A LIMITED TEMPORAL WINDOW FOR THE DERIVATION OF MULTILINEAGE REPOPULATING HEMATOPOIETIC PROGENITORS DURING EMBRYONAL STEM-CELL DIFFERENTIATION IN-VITRO

Embryonal stem cells have been shown to differentiate in vitro into al l hematopoietic lineages. This has been used successfully as one appro ach to the study of genetic events occurring during haematopoiesis, Ho wever, studies on the commitment of mesodermal precursors to the hemat opoietic lineage have been limited due to the inability to define a sy stem in which embryonal stem (ES) cells will give rise to primitive he matopoietic stem cells in vitro. Using a colony forming assay (CFU-A), we determined that the earliest time point at which primitive multili neage hematopoietic precursors can be detected during ES cell differen tiation in vitro in the absence of exogenous conditioned medium or str omal cell culture is 4 days. Lethally irradiated adult recipient mice that received differentiated ES cells from this time point survived fo r more than 3 weeks; and in two out three experiments, peripheral bloo d from these animals contained ES-derived progeny, Fluorescence activa ted cell sorting (FAGS) found ES-derived CD45(+) hematopoietic cells i n both lymphoid and myeloid compartments at 12 weeks posttransplantati on, suggesting that the population of day 4 differentiated ES cells co ntains primitive hematopoietic precursors. A preliminary RT-PCR analys is of gene expression around this time point suggests that there are v ery few hematopoietic cells present. This approach should prove useful in studies of genetic control of commitment to and maintenance of hem atopoietic lineages in vitro and in vivo. (C) 1996 by The American Soc iety of Hematology.