Hematopoietic Stem Cells Survive Warm Ischemia and the Bone Marrow Upregulates a Significant Number of Genes

Quiescent hematopoietic stem cells (HSCs) located in stem cell niches are characterized by a relative resistance to hypoxia. This study is focused primarily on maintainance of the repopulating ability of HSCs in structurally intact BM exposed to anoxia, lack of metabolic substrates and accumulation of metabolic waste products during a period of ischemia at three different temperatures. In the case of a warm ischemia at 37°C, changes in gene expression profile in the whole bone marrow has been also examined.

Murine congenic model C57Bl/6 Ly5.1/Ly5.2 was used in the experiments. Normal mice or mice recovering from a bone marrow damage induced either by cyclophosphamide or a sublethal irradiation were sacrified. Their BM was maintained in intact femurs at 37°C for different time periods up to 6 hours. For normal bone marrow, exposure to ischemia at 20°C and 4°C was also used for up to 20 and 48 hours, respectively. Afterwards, bone marrow cells were harvested and cells corresponding to a half of the femur were transplanted to sublethally (6 Gy) irradiated recipients in a competitive repopulation assay. Resulting chimerism was examined up to 6 months after transplantation to test for STRCs and LTRCs (Short and Long Term Repopulating Cells). Subpopulations of erythropoietic (Ter119+), B-lymphopoietic (B220+), granulo- and monocytopoietic (Gr-1/Mac+), and LSK (Lin-Sca-1+c-Kit+) bone marrow cells were analyzed for dead cells and apoptosis. Total RNA was isolated from bone marrow exposed to warm ischemia ranging 0 to 4 hours and dynamics of changes in its gene expression profile was determined by Illumina MouseRef8 BeadChip.

Repopulating ability of ischemic BM was fully preserved for 2 hour of the warm (37°C) ischemia and for 6 hours and 8 hours of 20°C and 4°C ischemia, respectively. There was no difference between STRCs and LTRCs in survival. STRCs and LTRCs from the bone marrow collected 2 days or 5 days after a single dose of cyclophosphamide exposed to warm ischemia showed decreased repopulating ability in comparison with those of normal mice. STRCs significantly prevailed over LTRCs in bone marrow collected 20 days after a sublethal irradiation and showed increased sensitivity to warm ischemia. B220+ cells were the most sensitive cells of the bone marrow to warm ischemia, LSK and Ter119 cells being the most resistant ones. Gene expression profile in bone marrow exposed to warm ischemia changed progressively over time. Despite the highly unfavorable metabolic conditions, hypoxia and lack of energy, a set of overexpressed genes equaled in number the one inhibited.

HSCs exposed to warm or cold ischemia maintain their repopulating ability for a considerable time. Bone marrow ischemia activates specific gene expression in paralel with supression of others. Supported by projects LC06044, MSM 0021620806 and the grant SVV-2010-254260507.

No relevant conflicts of interest to declare.