Identification of in vitro growth conditions for c-Kit-negative hematopoietic stem cells

Our laboratory recently identified a quiescent class of pluripotent hematopoietic stem cells (PHSCs) that are lineage negative (Lin(neg)), lack c-Kit, and are able to give rise to c-Kit-positive (c-Kit(pos)) PHSCs in vivo. This population fails to proliferate in vitro but has delayed reconstituting activity in vivo. In this study, we purified these cells to enrich for the PHSCs and we identified in vitro conditions capable of supporting their maturation. The c-Kit-negative (c- Kit(neg)) cells exhibited differential expression of Sca-1, CD34, CD43, CD45, and Thy 1.2. We purified the cells based on Sca-1, as it is expressed on active PHSCs. We detected pre- colony-forming unit spleen (pre-CFU-s) activity in both the Sca-1(neg) and Sca-1(pos) populations, indicating the presence of primitive PHSCs in both populations. However, our in vitro studies suggest that the Sca-1(pos) population is enriched for PHSCs. The in vitro systems that support the growth of these dormant cells include a modified long-term marrow culture and various stromal cell lines. In modified long-term bone marrow cultures, c-Kit(neg) cells gave rise to c-Kit(pos) PHSCs, with long-term reconstitution activity in vivo. Thus we have established an in vitro system to examine PHSC maturation that will allow us to study the mediators of the c-Kit(neg) to c- Kit(pos) transition. (C) 2003 by The American Society of Hematology.

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