Effect Of Hypoxic Exposure On Proliferation And Lineage Differentiation Of Bone Marrow Hematopoietic Stem Cells

Objective. Living at high altitude can cause increased production of red blood cells (RBCs). Excessive erythrocytosis may cause increased blood viscosity,increased vessel resistance, tissue hypoxia and various clinical symptoms. Predominant role of hypoxia- induced erythropoietin (EPO) for hypoxic polycythemia has been well established. However, the EPO level does not correlate well with the amount of RBC production at high altitude. In the adult, all mature blood cells are ultimately derived from hematopoietic stem cells (HSCs). Previous studies had shown that HSCs responsed to the stress of bleeding or infection to help in RBCs or WBCs recovery. It was uncertain whether HSCs compartment directly responds to hypoxic environment at high altitude and contribute to enhanced erythropoiesis. The aim of our study is to get at the truth.Methods. Lineage marker?Sca-1~+c-Kit~+ (LSK) HSC compartment in the bone marrow (BM) and the spleen of 0-28 days hypoxic mice were observed directly or indirectly. Normal LSK cells were then cultured under different conditions relating to high altitude (varying EPO levels, oxygen concentrations and BM culture supernatants) to investigate the causes of the HSC responses.Results. The mice exposed to hypoxic environment exhibited a marked increase of the LSK compartment in the bone marrow (BM) and the spleen that contains the HSC population, associated with enhanced proliferation of both active short-term repopulating activity HSCs (CD34+LSK ST-HSCs) and primitive long-term repopulating activity HSCs (CD34-LSK LT-HSCs). BM HSCs seemed to play a more important role in erythropoiesis at high altitude than spleen HSCs.According to our result, BM HSCs should contribute more to hypoxic hematopoiesis than spleen HSCs. One reason for this is that the BM and spleen displayed different time correlation of HSC expansion under high-altitude hypoxic exposure. On day 28 after hypoxic exposure, the BM LSK cell population significantly exceeded that at day 0 while the spleen LSK size returned to the value of normoxic mice. High-altitude erythrocytosis is a gradually developing event, so BM HSCs seem more important for this chronic process than spleen HSCs. The other reason was that the total number of LSK cells in BM from the whole body was 10-fold higher than that in the spleen both in and outside a hypoxic environment.Hypoxic mice periodically showed increased megakaryocyte-erythrocyte progenitors (MEPs) and reduced granulocyte-macrophage progenitors (GMPs) resulting from a lineage fate change of upstream HSCs, which was reflected by up-regulated erythroid lineage-specific GATA-1 mRNA and down-regulated myeloid-specific PU.1 mRNA expression in LSK subset. Further evidence is that LSK cells from hypoxic mice displayed more BFU-E and less CFU-GM frequency, as well as differentiating to more Ter119+ erythroid precursor after culturing.By culturing LSK cells under different conditions relating to high altitude, the following results were observed:(1) Not even the highest concentration of EPO could affect the size and fate of the Sca-1+ HSC population.(2) The average O2 concentration where most BM LSK cells are located is about 5% at sea level, and the O2 concentration likely decreases from 5% to about 2% in response to 6,000 m simulated hypoxic exposure. Our results showed that 2% O2 was not more efficient in promoting the expansion and erythroid commitment of LSK cells than 5% O2. Thus, the direct oxygen sensing mechanism is not be the principal cause of BM HSC reaction in vivo at high altitude,(3) BM culturing supernatant from hypoxic mice can induce the expansion and erythroid-priority differentiation of Sca-1+ HSC population. The expansion of HSCs was partly due to increasing interleukin 3 and interleukin 6 in the supernatant. Increasing interleukin 3 level also particated in promoting erythroid potential of HSCs.Conclusion. Our data collectively supported that during hypoxic exposure the priority of lineage choice was given to erythriosis even as early as in the HSC stage. Expansion of population and acceleration of erythroid lineage differentiation become predominant features of multipotent hematopoietic stem cells, resulting in more erythroid progenitors (EPO responsive cells, ERCs). In hypoxic mice, a series of altered cytokines in the bone marrow microenvironment seem to play import roles in influencing the population and fate of HSCs. This process has never been concerned in the past and may serve as one EPO-independent mechanism underlying the hematopoietic response to hypoxic exposure. This study may provide a new idea for treatment and prevention of high-altitude polycythemia.