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Hypoxia mediates low cell-cycle activity and increases the proportion of long-term reconstituting hematopoietic stem cells during in vitro culture
Linköping University, Department of Clinical and Experimental Medicine, Cell Biology. Linköping University, Faculty of Health Sciences.
Lund University.
Linköping University, Department of Clinical and Experimental Medicine. Linköping University, Faculty of Health Sciences.
Linköping University, Department of Medicine and Health Sciences, Radiation Physics . Linköping University, Faculty of Health Sciences.
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2010 (English)In: EXPERIMENTAL HEMATOLOGY, ISSN 0301-472X, Vol. 38, no 4, 301-310 p.Article in journal (Refereed) Published
Abstract [en]

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)-1 alpha. Materials and 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-1 alpha 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-1 alpha. Surprisingly, overexpression of constitutively active HIF-1 alpha 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-1 alpha, maintains HSCs by decreasing proliferation and favoring quiescence.

Place, publisher, year, edition, pages
2010. Vol. 38, no 4, 301-310 p.
National Category
Medical and Health Sciences
URN: urn:nbn:se:liu:diva-54780DOI: 10.1016/j.exphem.2010.01.005ISI: 000276054300005OAI: diva2:309839
Original Publication: Pernilla Eliasson, Matilda Rehn, Petter Hammar, Peter Larsson, Oksana Sirenko, Lee A Flippin, Jorg Cammenga and Jan-Ingvar Jönsson, Hypoxia mediates low cell-cycle activity and increases the proportion of long-term reconstituting hematopoietic stem cells during in vitro culture, 2010, EXPERIMENTAL HEMATOLOGY, (38), 4, 301-310. Copyright: Elsevier Science B.V., Amsterdam. Available from: 2010-04-09 Created: 2010-04-09 Last updated: 2011-04-27
In thesis
1. Live and Let Die: Critical regulation of survival in normal and malignant hematopoietic stem and progenitor cells
Open this publication in new window or tab >>Live and Let Die: Critical regulation of survival in normal and malignant hematopoietic stem and progenitor cells
2009 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

The hematopoietic stem cell (HSC) is characterized by its ability to self-renew and produce all mature blood cells throughout the life of an organism. This is tightly regulated to maintain a balance between survival, proliferation, and differentiation. The HSCs are located in specialized niches in the bone marrow thought to be low in oxygen, which is suggested to be involved in the regulation of HSC maintenance, proliferation, and migration. However, the importance of hypoxia in the stem cell niche and the molecular mechanisms involved remain fairly undefined. Another important regulator of human HSCs maintenance is the tyrosine kinase receptor FLT3, which triggers survival of HSCs and progenitor cells. Mutations in FLT3 cause constitutively active signaling. This leads to uncontrolled survival and proliferation, which can result in development of acute myeloid leukemia (AML). One of the purposes with this thesis is to investigate how survival, proliferation and self-renewal in normal HSCs are affected by hypoxia. To study this, we used both in vitro and in vivo models with isolated Lineage-Sca-1+Kit+ (LSK) and CD34-Flt3-LSK cells from mouse bone marrow. We found that hypoxia maintained an immature phenotype. In addition, hypoxia decreased proliferation and induced cell cycle arrest, which is the signature of HSCs with long term multipotential capacity. A dormant state of HSCs is suggested to be critical for protecting and preventing depletion of the stem cell pool. Furthermore, we observed that hypoxia rescues HSCs from oxidative stress-induced cell death, implicating that hypoxia is important in the bone marrow niche to limit reactive oxidative species (ROS) production and give life-long protection of HSCs. Another focus in this thesis is to investigate downstream pathways involved in tyrosine kinase inhibitor-induced cell death of primary AML cells and cell lines expressing mutated FLT3. Our results demonstrate an important role of the PI3K/AKT pathway to mediate survival signals from FLT3. We found FoxO3a and its target gene Bim to be key players of apoptosis in cells carrying oncogenic FLT3 after treatment with tyrosine kinase inhibitors. In conclusion, this thesis highlights hypoxic-mediated regulation of normal HSCs maintenance and critical effectors of apoptosis in leukemic cells expressing mutated FLT3.

Place, publisher, year, edition, pages
Linköping: Linköping University Electronic Press, 2009. 93 p.
Linköping University Medical Dissertations, ISSN 0345-0082 ; 1160
hematopoietic stem cells, hypoxia, self-renewal, survival, acute myeloid leukemia, FLT3
National Category
Biochemistry and Molecular Biology Hematology
urn:nbn:se:liu:diva-52932 (URN)978-91-7393-470-1 (ISBN)
Public defence
2009-12-11, Berzeliussalen, Hälsouniversitetet, Campus US, Linköpings universitet, Linköping, 09:00 (English)
On the day of the defence date the title of article II was "Hypoxia, via hypoxia-inducible factor (HIF)-1, mediates low cell cycle activity and preserves the engraftment potential of mouse hematopoietic stem cells" and one of the authors is no longer included in the article.Available from: 2010-01-19 Created: 2010-01-13 Last updated: 2011-04-27Bibliographically approved

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Eliasson, PernillaHammar, PetterLarsson, PeterJönsson, Jan-Ingvar
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