A number of recombinant cytokines have been shown to regulate the kinetics of the primitive hemopoietic progenitors. While expansion has been documented for hemopoietic cells, it is not yet known whether hemopoietic stem cells with long-term engrafting ability are expandable in culture. This proposal is designed to seek fundamental information regarding in vitro expansion of hemopoietic stem cells and progenitors. We will carry out parallel studies with murine and human hemopoietic cells harvested at different ontogenic stages. Suspension culture of FACS-sorted primitive progenitors will be carried out in the presence of combinations of early-acing cytokines under serum-containing and serum- free conditions.
Specific Aim #1 is to test the whether it is possible to expand murine hemopoietic stem cells in suspension culture. We will use a PCR-based assay for the identification of transplanted male cells.
Specific Aim #2 is to test whether human hemopoietic stem cells with long-term engrafting ability are expandable in culture. We will use in utero transplantation into pre-immune sheep as an assay for human stem cells.
Specific Aim #3 is to delineate the cytokine interactions supporting the optimal expansion of progenitors representing different developmental stages. We will use cell culture assays as well as in vivo engraftment assays for defining the expanded progenitor populations. An important aim here is to use the resulting information to guide the in vivo experiments described in Specific Aims # 1 and #2. Another major interest is to correlate the progenitors assayed in culture with the in vivo short-term and long-term engrafting progenitors in order to develop culture assay predictive of in vivo functions of the progenitors. We recently observed that IL-3 and IL-1 independently suppress early stages of B- and T-lymphopoiesis. We will test whether the inhibitor effects of IL-3 and IL-1 involve early states of all lymphohematopoiesis including self-renewal ability of the stem cells.
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