Hypoxic Culture Conditions Research Articles

Effect of simvastatin on the proliferation, apoptosis and migration of hypoxic A375 melanoma cells

Objective To evaluate the regulatory effect of simvastatin on the proliferation, apoptosis and migration of hypoxic A375 melanoma cells, and to explore their molecular mechanisms. Methods Under normoxic or hypoxic culture conditions, A375 cells were treated with 0.25, 0.5, 1, 2, 4, 8 μmol/L simvastatin (simvastatin groups) and dimethyl sulfoxide (DMSO, control group) separately for 48 hours. Cell counting kit-8 (CCK-8) assay, Transwell assay using immersion culture system and flow cytometry using annexin-V/propidium iodide (PI) staining were conducted to assess the proliferative and migratory activities and apoptosis of A375 cells, respectively. RT-PCR and Western blot analysis were performed to measure the mRNA and protein expression of hypoxia-inducible factor 1α (HIF-1α) , survivin and cyclin-dependent kinase inhibitor p27 in the simvastatin groups and control group, as well as in the HIF-1α-silenced hypoxic A375 cells. Results Under normoxic or hypoxic culture conditions, there were significant differences in the proliferative activities of A375 cells, proportion of apoptotic cells and number of migrating cells between the simvastatin groups and control groups (F= 95.84, 37.22 and 177.5 respectively, P < 0.001) . The hypoxic control group showed significantly higher cellular proliferative activity (1.391 ± 0.129) and higher number of migrating cells (322.550 ± 26.226) compared with the normoxic control group (0.807 ± 0.049, 125.583 ± 17.256 respectively, both P < 0.001) and hypoxia+ simvastatin group (0.685 ± 0.417, 115.167 ± 12.050 respectively, both P < 0.001) , but significantly lower proportion of apoptotic cells (6.167% ± 2.714%) compared with the normoxic control group (11.833% ±2.483%, P < 0.01) and hypoxia+ simvastatin group (17.833% ± 2.714%, P < 0.01) . Under the hypoxic condition, the simvastatin group showed significantly lower mRNA and protein expression of HIF-1α and survivin compared with the control group (all P < 0.05) , but significantly higher mRNA and protein expression of P27 compared with the control group (both P < 0.05) . Under the hypoxic condition, HIF-1α-silenced A375 cells showed significantly decreased mRNA and protein expression of survivin (t= 5.346 and 8.281 respectively, both P < 0.05) , but significantly increased mRNA and protein expression of p27 (t= 31.37 and 9.954 respectively, both P < 0.01) compared with the unsilenced cells. Conclusion Simvastatin can inhibit the proliferation and migration of hypoxic A375 cells, and promote their apoptosis, likely by activating the HIF signaling pathway. Key words: Melanoma; Cell line, tumor; Anoxia; Cell proliferation; Apoptosis; Cell migration assays; Survivin; Hypoxia inducible factor; Simvastatin

In Vitro Analysis of the Differentiation Capacity of Postmortally Isolated Human Chondrocytes Influenced by Different Growth Factors and Oxygen Levels.

In the present in vitro study, we analyzed the chondrogenic differentiation capacity of human chondrocytes postmortally isolated from unaffected knee cartilage by the addition of transforming growth factor-β1 (TGF-β1) and/or insulin-like growth factor-1 (IGF-1) and different oxygen levels. After 14 and 35 days, DNA concentrations and protein contents of Col1, Col2, aggrecan as well as glycosaminoglycans (GAGs) of chondrocytes cultivated as pellet cultures were analyzed. Additionally, expression rates of mesenchymal stem cell (MSC)-associated differentiation markers were assessed in monolayer cultures. All cultivated chondrocytes were found to be CD29+/CD44+/CD105+/CD166+. Chondrocytic pellets stimulated with TGF-β1 showed enhanced synthesis rates of hyaline cartilage markers and reduced expression of the non-hyaline cartilage marker Col1 under hypoxic culture conditions. Our results underline the substantial chondrogenic potential of human chondrocytes postmortally isolated from unaffected articular knee cartilage especially in case of TGF-β1 administration.

Energy metabolism in glioblastoma stem cells: PPARα a metabolic adaptor to intratumoral microenvironment.

Glioblastoma (GB), the most-common cancer in the adult brain, despite surgery and radio/ chemotherapy, is to date almost incurable. Many hypoxic tumors, including GB, show metabolic reprogramming to sustain uncontrolled proliferation, hypoxic conditions and angiogenesis. Peroxisome Proliferator-activated Receptors (PPAR), particularly the α isotype, have been involved in the control of energetic metabolism. Herein, we characterized patient-derived GB neurospheres focusing on their energetic metabolism and PPARα expression. Moreover, we used a specific PPARα antagonist and studied its effects on the energetic metabolism and cell proliferation/survival of GB stem cells. The results obtained demonstrate that tumor neurospheres are metabolically reprogrammed up-regulating glucose transporter, glucose uptake and glycogen and lipid storage, mainly under hypoxic culture conditions. Treatment with the PPARα antagonist GW6471 resulted in decreased cell proliferation and neurospheres formation. Therefore, PPARα antagonism arises as a potent new strategy as adjuvant to gold standard therapies for GB for counteracting recurrences and opening the way for pre-clinical trials for this class of compounds. When tumor neurospheres were grown in hypoxic conditions in the presence of different glucose concentrations, the most diluted one (0.25g/L) mimicking the real concentration present in the neurosphere core, PPARα increase/PPARγ decrease, increased proliferation and cholesterol content, decreased glycogen particles and LDs were observed. All these responses were reverted by the 72 h treatment with the PPARα antagonist.

Cytoplasmic overexpression of RNA-binding protein HuR is a marker of poor prognosis in meningioma, and HuR knockdown decreases meningioma cell growth and resistance to hypoxia.

HuR regulates cytoplasmic mRNA stability and translatability, and the HuR expression level has been shown to correlate with poor disease outcome in several cancer types; however, the prognostic value and potential pro-oncogenic properties of HuR in meningioma remain unclear. Thus, in the present study, we analysed 85 meningioma tissue samples to establish the relationship between HuR expression, tumour cell proliferation, and/or patient survival. In addition, we examined the anti-proliferative effects of HuR knockdown in two meningioma cell lines (IOMM-Lee and Ben-Men-1) and conducted transcriptome-wide analyses (IOMM-Lee cells) to elucidate the molecular consequences of HuR knockdown. The results of the present study showed HuR cytoplasmic expression to correlate positively with tumour grade (p = 1.2 × 10-8 ) and negatively with progression-free and overall survival (p = 0.01) time in human meningioma tissues. In vitro, siHuR-induced HuR knockdown was shown to reduce the growth of both Ben-Men-1 (p = 2 × 10-8 ) and IOMM-Lee (p = 4 × 10-9 ) cells. Transcriptome analyses revealed HuR knockdown in IOMM-Lee cells to deregulate the HIF1A signalling pathway (p = 1.5 × 10-6 ) and to up-regulate the expression of genes essential for the assembly of the cytoplasmic mRNA processing body, global genome nucleotide-excision repair, poly(A)-specific ribonuclease activity, the positive regulation of apoptosis and of cell cycle arrest, and the negative regulation of RNA splicing [p(FDR) < 0.001]. Interestingly, HuR knockdown under hypoxic culture conditions further potentiated the effects of HuR knockdown on cell growth, apoptosis, and HIF1A expression. We thus conclude that cytoplasmic HuR expression is a marker of poor prognosis in meningioma and that HuR is a promising potential therapeutic target for use in tumours refractory to standard therapies. Copyright © 2017 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Targeted CDX2 expression inhibits aggressive phenotypes of colon cancer cells in vitro and in vivo.

Loss of caudal type homeobox 2 (CDX2) is associated with the development of human colorectal cancer, while human telomerase reverse transcriptase (hTERT) frequently occurs in variety of human cancers. We investigated the effects of restoration of CDX2 expression using a hypoxia-inducible hTERT promoter-driven vector (pLVX-5HRE-hTERTp-CDX2-3FLAG) on colon cancer cell viability, cell cycle distribution, apoptosis, colony formation, invasion ability and xenograft tumor growth in nude mice. CDX2 overexpression significantly inhibited viability, colony formation, and the invasion and migration ability of LoVo cells, and induced cell cycle arrest and apoptosis in vitro, especially under hypoxic culture conditions. Overexpression of CDX2 under normoxic conditions significantly suppressed the expression of TGF-β, cyclin D1, uPA, MMP-9, MMP-2, and Bcl-2, and stimulated the expression of collagen IV, laminin-1, and Bax. Overexpression of CDX2 reduced colon cancer xenograft tumor formation in nude mice which was associated with downregulation of Ki-67. In conclusion, overexpression of CDX2 using a hypoxia-inducible hTERT promoter-driven vector suppressed malignant progression of colon cancer cells in vitro and in vivo. These results suggest that pLVX-5HRE-hTERTp-CDX2-3FLAG gene therapy may be a promising novel approach to treat colon cancer.

Hypoxia Epigenetically Confers Astrocytic Differentiation Potential on Human Pluripotent Cell-Derived Neural Precursor Cells.

SummaryHuman neural precursor cells (hNPCs) derived from pluripotent stem cells display a high propensity for neuronal differentiation, but they require long-term culturing to differentiate efficiently into astrocytes. The mechanisms underlying this biased fate specification of hNPCs remain elusive. Here, we show that hypoxia confers astrocytic differentiation potential on hNPCs through epigenetic gene regulation, and that this was achieved by cooperation between hypoxia-inducible factor 1α and Notch signaling, accompanied by a reduction of DNA methylation level in the promoter region of a typical astrocyte-specific gene, Glial fibrillary acidic protein. Furthermore, we found that this hypoxic culture condition could be applied to rapid generation of astrocytes from Rett syndrome patient-derived hNPCs, and that these astrocytes impaired neuronal development. Thus, our findings shed further light on the molecular mechanisms regulating hNPC differentiation and provide attractive tools for the development of therapeutic strategies for treating astrocyte-mediated neurological disorders.

Gene expression analysis of growth factor receptors in human chondrocytes in monolayer and 3D pellet cultures.

The main goal of cartilage repair is to create functional tissue by enhancing the in vitro conditions to more physiological in vivo conditions. Chondrogenic growth factors play an important role in influencing cartilage homeostasis. Insulin-like growth factor (IGF)-1 and transforming growth factor (TGF)-β1 affect the expression of collagen type II (Col2) and glycosaminoglycans (GAGs) and, therefore, the targeted use of growth factors could make chondrogenic redifferentiation more efficient. In the present study, human chondrocytes were postmortally isolated from healthy articular cartilage and cultivated as monolayer or 3D pellet cultures either under normoxia or hypoxia and stimulated with IGF-1 and/or TGF-β1 to compare the impact of the different growth factors. The mRNA levels of the specific receptors (IGF1R, TGFBR1, TGFBR2) were analyzed at different time points. Moreover, gene expression rates of collagen type 1 and 2 in pellet cultures were observed over a period of 5 weeks. Additionally, hyaline-like Col2 protein and sulphated GAG (sGAG) levels were quantified. Stimulation with IGF-1 resulted in an enhanced expression of IGF1R and TGFBR2 whereas TGF-β1 stimulated TGFBR1 in the monolayer and pellet cultures. In monolayer, the differences reached levels of significance. This effect was more pronounced under hypoxic culture conditions. In pellet cultures, increased amounts of Col2 protein and sGAGs after incubation with TGF-β1 and/or IGF-1 were validated. In summary, constructing a gene expression profile regarding mRNA levels of specific growth factor receptors in monolayer cultures could be helpful for a targeted application of growth factors in cartilage tissue engineering.

Protective Effects of Müller Glia Cells Towards Retinal Ganglion Cells

Müller glial cells carry out different tasks to warrant normal retinal functions. The aim of this study was to investigate if Müller cells also support retinal ganglion cells (RGC). RGC were cultured for 24 hours in the presence or absence of Müller glial cells under normoxic (20% O2, 5% CO2) or hypoxic (0.2% O2, 5% CO2, 94.8% N2) culture conditions. The number of vital RGC and the length of the newly developed neurites were evaluated. Under normoxic conditions, RGC vitality was significantly higher (p < 0.01) when cultured with Müller cells (62.85 ± 2.06%) than without (47.29 ± 2.83%). Under hypoxia, RGC vitality was significantly higher (p < 0.01) in co-cultures (41.07 ± 2.28%) than in homotypic RGC cultures (28.49 ± 2.16%). The maximum length of the newly developed neurites was found in the normoxic co-culture (90.7 ± 7.4 µm), but showed only a minor difference (p = 0.04) when compared to the normoxic homotypic RGC culture. Müller glial cells support RGC under normoxic and hypoxic culture conditions. Length of newly developed neurites and number of surviving RGC are both parameters to evaluate cell vitality.

Effects of Short Term Hypoxia-Preconditioning on Glial Phenotype Induction of Human Mesenchymal Stem Cells

Development of successful clinical treatments for peripheral nerve injury is limited due to the complications behind neural physiology. Human mesenchymal stem cells (hMSCs) have the ability to directly promote tissue repair and protect cells at the injury site. Studies have shown that hMSCs can be transplanted to improve nerve regeneration. Hypoxic culture condition has been proven to maintain the stemness of hMSCs for later differentiation. In this study, we investigated the effects of low oxygen (O2) (2% and 5% O2) pre-treatment and initial seeding density (500, 1000, and 2000 cells/cm2) on glial protein expression during glial differentiation of hMSCs. Results showed that the secretion of glial proteins was tunable by modifying the seeding density. Moreover, glial induction of hMSCs, characterized by the glial fibrillary acidic protein (GFAP) and S100β expressing phenotype, were enhanced by short-term hypoxia pretreatment. The significantly increased gene expression, including GFAP (10 folds in 2% O2, 25 folds in 5% O2), 2’,3’-Cyclic Nucleotide 3’ Phosphodiesterase (CNP) (600 folds in 2% O2, 800 folds in 5% O2), and neural growth factor receptor (NGFR) (4 folds in 5% O2), indicated that low oxygen, especially 5% O2 pretreated hMSCs had an improved potential for peripheral nerve regeneration.

Effect of hypoxia on the proliferation of porcine bone marrow-derived mesenchymal stem cells and adipose-derived mesenchymal stem cells in 2- and 3-dimensional culture

ObjectiveBone marrow-derived mesenchymal stem cells (MSCs) and adipose-derived mesenchymal stem cells (ASCs) currently represent a promising tool for the regeneration of large bony defects. Therefore, it is pivotal to find the best cell source within the body and the best conditions for in vitro cellular expansion. This study compared cellular response of MSCs and ASCs from a porcine animal in normoxic (21% O2) and hypoxic (2% O2) cell culture conditions via 2D and 3D experimental settings. Materials and methodsThe effect of constant exposure to hypoxia on primary pig stem cells was evaluated by two methods. First, a cumulative population doublings (cumPD) over a period of 40 days, a metabolic activity assay in both 2D and 3D beta-TCP-PHB scaffolds, followed by analysis of osteogenic differentiation potential in cell monolayers. ResultsOur results displayed enhanced cell culture proliferation in 2% O2 for both MSCs and ASCs, with impaired osteogenic differentiation of MSCs. The impact of constant hypoxia on porcine MSCs and ASCs exhibited a statistically significant decrease in osteogenic differentiation under hypoxic conditions with the MSCs. ConclusionsOur data suggest that MSCs and ASCs expanded in hypoxic culture conditions, might be more suitable for use in the clinical setting where large cell numbers are required. When differentiated in normoxic conditions, MSCs showed the highest osteogenic differentiation potential and might be the best choice of cells with consideration to bone repair.

HIF-1α Promotes Cbfbeta-SMMHC-Induced Acute Myeloid Leukemia Development

Acute myeloid leukemia (AML) develops by the acquisition of genomic alterations which initiate different pathways leading to full-blown malignancy. The type of genomic driver alteration governs AML biology and clinical disease course. Translocations involving the core-binding factor (CBF) complex such as t(8:21) and inv(16) represent distinct subgroups of AML genomic drivers. However, additional factors promoting CBF AML leukemogenesis remain largely unknown. Due to the hypoxic nature of the bone marrow the activation of the ubiquitous hypoxia-sensing pathway via HIF-1a might promote CBF leukemogenesis. Nevertheless, in different AML models HIF proteins were recently considered as oncogenic drivers and also as tumor suppressors. Therefore, we here aimed to clarify the role of HIF-1a in CBF AML.Initially, we investigated the impact of hypoxia-sensing pathway activation on the growth of the CBF AML cell lines Kasumi-1 [t(8:21)] and ME-1 [inv(16)]. Kasumi-1 cells showed increased growth after 24 as well as 48 hours at 3% compared to 21% oxygen conditions. In contrast, ME-1 cellular growth was not altered by hypoxic culture conditions. Next, we genetically modified the HIF-1a pathway in the CBF AML leukemogenesis context with a previously established conditional inv(16) mouse model (Cbfb+/56M). These mice express the Cbfbeta-SMMHC oncoprotein upon Cre-mediated recombination. In preliminary studies we could show neither vav-iCre mediated Hif-1a gain- nor loss-of-function altered steady-state hematopoiesis, which was recently confirmed by others (Milica Vukovic et al., Blood 2016). We proceeded by crossing conditional inv(16) with conditional mice overexpressing a HIF-1a variant (LSL-HIF1dPA) being insensitive to oxygen-dependent degradation. The conditional inv(16) and HIF1dPA alleles were simultaneously activated using the ubiquitous hematopoietic vav-iCre transgene. Strikingly, we observed accelerated leukemia development in inv16;HIF1dPA mice compared to inv(16) mice without genetic HIF-1a pathway alterations (median survival 68 vs. 116 days; Figure 1). In a complementary approach we crossed the vavi-Cre conditional inv(16) system into a conditional Hif-1a knock-out (KO) background [inv(16);Hif-1aKO mice]. Here we observed significantly delayed leukemia onset in inv(16);Hif-1aKO mice compared to inv(16) control mice (median survival 223 vs. 116 days; Figure 1). The gross leukemic phenotype with high peripheral leukocyte count, splenomegaly and liver infiltration did not differ between the groups. Finally, we analyzed deletion of the conditional Hif-1aKO allele of leukemic inv(16);Hif-1aKO mice and observed complete recombination resulting in a Hif-1a null constellation. Thus, AML in inv(16);Hif-1aKO mice did not develop from clones which evaded vav-iCre mediated Hif-1a deletion.In conclusion, HIF-1a represents a critical factor promoting murine inv(16) leukemogenesis and it will be worthwhile studying the impact of genetic or pharmacologic HIF-1a alteration in established inv(16) leukemia. [Display omitted] DisclosuresDührsen:Roche: Honoraria, Research Funding; Alexion Pharmaceuticals: Honoraria, Research Funding; Amgen: Honoraria, Research Funding. Göthert:Proteros: Consultancy; Ariad Pharmaceuticals: Consultancy; Bristol-Myers Squibb: Honoraria; Novartis: Honoraria.

Mitochondrial-Targeted Decyl-Triphenylphosphonium Enhances 2-Deoxy-D-Glucose Mediated Oxidative Stress and Clonogenic Killing of Multiple Myeloma Cells.

Therapeutic advances have markedly prolonged overall survival in multiple myeloma (MM) but the disease currently remains incurable. In a panel of MM cell lines (MM.1S, OPM-2, H929, and U266), using CD138 immunophenotyping, side population staining, and stem cell-related gene expression, we demonstrate the presence of stem-like tumor cells. Hypoxic culture conditions further increased CD138low stem-like cells with upregulated expression of OCT4 and NANOG. Compared to MM cells, these stem-like cells maintained lower steady-state pro-oxidant levels with increased uptake of the fluorescent deoxyglucose analog. In primary human MM samples, increased glycolytic gene expression correlated with poorer overall and event-free survival outcomes. Notably, stem-like cells showed increased mitochondrial mass, rhodamine 123 accumulation, and orthodox mitochondrial configuration while more condensed mitochondria were noted in the CD138high cells. Glycolytic inhibitor 2-deoxyglucose (2-DG) induced ER stress as detected by qPCR (BiP, ATF4) and immunoblotting (BiP, CHOP) and increased dihydroethidium probe oxidation both CD138low and CD138high cells. Treatment with a mitochondrial-targeting agent decyl-triphenylphosphonium (10-TPP) increased intracellular steady-state pro-oxidant levels in stem-like and mature MM cells. Furthermore, 10-TPP mediated increases in mitochondrial oxidant production were suppressed by ectopic expression of manganese superoxide dismutase. Relative to 2-DG or 10-TPP alone, 2-DG plus 10-TPP combination showed increased caspase 3 activation in MM cells with minimal toxicity to the normal hematopoietic progenitor cells. Notably, treatment with polyethylene glycol conjugated catalase significantly reduced 2-DG and/or 10-TPP-induced apoptosis of MM cells. Also, the combination of 2-DG with 10-TPP decreased clonogenic survival of MM cells. Taken together, this study provides a novel strategy of metabolic oxidative stress-induced cytotoxicity of MM cells via 2-DG and 10-TPP combination therapy.

Effect of the PI3K/AKT signaling pathway on hypoxia-induced proliferation and differentiation of bone marrow-derived mesenchymal stem cells.

Bone marrow-derived mesenchymal stem cell (BM-MSC) transplantation has been demonstrated to be an effective way of augmenting angiogenesis of ischemic tissue. The low oxygen conditions in ischemic tissue directly affect the biological behavior of engrafted cells. However, to date, the mechanism through which hypoxia regulates self-renewal, differentiation and paracrine function of BM-MSCs remains unclear. Clarification of this mechanism would be beneficial to the use of stem cell-based therapy. The PI3K/AKT pathway has been extensively investigated for its role in cell proliferation, cell transformation, paracrine function and angiogenesis. The present study aimed to analyze the role of PI3K/AKT pathway in hypoxia-induced proliferation of BM-MSCs and their differentiation into endothelial cells in vitro by the application of LY294002, a PI3K/AKT pathway inhibitor, with cells cultured in normoxia serving as a control. The results showed that rat BM-MSCs at passage 3 and 4 displayed only few phenotypical differences in the expression of surface antigens as detected by flow cytometry. When compared with the cells treated in normoxia, the proliferation of BM-MSCs in hypoxia was promoted, a greater number of cells expressed CD31 and a higher expression of vascular endothelial growth factor was observed after culture in hypoxic conditions. However, by inhibiting with LY294002, these changes induced by hypoxia were partly inhibited. In conclusion, the present study showed that the PI3K/AKT pathway served an important role in hypoxia-enhanced in vitro proliferation of BM-MSCs and their differentiation into endothelial cells and paracrine vascular endothelial growth factor.

Germinal Center Hypoxia Potentiates Immunoglobulin Class Switch Recombination.

Germinal centers (GCs) are anatomic sites where B cells undergo secondary diversification to produce high-affinity, class-switched Abs. We hypothesized that proliferating B cells in GCs create a hypoxic microenvironment that governs their further differentiation. Using molecular markers, we found GCs to be predominantly hypoxic. Compared to normoxia (21% O2), hypoxic culture conditions (1% O2) in vitro accelerated class switching and plasma cell formation and enhanced expression of GL-7 on B and CD4+ T cells. Reversal of GC hypoxia in vivo by breathing 60% O2 during immunization resulted in reduced frequencies of GC B cells, T follicular helper cells, and plasmacytes, as well as lower expression of ICOS on T follicular helper cells. Importantly, this reversal of GC hypoxia decreased Ag-specific serum IgG1 and reduced the frequency of IgG1+ B cells within the Ag-specific GC. Taken together, these observations reveal a critical role for hypoxia in GC B cell differentiation.

Hybrid Congregation of Islet Single Cells and Curcumin-Loaded Polymeric Microspheres as an Interventional Strategy to Overcome Apoptosis Associated with Pancreatic Islets Transplantation

Hypoxic or near-anoxic conditions that occur in the core of transplanted islets induce necrosis and apoptosis during the early stages after transplantation, primarily due to loss of vascularization during the isolation process. Moreover, secretion of various cytokines from pancreatic islets is detrimental to the viability of islet cells in vitro. In this study, we aimed to protect pancreatic islet cells against apoptosis by establishing a method for in situ delivery of curcumin to the pancreatic islets. Self-assembled heterospheroids composed of pancreatic islet cells and curcumin-loaded polymeric microspheres were prepared by the three-dimensional cell culture technique. Release of curcumin in the microenvironment of pancreatic islets promoted survival of the islets. In hypoxic culture conditions, which mimic the in vivo conditions after transplantation, viability of the islets was significantly improved, as indicated by a decreased expression of pro-apoptotic protein and an increased expression of anti-apoptotic protein. Additionally, oxidative stress-induced cell death was suppressed. Thus, unlike co-transplantation of pancreatic islets and free microspheres, which provided a wide distribution of microspheres throughout the transplanted area, the heterospheroid transplantation resulted in colocalization of pancreatic islet cells and microspheres, thereby exerting beneficial effects on the cells.

Hypoxia Suppresses Spontaneous Mineralization and Osteogenic Differentiation of Mesenchymal Stem Cells via IGFBP3 Up-Regulation.

Hypoxia has diverse stimulatory effects on human adipose-derived stem cells (ASCs). In the present study, we investigated whether hypoxic culture conditions (2% O2) suppress spontaneous mineralization and osteogenic differentiation of ASCs. We also investigated signaling pathways and molecular mechanisms involved in this process. We found that hypoxia suppressed spontaneous mineralization and osteogenic differentiation of ASCs, and up-regulated mRNA and protein expression of Insulin-like growth factor binding proteins (IGFBPs) in ASCs. Although treatment with recombinant IGFBPs did not affect osteogenic differentiation of ASCs, siRNA-mediated inhibition of IGFBP3 attenuated hypoxia-suppressed osteogenic differentiation of ASCs. In contrast, overexpression of IGFBP3 via lentiviral vectors inhibited ASC osteogenic differentiation. These results indicate that hypoxia suppresses spontaneous mineralization and osteogenic differentiation of ASCs via intracellular IGFBP3 up-regulation. We determined that reactive oxygen species (ROS) generation followed by activation of the MAPK and PI3K/Akt pathways play pivotal roles in IGFBP3 expression under hypoxia. For example, ROS scavengers and inhibitors for MAPK and PI3K/Akt pathways attenuated the hypoxia-induced IGFBP3 expression. Inhibition of Elk1 and NF-κB through siRNA transfection also led to down-regulation of IGFBP3 mRNA expression. We next addressed the proliferative potential of ASCs with overexpressed IGFBP3, but IGFBP3 overexpression reduced the proliferation of ASCs. In addition, hypoxia reduced the osteogenic differentiation of bone marrow-derived clonal mesenchymal stem cells. Collectively, our results indicate that hypoxia suppresses the osteogenic differentiation of mesenchymal stem cells via IGFBP3 up-regulation.

Differential effect of hypoxia on early endothelial–mesenchymal transition response to transforming growth beta isoforms 1 and 2

Angiogenesis is essential for mammalian development and tissue homeostasis, and is involved in several pathological processes, including tumor growth and dissemination. Many factors within the tissue microenvironment are known to modulate angiogenesis, including cytokines, such as transforming growth factor beta (TGFβ), and oxygen level. TGFβ exists in three different isoforms (1, 2 and 3), all of which (albeit in different contexts) might mediate angiogenesis and are able to induce endothelial–mesenchymal transition (EndoMT), a process involved in heart development, pathologic fibrosis and, as recently reported, in angiogenesis. Low oxygen level, referred to as hypoxia, has been independently shown to induce angiogenesis, modulate TGFβ signalling and promote EndoMT. However, how these phenomena might be interconnected to drive angiogenesis is rather unexplored. To begin addressing the potential contribution of TGFβ-induced EndoMT to angiogenesis, and to explore how microenvironmental hypoxia might influence these processes, we investigated the effect of TGFβ isoforms 1 and 2 on early EndoMT response in cultured adult endothelium under standard (21 %) and hypoxic (1 %) culture conditions. Our data indicates that EndoMT-like changes, such as an increase in expression and nuclear translocation of Snail, Slug and Zeb1, and reduction of VE-cadherin expression, occur in response to TGFβ1 and/or TGFβ2 as early as 6h after stimulation and might be enhanced by hypoxia in an isoform-specific manner. Further, hypoxia enhances canonical TGFβ signalling, and appears to be a key determinant of Snail's differential involvement in endothelial cell responses to TGFβ1 versus TGFβ2.

Effect of Hypoxia on Stemness and Differentiation of Dental Pulp Derived Stem Cells

Stem Cells are characterized by their properties of self renewal and multipotency. These stem cells are localized inside our body at specialized niches, which are known to have low oxygen tension. Dental pulp tissue found in the middle of the tooth is one such niche which experiences hypoxic conditions due to its anatomical location. However under in-vitro conditions, stem cells are cultured under normoxia condition (21% O2 concentration). It has been known since decades that low oxygen tension in tissue cultures has positive effect on the cell growth. In this study, we tested this hypothesis on in vitro isolated, expanded, Dental Pulp Stem Cells (DPSCs) grown under low oxygen. The comparative evaluation was done between DPSCs grown in normoxia (21% O2) and Hypoxia (3% O2) on the basis of proliferation, aging, maintenance of stemness and differentiation potential. Our study indicates that DPSCs when grown in hypoxia exhibited better renewal capacity, lesser population time and hence decreased differentiation. Thus, this study emphasizes that hypoxic culture conditions is better in vitro culture condition for expansion of DPSCs in large scale and maintenance of their stemness for therapeutic purpose.

NR2F2 regulates chondrogenesis of human mesenchymal stem cells in bioprinted cartilage.

Bioprinting as an advanced enabling technology has the capacity to construct tissues with respective anatomical structures. In order to maintain the precise printing resolution for anatomical tissue printing, cell seeding density in bioink is limited. Bone marrow derived mesenchymal stem cells (MSCs) are widely used for cartilage tissue engineering. However, the approach of ideal chondrogenic differentiation of MSCs without hypertrophy still remains elusive. Here, we reported NR2F2 plays a crucial role in MSC chondrogenesis in bioprinted cartilage. NR2F2 over-expressed MSCs showed significantly enhanced chondrogenesis and NR2F2 knockdown cells demonstrated the exactly opposite behavior. We evaluated the cells cultured in monolayer, 3D pellet, and bioprinted 3D scaffold. All observations were consistent among gene expression, biochemical analysis, histological assay, and biomechanical evaluation. The data also revealed possible involvement of NR2F2 in mechanism of MSC chondrogenic differentiation under hypoxic culture condition. Biotechnol. Bioeng. 2017;114: 208-216. © 2016 Wiley Periodicals, Inc.

Abstract 2799: Exosomes from hypoxic pancreatic cancer cells confer resistance to subsequent hypoxia insult

Abstract Pancreatic cancer (PC) remains one of the leading causes of cancer-associated deaths in the US due to its highly aggressive nature and lack of effective treatments. Pancreatic tumors are characterized by a dense, desmoplastic reaction and poor vasculature leading to reduced oxygen levels within the tumor microenvironment. It is believed that this encounter of pancreatic tumor cells to intratumoral hypoxic condition is a major cause of their persistent survival under a variety of stresses, increased aggressiveness and therapeutic-resistance. However, underlying molecular mechanisms responsible for PC's adaptation to hypoxia (along with enhanced growth and aggressiveness) remain elusive. In this study, we examined the role of exosomes, which are nanoscale biological vesicles with a lipid bilayer membrane, in imparting survival benefit to PC cells. For this, we first examined the effect of conditioned media (CM) from PC cells cultured under hypoxic (0.1% O2, H-CM) or normoxic (21% O2, N-CM) conditions on the growth of fresh PC cells cultured under hypoxic (0.1% O2) conditions. We found that H-CM conferred significant survival benefit to cancer cells under hypoxia as compared to N-CM. Thereafter, we isolated exosomes from N-CM (N-exo) or H-CM (H-exo) and used them to treat pancreatic cancer cells under hypoxic culture conditions. We observed that H-exo imparted a significantly higher survival advantage to cancer cells under hypoxia as compared to N-exo. Interestingly, protein quantification indicated the presence of higher levels of exosomes from cells cultured under hypoxic conditions as compared to those grown in normoxic environment. Western blot analysis with anti-CD9 and anti-CD63 antibodies confirmed enrichment of exosomes. Moreover, Dynamic Light Scattering-based size distribution suggested that H-exo were smaller in size compared to N-exo. Further studies are underway to identify the molecular determinants in exosomes responsible for inducing growth-promoting effects under hypoxia. Overall, our data provides the first evidence that hypoxic PC cells secrete exosomes that enhance survivability under hypoxic conditions. Citation Format: Mary C. Patton, Haseeb Zubair, Girijesh K. Patel, Seema Singh, Ajay P. Singh. Exosomes from hypoxic pancreatic cancer cells confer resistance to subsequent hypoxia insult. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2799.