ES細胞の試験管内分化とフローサイトメトリー

抄録

Embryonic stem (ES) cell lines are derived from the inner cell mass of blastocysts and possess a totipotency which is equivalent with that of epiblast cells. A part of the totipotency of ES cells can be revealed in vitro by using various methods like embryoid body formation in suspension cultures as well as stromal cell-dependent induction of differentiation. By combining with fluorescence activated cell sorting (FACS), in vitro differentiation of ES cells provides not only a therapeutic means to isolate cells intended for transplantation but also an experimental tool to study developmental process of a given cell lineage. This review summarizes a culture system of murine ES cells in which lateral mesoderm cells and their descendants such as endothelial cells and hematopoietic progenitors are sequentially isolated by FACS. Especially, ES cell clones that revealed activity of the Gata1 and Gata2 genes by expressing a GFP transgene as a marker enabled us to examine expression and function of these genes in the development of hematopoietic cell lineages. Expression of the Gata2 gene was already detected in a fraction of the earliest lateral mesoderm cells developed in a culture of the ES cells. The Gata2-expressing mesoderm cells purified by FACS exclusively contained primitive erythroid colony-forming cells (CFU-EryP). Expression of the Gata1 gene in mesoderm cells became detectable later than that of the Gata2 gene. CFU-EryP was highly enriched in the Gata1-expressing mesoderm cell fraction. Mesoderm cells which did not express the Gata1 gene contained high frequency of endothelial cell progenitors. Endothelial cells derived from these progenitors possessed a potential to give rise to definitive hematopoietic cells. The results suggested that the primitive hematopoietic cell lineage diverges directly from lateral mesoderm whereas the definitive hematopoietic cell lineage originates in the endothelium.