Hypoxia mediates low cell-cycle activity and increases the proportion of long-term–reconstituting hematopoietic stem cells during in vitro culture
Received 14 July 2009; received in revised form 12 January 2010; accepted 22 January 2010. published online 04 February 2010.
Objective
Recent evidence suggests that hematopoietic stem cells (HSCs) in the bone marrow (BM) are located in areas where the environment is hypoxic. Although previous studies have demonstrated positive effects by hypoxia, its role in HSC maintenance has not been fully elucidated, neither has the molecular mechanisms been delineated. Here, we have investigated the consequence of in vitro incubation of HSCs in hypoxia prior to transplantation and analyzed the role of hypoxia-inducible factor (HIF)−1α.
Materials and Methods
HSC and progenitor populations isolated from mouse BM were cultured in 20% or 1% O2, and analyzed for effects on cell cycle, expression of cyclin-dependent kinase inhibitors genes, and reconstituting ability to lethally irradiated mice. The involvement of HIF-1α was studied using methods of protein stabilization and gene silencing.
Results
When long-term FLT3−CD34− Lin−Sca-1+c-Kit+ (LSK) cells were cultured in hypoxia, cell numbers were significantly reduced in comparison to normoxia. This was due to a decrease in proliferation and more cells accumulating in G0. Moreover, the proportion of HSCs with long-term engraftment potential was increased. Whereas expression of the cyclin-dependent kinase inhibitor genes p21cip1, p27Kip1, and p57Kip2 increased in LSK cells by hypoxia, only p21cip1 was upregulated in FLT3−CD34−LSK cells. We could demonstrate that expression of p27Kip1 and p57Kip2 was dependent of HIF-1α. Surprisingly, overexpression of constitutively active HIF-1α or treatment with the HIF stabilizer agent FG-4497 led to a reduction in HSC reconstituting ability.
Conclusions
Our results imply that hypoxia, in part via HIF-1α, maintains HSCs by decreasing proliferation and favoring quiescence.
aExperimental Hematology Unit, Department of Clinical and Experimental Medicine, Linköping University, Linköping, Sweden
bLund Strategic Center for Stem Cell Biology and Cell Therapy, Lund University, Lund, Sweden
cRadiation Physics, Department of Medical and Health Sciences, Faculty of Health Sciences, Linköping University, Linköping, Sweden
Offprint requests to: Pernilla Eliasson, Ph.D., Experimental Hematology Unit, Department of Clinical and Experimental Medicine, Linköping University, SE-581 85 Linköping, Sweden
ABSTRACT