THE EFFECT OF CYTOKINES ON CD34(+) RH-123(HIGH) 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-123(high) (78.2 +/- 4.5%) and Rh-123(low) (21.8 +/- 3.6%). While 9.3 +/- 1.6% of the CD34(+)Rh-123(high) cells formed colonies in agar, only 0.4 +/- 0.2% of the CD34(+)Rh-123(low) population did so. However, the CD34(+)Rh-123(low) cells resulted in the greatest expansion of colony-forming cells (CFC) when cultured in liquid medium with different cytokine combinations. When the CD34(+)Rh-123(low) cells were cultured for 7 days with stem cell factor (SCF) and erythropoietin (Epo), the CD34(+)Rh-123(low) cells resulted in a 94-fold increase in CFC compared with a 2.5-fold increase from the CD34(+)Rh-123(high) 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-123(low) cells >28 days compared with only 21 days for the CD34(+)Rh-123(high) cells. Coculture of CD34(+)Rh-123(low) 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-123(high) cells. The duration of maintenance of CFC in liquid culture could be further enhanced by the addition of an antibody (Ah) directed against the binding site of the GM-CSF receptor. The addition of anti-GM-CSF receptor Ab to cultures of CD34(+)Rh-123(high) 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-123(high) and (low) cells. Although very few CFCs were present by 42 days in liquid cultures of CD34(+)Rh-123(high) tells, 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-123(low) cells, it was less dramatic as more CFC persisted even in the absence of Ab. The possible mechanism of this effect is discussed.