Xiao M, Broxmeyer H E, Horie M, Grigsby S, Lu L
Department of Medicine, (Hematology/Oncology), Indiana University School of Medicine, Indianapolis 46202-5121, USA.
Blood Cells. 1994;20(2-3):455-66; discussion 466-7.
Nonadherent, low-density T-lymphocyte-depleted (NALT-) CD34 cells from normal human cord blood were assessed in suspension culture for the effects of recombinant cytokines on their proliferation, differentiation, and generation of myeloid progenitor cells. In this cell population, 82% of cells expressed c-kit protein as assessed by in situ hybridization, and their cloning efficiency was 85% when cells were plated at low cell numbers with combinations of growth factors. CD34 cells were sorted as 1, 5, or 10 cell(s) per well and also at 5000 cells per dish to initiate stromal-free suspension cultures in the presence of steel factor (SLF), interleukin (IL)-1 alpha, and IL-3. Forty-eight percent of the wells started with a single CD34 cell were positive for growth after 14 days, and the wells contained greater than 5 x 10(3) cells by 21-28 days. Progenitors were assayed weekly with cultures initiated with 1 or 5000 cells. While the fold expansion of nucleated cells was greater in cultures initiated with one cell per well (> 5000 compared to 791-fold expansion for 5000 cells), the fold expansion of progenitors was greater than 5000 cells were used to initiate cultures. Under optimal conditions, there was, respectively, a 160-, 164-, and 57-fold output of high proliferative potential colony-forming cells, granulocyte-macrophage colony-forming units, and erythroid burst-forming units/granulocyte erythroid macrophage megakaryocyte colony-forming units within 1-3 weeks for cultures initiated with 5000 CD34 cells compared with respective fold increases of 29, 16, and 1, for single-initiated cultures. These results demonstrate the expansion capacity of single CD34 cord blood cells and demonstrate that factors in addition to SLF, IL-1 alpha, and IL-3 are necessary for optimal expansion of progenitors from single isolated CD34 cells.
对来自正常人脐带血的非贴壁、低密度T淋巴细胞去除（NALT-）CD34细胞进行悬浮培养，以评估重组细胞因子对其增殖、分化以及髓系祖细胞生成的影响。在该细胞群体中，通过原位杂交评估，82%的细胞表达c-kit蛋白，当细胞以低细胞数接种并与生长因子组合培养时，其克隆效率为85%。将CD34细胞按每孔1、5或10个细胞分选，也按每皿5000个细胞分选，在存在钢因子（SLF）、白细胞介素（IL）-1α和IL-3的情况下启动无基质悬浮培养。14天后，起始为单个CD34细胞的孔中有48%生长呈阳性，到21 - 28天时，孔中细胞数超过5×10³个。每周对起始接种1个或5000个细胞的培养物进行祖细胞检测。虽然每孔接种1个细胞起始的培养物中，有核细胞的扩增倍数更大（>5000倍，而接种5000个细胞的扩增倍数为791倍），但祖细胞的扩增倍数在接种5000个细胞起始的培养物中更大。在最佳条件下，接种5000个CD34细胞起始的培养物在1 - 3周内，高增殖潜能集落形成细胞、粒细胞-巨噬细胞集落形成单位以及红系爆式集落形成单位/粒细胞-红细胞-巨噬细胞-巨核细胞集落形成单位的产出分别为160倍、164倍和57倍，而单个细胞起始的培养物相应的倍数增加分别为29倍、16倍和1倍。这些结果证明了单个CD34脐带血细胞的扩增能力，并表明除了SLF、IL-1α和IL-3之外，其他因子对于从单个分离的CD34细胞中最佳扩增祖细胞是必需的。
