Kolodka T M, Garlick J A, Taichman L B
Department of Oral Biology and Pathology, State University of New York at Stony Brook, Stony Brook, NY 11794-8702, USA.
Proc Natl Acad Sci U S A. 1998 Apr 14;95(8):4356-61. doi: 10.1073/pnas.95.8.4356.
Epidermis is renewed by a population of stem cells that have been defined in vivo by slow turnover, label retention, position in the epidermis, and enrichment in beta1 integrin, and in vitro by clonogenic growth, prolonged serial passage, and rapid adherence to extracellular matrix. The goal of this study is to determine whether clonogenic cells with long-term growth potential in vitro persist in vivo and give rise to a fully differentiated epidermis. Human keratinocytes were genetically labeled in culture by transduction with a retrovirus encoding the lacZ gene and grafted to athymic mice. Analysis of the cultures before grafting showed that 21.1-27.8% of clonogenic cells with the capacity for >30 generations were successfully transduced. In vivo, beta-galactosidase (beta-gal) positive cells participated in the formation of a fully differentiated epithelium and were detected throughout the 40-week postgraft period, initially as loosely scattered clusters and later as distinct vertical columns. Viable cells recovered from excised grafts were seeded at clonal densities and 23.3-33.3% of the colonies thus formed were beta-gal positive. In addition, no evidence of transgene inactivation was obtained: all keratinocyte colonies recovered from grafted tissue that were beta-gal negative also lacked the lacZ transgene. These results show that cells with long-term growth properties in vitro do indeed persist in vivo and form a fully differentiated epidermis, thereby exhibiting the properties of stem cells.
表皮由一群干细胞更新，这些干细胞在体内可通过更新缓慢、标记保留、在表皮中的位置以及β1整合素富集来定义，在体外可通过克隆生长、长期连续传代以及快速黏附于细胞外基质来定义。本研究的目的是确定体外具有长期生长潜力的克隆细胞在体内是否持续存在并产生完全分化的表皮。通过用编码lacZ基因的逆转录病毒转导，在培养中对人角质形成细胞进行基因标记，然后移植到无胸腺小鼠体内。移植前对培养物的分析表明，具有超过30代能力的克隆细胞中有21.1 - 27.8%被成功转导。在体内，β-半乳糖苷酶（β-gal）阳性细胞参与形成完全分化的上皮，并且在移植后40周内都能检测到，最初是松散散布的簇状，后来是明显的垂直柱状。从切除的移植物中回收的活细胞以克隆密度接种，由此形成的菌落中有23.3 - 33.3%是β-gal阳性。此外，未获得转基因失活的证据：从移植组织中回收的所有β-gal阴性的角质形成细胞菌落也缺乏lacZ转基因。这些结果表明，体外具有长期生长特性的细胞确实在体内持续存在并形成完全分化的表皮，从而表现出干细胞的特性。
