Liu Bing, Hou Chun-Mei, Wu Ying, Zhang Shuang-Xi, Mao Ning
Department of Cell Biology, Institute of Basic Medical Sciences, Beijing 100850, China.
Sheng Wu Gong Cheng Xue Bao. 2003 May;19(3):312-6.
The hematopoietic system of the mouse arises from extraembryonic mesoderm that migrate through primitive streak to the presumptive yolk sac at day 7.0 of gestation. However, the mechanisms regulating mesoderm commitment to hematopoietic lineages remain poorly understood. Previous studies demonstrated that the development kinetics and growth factor responsiveness of hematopoietic precursors derived from embryonic stem cells (ES cells) is similar to that found in the yolk sac, indicating that the onset of hematopoiesis within the embryoid bodies (EBs) parallels that found in the embryo. Furthermore, in vitro differentiation of ES cells to hematopoietic cells is valuable for establishment of therapeutic clone against a variety of hematological disorders. Despite the identification of multipotential hematopoietic progenitors in EBs, a subset of more primitive progenitors, identical to the high proliferative potential colony-forming cells (HPP-CFC) derived from human and murine hematopoietic tissues, have not been clearly identified regarding particular their replating potential in vitro. HPP-CFC is among the most primitive hematopoietic multipotent precursors cultured in vitro. In this study, our aim was to investigate the in vitro and in vivo hematopoietic capacity of HPP-CFC within the day 12 EBs, rather than the expansion of more committed progenitors. In this study the HPP-CFC could be detected within EBs differentiated for 5 to 14 days of murine ES cells, but the development dynamics of the HPP-CFC differed greatly among distinct serum lots. Qualitatively HPP-CFC is capable of forming secondary colonies. As to our expectation the ES cells-derived HPP-CFC demonstrated similar regeneration capacity to those from yolk sac, giving rise to secondary granulocyte, erythrocyte, macrophage and mast cells, however largely differed from the counterparts of adult bone marrow. In addition, by RT-PCR ES cells-derived HPP-CFC were found to express transcription factors associated closely with stem cell proliferation including SCL, GATA-2 and AML1 as well as various receptors of hematopoietic growth factors such as c-kit, GM-CSF receptor and interleukin 3 receptor et al. Finally, in order to understand the in vivo hematopoietic capacity of the ES cells-derived HPP-CFC, spleen colony-forming unit (CFU-S) assay was performed. Nevertheless, typical CFU-S was not observed after transplantation of the day 12 EB cells or HPP-CFC colonies into lethally irradiated adult murine. In conclusion the HPP-CFC differentiated from murine ES cells displayed robust hematopoietic activity in vitro, however their in vivo reconstitution ability was not detected. The difference between in vitro and in vivo hematopoietic activities of ES cells-derived primitive hematopoietic precursors deserves further investigation.
小鼠的造血系统起源于胚外中胚层，在妊娠第7.0天，胚外中胚层通过原条迁移至假定的卵黄囊。然而，调节中胚层向造血谱系定向分化的机制仍知之甚少。先前的研究表明，胚胎干细胞（ES细胞）来源的造血前体细胞的发育动力学和生长因子反应性与卵黄囊中发现的相似，这表明胚状体（EBs）内造血的起始与胚胎中发现的相似。此外，ES细胞向造血细胞的体外分化对于建立针对多种血液系统疾病的治疗性克隆很有价值。尽管在EBs中已鉴定出多能造血祖细胞，但尚未明确鉴定出更原始祖细胞的一个亚群，该亚群与源自人和小鼠造血组织的高增殖潜能集落形成细胞（HPP-CFC）相同，尤其是其体外再接种潜能。HPP-CFC是体外培养的最原始的造血多能前体细胞之一。在本研究中，我们的目的是研究第12天的EBs内HPP-CFC的体外和体内造血能力，而不是更定向祖细胞的扩增。在本研究中，在小鼠ES细胞分化5至14天的EBs中可检测到HPP-CFC，但不同血清批次中HPP-CFC的发育动力学差异很大。定性地说，HPP-CFC能够形成次级集落。正如我们所预期的，ES细胞来源的HPP-CFC表现出与卵黄囊来源的HPP-CFC相似的再生能力，可产生次级粒细胞、红细胞、巨噬细胞和肥大细胞，但与成年骨髓来源的对应细胞有很大不同。此外，通过RT-PCR发现ES细胞来源的HPP-CFC表达与干细胞增殖密切相关的转录因子，包括SCL、GATA-2和AML1，以及造血生长因子的各种受体，如c-kit、GM-CSF受体和白细胞介素3受体等。最后，为了了解ES细胞来源的HPP-CFC的体内造血能力，进行了脾集落形成单位（CFU-S）测定。然而，将第12天的EB细胞或HPP-CFC集落移植到经致死剂量照射的成年小鼠后，未观察到典型的CFU-S。总之，从小鼠ES细胞分化而来的HPP-CFC在体外表现出强大的造血活性，但其体内重建能力未被检测到。ES细胞来源的原始造血前体细胞体外和体内造血活性的差异值得进一步研究。
