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

TY - JOUR

T1 - Hypoxia mediates low cell-cycle activity and increases the proportion of long-term-reconstituting hematopoietic stem cells during in vitro culture

AU - Eliasson, Pernilla

AU - Rehn, Matilda

AU - Hammar, Petter

AU - Larsson, Peter

AU - Sirenko, Oksana

AU - Flippin, Lee A

AU - Cammenga, Jörg

AU - Jönsson, Jan-Ingvar

PY - 2010/4

Y1 - 2010/4

N2 - 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.

AB - 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.

KW - Animals

KW - Cell Cycle/physiology

KW - Cell Hypoxia

KW - Cell Proliferation

KW - Cells, Cultured

KW - Gene Expression Regulation

KW - Hematopoietic Stem Cells/cytology

KW - Hypoxia-Inducible Factor 1, alpha Subunit/metabolism

KW - Mice

KW - Reverse Transcriptase Polymerase Chain Reaction

U2 - 10.1016/j.exphem.2010.01.005

DO - 10.1016/j.exphem.2010.01.005

M3 - Journal article

C2 - 20138114

SN - 0301-472X

VL - 38

SP - 301-310.e2

JO - Experimental Hematology

JF - Experimental Hematology

IS - 4

ER -