Hematopoietic commitment during embryonic stem cell differentiation in culture.

Abstract

We report that embryonic stem cells efficiently undergo differentiation in vitro to mesoderm and hematopoietic cells and that this in vitro system recapitulates days 6.5 to 7.5 of mouse hematopoietic development. Embryonic stem cells differentiated as embryoid bodies (EBs) develop erythroid precursors by day 4 of differentiation, and by day 6, more than 85% of EBs contain such cells. A comparative reverse transcriptase-mediated polymerase chain reaction proffle of marker genes for primitive endoderm (collagen a IV) and mesoderm (Brachyury) indicates that both cell types are present in the developing EBs as well in normal embryos prior to the onset of hematopoiesis. GATA-1, GATA-3, and vav are expressed in both the EBs and embryos just prior to and/or during the early onset of hematopoiesis, indicating that they could play a role in the early stages of hematopoietic development both in vivo and in vitro. The initial stages of hematopoietic development within the EBs occur in the absence of added growth factors and are not significantly influenced by the addition of a broad spectrum of factors, including interleukin-3 (IL-3), IL-1, IL.6, IL-11, erythropoietin, and Kit ligand. At days 10 and 14 of differentiation, EB hematopoiesis is significantly enhanced by the addition of both Kit ligand and ILH11 to the cultures. Kinetic analysis indicates that hematopoietic precursors develop within the EBs in an ordered pattern. Precursors of the primitive erythroid lineage appear first, approximately 24 h before precursors of the macrophage and definitive erythroid lineages. Bipotential neutrophil/macrophage and multilineage precursors appear next, and precursors of the mast cell lineage develop last. The kinetics of precursor development, as well as the growth factor responsiveness of these early cells, is similar to that found in the yolk sac and early fetal liver, indicating that the onset of hematopoiesis within the EBs parallels that found in the embryo.

Differentiation of murine embryonic stem cells induces progesterone receptor gene expression

“…We observed mRNA within 2-3 days for nestin (an early neural ectoderm marker) and brachyury (Brachy, an early mesoderm marker), whereas β-H1 globin (β-globin, a mesoderm marker) and α-fetoprotein (α-FP, an endoderm marker) mRNAs accumulated later (days 5-6). These patterns of expression are as previously reported for multilineage differentiation following LIF withdrawal [8,9,32,[38][39][40][41].…”

“…CCE mES cells were used for all studies except where indicated and were obtained from Dr. John Gearhart (Johns Hopkins University) with permission from Dr. Gordon Keller [31,32]. Plastic tissue culture dishes were pretreated with 0.1% porcine gelatin Type A (Sigma) in water for 30 min to 2 hrs at 37ºC.…”

Characterization of Dendritic Cells and Macrophages Generated by Directed Differentiation from Mouse Induced Pluripotent Stem Cells

“…Embryoid body-mediated differentiation to generate macrophages from mouse ES cells has been reported by several groups [31][32][33][34]. In the present study, we demonstrated efficient generation of functional macrophages from mouse iPS cells.…”

Yolk-sac hematopoiesis

“…Receptor numbers subsequently decrease as terminal differentiation proceeds. While erythropoietin transcripts have been detected in the mouse embryo at E7.5 and in the fetal liver at E14.5, the low levels of erythropoietin expression have hampered the spatial and temporal analysis of its expression during development [36][37][38]. A small percentage (7%-10%) of a supraphysiologic dose of erythropoietin can cross the mouse placenta just prior to birth (E19) [39].…”