JOURNAL OF HEMATOTHERAPY 7:127-139 (1998) Mary Ann Liebert, Inc. Effect of Stromal Cell Coculture on Progenitor Cell Expansion and Myeloid Effector Function In Vitro JANE L. LIESVELD, BETH A. MARTIN, ABIGAIL W. HARBOL, KAREN E. ROSELL, TODD J. BELANGER, SIGRUN E. REYKDAL, DINA L. PLEKAVICH, and CAMILLE N. ABBOUD ABSTRACT Stimulation of CD34+-enriched marrow or light density marrow with various growth factor com- binations can generate granulocyte progenitors and mature neutrophils in vitro. In this work, we have examined the influence of irradiated marrow stromal layers on growth factor-induced myeloid and early multipotential progenitor expansion from enriched marrow CD34+ progenitors. We have also explored whether the addition of early-acting growth factors known to enhance myelopoiesis in long-term culture, such as fibroblast growth factor (b-FGF), insulin growth factor (IGF-1), c-kit ligand or stem cell factor (SCF), and flk-2flt-3 ligand (FL), can lengthen survival of CD34+ prog- enitors in these cultures. Stromal cell coculture resulted in greater numbers of total cells and CFU- GM at day 7 and day 14, but with the addition of multiple growth factors, these effects of stromal cell coculture were diminished. At day 14, generally <1% of the expanded cells over stromal co- culture conditions were CD34+, with up to 90% demonstrating CD15 positivity. Culture of CD34+ cells in the presence of early-acting growth factors did not cause significant expansion of CD34+ cells over a 14-day life span, even in the presence of marrow stromal cells. These data suggest that although stromal cell coculture for a period up to 14 days can enhance expansion of total cell num- bers and CFU-GMs, stromal cell presence does not lead to expansion of CD34+ cells in these cul- tures and may diminish the number of clonogenic cells present when growth factors with differen- tiating capacity are present. Mature neutrophils harvested from such cultures are capable of chemotaxis, actin polymerization, and migration, suggesting a replete functional status. INTRODUCTION si°n of truly pluripotential, self-renewing hematopoietic cells remains undetermined (7). Such in vitro systems Several groups have reported on the feasibility of have the potential to offer a means by which cells at all expanding marrow, peripheral blood, or cord blood levels of hematopoietic maturation might be propagated progenitors in various in vitro settings from a starting in vitro, with the goal of using them in transplant settings population of enriched CD34+ cells or light density to diminish the duration of neutropenia after high-dose mononuclear cells through culture with various chemotherapy or total body irradiation or both. Also, such hematopoietic growth factors (1-5). Such expanded cul- systems might have the potential to increase numbers of tures also contain precursors of mature blood elements, cycling pluripotent hematopoietic stemlike cells available such as neutrophils, as well as a residual population of for retroviral-mediated transfer of genes, thus making in- CD34+ cells (6). Whether such systems result in expan- creased transduction efficiency possible. Hematology/Oncology Unit, Department of Medicine, University of Rochester School of Medicine & Dentistry, Rochester, NY 14642. 127