Expansion of human cord blood CD34(+)CD38(-) cells in ex vivo culture during retroviral transduction without a corresponding increase in SCID repopulating cell (SRC) frequency: dissociation of SRC phenotype and function

Current procedures for the genetic manipulation of hematopoietic stem cells are relatively inefficient due, in part, to a poor understanding of the co nditions for ex vivo maintenance or expansion of stem cells. We report impr ovements in the retroviral transduction of human stem cells based on the SC ID-repopulating cell (SRC) assay and analysis of Lin(-)CD34(+)CD38(-) cells as a surrogate measure of stem cell function. Based on our earlier study o f the conditions required for ex vivo expansion of Lin-CD34(+)CD38(-) cells and SRC, CD34(+)-enriched lineage-depleted umbilical cord blood cells were cultured for 2 to 6 days on fibronectin fragment in MGIN (MSCV-EGFP-Neo) r etroviral supernatant (containing 1.5% fetal bovine serum) and IL-6, SCF, F lt-3 ligand, and G-CSF, Both CD34(+)CD38(-) cells (20.8%) and CFC (26.3%) w ere efficiently marked. When the bone marrow of engrafted NOD/SCID mice was examined, 75% (12/16) contained multilineage (myeloid and B lymphoid) EGFP (+) human cells composing as much as 59% of the graft. Half of these mice r eceived a limiting dose of SRC, suggesting that the marked cells were deriv ed from a single transduced SRC, Surprisingly, these culture conditions pro duced a large expansion (166-fold) of cells with the CD34(+)CD38(-) phenoty pe (n = 20), However, there was no increase in SRC numbers, indicating diss ociation between the CD34(+)CD38(-) phenotype and SRC function. The underly ing mechanism involved apparent downregulation of CD38 expression within a population of cultured CD34(+)CD38(+) cells that no longer contained any SR C function. These results suggest that the relationship between stem cell f unction and cell surface phenotype may not be reliable for cultured cells. (C) 2000 by The American Society of Hematology.