The general utility of a novel, fluorescence-based procedure for assessing gene transfer and expression has been demonstrated using hematopoietic stem and progenitor cells. Lineage-depleted hematopoietic cells were isolated from the bone marrow or fetal livers of
acid sphingomyelinase-deficient mice, and retrovirally transduced with amphotropic or ecotropic vectors encoding a normal
acid sphingomyelinase (ASM)
cDNA. Anti-c-Kit
antibodies were then used to label stem- and progenitor-enriched cell populations, and the
Bodipy fluorescence was analyzed in each group after incubation with a
Bodipy-conjugated
sphingomyelin. Only cells expressing the functional ASM (ie, transduced) could degrade the
sphingomyelin, thereby reducing their
Bodipy fluorescence as compared with nontransduced cells. The usefulness of this procedure for the in vitro assessment of gene transfer into hematopoietic stem cells was evaluated, as well as its ability to provide an enrichment of transduced stem cells in vivo. To show the value of this method for in vitro analysis, the effects of retroviral transduction using ecotropic versus amphotropic vectors, various
growth factor combinations, and adult bone marrow versus fetal liver stem cells were assessed. The results of these studies confirmed the fact that ecotropic vectors were much more efficient at transducing murine stem cells than amphotropic vectors, and that among the three most commonly used
growth factors (
stem cell factor [SCF] and
interleukins 3 and 6 [IL-3 and IL-6]), SCF had the most significant effect on the transduction of stem cells, whereas
IL-6 had the most significant effect on progenitor cells. In addition, it was determined that fetal liver stem cells were only approximately twofold more "transducible" than stem cells from adult bone marrow.
Transplantation of
Bodipy-selected bone marrow cells into lethally irradiated mice showed that the number of spleen colony-forming units that were positive for the retroviral vector (as determined by polymerase chain reaction) was 76%, as compared with 32% in animals that were transplanted with cells that were nonselected. The methods described within this manuscript are particularly useful for evaluating hematopoietic stem cell gene transfer in vivo because the marker gene used in the procedure (ASM) encodes a naturally occurring mammalian
enzyme that has no known adverse effects, and the fluorescent compound used for selection (
Bodipy sphingomyelin) is removed from the cells before
transplantation.