HEMATOPOIETIC GROWTH FACTOR REQUIREMENTS FOR STEM CELL SURVIVAL

Abstract

Constitutive hematopoiesis is supported by the proliferation and differentiation of a limited number of pluripotential stem cells (PSCs). This process is tightly regulated by hematopoietic growth factors (HGFs) and the microenvironment. While it has been shown that HGFs alone or in combination promote the survival of progenitor/stem cells through proliferation and differentiation, little is known how HGFs regulate stem cell survival. Bone marrow cells (BMCs) were separated by countercurrent centrifugal elutriation (CCE) (separates cells on the basis of size and density) at a flow rate of 27 mls/min (CCE-27) to isolate primitive progenitors. Enrichment of primitive progenitors was examined by the ability of these cells to proliferate and differentiate in soft agar colony assays in response to single (SLF, IL-3, GMCSF, G-CSF, and M-CSF) versus multiple HGFs [(IL-3 + SLF) (GM-CSF + SLF)]. CCE-27 showed little or no growth with single HGFs (characteristic of committed cells) but contained progenitors that respond to multiple HGF combinations (characteristic of primitive progenitors). This response was specifically high when stimulated with the combination SLF + IL-3 (SLF/IL-3). A novel in vitro soft agar colony assay was developed to valuate the effects of cytokines (known to effect hematopoietic progenitor growth) on survival of CCE-27 IL-3/SLF-responsive progenitors. CCE-27 cells showed an absolute growth requirement for SLF at 0, 24, and 48 ours which gradually declined by 72 and 96 hours. IL-3 showed a reduced by statistically significant effect on survival when compared to medium controls. It was determined that the effect of HGFs (SLF, IL-3 and G-CSF) to promote the survival of CCE-27 was direct using a novel single cell survival assay. Also, CCE-27 cells were further enriched for cells that express c-kit (receptor for SLF). While SLF best promoted survival of the SLF/IL-3 responsive CCE-27-c-kit cells after 24 and 48 hours in liquid culture, IL-3 and G-CSF had little or no effect on single stem/progenitor cell survival. SLF induced significant proliferation as measured by 3H-thymidine incorporation of CCE-27 and Lin- CCE-27 (mature B cells and granulocytes depleted) at all times examined suggesting that survival may have been a consequence of cell division. However, SLF supported the survival of progenitor cells in the absence of cell division using the mitotic inhibitors nocodazole and aphidicolin. HGF survival requirements of pluripotent short-term reconstituting cells (STRCs) and long-term reconstituting cells (LTRCs) contained in the CCE-27 bone marrow fraction were determined by transplantation of SLF- or IL-3- treated CCE-27 [C57BL/6 (Ly5.2 donor)] cells into lethally irradiated recipient mice [C57BL/6 (Ly5.1 host)]. SLF promoted the survival of STRCs and LTRCs with trilineage reconstitution potential in vivo. In conclusion, these data show that 1) SLF and IL-3 to a lesser extent can directly promote survival of SLF/IL-3 responsive hematopoietic stem/progenitor cells in vitro; 2) SLF in vitro can support survival of STRCs and LTRCs in vivo.