Studies of differentiation mechanisms of hematopoietic stem cells using cytokine-receptor transgenic mice

粒细胞巨噬细胞集落刺激因子(GM-CSF)通过表达于靶细胞上的细胞表面受体刺激髓系祖细胞的增殖和成熟。为了分析hGMR受体表达与造血细胞分化的关系，利用hGMRα和βcDNA构建了表达hGMR的转基因小鼠(TG小鼠)。HGM-CSF支持多种类型的集落形成，包括原始细胞集落、红系集落、巨核细胞集落和混合造血集落。给TG小鼠注射hGM-CSF后，其外周血中中性粒细胞、单核细胞、嗜酸性粒细胞、NK细胞及红细胞数量均明显增加。为了阐明GM-CSFR信号在红细胞生成中是否可以补偿促红细胞生成素受体(EPOR)信号，我们接下来产生了双突变小鼠，HGM-CSFR+/+和EPOR-/-。尽管双突变小鼠具有胚胎致死性，但hGM-CSF从双突变小鼠的第14天胎肝细胞中诱导出大量的红系集落。我们还产生了普遍表达野生型人G-CSFR和野生型小鼠G-CSFR的TG小鼠。在骨髓和脾细胞的甲基纤维素集落培养中，G-CSF不仅能促进粒细胞的增殖和分化，还能促进巨噬细胞、巨核细胞、肥大细胞、红系和更原始的造血祖细胞在有血清和无血清培养中的增殖和分化。这些结果表明，当细胞表面存在功能性的GM-CSFR或G-CSFR时，这两种细胞因子都不会诱导对中性粒细胞和巨噬细胞的专一承诺。用转基因小鼠进行的体外和体内研究表明，当受体表达时，hGM-CSF或hG-CSF等细胞因子支持各种造血祖细胞的生长，但不改变他们的承诺程序，因此倾向于“随机模型”而不是“确定性模型”。

Granulocyte-macrophage colony stimulating factor (GM-CSF) stimulates proliferation and maturation of myeloid progenitor cells through cell surface receptors expressed on target cells. To analyze the relationship between receptor expression and differentiation of hemopoietic cells, transgenic mice (Tg mice) which express hGMR at all stages of hemopoitic cell development were generated using hGMR α and β cDNAs. hGM-CSF supported colony formation of various types including blast cell, erythroid (E), megakaryocyte (Meg), and mixed hematopoietic colonies. Administration of hGM-CSF to Tg mice resulted in increase of not only neutrophils, monocyte, eosinophils, NK cells but also erythrocytes in their peripheral blood. To clalify whether GM-CSFR signaling can compensate erythropoietin receptor (EPOR) signaling in erythropoiesis, we next generated double mutant mice, hGM-CSFR+/+ and EPOR-/-. Although double mutant mice were embryonic lethal, hGM-CSF induced a large number of erythroid colonies from day 14 fetal liver cells of double mutant mice. We also generated Tg mice that had ubiquitous expressions of wild type human G-CSFR, wild type murine G-CSFR. In methylcellulose colony assay of bone marrow and spleen cells G-CSF have an effect on promoting the proliferation and differentiation of not only granulocyte but also macrophage, megakaryocyte, mast cells, erythroid and more primitive hematopoietic progenitors in both serum-containing and -free culture. These results shows that when functional GM-CSFR or G-CSFR are present on the cell surface, both cytokines does not induce exclusive commitment to the neutrophil and macrophage lineages. In vitro and in vivo studies using the Tg mice indicated that cytokines such as hGM-CSF or hG-CSF supported the growth of various hematopoietic progenitors when the receptor was expressed, but did not alter their commitment program, thus favoring the "stochastic model" rather than the "deterministic model".