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Blood, Vol. 93 No. 1 (January 1), 1999:
pp. 80-86
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
Shai Erlich,
Silvia R.P. Miranda,
Jan W.M. Visser,
Arie Dagan,
Shimon Gatt, and
Edward H. Schuchman
From the Department of Human Genetics and Institute for Gene Therapy
and Molecular Medicine, Mount Sinai School of Medicine, New York, NY;
the Laboratory of Stem Cell Biology, Lindsley F. Kimball Research
Institute, New York Blood Center, New York, NY; and the Department of
Biochemistry, Hebrew University Hadassah School of Medicine,
Jerusalem, Israel.
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.
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