) containing a mixture of tumor cells and MPC1cE (A) and tumor cells only (B) 5 days after subcutaneous implantation in rat,MPC1cE: rat immortalized mesenchymal progeni