| Background and Objection:Mesenchymal stem cells are capable of osteogenic, chondrogenic, and adipogenic differentiation. Mesenchymal stem cells attracted more and more attention in recent years.The main research focus on tissue engineering, stem cell therapy for ischemic disease. Stem cell therapy appears brought a new way to cure many diseases. Adipose-derived stromal cells offer distinct advantages over bone marrow-derived mesenchymal stem cells because they are readily accessible, plentiful, and expandable. Therefore, the accessibility, abundance, and multilineage differentiation capacity of adipose-derived stem cells has stimulated tremendous interest in using this cell population for regeneration and replacement of mesenchymal-derived tissues such as bone, cartilage, and muscle.The oxygen concentration in the microenvironment of stem cells plays an important role in controlling stem cell potency, proliferation, and differentiation ability. Stem cells that are normally cultured at ambient air(21%02) in an in vitro environment differ in their exposure to the concentration of oxygen, compared with their natural physiological niches where they reside and function. Mesenchymal stem cells maintained in a "niche" that the oxygen about2%-8%, even lower. During cell transplantation procedures to treat conditions such as myocardial infarction (MI), the cultured stem cells encounter a sudden shortage in oxygen availability when transplanted into an ischemic heart tissue. A growing body of evidence attributes failure of stem cell therapy to the extensive loss of transplanted stem cells, upon introducing them to such a harsh ischemic environment, which is high in inflammation factors and free radicals generated by oxidative stress. Varying the oxygen exposure level while culturing the stem cells may play a major role in determining the survival of the cell as it is an essential adaptive factor for sustaining many of the energy-driven cellular processes along with playing an important role in cell signaling mechanisms that determine the course of a cell’s fate.Recent advances in stem cell-based regenerative therapies including cell therapy and tissue engineering, provide promise as novel therapies to repair and restore tissue function, after disease or trauma. However, the usefulness of stem cell therapies seems to be limited by poor cell survival after implantation. For example, more than90%of implanted cells are lost within the first4days after transplantation to the heart。To improve the survival of stem cells are required. In this regard, preconditioning is considered to be one of the most promising strategies to promote stem cell survival and function in hostile ischemic environments. Although some researchers have tried to add or transfect growth factors to improve the results, However, transfected growth factors are not safe enough to use clinically, and exogenous growth factors have a short half-life, are easily degradable in vivo and cannot be maintained for long time. Therefore preconditioning occurs when cells are able to survive a lethal insult due to prior activation of survival mechanisms by a sub-lethal stimulus, and this is one method shown to improve stem cell survival in harsh environments. Multiple studies have demonstrated that both pharmacological preconditioning and genetic manipulation improves the survival of bone marrow-derived mesenchymal stem cells after implantation into the infarcted heart.Preconditioning with hypoxia is another clinically viable way in which cells can be protected.The present study demonstrates that preconditioning ASCs with hypoxia increases cell survival via a HIF-1a/VEGF/bFGF mediated pathway. Further to this, we found that HP-ASC increases their paracrine effects in promoting endothelial cell survival and proangiogenic responses in vitro, both of which are important elements of efficacy for stem cell-based regenerative therapies. Although ASCs failed to like SVF are widely used in clinical, but it is expected to further research to more benefit the treatment of patients with ischemic diseases.We believe that HP will be a helpful tool for successful cell-based therapies in the near future.Methods and materials1. Isolation and preparation of ASCsHuman adipose tissue (lipoaspirate) of healthy22-to45-year-old donors was obtained from elective liposuction procedures under anaesthesia after informed and written consent was received, as set down in the guidelines approved by the Ethics Committee on the Use of Human Subjects (Southern Medical University and Nan Fang Hospital). Male to Female ratio1:2.The stem cells were isolated from the fatty portion of the liposuction aspirates by using the procedure reported by Zuk et al.#with some modifications. Briefly, the fat tissue was excised, finely minced, digested with0.125%collagenase I (Sigma-Aldrich) and vigorously shaken for30min at37℃in a50-ml centrifuge tube. An equal volume of Dulbecco’s Modified Eagle Medium (DMEM; Gibco) with10%foetal bovine serum (FBS; Hyclone) was then added to neutralize the collagenase. The tissue mixture was passed through a100-mm nylon mesh filter to remove the large, undigested tissue fragments, after which the cell suspension was centrifuged at1200g for5min. Pellets were resuspended in red blood cell lysis buffer (Sigma-Aldrich) and incubated for5min at room temperature. After being washed, filtered cells were cultivated in the DMEM supplemented with10%FBS andl%penicillin-streptomycin (Sigma-Aldrich). The cells were maintained at37℃in5%CO2until use. The medium was replaced every3days, and the non-adherent cells were discarded. Cells were passaged at a ratio of1:3per week. Only cells that had been cultured for three passages were used in this study.2. Hypoxia and normoxia preconditioning protocolFor HP, cells were subcultured1:2and cultured for a total of two passages under normoxic conditions. When the ASCs reached confluence, they were passaged at a ratio of1:2in100-mm culture dishes. Fresh complete medium was then added. HP was achieved with a humidified atmosphere inside a tri-gas incubator (Thermo) containing5%CO2,1%O2and94%N2for48h. For normoxia preconditioning (NP), cells were cultured in an incubator containing5%CO2and21%O2for48h.3. The biology detection of hypoxia preconditioned ASCsWe compared the two groups:hypoxia preconditioned ASCs and normoxia ASCs:Cell proliferation, viability, cell cycle assay, apoptosis. Flow cytometry analysis of cell surface markers. Differentiation assays were carried out at the end of the HP or NP to determine whether both cell populations retained their multi-lineage potential. Both groups were differentiated under the same oxygen conditions (21%O2)..adipogenesis was assessed by oil red O staining after1week of adipogenic differentiation, chondrogenesis was assessed by Alcian blue and osteogenic differentiation were performed at3weeks. Matrix mineralization was quantified by alizarin red staining.All of this measurement is try to clear whether hypoxia preconditioned changed the multiple potential of ASCs4.The mechanism of hypoxia preconditioned ASCs enhance angiogenic potentialASCs were cultured for a total of two passages under normoxic conditions. When the ASCs reached confluence, they were passaged at a ratio of1:2in100-mm culture dishes. The medium was then changed to fresh DMEM. ASCs were exposed to HP or NP for48h. After that, the conditioned media (CM) of ASCs derived from normoxic (nor-CM) and hypoxic (hypo-CM) conditions were collected, centrifuged at256g at4℃for10min, recollected and kept at-80℃until the experiment was performed.The levels of vascular endothelial growth factor (VEGF), basic fibroblast growth factor (bFGF) and hepatocyte growth factor (HGF) proteins in nor-CM and hypo-CM were measured by using a human ELISA kit (R&D) according to the manufacturer’s instructions. qRT-PCR Measured of the effect of hypoxia on growth factor mRNA For the measurement of cell migration, confluent HUVECs were kept in a serum-free medium in a six-well plate for4h and wounded with a plastic micropipette tip with a large orifice. After washing, the medium was replaced with either nor-CM or hypo-CM with neutralizing antibodies against VEGF (10μg/ml anti-VEGF, R&D) or with neutralizing antibodies against bFGF (5μg/ml anti-bFGF, R&D). The effect of the CM on capillary-like tube formation in vitro on Matrigel (BD Bioscience) was evaluated. The formation of vascular-like structures by HUVECs on growth-factor-reduced Matrigel was performed。5. Statistical analysisThe student’s t-test was used to compare data between the two experimental groups. A one-way analysis of variance test was used to compare data among the three experimental groups. Data were expressed as the mean±SD. A p-value of less than0.05was considered statistically significant. The SPSS software package version13.0(SPSS, USA) was used for the statistical tests.6. Results 1.After ASCs were exposed to HP for48h, they remained fully viable, similar to NP control ASCs The morphological appearance of ASCs is unchanged after HP compared with NP.2. Furthermore, exposure to hypoxic conditions did not negatively affect cell survival and proliferation. On the contrary, hypoxia-exposed cells showed a high proliferation rate compared with normoxia-exposed cells. A different percentage of cells in the S phases of the cell cycle was observed in the culture conditions, the HP-ASCs showed higher vitality than NP-ASCs at24h and48h and lower apoptosis in apoptosis assays. This demonstrates that HP-ASCs had an increased capacity to proliferate, even in severe microenvironmental conditions such as low-serum medium. Subjecting them to flow cytometric analysis revealed that NP-ASCs expressed CD90(99.93±0.1%) and CD105(99.92±0.12%), but did not express CD31(0.21±0.14%). Similarly, HP-ASCs expressed CD90(99.93±0.10%) and CD105(99.94±0.04%), but showed low expression of CD31(0.52±0.38%). No obvious differences were observed in the expression of surface antigens (p>0.05). These results indicate that neither the morphological features nor the natural cell surface characteristics of ASCs were altered by HP. We also observed the same result in the chondrogenic, osteogenic, adipogenic differentiation.These results demonstrated that hypoxia preconditioned in ASCs under21%oxygen condition maitained multi-lineage potential in vitro.3. To determine the mechanisms responsible for the observed protection afforded to ASCs by HP, we evaluated gene and protein expression levels of several cytoprotective molecules. Hypoxia inducible factor-1alpha (HIF-la) protein expression was up-regulated in ASCs after HP. Furthermore, compared with NP-ASCs, HP-ASCs expressed significantly increased mRNA levels of the HIF-la downstream target genes VEGF and bFGF; mRNA levels of HGF, however, were not affected by this hypoxic stress. Expression and secretion of growth factors were confirmed by ELISA. HP significantly increased the secretion of VEGF and bFGF HP did not, however, increase HGF secretion significantly, suggesting that under these conditions, the protective effects of HP on ASCs is at least in part related to the production of VEGF and bFGF.4. Compared with nor-CM, hypo-CM significantly increased the viability of HUVECs, also they showed significantly lower apoptosis rates.5.We observed that the migration of HUVECs was enhanced in the hypo-CM, then we neutralized VEGF and bFGF by using specific antibodies and then investigated the migration of HUVECs. The migration of HUVECs was slightly decreased by the addition of neutralizing antibodies against bFGF, but was significantly decreased by the addition of neutralizing antibodies against VEGF. In the normoxia group we abserved the same result. We compared the hypo-CM and nor-CM groups adding the neutralizing antibodies against VEGF or bFGF.The showed no significant difference between the two groups. Therefore, VEGF and bFGF may, at least partly, play a role in the enhancement of angiogenesis by hypoxia.6. To prove that hypoxia treatment did indeed stimulate the production of angiogenic growth factors by ASCs, we tested the ability of hypo-CM to up-regulate the formation of capillary-like structures in HUVECs on Matrigel in vitro. Hypo-CM stimulated the formation of capillary-like structures, whereas anti-VEGF and anti-bFGF in the CM inhibited these structures. We observed the same result in the nor-CM.These data confirmed that hypoxia induces the production of angiogenic factors like VEGF and bFGF by ASCs, which in turn stimulate the formation of capillary-like structures by endothelial cells in vitro.7. DiscussionRecent advances in stem cell-based regenerative therapies including cell therapy and tissue engineering, provide promise as novel therapies to repair and restore tissue function, after disease or trauma. However, the usefulness of stem cell therapies seems to be limited by poor cell survival after implantation. For example, more than90%of implanted cells are lost within the first4days after transplantation to the heart。 To improve the survival of stem cells are required. Although some researchers like lee and lu et al have tried to add or transfect growth factors to improve the results, also get the satisfaction result. In this regard, preconditioning is considered to be one of the most promising strategies to promote stem cell survival and function in hostile ischemic environments. However, transfected growth factors are not safe enough to use clinically, and exogenous growth factors have a short half-life, are easily degradable in vivo and cannot be maintained for long time. Nevertheless, these approaches have limitations, such as the tempo-spatial release control for the growth factor co-delivery and a potential risk of immunogenicity or mutagenesis in case of the genetic modifications. Oxygen concentrations range from4to14%in highly perfused organs (such as lung, liver, kidneys and heart) but are very low in bone marrow (0-4%). Importantly, oxygen concentrations are typically less than3%in adipose tissue. Adipose-derived stromal cells offer distinct advantages over bone marrow-derived mesenchymal stem cells because they are readily accessible, plentiful, and expandable. Therefore, the accessibility, abundance, and multilineage differentiation capacity of adipose-derived stem cells has stimulated tremendous interest in using this cell population for regeneration medicine.Recently, stem/progenitor cells of mesenchymal lineage have been discovered in fat tissue. Among these cells, ASCs can be easily expanded and differentiated into a variety of cells not only along the mesenchymal lineage, but also along hematopoietic and neuronal lineages. Since adipose tissue is abundant and can be obtained repeatedly under local anaesthesia with a minimum of patient discomfort, ASCs have enormous potential as an autologous cell source for regenerative therapy. Tissue damage is often linked to ischaemia and hypoxia. For this reason, it is important to understand the reaction of ASCs to hypoxia, but also because hypoxia can be used to stimulate an increase in angiogenesis of MSCs via secreted factors.In the present study, we revealed through fluorescence-activated cell sorting analysis that HP-ASCs have cell surface markers that are indistinguishable from those of NP-ASCs. HP-ASCs are positive for mesenchymal cell markers such as CD90and CD105and negative for endothelial cell marker CD31, but there tends to be an increased percentage of CD31-positive cells. We also found that the morphological features of HP-ASCs were not clearly different from those of NP-ASCs. Here we confirm that ASCs have the identical morphological features and phenotype when they undergo the process of HP. We also demonstrated that ASCs exhibit a pro-propogation response to hypoxic stress, more percentage of cells in the S phases of the cell cycle was observed in the hypoxia preconditioned, as well as decreased apoptosis even in severe microenvironmental conditions such as low-serum medium Furthermore, hypoxia preconditioned ASCs showed multi-lineage potential differentiation.Hypoxia has been known to regulate several cellular processes and signal transductions via the expression of HIF-1, which is regulated by the cellular O2concentration and determines the transcriptional activity of HIF-1α.Our study showed that there is a significant increase in HIF-1α and its downstream target genes VEGF and bFGF in the hypoxia group, two important angiogenesis factors in the early stage of vascular formation. Similar up-regulation of basal VEGF mRNA by HP was found in MSCs derived from bone marrow. We studied the levels of VEGF, bFGF and HGF in the CM of ASCs. These three molecules have been shown to play important roles in angiogenesis and tissue regeneration and are known to be secreted by ASCs. To our surprise, HP-ASCs secreted a significantly higher amount of VEGF and bFGF than NP-ASCs, although the levels of HGF did not differ between these two groups. In previous research, ASCs have been shown to promote the proliferation and viability of endothelial cells. Hence, we studied the effects of the CM from HP-ASCs and NP-ASCs on HUVECs. We found that hypo-CM enhanced HUVEC proliferation and inhibited apoptosis more efficiently than nor-CM did. Antibodies against VEGF and bFGF can inhibit the formation of capillary-like structures and the migration of. HUVECs. The results suggest that VEGF and bFGF are two major cytokines promoting the repair of vessels and protecting them from injury, although other molecules may also play important roles.Endogenous and environmental factors, such as those involved in the inflammatory response, may contribute to cell death. Thus, improving grafted cell survival after transplantation is critical for enhancing the efficacy and efficiency of stem cell therapy. Several strategies have been proposed to improve the survival of transplanted therapeutic stem cells. In order to better apply ASCs to patients, it is crucial to improve the angiogenic effect of these cells. ASCs produce many angiogenic and anti-apoptotic growth factors, and their secretion is significantly enhanced by hypoxia. HP is a novel strategy to make stem cells resistant to the ischaemic environment they encounter after transplantation into injured tissue; this strategy improves survival of both the transplanted cells and the host cells at the injury site. HP-ASCs may have great potential in regenerative cell therapy for ischaemic disease in the clinical setting. We believe that HP will be a helpful tool for successful cell-based therapies in the near future.The low oxygen concentration tension is an important part of the stem cells microenvironment conducive signal to provide stem cell maintenance related functions. Studies have shown that mesenchymal stem cells cultured under physiological oxygen tension range between allowing promote their proliferation. Lennon used less than5%of the oxygen concentration in the culture of mesenchymal stem cells of mice, found that the rate of proliferation relative under normoxic culture about40%more. The experiments Yang and Valorani get the similar conclusion. Hypoxia preconditioned has not broad prospects for mesenchymal stem cells in cell therapy, tissue engineering machine. Because the the hypoxia preconditioned oxygen concentration, and time is not the formation of a broad consensus. Oxygen concentration from0.5%to10%, the processing time from24h to72h.Not entirely consistent with the results thus obtained, but the research results show that hypoxia preconditioned mesenchymal stem cells can promote the biological activity of the mesenchymal stem cells, but there is considerable controversy for cell differentiation potential.This experiment to take oxygen concentration of1%the pretreatment fat-derived stem cells, the cells closer to the body’s normal physiological environment.This experiment confirmed the hypoxic preconditioning promote fat-derived stem cell proliferation, cell activity, and can maintain adipose-derived stem cells, mesenchymal stem cell characteristics, for adipose-derived stem cells into the body to play better biological effects to provide a more favorable the condition, however, the hypoxia preconditioned oxygen concentration of adipose-derived stem cells and the processing time requires further in-depth research and specification to provide new experimental and theoretical basis, so as to improve the efficiency of stem cell transplantation.Conclusion:1. Hypoxia preconditioned human adipose-derived stem cells have similar morphological characteristics to adipose-derived stem cells cultured with normal oxygen concentration. Hypoxia preconditioned human adipose-derived stem cells compared with normoxic cultured human adipose-derived stem cells have a stronger proliferation rate, a higher ratio in the proliferation of the cells, with fewer apoptotic cells in low serum environment, higher biological activity.Hypoxia preconditioned human adipose-derived stem cells remains high expression the CD90/CD105and other mesenchymal stem cell surface markers. Low expression of CD31and endothelial cells logo. And has adipogenic, osteogenic, chondrogenic differentiation potential.2, Hypoxia preconditioned human adipose-derived stem cells than normoxic cultured human adipose-derived stem cells secrete more angiogenic factors such as vascular endothelial growth factor, basic fibroblast growth factor. QRT-PCR results confirmed the hypoxic environment induced hypoxia inducible factor HIF-1α expression increased in turn led to its downstream angiogenic factors:vascular endothelial growth factor and basic fibroblast growth factor gene expression increased. Hepatocyte growth factor basically have not been hypoxic preconditioning effect of altered expression. These results suggest that the protective effect of hypoxic preconditioning on human adipose-derived stem cells, at least in part due to hypoxia caused by vascular endothelial growth factor and basic fibroblast growth factor increased secretion caused.3,The condition medium of Hypoxia preconditioned human adipose-derived stem cells compared with normoxic culture medium to better promote the activity of human umbilical vein endothelial cells and reduced apoptosis.4.Hypoxia preconditioned human adipose-derived stem cell conditioned medium can be compared with normoxic medium to better promote the migration of human umbilical vein endothelial cells, the use of anti-vascular endothelial growth factor, basic fibroblast growth factor antibody in hypoxia Group culture medium and after its migration by significantly inhibited. Similarly, we also observed in normoxia medium to anti-vascular endothelial growth factor and basic fibroblast growth factor antibody can inhibit endothelial cell migration.Hypoxic preconditioning of human adipose-derived stem cell conditioned medium can be compared with normoxic medium to better promote human umbilical vein endothelial cells in Matrigel into a tube, and the use of anti-vascular endothelial growth factor, basic as fibroblast growth factor antibody into a tube and after effect of weakening.Hypoxa preconditioned is an efficient and be able applied to the clinical preprocessing. Characteristics of mesenchymal stem cells and mesenchymal stem cells can be well maintained by secreting angiogenic factor to improve local survival and the purpose of promoting functional recovery of the surrounding tissue, In the near future is bound to become an effective means to stem cell therapy. |
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