Keller G, Kennedy M, Papayannopoulou T, Wiles M V
National Jewish Center, Denver, Colorado 80206.
Mol Cell Biol. 1993 Jan;13(1):473-86. doi: 10.1128/mcb.13.1.473-486.1993.
We report that embryonic stem cells efficiently undergo differentiation in vitro to mesoderm and hematopoietic cells and that this in vitro system recapitulates days 6.5 to 7.5 of mouse hematopoietic development. Embryonic stem cells differentiated as embryoid bodies (EBs) develop erythroid precursors by day 4 of differentiation, and by day 6, more than 85% of EBs contain such cells. A comparative reverse transcriptase-mediated polymerase chain reaction profile of marker genes for primitive endoderm (collagen alpha IV) and mesoderm (Brachyury) indicates that both cell types are present in the developing EBs as well in normal embryos prior to the onset of hematopoiesis. GATA-1, GATA-3, and vav are expressed in both the EBs and embryos just prior to and/or during the early onset of hematopoiesis, indicating that they could play a role in the early stages of hematopoietic development both in vivo and in vitro. The initial stages of hematopoietic development within the EBs occur in the absence of added growth factors and are not significantly influenced by the addition of a broad spectrum of factors, including interleukin-3 (IL-3), IL-1, IL-6, IL-11, erythropoietin, and Kit ligand. At days 10 and 14 of differentiation, EB hematopoiesis is significantly enhanced by the addition of both Kit ligand and IL-11 to the cultures. Kinetic analysis indicates that hematopoietic precursors develop within the EBs in an ordered pattern. Precursors of the primitive erythroid lineage appear first, approximately 24 h before precursors of the macrophage and definitive erythroid lineages. Bipotential neutrophil/macrophage and multilineage precursors appear next, and precursors of the mast cell lineage develop last. The kinetics of precursor development, as well as the growth factor responsiveness of these early cells, is similar to that found in the yolk sac and early fetal liver, indicating that the onset of hematopoiesis within the EBs parallels that found in the embryo.
我们报告称，胚胎干细胞能在体外高效分化为中胚层和造血细胞，且该体外系统概括了小鼠造血发育的第6.5至7.5天。分化为胚状体（EBs）的胚胎干细胞在分化第4天时发育出红系前体细胞，到第6天时，超过85%的EBs含有此类细胞。对原始内胚层（胶原蛋白αIV）和中胚层（短尾型）标记基因进行的比较性逆转录酶介导的聚合酶链反应分析表明，在造血开始之前，这两种细胞类型在发育中的EBs以及正常胚胎中均存在。GATA - 1、GATA - 3和vav在造血开始前及/或早期在EBs和胚胎中均有表达，表明它们可能在体内和体外造血发育的早期阶段发挥作用。EBs内造血发育的初始阶段在未添加生长因子的情况下发生，并且不受包括白细胞介素 - 3（IL - 3）、IL - 1、IL - 6、IL - 11、促红细胞生成素和Kit配体在内的多种因子添加的显著影响。在分化的第10天和第14天，向培养物中添加Kit配体和IL - 11可显著增强EBs的造血功能。动力学分析表明，造血前体细胞在EBs内按有序模式发育。原始红系谱系的前体细胞最先出现，大约比巨噬细胞和确定红系谱系的前体细胞早24小时。双潜能中性粒细胞/巨噬细胞和多谱系前体细胞随后出现，肥大细胞谱系的前体细胞最后发育。前体细胞发育的动力学以及这些早期细胞对生长因子的反应性与卵黄囊和早期胎儿肝脏中的情况相似，表明EBs内造血的开始与胚胎中的情况相似。
