Novotny J R, Duehrsen U, Welch K, Layton J E, Cebon J S, Boyd A W
Lions Clinical Cancer Research Laboratory, Walter and Eliza Hall Institute of Medical Research, PO Royal Melbourne Hospital, Australia.
Exp Hematol. 1990 Aug;18(7):775-84.
We report a human bone marrow culture technique that initially parallels the murine Whitlock/Witte culture system. As in the murine system, B cells predominate over other cell types, and all differentiation stages from pre-B to plasma cell are observed. Although these human long-term cultures pass through stages resembling phases I to III of murine Whitlock/Witte cultures, no outgrowth of nonadherent cells was seen after cultures had reached the "crisis" phase unless Epstein-Barr virus (EBV)-transformants appeared. The stromal cells persisted well beyond crisis, but they could not be maintained and passaged as cell lines, limiting their use in molecular analysis. Transfection of these stromal cells with plasmid DNA containing the simian virus 40 (SV40) early region yielded 124 cloned cell lines. Analysis of these lines showed that all expressed SV40 large T antigen, but they retained most phenotypic markers found on non-transformed stromal cells. When adherent and T-cell-depleted bone marrow cells were cultured on either nontransformed stromal layers or transformed cell lines they proliferated actively and soon yielded predominantly lymphoid nonadherent populations. Moreover, prolonged survival of acute lymphoblastic leukemia cells of pre-B phenotype was regularly achieved on both normal and transformed adherent cell layers. Although the liquid culture system favored lymphocytes, transformed stroma supported colony formation by both human and murine hemopoietic progenitors when marrow was added in agar medium. This was not explained by colony-stimulating factor (CSF) production, because striking heterogeneity in the levels of granulocyte CSF (G-CSF) and granulocyte-macrophage CSF (GM-CSF) secretion by the lines was noted. Some lines that did not produce detectable CSF demonstrated good support of fresh bone marrow growth and acute lymphoblastic leukemia (ALL) cell survival. The heterogeneity of these cell lines and their capacity to support hemopoiesis suggest that they will be useful in studying the molecular basis of in vitro lymphohemopoiesis in man.
我们报告了一种人类骨髓培养技术，该技术最初与小鼠惠特洛克/维特培养系统相似。与小鼠系统一样，B细胞在其他细胞类型中占主导地位，并且可以观察到从前B细胞到浆细胞的所有分化阶段。尽管这些人类长期培养物经历了类似于小鼠惠特洛克/维特培养物I至III期的阶段，但在培养物达到“危机”阶段后，除非出现爱泼斯坦-巴尔病毒（EBV）转化体，否则未观察到非贴壁细胞的生长。基质细胞在危机后持续存在，但它们不能作为细胞系进行维持和传代，这限制了它们在分子分析中的应用。用含有猿猴病毒40（SV40）早期区域的质粒DNA转染这些基质细胞，产生了124个克隆细胞系。对这些细胞系的分析表明，所有细胞系都表达SV40大T抗原，但它们保留了未转化基质细胞上发现的大多数表型标记。当将贴壁且去除T细胞的骨髓细胞培养在未转化的基质层或转化细胞系上时，它们会积极增殖，并很快产生主要为淋巴样非贴壁群体。此外，在正常和转化的贴壁细胞层上，前B表型的急性淋巴细胞白血病细胞都能定期实现长期存活。尽管液体培养系统有利于淋巴细胞生长，但当在琼脂培养基中加入骨髓时，转化的基质支持人和小鼠造血祖细胞形成集落。这不能用集落刺激因子（CSF）的产生来解释，因为注意到这些细胞系在粒细胞CSF（G-CSF）和粒细胞-巨噬细胞CSF（GM-CSF）分泌水平上存在显著异质性。一些不产生可检测CSF的细胞系对新鲜骨髓生长和急性淋巴细胞白血病（ALL）细胞存活表现出良好的支持作用。这些细胞系的异质性及其支持造血的能力表明，它们将有助于研究人类体外淋巴细胞生成的分子基础。
