Rios M, Williams D A
Howard Hughes Medical Institute, Harvard Medical School, Boston, Massachusetts 02115.
J Cell Physiol. 1990 Dec;145(3):434-43. doi: 10.1002/jcp.1041450307.
Hematopoietic stem cells interact with a complex microenvironment both in vivo and in vitro. In association with this microenvironment, murine stem cells are maintained in vitro for several months. Fibroblast-like stromal cells appear to be important components of the microenvironment, since several laboratories have demonstrated that cloned stromal cell lines support hematopoiesis in vitro. The importance of the tissue of origin of such cell lines remains unknown, since systematic generation of stromal cell lines from adult tissues has never been accomplished. In addition, the capacity of stromal cell lines to support reconstituting stem cell has not been examined. We have previously described an efficient and rapid method for the immortalization of primary bone marrow stromal cell lines (Williams et al., Mol. Cell. Biol. 8:3864-3871, 1988) which can be used to systematically derive cell lines from multiple tissues of the adult mouse. Here we report the immortalization of primary murine lung, kidney, skin, and bone marrow stromal cells using a recombinant retrovirus vector (U19-5) containing the simian virus large T antigen (SV40 LT) and the neophosphotransferase gene. The interaction of these stromal cells with factor-dependent cells Patterson-Mix (FDCP-Mix), colony forming units-spleen (CFU-S), and reconstituting hematopoietic stem cells was studied in order to analyze the ability of such lines to support multipotent stem cells in vitro. These studies revealed that stromal cell lines from these diverse tissues were morphologically and phenotypically similar and that they quantitatively bound CFU-S and FDCP-Mix cells equally well. However, only those cell lines derived from bone marrow-supported maintenance of day 12 CFU-S in vitro. One lung-derived stromal cell line, ULU-3, supported the survival of day 8 CFU-S, but not the more primitive CFU-S12. A bone marrow-derived stromal cell line, U2, supported the survival of long-term reconstituting stem cells for up to 3 weeks in vitro as assayed by reconstitution 1 year post-transplant. These studies suggest that adherence of HSC to stromal cells is necessary but not sufficient for maintenance of these stem cell populations and that bone marrow provides specific signals relating to hematopoietic stem cell survival and proliferation.
造血干细胞在体内和体外均与复杂的微环境相互作用。与这种微环境相关联，小鼠干细胞可在体外维持数月。成纤维细胞样基质细胞似乎是微环境的重要组成部分，因为多个实验室已证明克隆的基质细胞系可在体外支持造血。此类细胞系的组织来源的重要性仍然未知，因为从未实现过从成年组织系统地生成基质细胞系。此外，基质细胞系支持重建造血干细胞的能力尚未得到研究。我们之前描述了一种高效快速的方法，用于使原代骨髓基质细胞系永生化（Williams等人，《分子与细胞生物学》8:3864 - 3871，1988），该方法可用于从成年小鼠的多个组织系统地获得细胞系。在此我们报告使用含有猿猴病毒大T抗原（SV40 LT）和新磷酸转移酶基因的重组逆转录病毒载体（U19 - 5）使原代小鼠肺、肾、皮肤和骨髓基质细胞永生化。研究了这些基质细胞与因子依赖性细胞帕特森 - 混合细胞（FDCP - Mix）、脾集落形成单位（CFU - S）和重建造血干细胞之间的相互作用，以分析此类细胞系在体外支持多能干细胞的能力。这些研究表明，来自这些不同组织的基质细胞系在形态和表型上相似，并且它们定量结合CFU - S和FDCP - Mix细胞的能力相同。然而，只有那些源自骨髓的细胞系在体外支持第12天的CFU - S的维持。一个源自肺的基质细胞系ULU - 3支持第8天CFU - S的存活，但不支持更原始的CFU - S12。一个源自骨髓的基质细胞系U2支持长期重建造血干细胞在体外存活长达3周，移植后1年的重建分析表明了这一点。这些研究表明，造血干细胞与基质细胞的黏附对于维持这些干细胞群体是必要的，但并不充分，并且骨髓提供了与造血干细胞存活和增殖相关的特定信号。
