Derivation of hematopoietic stem cells from murine embryonic stem cells.
A stem cell is defined as a cell with the capacity to both self-renew and generate multiple differentiated progeny. Embryonic stem cells (ESC) are derived from the blastocyst of the early embryo and are pluripotent in differentiative ability. Their vast differentiative potential has made them the focus of much research centered on deducing how to coax them to generate clinically useful cell types. The successful derivation of hematopoietic stem cells (HSC) from mouse ESC has recently been accomplished and can be visualized in this video protocol. HSC, arguably the most clinically exploited cell population, are used to treat a myriad of hematopoietic malignancies and disorders. However, many patients that might benefit from HSC therapy lack access to suitable donors. ESC could provide an alternative source of HSC for these patients. The following protocol establishes a baseline from which ESC-HSC can be studied and inform efforts to isolate HSC from human ESC. In this protocol, ESC are differentiated as embryoid bodies (EBs) for 6 days in commercially available serum pre-screened for optimal hematopoietic differentiation. EBs are then dissociated and infected with retroviral HoxB4. Infected EB-derived cells are plated on OP9 stroma, a bone marrow stromal cell line derived from the calvaria of M-CSF-/- mice, and co-cultured in the presence of hematopoiesis promoting cytokines for ten days. During this co-culture, the infected cells expand greatly, resulting in the generation a heterogeneous pool of 100 s of millions of cells. These cells can then be used to rescue and reconstitute lethally irradiated mice.
McKinney-Freeman
Shannon
S
Children's Hospital, Harvard Stem Cell Institute, Harvard Medical School, Boston, MA, USA.
Daley
George
G
D000818:Animals; D002454:Cell Differentiation; D002460:Cell Line; D002469:Cell Separation / Q000379:methods*; D018920:Coculture Techniques; D053595:Embryonic Stem Cells / Q000166:cytology*; D018380:Hematopoietic Stem Cell Transplantation / Q000379:methods; D006412:Hematopoietic Stem Cells / Q000166:cytology*; D051379:Mice; D018345:Mice, Knockout; D017154:Stromal Cells / Q000166:cytology