We have continued to purify and characterize a unique hematopoietic stem cell population that is characterized by the lack of c-kit expression (receptor for stell factor, SLF). This is in contrast to all previously purified and characterized pluripotential stem cells (PHSCs) which express c-kit. We have recently placed this cell population in the c-kit maturation pathway in bone marrow cell development. In this regard, we have demonstrated that the expression of c-kit is induced on c-kit- negative cells in vivo using bone marrow transplantation assays and bone marrow cells obtained from congenic mouse strains that are allelic with respect to the Ly-5 locus (CD-45) which can be used to distinguish host and donor resconstitution. We have also demonstrated that the in vivo administration of 5-fluorouracil results in a rapid loss of greater that 95% of the c-kit-positive cell population after 48 hours which is induced on c-kit-negative cells as the mice recover. Using flow cytometry and antibodies that recognize cell surface antigens we demonstrated that the c-kit-negative PHSCs lack the expression of antigens detected on committed hematopoietic cells including GR-1 (granulocytes), F4/80 (macrophages), Ter-119 (erythrocytes), B220 (B- cells), CD-19 (B-cells), CD-3 (T-cells) - thus far considered lineage- negative. However, some of these cells are characterized by the presence of Sca-1 which is often found co-expressed with c-kit on PHSCs, and some are characterized by the absence of Sca-1 expression. We have proposed that steady state hematopoiesis is supported by PHSCs that express c-kit and that c-kit negative cells are recruited into this actively contributing pool of stem cells to sustain hematopoietic growth and development. Future studies are aimed at determining what regulates c- kit expression on c-kit-negative cells. In other studies, we have developed an in vivo model to determine the frequency and efficiency of gene transfer to human cells by transplanting human hematopoietic cells into severe combined immuno- deficient (SCID) mice. Specifically, we defined the conditions for engraftment of SCID mice with human bone marrow or cord blood cells cultured in vitro for six days in the presence of hematopoietic growth factors before transfer to irradiated SCID recipients. While the engraftment of human cells was dependent on the route of administration, it was not significantly affected by irradiation of recipients or the administration of human cytokines following transfer of cells. Human progenitor cells were detected in the bone marrow and spleen of SCID recipients up to seven weeks following gene transfer. This model represents a rapid in vivo (pre-clinical) assay to evaluate gene transfer and expression to primitive human cord blood and bone marrow progenitor cells.

Agency
National Institute of Health (NIH)
Institute
National Cancer Institute (NCI)
Type
Intramural Research (Z01)
Project #
1Z01BC010001-02
Application #
6161093
Study Section
Special Emphasis Panel (LLB)
Project Start
Project End
Budget Start
Budget End
Support Year
2
Fiscal Year
1997
Total Cost
Indirect Cost
Name
National Cancer Institute Division of Basic Sciences
Department
Type
DUNS #
City
State
Country
United States
Zip Code
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