RETENTION OF QUIESCENT HEMATOPOIETIC-CELLS WITH HIGH PROLIFERATIVE POTENTIAL DURING EX-VIVO STEM-CELL CULTURE

Human CD34(+)/Thy-1(+)/Lin(-) hematopoietic cells purified from bone m arrow (BM) or mobilized peripheral blood (MPB) are highly enriched for pluripotent stem cells, Ex vivo expansion of this population is propo sed as a means of providing accelerated short-term, as well as long-te rm, engraftment after myeloablative therapy. Here we demonstrate that primitive quiescent cells are retained in bulk expansion cultures of C D34(+)/Thy-1(+)/Lin(-) cells and that the cell production capacity of the expanded cell product can largely be attributed to cells exhibitin g quiescent behavior during culture. CD34(+)/Thy-1(+)/Lin(-) cells fro m adult BM or MPB were labeled with the fluorescent membrane dye PKH26 , followed by in vitro culture of 10(4) cells on a murine stromal laye r in the presence of interleukin (IL)-3, IL-6, c-kit ligand (KL), and leukemia inhibitory factor (LIF). With each subsequent cell division, PKH26 fluorescence is reduced by roughly half, which allows tracking o f the number of cell divisions. Progenitor cells present after a a-wee k expansion period were sorted into CD34(+)/Lin(-)/dye(bright) and CD3 4(+)/Lin(-)/dye(dim) fractions and then cultured in a 4-week single-ce ll proliferation assay to characterize the proliferative capacity of e ach group. Fifty-nine percent of progenitors remaining dye(bright) aft er bulk culture (four or fewer cell divisions) were observed to prolif erate in single cell culture, and produced an average of 1,780 cells p er plated cell. In contrast, only 26% of dye(dim) (more than four divi sions) progenitors were observed to proliferate and displayed a lower average proliferative capacity of 225 cells per plated cell. Similar b ehaviors were observed after a second consecutive cycle of bulk cultur e, indicating that quiescent cells with high proliferative capacity ex isted in culture for at least 4 weeks. Single CD34(+)/Lin(-)/dye(brigh t) progenitors purified from bulk cultures were observed to produce as many as 1,000 CD34 positive progeny during single cell culture, and t hese progeny included multilineage colony forming cells. These data de monstrate that among CD34 positive cells recovered after in vitro bulk culture, a higher proliferative capacity correlated with quiescent be havior. The described culture method provides quantitation of the cell producing capacity of individual cells in hematopoietic cell mixtures and may prove useful for predicting engrafting potential in products intended for cellular therapy. (C) 1996 by The American Society of Hem atology.