Nishikawa S I, Nishikawa S, Hirashima M, Matsuyoshi N, Kodama H
Department of Molecular Genetics, Faculty of Medicine, Kyoto University, Kyoto, Japan.
Development. 1998 May;125(9):1747-57. doi: 10.1242/dev.125.9.1747.
Totipotent murine ES cells have an enormous potential for the study of cell specification. Here we demonstrate that ES cells can differentiate to hemopoietic cells through the proximal lateral mesoderm, merely upon culturing in type IV collagen-coated dishes. Separation of the Flk1+ mesoderm from other cell lineages was critical for hemopoietic cell differentiation, whereas formation of the embryoid body was not. Since the two-dimensionally spreading cells can be monitored easily in real time, this culture system will greatly facilitate the study of the mechanisms involved in the cell specification to mesoderm, endothelial, and hemopoietic cells. In the culture of ES cells, however, lineages and stages of differentiating cells can only be defined by their own characteristics. We showed that a combination of monoclonal antibodies against E-cadherin, Flk1/KDR, PDGF receptor(alpha), VE-cadherin, CD45 and Ter119 was sufficient to define most intermediate stages during differentiation of ES cells to blood cells. Using this culture system and surface markers, we determined the following order for blood cell differentiation: ES cell (E-cadherin+Flk1-PDGFRalpha-), proximal lateral mesoderm (E-cadherin-Flk1+VE-cadherin-), progenitor with hemoangiogenic potential (Flk1+VE-cadherin+CD45-), hemopoietic progenitor (CD45+c-Kit+) and mature blood cells (c-Kit-CD45+ or Ter119+), though direct differentiation of blood cells from the Flk1+VE-cadherin- stage cannot be ruled out. Not only the VE-cadherin+CD45- population generated from ES cells but also those directly sorted from the yolk sac of 9.5 dpc embryos have a potential to give rise to hemopoietic cells. Progenitors with hemoangiogenic potential were identified in both the Flk1+VE-cadherin- and Flk1+VE-cadherin+ populations by the single cell deposition experiment. This line of evidence implicates Flk1+VE-cadherin+ cells as a diverging point of hemopoietic and endothelial cell lineages.
全能性小鼠胚胎干细胞在细胞特化研究方面具有巨大潜力。在此我们证明，仅仅通过在IV型胶原包被的培养皿中培养，胚胎干细胞就能通过近侧外侧中胚层分化为造血细胞。将Flk1⁺中胚层与其他细胞谱系分离对于造血细胞分化至关重要，而胚状体的形成则并非如此。由于二维铺展的细胞易于实时监测，该培养系统将极大地促进对中胚层、内皮细胞和造血细胞特化相关机制的研究。然而，在胚胎干细胞培养中，分化细胞的谱系和阶段只能通过其自身特征来定义。我们表明，针对E-钙黏蛋白、Flk1/KDR、血小板衍生生长因子受体（α）、VE-钙黏蛋白、CD45和Ter119的单克隆抗体组合足以定义胚胎干细胞向血细胞分化过程中的大多数中间阶段。利用该培养系统和表面标志物，我们确定了血细胞分化的以下顺序：胚胎干细胞（E-钙黏蛋白⁺Flk1⁻血小板衍生生长因子受体α⁻）、近侧外侧中胚层（E-钙黏蛋白⁻Flk1⁺VE-钙黏蛋白⁻）、具有血管生成潜力的祖细胞（Flk1⁺VE-钙黏蛋白⁺CD45⁻）、造血祖细胞（CD45⁺c-Kit⁺）和成熟血细胞（c-Kit⁻CD45⁺或Ter119⁺），不过不能排除血细胞从Flk1⁺VE-钙黏蛋白⁻阶段直接分化的可能性。不仅胚胎干细胞产生的VE-钙黏蛋白⁺CD45⁻群体，而且直接从9.5天胚龄胚胎的卵黄囊中分选出来的那些群体都具有产生造血细胞的潜力。通过单细胞沉积实验在Flk1⁺VE-钙黏蛋白⁻和Flk1⁺VE-钙黏蛋白⁺群体中都鉴定出了具有血管生成潜力的祖细胞。这一系列证据表明Flk1⁺VE-钙黏蛋白⁺细胞是造血细胞和内皮细胞谱系的一个分歧点。
