Trevisan M, Yan X Q, Iscove N N
Ontario Cancer Institute, Toronto, Canada.
Blood. 1996 Dec 1;88(11):4149-58.
This investigation was directed at separating long-term reconstituting (LTR) stem cells in normal murine marrow from hematopoietic precursors detectable in short-term assays in vitro and in vivo, and then at determining whether purified LTR cells could themselves form colonies in culture. To do so, it was first necessary to identify culture conditions that would induce their growth while preserving their long-term reconstituting capacity. Marrow was cultured with various cytokines in liquid suspension for 4 days, after which the surviving LTR activity was quantitated in a competitive in vivo assay. Activity was preserved near input levels with combined murine c-kit ligand (KL), interleukin-1 (IL-1), IL-6, and IL-11. When the cultures also included tritiated or unlabeled thymidine, LTR potential was eliminated, indicating that essentially all LTR cells were induced into cell cycle with these cytokines. To purify them, marrow was sorted on the basis of Ly6A expression and Rhodamine 123 retention. The Ly6AhiRh123ls fraction contained 85% of total recovered LTR activity but only 1% of the recovered cells measured by multilineage colony formation in spleens or in vitro. This fraction was cultured in methyl cellulose with KL, IL-1, IL-6, and IL-11 for 4 to 6 days, after which colonies were isolated and injected into mice. High levels of permanent reconstitution were achievable in sublethally irradiated W41/W41 mice after the injection of a single reconstituting unit, and limiting dilution analysis estimated the frequency of multilineage LTR at 1 in 11,200 unpurified adult marrow cells. In either lethally irradiated normal or sublethally irradiated W41/W41 mice, 1-year lymphomyeloid reconstitutions were obtained from 1 in 65 to 84 colonies of 2 to 16 dispersed cells, but not from larger colonies or those with clumped cells. The results establish that resting marrow LTR cells can be separated from almost all of the more advanced clonogenic cells that are still pluripotential, can be induced to cycle in culture by defined cytokines with preservation of their reconstituting potential, and can be manipulated and assayed efficiently at single-cell and colony levels.
本研究旨在从正常小鼠骨髓中分离出长期重建造血（LTR）干细胞，这些干细胞在体外和体内短期检测中是难以检测到的造血前体细胞，然后确定纯化后的LTR细胞本身是否能在培养中形成集落。为此，首先需要确定能够诱导其生长同时保留其长期重建造血能力的培养条件。将骨髓与各种细胞因子在液体悬浮液中培养4天，之后在竞争性体内检测中对存活的LTR活性进行定量分析。联合使用小鼠c-kit配体（KL）、白细胞介素-1（IL-1）、IL-6和IL-11时，活性保持在接近输入水平。当培养物中还含有氚标记或未标记的胸腺嘧啶核苷时，LTR潜能被消除，这表明基本上所有LTR细胞都被这些细胞因子诱导进入细胞周期。为了纯化它们，根据Ly6A表达和罗丹明123保留情况对骨髓进行分选。Ly6AhiRh123ls组分包含了总回收LTR活性的85%，但通过脾内或体外多谱系集落形成检测，其回收细胞仅占1%。将该组分在含有KL、IL-1、IL-6和IL-11的甲基纤维素中培养4至6天，之后分离集落并注射到小鼠体内。在亚致死剂量照射的W41/W41小鼠中，注射单个重建造血单位后可实现高水平的长期造血重建，极限稀释分析估计多谱系LTR在未纯化的成年骨髓细胞中的频率为1/11,200。在致死剂量照射的正常小鼠或亚致死剂量照射的W41/W41小鼠中，从2至16个分散细胞形成的65至84个集落中的1个可获得1年的淋巴细胞和髓细胞重建，但从较大集落或细胞聚集的集落中则无法获得。这些结果表明，静止的骨髓LTR细胞可以与几乎所有仍具有多能性的更晚期克隆形成细胞分离，可以通过特定细胞因子在培养中诱导其进入细胞周期并保留其重建造血潜能，并且可以在单细胞和集落水平上进行有效操作和检测。
