Derivation of Hematopoietic Stem Cells from Embryonic Stem Cells.

Despite the significant in vitro blood-forming potential of murine embryonic stem cells (ESCs), deriving hematopoietic stem cells (HSCs) that can reconstitute irradiated mice has proven to be challenging. Previously, we successfully engrafted lethally irradiated adult mice with ESCs engineered to ectopically express the homeodomain gene hoxB4. In engrafted animals, blood reconstitution showed a myeloid predominance, likely due to an inability to fully pattern the adult HSC from these embryonic populations. Recently, we have investigated cdx4, a caudal-related homeobox gene whose function has been linked to blood development in the zebrafish. During in vitro differentiation of murine ESCs, cdx4 is expressed during a very narrow time interval on day 3, coincident with the specification of hematopoietic mesoderm. To further characterize the function of cdx4 in mouse hematopoiesis, we have established a tetracycline-inducible murine embryonic stem cell line. When cdx4 expression is conditionally induced over a protracted period from day 2 and 6, we observe a marked enhancement of hemangioblast formation as well as significant increases in primitive and definitive hematopoietic colonies. Cdx4 acts to induce a broad array of hox genes, including a modest elevation in hoxb4. Co-expression of cdx4 and hoxb4 promotes robust expansion of hematopoietic blasts on supportive OP9 stromal cultures. When injected intravenously into lethally-irradiated mice, these cell populations provide robust radio-protection, and reconstitute high-level lymphoid-myeloid donor chimerism. Marrow from engrafted primary animals can be transplanted into irradiated secondary mice. B220+ splenic lymphoid cells and Mac-1/Gr-1+ marrow myeloid cells purified from primary and secondary mice show multiple common sites of retroviral integration, thereby proving the derivation of long-term hematopoietic stem cells from embryonic stem cells in vitro. Our data support a central role for the cdx4-hox gene pathway in specifying murine HSC development, and establish a robust system for hematopoietic reconstitution from ESCs. We have coupled techniques for generating ESCs by nuclear transfer with these methods for blood reconstitution to model the treatment of genetic disorders of the bone marrow.