Matilda Rehn

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. 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-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.

HIF-2 alpha maintains an undifferentiated state in neural crest-like human neuroblastoma tumor-initiating cells

High hypoxia-inducible factor-2α (HIF-2α) protein levels predict poor outcome in neuroblastoma, and hypoxia dedifferentiates cultured neuroblastoma cells toward a neural crest-like phenotype. Here, we identify HIF-2α as a marker of normoxic neural crest-like neuroblastoma tumor-initiating/stem cells (TICs) isolated from patient bone marrows. Knockdown of HIF-2α reduced VEGF expression and induced partial sympathetic neuronal differentiation when these TICs were grown in vitro under stem cell-promoting conditions. Xenograft tumors of HIF-2α-silenced cells were widely necrotic, poorly vascularized, and resembled the bulk of tumor cells in clinical neuroblastomas by expressing additional sympathetic neuronal markers, whereas control tumors were immature, well-vascularized, and stroma-rich. Thus, HIF-2α maintains an undifferentiated state of neuroblastoma TICs. Because low differentiation is associated with poor outcome and angiogenesis is crucial for tumor growth, HIF-2α is an attractive target for neuroblastoma therapy.

Hypoxic induction of vascular endothelial growth factor regulates murine hematopoietic stem cell function in the low-oxygenic niche.

Hypoxia is emerging as an important characteristic of the hematopoietic stem cell (HSC) niche, but the molecular mechanisms contributing to quiescence, self-renewal, and survival remain elusive. Vascular endothelial growth factor A (VEGFA) is a key regulator of angiogenesis and hematopoiesis. Its expression is commonly regulated by hypoxia-inducible factors (HIF) that are functionally induced in low-oxygen conditions and that activate transcription by binding to hypoxia-response elements (HRE). Vegfa is indispensable for HSC survival, mediated by a cell-intrinsic, autocrine mechanism. We hypothesized that a hypoxic HSC microenvironment is required for maintenance or up-regulation of Vegfa expression in HSCs and therefore crucial for HSC survival. We have tested this hypothesis in the mouse model Vegfa(δ/δ), where the HRE in the Vegfa promoter is mutated, preventing HIF binding. Vegfa expression was reduced in highly purified HSCs from Vegfa(δ/δ) mice, showing that HSCs reside in hypoxic areas. Loss of hypoxia-regulated Vegfa expression increases the numbers of phenotypically defined hematopoietic stem and progenitor cells. However, HSC function was clearly impaired when assessed in competitive transplantation assays. Our data provide further evidence that HSCs reside in a hypoxic microenvironment and demonstrate a novel way in which the hypoxic niche affects HSC fate, via the hypoxia-VEGFA axis.

HIF-1α can act as a tumor suppressor gene in murine Acute Myeloid Leukemia.

Self-renewal of hematopoietic stem cells (HSCs) and leukemia-initiating cells (LICs) has been proposed to be influenced by low oxygen tension (hypoxia). This signaling, related to the cellular localization inside the bone marrow niche and/or influenced by extrinsic factors, promotes the stabilization of hypoxia-inducible factors (HIFs). Whether HIF-1α can be used as a therapeutic target in the treatment of myeloid malignancies remains unknown. We have used 3 different murine models to investigate the role of HIF-1α in acute myeloid leukemia (AML) initiation/progression and self-renewal of LICs. Unexpectedly, we failed to observe a delay or prevention of disease development from hematopoietic cells lacking Hif-1α. In contrast, deletion of Hif-1α resulted in faster development of the disease and an enhanced leukemia phenotype in some of the investigated models. Our results therefore warrant reconsideration of the role of HIF-1α and, as a consequence, question its generic therapeutic usefulness in AML.

Molecular and cellular effects of oncogene cooperation in a genetically accurate AML mouse model

Biallelic CEBPA mutations and FMS-like tyrosine kinase receptor 3 (FLT3) length mutations are frequently identified in human acute myeloid leukemia (AML) with normal cytogenetics. However, the molecular and cellular mechanisms of oncogene cooperation remain unclear because of a lack of disease models. We have generated an AML mouse model using knockin mouse strains to study cooperation of an internal tandem duplication (ITD) mutation in the Flt3 gene with commonly observed CCAAT/enhancer binding protein alpha (C/EBPα) mutations. This study provides evidence that FLT3 ITD cooperates in leukemogenesis by enhancing the generation of leukemia-initiating granulocyte-monocyte progenitors (GMPs) otherwise prevented by a block in differentiation and skewed lineage priming induced by biallelic C/EBPα mutations. These cellular changes are accompanied by an upregulation of hematopoietic stem cell and STAT5 target genes. By gene expression analysis in premalignant populations, we further show a role of FLT3 ITD in activating genes involved in survival/transformation and chemoresistance. Both multipotent progenitors and GMP cells contain the potential to induce AML similar to corresponding cells in human AML samples showing that this model resembles human disease.

Hematopoietic stem cells are regulated by Cripto, as an intermediary of HIF‐1α in the hypoxic bone marrow niche

Cripto has been known as an embryonic stem (ES)‐ or tumor‐related soluble/cell membrane protein. In this study, we demonstrated that Cripto has a role as an important regulatory factor for hematopoietic stem cells (HSCs). Recombinant Cripto sustained the reconstitution ability of HSCs in vitro. Flow cytometry analysis uncovered that GRP78, one of the candidate receptors for Cripto, was expressed on a subset of HSCs and could distinguish dormant/myeloid‐biased HSCs and active/lymphoid‐biased HSCs. Cripto is expressed in hypoxic endosteal niche cells where GRP78+ HSCs mainly reside. Proteomics analysis revealed that Cripto‐GRP78 binding stimulates glycolytic metabolism‐related proteins and results in lower mitochondrial potential in HSCs. Furthermore, conditional knockout mice for HIF‐1α, a master regulator of hypoxic responses, showed reduced Cripto expression and decreased GRP78+ HSCs in the endosteal niche area. Thus, Cripto‐GRP78 is a novel HSC regulatory signal mainly working in the hypoxic niche.

Hypoxic induction of vascular endothelial growth factor regulates erythropoiesis but not hematopoietic stem cell function in the fetal liver.

Hypoxia is an important factor in the hematopoietic stem cell (HSC) niche in the bone marrow, but whether it also plays a role in the regulation of fetal liver (FL) HSCs is unclear. Vascular endothelial growth factor A (VEGFA) is essential for adult HSC survival, and hypoxic induction of VEGFA in adult HSCs is required for proper function. Loss of hypoxia-regulated VEGFA expression increases the number of phenotypically defined hematopoietic stem and progenitor cells in the FL, but whether stem cell function is affected in FL HSCs has not, to our knowledge, been assessed. We show that fetal erythropoiesis is severely impaired when hypoxic induction of VEGFA is lacking. FL HSCs deficient for hypoxia-induced VEGFA expression have normal HSC function, arguing against a hypoxic FL HSC niche. However, after adaptation of FL HSCs to the bone marrow microenvironment, FL HSCs lose their function, as measured by serial transplantation.

(H)if blood stem cells gO 2 sleep: What can wake them up?

Comment on: Rehn M, et al. Blood 2011; 118:1534-43.