HEMATOPOIETIC COMMITMENT DURING EMBRYONIC STEM-CELL DIFFERENTIATION IN CULTURE
HEMATOPOIETIC COMMITMENT DURING EMBRYONIC STEM-CELL DIFFERENTIATION IN CULTURE
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DOI：
10.1128/mcb.13.1.473
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发表时间：
1993-01-01
影响因子：
5.3
通讯作者：
WILES, MV
中科院分区：
文献类型：
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作者：
KELLER, G;KENNEDY, M;WILES, MV
我们报告说，胚胎干细胞有效地进行分化，在体外中胚层和造血细胞，在体外系统重演6.5至7.5天的小鼠造血发育。分化为胚状体（EB）的胚胎干细胞在分化的第4天发育成红系前体，并且在第6天，超过85%的EB含有这样的细胞。原始内胚层（胶原蛋白α IV）和中胚层（Brachyury）的标记基因的比较逆转录酶介导的聚合酶链反应谱表明，这两种细胞类型存在于发育中的EB以及正常胚胎造血发生之前。加塔-1、加塔-3和vav在造血早期发生之前和/或期间在EB和胚胎中表达，表明它们在体内和体外造血发育的早期阶段中发挥作用。EB内造血发育的初始阶段发生在没有添加生长因子的情况下，并且不受添加广谱因子（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）的显著影响。在分化的第10天和第14天，通过向培养物中添加Kit配体和IL-11，EB造血显著增强。动力学分析表明，造血前体细胞在EB内以有序的模式发育。原始红细胞谱系的前体首先出现，比巨噬细胞和定形红细胞谱系的前体早约24小时。接下来出现双能中性粒细胞/巨噬细胞和多谱系前体细胞，肥大细胞谱系的前体细胞最后发育。前体发育的动力学，以及这些早期细胞的生长因子的反应性，是类似的发现在卵黄囊和早期胎肝，这表明，在EB内的造血的发病与发现在胚胎中。
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.