Isolation of hematopoietic stem cells from mouse embryonic stem cells.
This unit describes a protocol for the isolation of cells from murine embryonic stem cells with hematopoietic stem cell activity, defined by the ability to reconstitute, long term, multiple lineages of the hematopoietic system of lethally irradiated mice. The protocol subjects hematopoietic progenitors specified in differentiating embryoid bodies to ectopic HoxB4 expression (delivered via retroviral infection), followed by coculture and expansion on OP9 stromal cells in the presence of hematopoietic cytokines for 10 days. The protocol results in the generation of hundreds of millions of cells that can rescue mice from lethal irradiation. Although little is known about the phenotype and frequency of the actual hematopoietic stem cell-like cell within the population of cells generated by this protocol, the protocol establishes a system in which these cells can be further studied and the results ultimately translated to the human system.
McKinney-Freeman
Shannon L
SL
Division of Pediatric Hematology/Oncology, Children's Hospital, Boston, Massachusetts, USA.
Naveiras
Olaia
O
Daley
George Q
GQ
D000818:Animals; D002454:Cell Differentiation; D049109:Cell Proliferation; D002469:Cell Separation / Q000379:methods*; D002478:Cells, Cultured; D053595:Embryonic Stem Cells / Q000166:cytology*; D049452:Green Fluorescent Proteins / Q000378:metabolism; D006412:Hematopoietic Stem Cells / Q000166:cytology*; D018398:Homeodomain Proteins / Q000378:metabolism; D051379:Mice; D012190:Retroviridae; D033581:Stem Cell Transplantation; D017154:Stromal Cells; D014157:Transcription Factors / Q000378:metabolism
D018398:Homeodomain Proteins; C513262:Hoxb4 protein, mouse; D014157:Transcription Factors; D049452:Green Fluorescent Proteins