Despite intense efforts to develop ex vivo culture systems that support hemopoietic stem cell (HSC) expansion most if not all culture systems lead to loss rather than expansion of HSC. To obtain a net increase in HSC number or HSC expansion) symmetrical cell visions are needed, i.e. both daughter cells retain HSC characteristics. If one of the 2 daughter cells non net expansion of HSC will be seen. Lack of HSC expansion may therefore be due to HSC death (apoptosis), HSC commitment/differentiation, or insufficient HSC proliferation. We have developed an in vitro assay that can evaluate the fate of a single primitive hemopoietic progenitor capable of generating multiple daughter cells that can initiate long-term myeloid and lymphoid cultures, also determined Myeloid-Lymphoid-Initiating Cells of ML-IC. Using vital dyes, we have shown that ML-IC can undergo self-renewing cell divisions in vitro, therefore fulfilling the two minimal criteria of HSC, namely self-renewal and multi-lineage potential. Finally, we have shown that same conditions that support ML-IC growth also support high levels of Murine-Leukemia (MuL) viral gene transfer in ML-IC. This should now allow us to examine the mechanism underlying lack of ex vivo expansion of HSC. Specifically, we will assess the contribution of cell death, differentiation or poor proliferation to overall poor HSC expansion. This will be accomplished by evaluating the effect of over-expression of anti-apoptosis genes, genes that alter cell fate decision making processes, and genes that enhance cell proliferation on the number of repopulating cells. We will also determine the same division requirement for MuL virus and HIV based gene transfer in HSC. These studies will help to define optimal conditions for ex vivo HSC expansion and transduction. Finally, these studies will help to determine the relationship between in vitro defined stem cells, or ML-IC, and in vivo defined stem cells, or SRC.
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