Capel B, Hawley R, Covarrubias L, Hawley T, Mintz B
Institute for Cancer Research, Fox Chase Cancer Center, Philadelphia, PA 19111.
Proc Natl Acad Sci U S A. 1989 Jun;86(12):4564-8. doi: 10.1073/pnas.86.12.4564.
Mice were repopulated with small numbers of retrovirally marked hematopoietic cells operationally definable as totipotent hematopoietic stem cells, without engraftment of cells at later stages of hematopoiesis, in order to facilitate analysis of stem cell clonal histories. This result depended upon the use of unirradiated W/Wv newborn recipients. Before transplantation, viral integration markers were introduced during cocultivation of fetal liver or bone marrow cells with helper cell lines exporting defective recombinant murine retroviruses of the HHAM series. Omission of selection in culture [although the vector contained the bacterial neomycin-resistance (neo) gene] also limited the proportion of stem cells that were virally labeled. Under these conditions, engraftment was restricted to a small population of marked and unmarked normal donor stem cells, due to their competitive advantage over the corresponding defective cells of the mutant hosts. A relatively simple and coherent pattern emerged, of one or a few virally marked clones, in contrast to previous studies. In order to establish the totipotent hematopoietic stem cell identity of the engrafted cells, tissues were sampled for viral and inbred-strain markers for periods close to one year after transplantation. The virally labeled clones were characterized as stem cell clones by their extensive self-renewal and by formation of the wide range of myeloid and lymphoid lineages tested. Results clearly documented concurrent contributions of cohorts of stem cells to hematopoiesis. A given stem cell can increase or decrease its proliferative activity, become completely inactive or lost, or become active after a long latent period. The contribution of a single clone present in a particular lineage was usually between 5% and 20%.
用少量经逆转录病毒标记的造血细胞对小鼠进行再移植，这些细胞在操作上可定义为全能造血干细胞，且未植入造血后期的细胞，以便于分析干细胞的克隆历史。这一结果依赖于使用未受辐射的W/Wv新生受体。在移植前，在胎肝或骨髓细胞与输出HHAM系列缺陷重组鼠逆转录病毒的辅助细胞系共培养期间引入病毒整合标记。培养过程中不进行选择（尽管载体含有细菌新霉素抗性基因）也限制了被病毒标记的干细胞比例。在这些条件下，由于标记和未标记的正常供体干细胞相对于突变宿主相应的缺陷细胞具有竞争优势，移植仅限于一小部分标记和未标记的正常供体干细胞。与先前的研究相比，出现了一种相对简单且连贯的模式，即一个或几个病毒标记的克隆。为了确定移植细胞的全能造血干细胞身份，在移植后接近一年的时间内对组织进行采样，检测病毒和近交系标记。通过广泛的自我更新以及形成所测试的广泛的髓系和淋巴系，将病毒标记的克隆鉴定为干细胞克隆。结果清楚地证明了干细胞群体对造血的同时贡献。一个给定的干细胞可以增加或降低其增殖活性，变得完全无活性或丢失，或者在长时间潜伏期后变得活跃。特定谱系中单个克隆的贡献通常在5%至20%之间。
