Studies of quiescence of hematopoietic stem cells in non-human primates

Abstract

Well documented differences exist between murine and primate hematopoiesis, therefore we examined the quiescence of PHSC in vivo by continuous oral bromodeoxyuridine (BrdU) administration to baboons in unperturbed and perturbed states. CD34+ cells from bone marrow (BM) or peripheral blood (PB) were fractionated based upon relative fluorescence of Hoechst (Ho) and Rhodamine (Rho). HoLow/RhoLow cells contained the highest frequency of cobblestone area-forming cells (CAFCs). Within 1 week of BrdU feeding more than 90% of granulocytes but only 2% of HoLow/RhoLow cells were BrdU+. The BrdU labeling HoLow/RhoLow cells never exceeded 26% and 44% despite 52 and 61 weeks of BrdU feeding respectively during steady state. Granulocytes (>90%) from one of these animals remained BrdU labeled despite withdrawal of BrdU for more than 1 month, confirming that indeed granulocytes are derived from previously labeled progenitors. To determine the cell cycle status of HSC during stress, two additional baboons were given G-CSF alone (100μg/kg × 5 days), one baboon was given SCF (25 μg/kg/day, day 1–8) and G-CSF (100 μg/kg/day day 4–8), and one baboon was given thrombopoietin alone (TPO, 2.5 μg/kg/day × 14 days). During this period these animals were also fed with BrdU continuously. Following administration of G-CSF alone 18% of the HoLow/RhoLow cells were BrdU+ in the mobilized peripheral blood (MPB). SCF alone resulted in only 3% of HoLow/RhoLow cells undergoing cell division, however, when G-CSF was combined with SCF, 34% of HoLow/RhoLow cells acquired BrdU labeling. Although TPO did not effectively mobilize HSC, 27% of BM HoLow/RhoLow cells were labeled with BrdU after 6 days. Since the majority of HSCs in steady state BM were quiescent, these findings are consistent with the clonal succession hypothesis. Our studies provide a direct evidence in a large animal model that hematopoietic stress induced by growth factors leads to a rapid increase in the proportion of cycling HSCs within few days. The presence of BrdU+ cells in MPB suggests that PHSCs cycle in the BM following cytokine administration and then mobilize into the PB where they re-enter the quiescent phase. Indeed, a significant proportion of HoLow/RhoLow cells present in MPB were not labeled with BrdU, which suggest that HSC mobilization is independent of cell cycle status. The quiescence of PHSC in baboons and the potential of growth factors to perturb the quiescence may provide insights regarding human clinical therapies involving HSC. These studies likely more closely portray the behavior of human PHSCs than studies reported in mice.