Ex Vivo Expansion and Differentiation of CD34+ Cells Into Red Blood Cell Precursors
A. Caraux, G. Stoney, B. Kern, G. Molineux, R. Briddell
Amgen Ine., Thousands Oaks, CA, U.S.A.
Infusion of expanded CD34+ cells has been reported to abrogate post myeloablative chemotherapy neutropenia. This effect is related to ex-vivo expansion and differentiation of CD34+ cells into neutrophil precursors. When injected in vivo, expanded cells are capable of rapidly differentiating into mature neutrophils.
In these studies, we have assessed the potential of r-metHuSCF (S), PEG-rHuMGDF (M) and escalating doses of either rHuEPO (E) or darbopoietin (NESP, N) to expand cell numbers and promote the differentiation of human CD34+ cells into the erythroid lineage. CD34+ selected peripheral blood cells (>95% pure) were cultured for 10 days in teflon bags containing a defined medium supplemented with various combinations of S (100ng/mL), M (100ng/mL) and E or N at varying concentrations (10, 100; or 1000 ng/mL). Cultures containing S+M promoted the expansion of much lower numbers of nucleated cells (4-fold) compared with cultures containing optimal levels of either combination of S+M+E (51-fold) or S+M+N (42-fold). Previous reports have indicated that cultures containing S+M + rmetHuG-CSF (G) resulted in the expansion and differentiation of CD34+ cells to promyelocytes. In our studies, control cultures containing S+M+G all at 100ng/mL resulted in a 22-fold expansion of predominantly myeloid nucleated cells.
The expanded cells were harvested and cultured in a methylcellulose-based progenitor cell assay system. Control expansion cultures containing only S+M promoted a 0.3-fold expansion of BFU-E; cultures containing optimal levels of EPO and NESP promoted a 28.8-fold and 14.0-fold expansion of BFU-E respectively. However, under these conditions, the production of GM-CFC was reduced.
The expanded cells were also phenotyped for lineage markers using monoclonal antibodies specific for CD3, CD14, CD15, CD34, CD36, CD61 and CD71. Cultures containing S+M+E or S+M+N resulted in the differentiation of CD34+ cells into early and late red blood cell precursors as measured by expression of CD36 (75.6% and 62.7% respectively for S+M+E and S+M+N) and CD71 (84.9 % and 81.8 % respectively for S+M+E and S+M+N) but not into lymphoid (CD3), monocytic (CDI4), granulocytic (CDI5) or megakaryocytic (CD61) lineages. CD34+ cell numbers remained fairly constant in cultures supplemented with S+M+E (1.35 fold) or with S+M+N (0.95 fold). In cultures containing r-metHuSCF and PEG-rHuMGDF with rHuEPO or darbopoietin (NESP), we detected higher numbers of erythroid precursors as compared with control cultures.
These methods may prove useful for the production of large quantities of mature red blood cell precursors for therapeutic use and cellular support in high-dose chemotherapy settings.