Umeda Katsutsugu, Heike Toshio, Yoshimoto Momoko, Shiota Mitsutaka, Suemori Hirofumi, Luo Hong Yuan, Chui David H K, Torii Ryuzo, Shibuya Masabumi, Nakatsuji Norio, Nakahata Tatsutoshi
Department of Pediatrics, Graduate School of Medicine, Kyoto University, 54 Kawahara-cho, Shogoin, Sakyo-ku, Kyoto 606-8507, Japan.
Development. 2004 Apr;131(8):1869-79. doi: 10.1242/dev.01065.
Although information about the development of primitive and definitive hematopoiesis has been elucidated in murine embryos and embryonic stem (ES) cells, there have been few in vitro studies of these processes in primates. In this study, we investigated hematopoietic differentiation from cynomolgus monkey ES cells grown on OP9, a stromal cell line deficient in macrophage colony-stimulating factor. Primitive erythrocytes (EryP) and definitive erythrocytes (EryD) developed sequentially from ES cells in the culture system; this was confirmed by immunostaining and reverse transcriptase-polymerase chain reaction (RT-PCR) analysis of embryonic, fetal and adult globin genes. EryP were detected on day 8 without exogenous erythropoietin (EPO), whereas EryD appeared on day 16 and had an indispensable requirement for exogenous EPO. RT-PCR analysis of the cultures revealed a sequential expression of genes associated with primitive and definitive hematopoietic development that was equivalent to that seen during primate ontogeny in vivo. Vascular endothelial growth factor (VEGF) increased, in a dose-dependent manner, not only the number of floating hematopoietic cells, but also the number of adherent hematopoietic cell clusters containing CD34-positive immature progenitors. In colony assays, exogenous VEGF also had a dose-dependent stimulatory effect on the generation of primitive erythroid colonies. More efficient primitive and definitive erythropoiesis was induced by re-plating sorted CD34-positive cells. Thus, this system reproduces early hematopoietic development in vitro and can serve as a model for analyzing the mechanisms of hematopoietic development in primates.
尽管有关原始造血和定型造血发育的信息已在小鼠胚胎和胚胎干细胞（ES细胞）中得到阐明，但在灵长类动物中对这些过程进行的体外研究却很少。在本研究中，我们调查了在缺乏巨噬细胞集落刺激因子的基质细胞系OP9上生长的食蟹猴ES细胞的造血分化情况。在培养系统中，原始红细胞（EryP）和定型红细胞（EryD）从ES细胞中依次发育；这通过对胚胎、胎儿和成人珠蛋白基因的免疫染色和逆转录聚合酶链反应（RT-PCR）分析得到了证实。在没有外源性促红细胞生成素（EPO）的情况下，第8天检测到EryP，而EryD在第16天出现，并且对外源性EPO有不可或缺的需求。对培养物的RT-PCR分析揭示了与原始造血和定型造血发育相关基因的顺序表达，这与在灵长类动物体内个体发育过程中所见的情况相当。血管内皮生长因子（VEGF）不仅以剂量依赖的方式增加了漂浮造血细胞的数量，还增加了含有CD34阳性未成熟祖细胞的贴壁造血细胞簇的数量。在集落测定中，外源性VEGF对原始红系集落的生成也有剂量依赖的刺激作用。通过重新接种分选的CD34阳性细胞可诱导更有效的原始造血和定型造血。因此，该系统在体外重现了早期造血发育，可作为分析灵长类动物造血发育机制的模型。
