Induction of differentiation of human embryonic stem cells into functional hair-cell-like cells in the absence of stromal cells

Human embryonic stem cells (hESCs) provide an important means to effectively study soluble and cell-bound mediators that regulate development of early blood and endothelial cells in a human model system. Here, several complementary methods are used to demonstrate canonical Wnt signaling is important for development of hESC-derived cells with both hematopoietic and endothelial potential. Analyses using both standard flow cy-tometry, as well the more detailed high-throughput image scanning flow cytometry, characterizes sequential development of distinct early developing CD34brightCD31+Flk1+ cells and a later population of CD34dimCD45+ cells. While the CD34brightCD31+Flk1+ have a more complex morphology and can develop into both endothelial cells and hematopoietic cells, the CD34dimCD45+ cells have a simpler morphology and give rise to only hematopoietic cells. Treatment with dickkopf1 to inhibit Wnt signaling results in a dramatic decrease in development of cells with hematoendothelial potential. In addition, activation of the canonical Wnt signaling pathway in hESCs by coculture with stromal cells that express Wnt1, but not use of noncanonical Wnt5-expressing stromal cells, results in an accelerated differentiation and higher percentage of CD34brightCD31+Flk1+ cells at earlier stages of differentiation. These studies effectively demonstrate the importance of canonical Wnt signaling to mediate development of early hematoendothelial progenitors during human development.

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A Novel Method for Efficient Production of Multipotential Hematopoietic Progenitors from Human Embryonic Stem Cells by Co-Culture with Murine Fetal Liver-Derived Stromal Cells.

Blood ◽

10.1182/blood.v106.11.4214.4214 ◽

2005 ◽

Vol 106 (11) ◽

pp. 4214-4214

Author(s):

Feng Ma ◽

Dan Wang ◽

Sachiyo Hanada ◽

Hirohide Kawasaki ◽

Yuji Zaike ◽

...

Keyword(s):

Stem Cells ◽

Embryonic Stem Cells ◽

Human Embryonic Stem Cells ◽

Stromal Cells ◽

Blood Cells ◽

Fetal Liver ◽

Hematopoietic Cells ◽

Embryonic Stem ◽

Efficient Production ◽

Hematopoietic Progenitors

Abstract Human embryonic stem cells provide a unique tool to study early events occurring in the development of human embryonic hematopoiesis, and their totipotent capability indicates a potent clinical application based on the cellular therapy and the evaluation of drug effects on hematopoietic and blood cells. To achieve efficient production of hematopoietic cells from human embryonic stem cells, we attempted to reproduce the circumstance surrounding embryonic hematopoietic cells in vitro. Since fetal liver is the predominant source of hematopoietic and blood cells in mammalian embryogenesis, we established stromal cells from mouse fetal liver at days 14 to 15 of gestation. In the co-culture of human embryonic stem cells with the established stromal cells, a number of hematopoietic progenitors were generated at around day 14 of co-culture, and this hematopoietic activity was highly enriched in the cobble stone-like cells under the stromal layer. Most of the cobble stone-like cells collected expressed CD34 and contained a variety of hematopoietic colony-forming cells, especially multilineage colony-forming cells, at a high frequency. The multipotential hematopoietic progenitors in the cobble stone-like cells produced all types of mature blood cells, including adult type hemoglobin-synthesizing erythrocytes and tryptase and chymase-bouble positive mast cells in the suspension cultiue with a cytokine cocktail. The developed co-culture system of human embryonic stem cells should offer a novel source for hematopoietic and blood cells applicable to cellular therapies and drug screening.

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