Fellous Tariq G, McDonald Stuart A C, Burkert Julia, Humphries Adam, Islam Shahriar, De-Alwis Nemantha M W, Gutierrez-Gonzalez Lydia, Tadrous Paul J, Elia George, Kocher Hemant M, Bhattacharya Satyajit, Mears Lisa, El-Bahrawy Mona, Turnbull Douglas M, Taylor Robert W, Greaves Laura C, Chinnery Patrick F, Day Christopher P, Wright Nicholas A, Alison Malcolm R
Centre for Diabetes and Metabolic Medicine, Barts and The London School of Medicine and Dentistry, Queen Mary University of London, London, United Kingdom.
Stem Cells. 2009 Jun;27(6):1410-20. doi: 10.1002/stem.67.
Methods for lineage tracing of stem cell progeny in human tissues are currently not available. We describe a technique for detecting the expansion of a single cell's progeny that contain clonal mitochondrial DNA (mtDNA) mutations affecting the expression of mtDNA-encoded cytochrome c oxidase (COX). Because such mutations take up to 40 years to become phenotypically apparent, we believe these clonal patches originate in stem cells. Dual-color enzyme histochemistry was used to identify COX-deficient cells, and mutations were confirmed by microdissection of single cells with polymerase chain reaction sequencing of the entire mtDNA genome. These techniques have been applied to human intestine, liver, pancreas, and skin. Our results suggest that the stem cell niche is located at the base of colonic crypts and above the Paneth cell region in the small intestine, in accord with dynamic cell kinetic studies in animals. In the pancreas, exocrine tissue progenitors appeared to be located in or close to interlobular ducts, and, in the liver, we propose that stem cells are located in the periportal region. In the skin, the origin of a basal cell carcinoma appeared to be from the outer root sheath of the hair follicle. We propose that this is a general method for detecting clonal cell populations from which the location of the niche can be inferred, also affording the generation of cell fate maps, all in human tissues. In addition, the technique allows analysis of the origin of human tumors from specific tissue sites.
目前尚无用于追踪人体组织中干细胞子代谱系的方法。我们描述了一种检测单个细胞子代扩张的技术，这些子代细胞包含影响线粒体DNA（mtDNA）编码的细胞色素c氧化酶（COX）表达的克隆性mtDNA突变。由于此类突变需要长达40年才会在表型上显现出来，我们认为这些克隆斑起源于干细胞。采用双色酶组织化学法鉴定COX缺陷细胞，并通过对单个细胞进行显微切割，对整个mtDNA基因组进行聚合酶链反应测序来确认突变。这些技术已应用于人体的肠道、肝脏、胰腺和皮肤。我们的结果表明，干细胞龛位于结肠隐窝底部以及小肠潘氏细胞区域上方，这与动物动态细胞动力学研究结果一致。在胰腺中，外分泌组织祖细胞似乎位于小叶间导管内或其附近，而在肝脏中，我们认为干细胞位于门静脉周围区域。在皮肤中，基底细胞癌的起源似乎来自毛囊外根鞘。我们认为这是一种检测克隆细胞群体的通用方法，据此可以推断出龛的位置，还能生成细胞命运图谱，所有这些均适用于人体组织。此外，该技术还能分析源自特定组织部位的人类肿瘤的起源。
