The development of in vitro conditions that promote a numerical expansion of hematopoietic stem cells (HSCs) with long-term reconstituting ability has been a long-standing goal in experimental hematology. In previous studies, we showed that input numbers of such cells, i.e., competitive repopulating units (CRU), could be maintained for 2 weeks when purified Sca-1+Lin-WGA+ adult bone marrow (BM) cells were cultured in serum-free and stromal cell-free cultures containing Steel factor (SF), interleukin 6 (IL-6), and erythropoietin (Epo). In separate studies, we showed that limiting numbers of purified fetal liver (FL) cells that are highly enriched for CRU display a higher proliferative and self-renewal potential in vivo compared with similar cells purified from adult BM. These findings prompted us to explore the possibility of achieving a numerical expansion of purified FL cells in culture. Although we observed an extensive increase in the number of nucleated FL cells in all culture conditions tested, none of the cultures, including cultures in serum-containing medium and cocultures on a preestablished feeder layer of BM stromal cells of S17 cells, sustained the expected expansion or even supported the maintenance of input numbers of FL CRU. Single cell cultures showed that the production of nucleated cells by purified Sca-1++Lin·-AA4.1+ FL cells stayed behind that of purified Sca-1+Lin-WGA+ adult BM cells. Taken together, our results show that a variety of culture conditions tested, including conditions that support maintenance and limited expansion of adult BM CRU do not support the production of repopulating stem cells from FL. Because such expansion can be observed in vivo, this system appears useful to search for novel culture conditions and - perhaps yet unidentified - cytokines or other microenvironment-related factors that may be required for FL CRU to prevent loss of repopulation potential in vitro and allow these cells to exhibit their expected self-renewal potential.
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