Fluorescence-based selection of gene-corrected hematopoietic stem and progenitor cells from acid sphingomyelinase-deficient mice: Implications for Niemann-Pick disease gene therapy and the development of improved stem cell gene transfer procedures

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 f rom the bone marrow or fetal livers of acid sphingomyelinase-deficient mice , and retrovirally transduced with amphotropic or ecotropic vectors encodin g a normal acid sphingomyelinase (ASM) cDNA. Anti-c-Kit antibodies were the n used to label stem- and progenitor-enriched cell populations, and the Bod ipy fluorescence was analyzed in each group after incubation with a Bodipy- conjugated sphingomyelin. Only cells expressing the functional ASM (ie, tra nsduced) could degrade the sphingomyelin, thereby reducing their Bodipy flu orescence as compared with nontransduced cells. The usefulness of this proc edure for the in vitro assessment of gene transfer into hematopoietic stem cells was evaluated, as well as its ability to provide an enrichment of tra nsduced stem cells in vivo. To show the value of this method for in vitro a nalysis, the effects of retroviral transduction using ecotropic versus amph otropic vectors, various growth factor combinations, and adult bone marrow versus fetal liver stem cells were assessed. The results of these studies c onfirmed the fact that ecotropic vectors were much more efficient at transd ucing 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 transd uction of stem cells, whereas IL-6 had the most significant effect on proge nitor cells. In addition, it was determined that fetal liver stem cells wer e only approximately twofold more "transducible" than stem cells from adult bone marrow. Transplantation of Bodipy-selected bone marrow cells into let hally 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 transpla nted with cells that were nonselected. The methods described within this ma nuscript are particularly useful for evaluating hematopoietic stem cell gen e transfer in vivo because the marker gene used in the procedure (ASM) enco des a naturally occurring mammalian enzyme that has no known adverse effect s, and the fluorescent compound used for selection (Bodipy sphingomyelin) i s removed from the cells before transplantation. (C) 1999 by The American S ociety of Hematology.