In vivo selection of human embryonic stem cell-derived cells expressing methotrexate-resistant dihydrofolate reductase.
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-2Rgammac(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.
Gori
J L
JL
Gene Therapy Program, Department of Genetics, Cell Biology and Development, Institute of Human Genetics, University of Minnesota, Minneapolis, MN 55455, USA.
Tian
X
X
Swanson
D
D
Gunther
R
R
Shultz
L D
LD
McIvor
R S
RS
Kaufman
D S
DS
D000818:Animals; D001853:Bone Marrow; D002454:Cell Differentiation; D002460:Cell Line; D004351:Drug Resistance; D053595:Embryonic Stem Cells / Q000378:metabolism*; D015316:Genetic Therapy / Q000379:methods; D006085:Graft Survival; D006801:Humans; D008727:Methotrexate / Q000494:pharmacology*; D051379:Mice; D016688:Mice, Inbred NOD; D016513:Mice, SCID; D033581:Stem Cell Transplantation / Q000379:methods*; D013724:Teratoma / Q000235:genetics; D013762:Tetrahydrofolate Dehydrogenase / Q000235:genetics* / Q000378:metabolism
D013762:Tetrahydrofolate Dehydrogenase; D008727:Methotrexate