Physiological Hypoxia Enhances Stemness Preservation, Proliferation, and Bidifferentiation of Induced Hepatic Stem Cells.
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ABSTRACT:
SUBMITTER: Zhi X
PROVIDER: S-EPMC5831960 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:The aim of the study was to obtain the highest number of multipotent adipose-derived mesenchymal stem cells (ADMSCs) by using culture conditions which favour cell expansion without loss of mesenchymal stem cells (MSC)-like properties. Based on the assumption that stem cells reside in niches characterized by hypoxic condition, we investigated if the low oxygen tension may improve the proliferation and stemness of ADMSCs. Intact adipose tissue was resected from eight subjects, and the stromal vascular fraction was obtained by using type II collagenase. The heterogeneity of cellular lineages was confirmed by immunophenotypic analysis that showed the presence of leukocytes (CD45+), endothelial cells (CD34+), and pericytes (CD140+). The immunophenotype of confluent ADMSCs was similar to that of bone marrow-derived MSCs, except for the expression of CD34, which was variable (donor-dependent) and inversely correlated to the CD36 expression. ADMSCs showed a high clonal efficiency (94.5 ± 1 %) and were able to generate osteoblastic, chondrocytic and adipocytic lineages. ADMSCs were cultured under normoxic (21 % O2) and hypoxic (1 % O2) conditions, and we found that hypoxia significantly favoured ADMSC proliferation and preserved the expression of stemness genes, i.e. Nanog and Sox2. Since hypoxia reflects the microenvironment in which ADMSCs must proliferate and differentiate, the culture in hypoxic condition allows to better understand the biology of these cells and their regenerative potential. Low oxygen concentrations promote cell proliferation and stemness, thus enriching the pool of cells potentially able to differentiate into multi-lineages, and extending the possibility of a long-term expansion.
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Project description:BackgroundNeural stem cells (NSCs) represent an optimal tool for studies and therapy of neurodegenerative diseases. We recently established a v-myc immortalized human NSC (IhNSC) line, which retains stem properties comparable to parental cells. Oxygen concentration is one of the most crucial environmental conditions for cell proliferation and differentiation both in vitro and in vivo. In the central nervous system, physiological concentrations of oxygen range from 0.55 to 8% oxygen. In particular, in the in the subventricular zone niche area, it's estimated to be 2.5 to 3%.Methodology/principal findingsWe investigated in vitro the effects of 1, 2.5, 5, and 20% oxygen concentrations on IhNSCs both during proliferation and differentiation. The highest proliferation rate, evaluated through neurosphere formation assay, was obtained at 2.5 and 5% oxygen, while 1% oxygen was most noxious for cell survival. The differentiation assays showed that the percentages of beta-tubIII+ or MAP2+ neuronal cells and of GalC+ oligodendrocytes were significantly higher at 2.5% compared with 1, 5, or 20% oxygen at 17 days in vitro. Mild hypoxia (2.5 to 5% oxygen) promoted differentiation into neuro-oligodendroglial progenitors as revealed by the higher percentage of MAP2+/Ki67+ and GalC+/Ki67+ residual proliferating progenitors, and enhanced the yield of GABAergic and slightly of glutamatergic neurons compared to 1% and 20% oxygen where a significant percentage of GFAP+/nestin+ cells were still present at 17 days of differentiation.Conclusions/significanceThese findings raise the possibility that reduced oxygen levels occurring in neuronal disorders like cerebral ischemia transiently lead to NSC remaining in a state of quiescence. Conversely, mild hypoxia favors NSC proliferation and neuronal and oligodendroglial differentiation, thus providing an important advance and a useful tool for NSC-mediated therapy of ischemic stroke and neurodegenerative diseases like Parkinson's disease, multiple sclerosis, and Alzheimer's disease.
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Project description:Two important criteria of an ideal biomaterial in the field of stem cells research are to regulate the cell proliferation without the loss of its pluripotency and to direct the differentiation into a specific cell lineage when desired. The present study describes the influence of TiO2 nanofibrous surface structures on the regulation of proliferation and stemness preservation of adipose-derived stem cells (ADSCs). TiO2 nanofiber arrays were produced in situ onto Ti-6Al-4V substrate via a thermal oxidation process and the successful fabrication of these nanostructures was confirmed by field emission scanning electron microscopy (FESEM), energy dispersive spectrometer (EDS), X-ray diffractometer (XRD), and contact angle measurement. ADSCs were seeded on two types of Ti-6Al-4V surfaces (TiO2 nanofibers and flat control), and their morphology, proliferation, and stemness expression were analyzed using FESEM, AlamarBlue assay, flow cytometry, and quantitative real-time polymerase chain reaction (qRT-PCR) after 2 weeks of incubation, respectively. The results show that ADSCs exhibit better adhesion and significantly enhanced proliferation on the TiO2 nanofibrous surfaces compared to the flat control surfaces. The greater proliferation ability of TiO2 nanofibrous surfaces was further confirmed by the results of cell cycle assay. More importantly, TiO2 nanofibrous surfaces significantly upregulate the expressions of stemness markers Sox-2, Nanog3, Rex-1, and Nestin. These results demonstrate that TiO2 nanofibrous surfaces can be used to enhance cell adhesion and proliferation while simultaneously maintaining the stemness of ADSCs, thereby representing a promising approach for their potential application in the field of bone tissue engineering as well as regenerative therapies.
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Project description:BackgroundAdipose tissue-derived stem cells (ASCs) have been recently isolated from human subcutaneous adipose tissue. ASCs may be useful in regenerative medicine as an alternative to bone marrow-derived stem cells. Changes in the oxygen concentration influence physiological activities, such as stem cell proliferation. However, the effects of the oxygen concentration on ASCs remain unclear. In the present study, the effects of hypoxia on ASC proliferation were examined.MethodsNormal human adipose tissue was collected from the lower abdomen, and ASCs were prepared with collagenase treatment. The ASCs were cultured in hypoxic (1%) or normoxic (20%) conditions. Cell proliferation was investigated in the presence or absence of inhibitors of various potentially important kinases. Hypoxia inducible factor (HIF)-1α expression and MAP kinase phosphorylation in the hypoxic culture were determined with western blotting. In addition, the mRNA expression of vascular endothelial growth factor (VEGF) and fibroblast growth factor (FGF)-2 in hypoxic or normoxic conditions were determined with real-time RT-PCR. The effects of these growth factors on ASC proliferation were investigated. Chromatin immunoprecipitation (ChIP) of the HIF-1α-binding hypoxia responsive element in FGF-2 was performed. HIF-1α was knocked down by siRNA, and FGF-2 expression was investigated.ResultsASC proliferation was significantly enhanced in the hypoxic culture and was inhibited by ERK and Akt inhibitors. Hypoxia for 5-15 minutes stimulated the phosphorylation of ERK1/2 among MAP kinases and induced HIF-1α expression. The levels of VEGF and FGF-2 mRNA and protein in the ASCs were significantly enhanced in hypoxia, and FGF-2 increased ASC proliferation. The ChIP assay revealed an 8-fold increase in the binding of HIF-1α to FGF-2 in hypoxia. HIF-1α knockdown by siRNA partially inhibited the FGF-2 expression of ASCs induced by hypoxia.ConclusionASC proliferation was enhanced by hypoxia. HIF-1α activation, FGF-2 production, and the ERK1/2 and Akt pathway were involved in this regulatory mechanism.
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Project description:Hypoxic condition influences biological responses in various cell types. However, a hypoxic regulating osteogenic differentiation remains controversy. Here, an influence of short-term culture in hypoxic condition on osteogenic marker gene expression by retinoic acid-treated murine gingival fibroblast-derived induced pluripotent stem cells (RA-miPS) was investigated. Results demonstrated that hypoxic condition significantly upregulated Vegf, Runx2, Osx, and Ocn mRNA expression by RA-miPS in normal culture medium at day 3. Further, desferrioxamine significantly downregulated pluripotent marker (Nanog and Oct4) and enhanced osteogenic marker (Runx2, Osx, Dlx5, and Ocn) gene expression as well as promoted in vitro mineral deposition. However, the effect of cobalt chloride on osteogenic differentiation of RA-miPS was not robust. In summary, the results imply that hypoxic condition may be useful in the enhancement of osteogenic differentiation in RA-miPS.Electronic supplementary materialSupplementary material is available for this article at 10.1007/s13770-016-9127-9 and is accessible for authorized users.
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Project description:Standard culture of human induced pluripotent stem cells (hiPSCs) requires basic Fibroblast Growth Factor (bFGF) to maintain the pluripotent state, whereas hiPSC more closely resemble epiblast stem cells than true naïve state ES which requires LIF to maintain pluripotency. Here we show that chemokine (C-C motif) ligand 2 (CCL2) enhances the expression of pluripotent marker genes through the phosphorylation of the signal transducer and activator of transcription 3 (STAT3) protein. Moreover, comparison of transcriptomes between hiPSCs cultured with CCL2 versus with bFGF, we found that CCL2 activates hypoxia related genes, suggesting that CCL2 enhanced pluripotency by inducing a hypoxic-like response.Further, we show that hiPSCs cultured with CCL2 can differentiate at a higher efficiency than culturing withjust bFGF and we show CCL2 can be used in feeder-free conditions [corrected]. Taken together, our finding indicates the novel functions of CCL2 in enhancing its pluripotency in hiPSCs.
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Project description:Secretome derived from human amniotic fluid stem cells (AFSC-S) is rich in soluble bioactive factors (SBF) and offers untapped therapeutic potential for regenerative medicine while avoiding putative cell-related complications. Characterization and optimal generation of AFSC-S remains challenging. We hypothesized that modulation of oxygen conditions during AFSC-S generation enriches SBF and confers enhanced regenerative and cardioprotective effects on cardiovascular cells. We collected secretome at 6-hourly intervals up to 30 h following incubation of AFSC in normoxic (21%O2, nAFSC-S) and hypoxic (1%O2, hAFSC-S) conditions. Proliferation of human adult cardiomyocytes (hCM) and umbilical cord endothelial cells (HUVEC) incubated with nAFSC-S or hAFSC-S were examined following culture in normoxia or hypoxia. Lower AFSC counts and richer protein content in AFSC-S were observed in hypoxia. Characterization of AFSC-S by multiplex immunoassay showed higher concentrations of pro-angiogenic and anti-inflammatory SBF. hCM demonstrated highest proliferation with 30h-hAFSC-S in hypoxic culture. The cardioprotective potential of concentrated 30h-hAFSC-S treatment was demonstrated in a myocardial ischemia-reperfusion injury mouse model by infarct size and cell apoptosis reduction and cell proliferation increase when compared to saline treatment controls. Thus, we project that hypoxic-generated AFSC-S, with higher pro-angiogenic and anti-inflammatory SBF, can be harnessed and refined for tailored regenerative applications in ischemic cardiovascular disease.
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Project description:Hypoxia is a characteristic of solid tumors especially Glioblastoma and is critical to chemoresistance. Cancer stem cells present in hypoxic niches are known to be a major cause of the progression, metastasis and relapse. We tried to identify synergistic combinations of drugs effective in both hypoxia and normoxia in tumor cells as well as in cancer stem cells. Since COX-2 is over-expressed in subset of glioblastoma and is also induced in hypoxia, we studied combinations of a prototype Cyclooxygenase (COX-2) inhibitor, NS-398 with various drugs (BCNU, Temozolomide, 2-Deoxy-D-glucose and Cisplatin) for their ability to abrogate chemoresistance under both severe hypoxia (0.2% O2) and normoxia (20% O2) in glioma cells. The only effective combination was of NS-398 and BCNU which showed a synergistic effect in both hypoxia and normoxia. This synergism was evident at sub-lethal doses for either of the single agent. The effectiveness of the combination resulted from increased pro- apoptotic and decreased anti-apoptotic molecules and increased caspase activity. PGE2 levels, a manifestation of COX-2 activity were increased during hypoxia, but were reduced by the combination during both hypoxia and normoxia. The combination reduced the levels of epithelial-mesenchymal transition (EMT) markers. It also resulted in a greater reduction of cell migration. While single drugs could reduce the number of gliomaspheres, the combination successfully abrogated their formation. The combination also resulted in a greater reduction of the cancer stem cell marker CD133. This combination could be a prototype of possible therapy in a tumor with a high degree of hypoxia like glioma.
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Project description:The physiological stresses that diminish tissue stem-cell characteristics remain largely unknown. We previously reported that type I interferon (IFN), which is essential for host antiviral responses, is a physiological stressor for hematopoietic stem cells (HSCs) and small intestinal stem cells (ISCs) and that interferon regulatory factor-2 (IRF2), which attenuates IFN signaling, maintains their stemness. Here, using a dextran sodium sulfate (DSS)-induced colitis model, we explore the role of IRF2 in maintaining colonic epithelial stem cells (CoSCs). In mice with a conditional Irf2 deletion in the intestinal epithelium (hereafter Irf2ΔIEC mice), both the number and the organoid-forming potential of CoSCs were markedly reduced. Consistent with this finding, the ability of Irf2ΔIEC mice to regenerate colon epithelium after inducing colitis was severely impaired, independently of microbial dysbiosis. Mechanistically, CoSCs differentiated prematurely into transit-amplifying (TA) cells in Irf2ΔIEC mice, which might explain their low CoSC counts. A similar phenotype was induced in wild-type mice by repeated injections of low doses of poly(I:C), which induces type I IFN. Collectively, we demonstrated that chronic IFN signaling physiologically stresses CoSCs. This study provides new insight into the development of colitis and molecular mechanisms that maintain functional CoSCs throughout life.
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