Progressive lineage analysis by cell sorting and culture identifies FLK1+VEcadherin+ cells at a diverging point of endothelial and hemopoietic lineages

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Abstract
Totipotent murine ES cells h ave an enormous potential for the study of cell specification. He re we demonstrate that ES cells can diffe rentiate to hemopoietic cells th rough the proximal lateral mesoderm, me rely upon culturing in type IV collagen-coated dishes. Separation of the Flk1+ mesoderm f rom other cell lineages was critical for hemopoietic cell diffe rentiation, whe reas formation of the embryoid body was not. Since the two-dimensionally spreading cells can be monito red easily in real time, this culture system will g reatly facilitate the study of the mechanisms i nvolved in the cell specification to mesoderm, endothelial, and hemopoietic cells. In the cultu re of ES cells, how ever, lineages and stages of diffe rentiating cells can only be defined by their own characteristics. We showed that a combination of monoclonal antibodies against E-cadherin, Flk1/KDR, PDGF recepto rα, VEcadherin, CD45 and Ter119 was suf ficient to define most intermediate stages during diffe rentiation of ES cells to blood cells. Using this cultu re system and surface markers, we determined the following order for blood cell differentiation: ES cell (E-cadherin+Flk1™PDGFRα™), proximal lateral mesoderm (E-cadherin™ Flk1+VEcadherin™ ), progenitor with hemoangiogenic potential (Flk1+VE-cadherin+CD45™ ), hemopoietic p rogenitor (CD45+c-Kit+) and mature blood cells (c-Kit™ CD45+ or Ter119+), though direct differentiation of blood cells f rom the Flk1+VE-cadherin™ stage cannot be ruled out. Not only the VE-cadherin+CD45™ population generated f rom ES cells but also those di rectly sorted f rom the yolk sac of 9.5 dpc embryos h ave a potential to give rise to hemopoietic cells. P rogenitors with hemoangiogenic potential were identified in both the Flk1+VE-cadherin™and Flk1+VEcadherin+populations by the single cell deposition experiment. This line of evidence implicates Flk1+VEcadherin+ cells as a diverging point of hemopoietic and endothelial cell lineages.