Peripheral blood accessory cells modulate committed colony-forming units but not 5-week cobblestone-area-forming cell outgrowth from CD34+ cells.

Abstract

While cellular modulation in vitro of committed hematopoietic stem cell (HSC) growth has been known for some time, less is known about the effect of accessory cells (AC) on the growth of more immature HSC. We have examined the effect of peripheral blood (PB) AC on hematopoiesis by coculturing enriched PB CD34+ cells (>96% pure) with different quantities of CD34 cells (<1% contamination) harvested from 10 breast cancer patients. As expected colony growth was predominantly present in the CD34+ fractions, in which colony forming units granulocyte-macrophage (CFU-GM) varied between 89-3289/10(5) (median 1422/10(5) seeded cells) and week 5 cobblestone area forming cells (CAFC) between 64-1330/10(5) (median 427/10(5) seeded cells). Few CFU-GM (0-27/10(5) seeded cells) and no week 5 CAFC (0-1/10(5) seeded cells) were present in the CD34 fractions. The addition of PB CD34 cells to cultures of CD34+ cells resulted in a considerable variation in the cloning efficiency at the CFU-GM level, and the extent of modulation within the single patient was inconsistent between the different CD34+/CD34 cell mixtures. Overall the stimulatory effect was more pronounced than inhibition and on average the CFU-GM formation per CD34+ cell seeded increased 3 fold (stimulatory effect ranged between 3-17 fold and decreases between 2-10 fold). In contrast, the cloning efficiency at the week 5 CAFC level of differentiation remained unaffected by the addition of different amounts of CD34 cells (the stimulatory effect was maximally 3-fold and inhibition 3-fold). We conclude that while the CFU assay is modulated by the presence of AC, the CAFC assay is more robust and can be employed as a reliable and reproducible tool for HSC measurement.