Cicuttini F M, Martin M, Salvaris E, Ashman L, Begley C G, Novotny J, Maher D, Boyd A W
Lions Clinical Cancer Research Laboratory, Walter and Eliza Hall Institute of Medical Research, PO Royal Melbourne Hospital, Victoria, Australia.
Blood. 1992 Jul 1;80(1):102-12.
We describe the development of a human bone marrow (BM) culture system which allows study of the interaction of stromal cell lines (SCL) and highly purified hematopoietic progenitor cells. Normal BM stromal cells were electroporated with a plasmid containing the simian virus 40 (SV40) large T antigen (SV40 T Ag) under the control of a synthetic metallothionein promoter (MT4); this construct is designated MT4 SV40 T Ag. SCL in which the rate of proliferation could be controlled by altering the zinc (Zn) concentration were characterized, demonstrating that the SCL were heterogeneous with respect to G-CSF and GM-CSF production. Suppression of SCL proliferation on removal of Zn made it possible to use these lines in coculture with purified CD34+ progenitor cells from umbilical cord blood. The ability to control proliferation of SCL has allowed us to maintain the survival and expansion of colony-forming cells in culture for up to 2 months. These lines have enabled us to test for stromal cell characteristics at a clonal level and provided us with a tool to analyze the events leading to lineage commitment and hematopoietic differentiation, as demonstrated by suppression of hematopoiesis by an antibody directed against the c-kit molecule.
我们描述了一种人类骨髓（BM）培养系统的开发，该系统允许研究基质细胞系（SCL）与高度纯化的造血祖细胞之间的相互作用。用含有在合成金属硫蛋白启动子（MT4）控制下的猿猴病毒40（SV40）大T抗原（SV40 T Ag）的质粒对正常BM基质细胞进行电穿孔；该构建体命名为MT4 SV40 T Ag。对通过改变锌（Zn）浓度可控制增殖速率的SCL进行了表征，表明这些SCL在粒细胞集落刺激因子（G-CSF）和粒细胞-巨噬细胞集落刺激因子（GM-CSF）产生方面具有异质性。去除锌后SCL增殖的抑制使得能够将这些细胞系与来自脐带血的纯化CD34+祖细胞共培养。控制SCL增殖的能力使我们能够在培养中维持集落形成细胞的存活和扩增长达2个月。这些细胞系使我们能够在克隆水平上测试基质细胞特征，并为我们提供了一种工具来分析导致谱系定向和造血分化的事件，如通过针对c-kit分子的抗体抑制造血所证明的那样。
