Hypoxia mediates low cell-cycle activity and increases the proportion of long-term-reconstituting hematopoietic stem cells during in vitro culture.
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)-1alpha.


METHODS
HSC and progenitor populations isolated from mouse BM were cultured in 20% or 1% O(2), 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-1alpha 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 G(0). Moreover, the proportion of HSCs with long-term engraftment potential was increased. Whereas expression of the cyclin-dependent kinase inhibitor genes p21(cip1), p27(Kip1), and p57(Kip2) increased in LSK cells by hypoxia, only p21(cip1) was upregulated in FLT3(-)CD34(-)LSK cells. We could demonstrate that expression of p27(Kip1) and p57(Kip2) was dependent of HIF-1alpha. Surprisingly, overexpression of constitutively active HIF-1alpha 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-1alpha, maintains HSCs by decreasing proliferation and favoring quiescence.
Experimental Hematology Unit, Department of Clinical and Experimental Medicine, Linköping University, Linköping, Sweden. pernilla.m.eliasson@liu.se <pernilla.m.eliasson@liu.se>
Pernilla
P
Eliasson
Matilda
M
Rehn
Petter
P
Hammar
Peter
P
Larsson
Oksana
O
Sirenko
Lee A
LA
Flippin
Jörg
J
Cammenga
Jan-Ingvar
JI
Jönsson
D000818:Animals; D002453:Cell Cycle / Q000502:physiology; D015687:Cell Hypoxia; D049109:Cell Proliferation; D002478:Cells, Cultured; D005786:Gene Expression Regulation; D006412:Hematopoietic Stem Cells / Q000166:cytology* / Q000378:metabolism / Q000502:physiology*; D051795:Hypoxia-Inducible Factor 1, alpha Subunit / Q000378:metabolism*; D051379:Mice; D020133:Reverse Transcriptase Polymerase Chain Reaction
D051795:Hypoxia-Inducible Factor 1, alpha Subunit