Different in vivo repopulating activities of purified hematopoietic stem cells before and after being stimulated to divide in vitro with the same kinetics

Objective The Hoechst 33342-effluxing side population (SP) of adult mouse bone marrow (BM) contains most of the hematopoietic stem cells (HSCs). Here we measured the HSC content of specific subsets of SP cells and then used a highly HSC-enriched fraction to investigate the effect of different growth factors on the initial rate of HSC proliferation in vitro and the accompanying maintenance (or loss) of HSCs in the first-division progeny. Materials and methods Staining with Rhodamine-123 (Rho) was used to subfractionate lineage marker–negative (lin−) SP cells. Cells were assayed for HSCs by examining their ability to generate sustained (>4 months) multi-lineage lympho-myeloid clones in irradiated hosts. Cultures of single lin− Rho− SP cells were used to monitor growth factor effects on HSC proliferation and function. Results More than 40% of mice injected with single lin− Rho− SP cells showed long-term lympho-myeloid reconstitution. Some clones peaked within 8 weeks but others developed more slowly apparently unrelated to the pattern of lineage representation. 3/3 clones tested repopulated secondary mice. Either Steel factor + interleukin-11 (± flt3-ligand) or Steel factor + thrombopoietin stimulated at least 75% of single lin−Rho− SP cells to divide in vitro with the same synchronous kinetics. However, in the first cocktail, the frequency of HSCs among the first-division doublets was preserved but in the latter it was greatly diminished. Conclusion Exogenous growth factors can differentially affect the ability of HSCs to execute a self-renewal division within a single cell cycle even when the kinetics of proliferation are the same.