Human embryonic stem cells (hESCs) provide a novel source of hematopoietic and other cell populations suitable for gene therapy applications. Preclinical studies to evaluate engraftment of hESC-derived hematopoietic cells transplanted into immunodeficient mice demonstrate only limited repopulation. Expression of a drug-resistance gene, such as Tyr22-dihydrofolate reductase (Tyr22-DHFR), coupled to methotrexate (MTX) chemotherapy has the potential to selectively increase the engraftment of gene-modified, hESC-derived cells in mouse xenografts. Here, we describe the generation of Tyr22-DHFR-GFP-expressing hESCs that maintain pluripotency, produce teratomas and can differentiate into MTXr-hemato-endothelial cells. We demonstrate that MTX administered to nonobese diabetic/severe combined immunodeficient/IL-2Rγc null (NSG) mice after injection of Tyr22-DHFR-hESC-derived cells significantly increases human CD34 and CD45 cell engraftment in the bone marrow (BM) and peripheral blood of transplanted MTX-treated mice. These results demonstrate that MTX treatment supports selective, long-term engraftment of Tyr22-DHFR cells in vivo, and provides a novel approach for combined human cell and gene therapy.
Bibliographical noteFunding Information:
This work was supported by National Institutes of Health Grants CA060803 (RSM), HL77923 (DSK) and CA34196 (LDS). We thank Andrea Mohs, Melinda Hexum, Katie Hill, Julie Morris, Jeremy Allred and Jon Linehan for assistance with the studies.
- Dihydrofolate reductase
- Embryonic stem cells