Directed differentiation of human embryonic stem cells as spin embryoid bodies and a description of the hematopoietic blast colony forming assay.
This unit describes a protocol for the differentiation of human embryonic stem cells (hESCs). To generate spin embryoid bodies (EBs), known numbers of hESCs are deposited into low-attachment, round-bottomed 96-well plates in a serum-free medium supplemented with growth factors. The cells are then aggregated by centrifugation, initiating formation of EBs of uniform size. The spin EBs generated using this technique differentiate efficiently and synchronously along the lineages preferentially induced by the combinations of growth factors to which the cells are exposed. The 96-well format permits an assessment of the effects of different combinations of growth factors in the same experiment, facilitating the optimization of differentiation conditions for any given cell type. Up to 40 plates can be set up within a couple of hours by one experimenter, and aliquots of the differentiating EBs can be harvested at intervals and subjected to analyses using a variety of techniques.
Ng
Elizabeth S
ES
Monash Immunology and Stem Cell Laboratories, Monash University, Clayton, Victoria, Australia.
Davis
Richard P
RP
Hatzistavrou
Tanya
T
Stanley
Edouard G
EG
Elefanty
Andrew G
AG
D000818:Animals; D018929:Cell Culture Techniques / Q000379:methods; D002454:Cell Differentiation; D049109:Cell Proliferation; D002478:Cells, Cultured; D003114:Colony-Forming Units Assay / Q000379:methods*; D004622:Embryo, Mammalian / Q000166:cytology*; D053595:Embryonic Stem Cells / Q000166:cytology*; D006412:Hematopoietic Stem Cells / Q000166:cytology*; D006801:Humans; D008747:Methylcellulose; D051379:Mice
D008747:Methylcellulose