Hematoendothelial Differentiation of Human Embryonic Stem Cells

Abstract

Human embryonic stem cells (hESCs) represent a unique population of cells capable of self‐renewal and differentiation into all types of somatic cells, including hematopoietic and endothelial cells. Since the pattern of hematopoietic and endothelial development observed in the embryo can be reproduced using ESCs differentiated in culture, hESCs can be used as a model for studies of specification and diversification of hematoendothelial progenitors. In addition, hESCs can be seen as a scalable source of hematopoietic and endothelial cells for in vitro studies. This unit describes a method for efficient differentiation of hESCs into hematopoietic progenitors and endothelial cells through coculture with mouse OP9 bone marrow stromal cells, as well as an approach for their analysis and isolation. Support protocols are provided for culture of mouse embryonic fibroblasts, evaluation of hematopoietic and endothelial differentiation by flow cytometry and colony‐forming assay, removal of OP9 cells, and propagation of hESC‐derived endothelial cells. Curr. Protoc. Cell Biol. 36:23.6.1‐23.6.28. © 2007 by John Wiley & Sons, Inc.

Generation of mature human myelomonocytic cells through expansion and differentiation of pluripotent stem cell–derived lin–CD34+CD43+CD45+ progenitors

“…The mouse bone marrow stromal cell line OP9 was obtained from Toru Nakano ( hESC and hiPSC differentiation in OP9 coculture and expansion of myeloid progenitors. Hematopoietic differentiation was induced by transferring the hESCs or hiPSCs onto OP9 feeders as we have previously described in detail (11,41). On day 9 of coculture, differentiated hESCs/hiPSCs were harvested by treatment with collagenase IV (Invitrogen; 1 mg/ml in α-MEM) for 20 minutes at 37°C, followed by treatment with 0.05% Trypsin-0.5 mM EDTA (Invitrogen) for 15 minutes at 37°C.…”

Dynamic Expression of Specific miRNAs during Erythroid Differentiation of Human Embryonic Stem Cells

“…Hematopoietic differentiation of hESC on OP9 feeder cells was induced in α-MEM containing 10% FBS and 100 μM monothioglycerol (MTG, Sigma, USA) with complete medium change on day 1 followed by a half-medium change on days 4, 6, and 8. Finally on day 10, CD34+ hematopoietic cells were isolated for further differentiation into erythroid cells (Vodyanik and Slukvin, 2007;Vodyanik et al, 2005).…”

In vitro and in vivo differentiation of human embryonic stem cells into retina‐like organs and comparison with that from mouse pluripotent epiblast stem cells

“…For the induction of specific cell lineages from ES cells, purified growth and/or differentiation factors or factorproducing feeder or stromal cells have been used (Chadwick et al, 2003;Cohen et al, 2007;Green et al, 2003;Kaufman et al, 2001;Kehat et al, 2001;Lavon et al, 2006;Levenberg et al, 2002;Mummery et al, 2003;Rambhatla et al, 2003;Sottile et al, 2003;Vodyanik and Slukvin, 2007;Zhang et al, 2001). In some cases, very complex culture strategies have been used, including the sequential addition and withdrawal of factors and purification of specific cell populations during the culture period (Fang et al, 2006;Wang et al, 2005).…”