In Vivo Selection of Human Hematopoietic Cells in a Xenograft Model Using Combined Pharmacologic and Genetic Manipulations

Abstract

Strategies that increase the ability of human hematopoietic stem and progenitor cells to repair alkylator-induced DNA damage may prevent the severe hematopoietic toxicity in patients with cancer undergoing high-dose alkylator therapy. In the context of genetic diseases, this approach may allow for selection of small numbers of cells that would not otherwise have a favorable growth advantage. No studies have tested this approach in vivo using human hematopoietic stem and progenitor cells. Human CD34⁺ cells were transduced with a bicistronic oncoretrovirus vector that coexpresses a mutant form of O⁶-methylguanine DNA methyltransferase (MGMT^P140K) and the enhanced green fluorescent protein (EGFP) and transplanted into nonobese diabetic/severe combined immunodeficient (NOD/SCID) mice. Mice were either not treated or treated with O⁶-benzylguanine (6BG) and 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU). At 8-weeks postinjection, a 2- to 8-fold increase in the percentage of human CD45⁺EGFP⁺ cells in 6BG/BCNU-treated versus nontreated mice was observed in the bone marrow and was associated with increased MGMT^P140K-repair activity. Functionally, 6BG/BCNU-treated mice demonstrated multilineage differentiation in vivo, although some skewing in the maturation of myeloid and B cells was observed in mice transplanted with granulocyte-colony stimulating factor (G-CSF)-mobilized peripheral blood compared to umbilical cord blood. Expansion of human cells in 6BG/BCNU-treated mice was observed in the majority of mice previously transplanted with transduced umbilical cord blood cells. In addition, a significant increase in the number of EGFP⁺ progenitor colonies in treated versus nontreated mice were observed in highly engrafted mice indicating that selection and maintenance of human progenitor cells can be accomplished by expression of MGMT^P140K and treatment with 6BG/BCNU.