Thrombopoietin mediated regulation of murine hematopoietic progenitors

A number of studies have suggested that primitive hematopoietic cells respond to thrombopoietin (TPO) and that this growth factor has a non-redundant role in regulating early stages of hematopoiesis. The present study was designed to investigate more precisely the effects of an absence of TPO-mediated signaling in vivo on the generation/maintenance of specific subsets of primitive hematopoietic cells and to evaluate their ability to generate derivative progenitors in response to various growth factors in vitro. Initial tests confirmed that colony-forming cell (CFC) frequencies in the marrow of both types of mutant mice were reduced (~2-fold) and showed that longterm culture-initiating cell (LTC-IC) numers were even further reduced (~3-fold). The marrow of tpo[sup -/-] and c-mpl[sup -/-] mice also contained fewer Sca-1⁺(S⁺) lineage marker-negative (lin⁻) Hoechst 33342-stained side population (SP) and S⁺ c-kit⁺ L⁻ (S⁺K⁺L⁻) cells (~2-fold). The CFC output per LTC-IC from tpo[sup -/-] and c-mpl[sup -/-] mice was also reduced (~3-fold) as shown by both limiting dilution assays and single cell LTC-IC cultures. Similarly, 10-day suspension cultures of S⁺K⁺L⁻ and S⁺L⁻ SP cells from tpo[sup -/-] and c-mpl[sup -/-] mice produced 3- 5-fold fewer CFCs, regardless of the growth factors added. Single cell cultures showed this was due to a decreased number of mutant clones containing CFCs. Addition of either TPO or interleukin-11 to Steel factor (SF) and flt3-ligand (FL) in cultures of tpo[sup -/-] S⁺K⁺L⁻ cells also failed to enhance the generation of LTC-IC activity in spite of an ability of IL-11 to elicit such a response. The most significant findings from these studies are: 1) in vivo TPO-mediated signaling is required for the generation of normal sized compartments of several stages of early hematopoietic progenitors with the most profound influence seen on the more primitive cells; and 2) in the presence of SF and FL in vitro, TPO does not enhance the output of primitive progenitors from +/+ cells beyond that obtained by the activation of gp130.Medicine, Faculty ofMedicine, Department ofExperimental Medicine, Division ofGraduat