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The effect of cytokines on CD34+ Rh-123high and low progenitor cells from human umbilical cord blood.

In this study, we show how Rhodamine-123 (Rh-123), as in other hematopoietic populations, can be used to define functionally distinct progenitor cells from human umbilical cord blood (HUCB). CD34+ cells were subdivided into Rh-123high (78.2 +/- 4.5%) and Rh-123low (21.8 +/- 3.6%). While 9.3 +/- 1.6% of the CD34+Rh-123high cells formed colonies in agar, only 0.4 +/- 0.2% of the CD34+Rh-123low population did so. However, the CD34+Rh-123low cells resulted in the greatest expansion of colony-forming cells (CFC) when cultured in liquid medium with different cytokine combinations. When the CD34+Rh-123low cells were cultured for 7 days with stem cell factor (SCF) and erythropoietin (Epo), the CD34+Rh-123low cells resulted in a 94-fold increase in CFC compared with a 2.5-fold increase from the CD34+Rh-123high cells. The combination of SCF and Epo or granulocyte-macrophage colony-stimulating factor (GM-CSF) supported the production and maintenance of CFC from CD34+Rh-123low cells > 28 days compared with only 21 days for the CD34+Rh-123high cells. Coculture of CD34+Rh-123low cells with stromal cell line 11 (SCL11) demonstrated that long-term culture initiating cells (LTCIC) were present within this population, as CFC could be recovered for > 10 weeks compared with < 6 weeks in cocultures with CD34+Rh-123high cells. The duration of maintenance of CFC in liquid culture could be further enhanced by the addition of an antibody (Ab) directed against the binding site of the GM-CSF receptor. The addition of anti-GM-CSF receptor Ab to cultures of CD34+Rh-123high and low cells supplemented with SCF, interleukin-3 (IL-3), and IL-6 resulted in an initial 10-fold decrease in CFC in cultures of both the CD34+Rh-123high and low cells. Although very few CFCs were present by 42 days in liquid cultures of CD34+Rh-123high cells, the number of CFCs in these cultures was significantly increased when anti-GM-CSF receptor Ab was added. Although this effect was also observed in cultures of CD34+Rh-123low cells, it was less dramatic as more CFC persisted even in the absence of Ab. The possible mechanism of this effect is discussed.