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 all hematopoietic lineages. This has been used successfully as one approach to the study of genetic events occurring during haematopoiesis. However, studies on the commitment of mesodermal precursors to the hematopoietic lineage have been limited due to the inability to define a system in which embryonal stem (ES) cells will give rise to primitive hematopoietic stem cells in vitro. Using a colony forming assay (CFU-A), we determined that the earliest time point at which primitive multilineage hematopoietic precursors can be detected during ES cell differentiation in vitro in the absence of exogenous conditioned medium or stromal cell culture is 4 days. Lethally irradiated adult recipient mice that received differentiated ES cells from this time point survived for more than 3 weeks; and in two out three experiments, peripheral blood from these animals contained ES-derived progeny. Fluorescence activated cell sorting (FACS) found ES-derived CD45+ hematopoietic cells in both lymphoid and myeloid compartments at 12 weeks posttransplantation, suggesting that the population of day 4 differentiated ES cells contains primitive hematopoietic precursors. A preliminary RT-PCR analysis of gene expression around this time point suggests that there are very few hematopoietic cells present. This approach should prove useful in studies of genetic control of commitment to and maintenance of hematopoietic lineages in vitro and in vivo.
Hole
N
N
Institute of Cell Animal and Population Biology, University of Edinburgh, Scotland.
Graham
G J
GJ
Menzel
U
U
Ansell
J D
JD
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002454:Cell Differentiation; D002478:Cells, Cultured; D003114:Colony-Forming Units Assay; D017931:DNA Primers / Q000737:chemistry; D015870:Gene Expression; D006410:Hematopoiesis; D006412:Hematopoietic Stem Cells / Q000166:cytology*; D051379:Mice; D008969:Molecular Sequence Data; D012333:RNA, Messenger / Q000235:genetics; D011828:Radiation Chimera; D013997:Time Factors
D017931:DNA Primers; D012333:RNA, Messenger