Lu L, Ge Y, Li Z H, Keeble W, Kabat D, Bagby G C, Broxmeyer H E, Hoatlin M E
Department of Medicine (Hematology/Oncology), Indiana University School of Medicine, Indianapolis 46202-5121, USA.
Blood. 1996 Jan 15;87(2):525-34.
To test whether an enforced expression of a lineage-specific cytokine receptor would influence the proliferation/differentiation of hematopoietic stem/progenitor cells, retroviral vectors containing the human erythropoietin receptor (hEpoR) gene were used to transduce the hEpoR gene into phenotypically sorted subsets of cells. CD34 , CD34++CD33-, and CD34++CD33+ populations of human cord blood, highly enriched for hematopoietic stem/progenitor cells, were sorted and plated as single cells per well in methylcellulose culture medium containing early acting growth factors in the presence or absence of Epo. The hEpoR gene was efficiently transduced into single high proliferative potential colony-forming cells (HPP-CFC) and multipotential (colony-forming unit granulocyte, erythroid, monocyte, megakaryocyte [CFU-GEMM]), erythroid (burst-forming unit-erythroid [BFU-E]), and granulocyte-macrophage (colony-forming unit-granulocyte-macrophage [CFU-GM]) progenitor cells. As expected in cultures grown in the absence of Epo, no BFU-E or CFU-GEMM colonies grew. In the presence of Epo, the hEpoR-gene transduced cells formed significantly more CFU-GEMM and BFU-E colonies than did the controls. A significant decrease in HPP-CFC colonies was also observed under these conditions. Little or no effect of hEpoR gene transduction was apparent in the numbers of CFU-GM colonies formed in the presence or absence of Epo. All of the above results were similar whether the cell populations assessed were CD34 or their CD33- or CD33+ subsets plated in the presence of growth factors at 200 cells/mL or after limiting dilution at 2 cells/well. These results suggest that the profile of detectable stem/progenitors can be altered by retrovirus-mediated expression of the hEpoR gene.
为了检测谱系特异性细胞因子受体的强制表达是否会影响造血干/祖细胞的增殖/分化，使用含有人类促红细胞生成素受体（hEpoR）基因的逆转录病毒载体将hEpoR基因转导到经表型分选的细胞亚群中。对富含造血干/祖细胞的人脐带血CD34⁺、CD34⁺⁺CD33⁻和CD34⁺⁺CD33⁺群体进行分选，并在含有早期作用生长因子的甲基纤维素培养基中，以每孔单细胞的形式接种，分别置于有或无促红细胞生成素（Epo）的条件下培养。hEpoR基因被有效地转导到单个高增殖潜能集落形成细胞（HPP-CFC）、多能（粒系、红系、单核系、巨核系集落形成单位[CFU-GEMM]）、红系（爆式红系集落形成单位[BFU-E]）和粒系-巨噬系（粒系-巨噬系集落形成单位[CFU-GM]）祖细胞中。正如在无Epo培养条件下所预期的那样，没有BFU-E或CFU-GEMM集落生长。在有Epo的情况下，hEpoR基因转导的细胞形成的CFU-GEMM和BFU-E集落明显多于对照组。在这些条件下，还观察到HPP-CFC集落显著减少。无论评估的细胞群体是CD34⁺，还是其CD33⁻或CD33⁺亚群，在有生长因子存在的情况下以200个细胞/毫升接种，或在有限稀释后以2个细胞/孔接种，上述所有结果都是相似的。这些结果表明，hEpoR基因的逆转录病毒介导表达可以改变可检测到的干/祖细胞的概况。
