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）通过靶细胞表面表达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hGMR α和hGMR β cDNA构建了在造血细胞发育的各个阶段表达hGMR的转基因小鼠（Tg小鼠）。hGM-CSF支持各种类型的集落形成，包括原始细胞、红系细胞（E）、巨核细胞（Meg）和混合造血集落。对Tg小鼠施用hGM-CSF不仅导致其外周血中的中性粒细胞、单核细胞、嗜酸性粒细胞、NK细胞的增加，而且还导致红细胞的增加。为了阐明GM-CSFR信号传导是否可以补偿红细胞生成中的促红细胞生成素受体（EPOR）信号传导，我们接下来产生双突变小鼠，hGM-CSFR+/+和EPOR-/-。虽然双突变小鼠是胚胎致死的，hGM-CSF诱导大量的红系集落从双突变小鼠胎肝细胞的第14天。我们还产生了具有野生型人G-CSFR、野生型鼠G-CSFR的普遍表达的Tg小鼠。在骨髓和脾细胞甲基纤维素集落实验中，G-CSF不仅对粒细胞有促进作用，而且对巨噬细胞、巨核细胞、肥大细胞、红系细胞和更多的原始造血祖细胞在含血清和无血清培养中均有促进增殖和分化的作用。这些结果表明，当功能性GM-CSFR或G-CSFR存在于细胞表面上时，两种细胞因子均不诱导对嗜中性粒细胞和巨噬细胞谱系的排他性定型。使用Tg小鼠的体外和体内研究表明，当受体表达时，细胞因子如hGM-CSF或hG-CSF支持各种造血祖细胞的生长，但不改变它们的定型程序，因此有利于“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".