In vivo selection of human embryonic stem cell-derived cells expressing methotrexate-resistant dihydrofolate reductase

Abstract

SUMMARY 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 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-2Rγcnull (NSG) mice after injection of Tyr22-DHFR-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.

Vascular niche promotes hematopoietic multipotent progenitor formation from pluripotent stem cells

“…Previous work from several groups shows that hematoendothelial precursors specified toward hematopoietic fate by coculture with growth factors alone (23)(24)(25) or with stromal cell support (2,26) give rise to phenotypic but primitive hematopoietic progenitors that lack robust, long-term multilineage engraftment potential.…”

Selectively weakened binding of methotrexate by human dihydrofolate reductase allows rapid ex vivo selection of mammalian cells

“…Hematopoietic cell lineages, such as more differentiated progenitor cells, were considerably enriched and continued to proliferate following MTX selection. These variants could potentially be applied to hematopoietic cell gene therapy to protect BM cells from MTX cytotoxicity during chemotherapy as recently shown for embryonic stem cells (Gori et al, 2009), and could increase the clinical index of the inhibitor. …”

A Multidrug-resistant Engineered CAR T Cell for Allogeneic Combination Immunotherapy

“…Combination therapy using chemo- and cell-based therapies represents an appealing strategy to improve the efficiency and persistence of treatment as demonstrated for stem cell adoptive therapy. 31 , 32 , 33 The potential advantage of this approach for CAR T-cell immunotherapies relies on the well-known ability of certain chemotherapies to increase the persistence and activity of effector T cells, 30 increase their tumor trafficking, 34 , 35 modulate immunosupressive factors 36 and for some of them including PNAs, directly affect tumor cell viability. 6 While highly promising, the development of combination therapies have been dampened by the cytotoxic properties of chemotherapy toward immunotherapeutic cellular agents.…”