Identification of culture conditions that support expansion or even long-term maintenance of in vivo repopulating human hematopoietic stem cells is still a major challenge. We report here that human hematopoietic stem cells capable of repopulating NOD/ SCID mice can be maintained after an extremely long period of suspension cultures. Using a combination of FLT3 Ligand (FL), Stem Cell Factor (SCF), Thrombopoietin (TPO) and IL6 we cultured cord blood (CB) CD34 cells for up to 12 weeks and transplanted their progeny into sublethally irradiated NOD/SCID mice. Bone marrow engraftment was considered successful when recipients animals contained measurable numbers of human CD45", CD7Tand Glycophorin-A (GpA) cells 8 weeks after transplantation. When cells were cultured with FL+SCF+TPO+IL6 for 12 weeks all of the recipients were successfully engrafted and human CD45++CD71++GpA+ represented 4.3 to 22.4% of bone marrow cells. When cells were cultured with FL+ SCF+ TPO and IL3 in the place of IL6 we observed an even better expansion of cells and a similar clonogenic progenitor output in the first 8 weeks of ex vivo cultures. However, more primitive LTC-IC output increased up to week 6 with the growth factor combination containing IL3 and then decreased and disappeared, while with the growth factor combination containing IL6 LTC-IC kept increasing up to week 12 and further. Even more strikingly, cells cultured for 4 weeks with the four-factor combination containing IL3 less efficiently engrafted NOD/SCID mice, both as measured by frequency of positive recipients (4 out of 10) and percentage of engrafted human cells (maximum 2%). Repopulation of NOD/SCID mice was no longer observed when ex-vivo expansion was performed for 6 weeks. This study provides some evidence that many, but not all, so called "early acting" cytokines, can sustain long term maintenance and even expansion of human primitive in vivo repopulating stem cells. In particular, in the culture conditions employed in this study, the presence of IL3 greatly reduces the repopulating potential of expanded CD34 cord blood cells.
|Issue number||11 PART II|
|Publication status||Published - 2000|
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