Hypoxic preconditioning improves survival of cardiac progenitor cells: role of stromal cell derived factor-1α-CXCR4 axis

Background

Cardiac progenitor cells (CPCs) have been shown to be suitable in stem cell therapy for resurrecting damaged myocardium, but poor retention of transplanted cells in the ischemic myocardium causes ineffective cell therapy. Hypoxic preconditioning of cells can increase the expression of CXCR4 and pro-survival genes to promote better cell survival; however, it is unknown whether hypoxia preconditioning will influence the survival and retention of CPCs via the SDF-1α/CXCR4 axis.

Methods and Results

CPCs were isolated from adult mouse hearts and purified by magnetic activated cell sorting using c-kit magnetic beads. These cells were cultured at various times in either normoxic or hypoxic conditions, and cell survival was analyzed using flow cytometry and the expression of hypoxia-inducible factor-1α (HIF-1α), CXCR4, phosphorylated Akt and Bcl-2 were measured by Western blot. Results showed that the expression of pro-survival genes significantly increased after hypoxia treatment, especially in cells cultured in hypoxic conditions for six hours. Upon completion of hypoxia preconditioning from c-kit+ CPCs for six hours, the anti-apoptosis, migration and cardiac repair potential were evaluated. Results showed a significant enhancement in anti-apoptosis and migration in vitro, and better survival and cardiac function after being transplanted into acute myocardial infarction (MI) mice in vivo. The beneficial effects induced by hypoxia preconditioning of c-kit+ CPCs could largely be blocked by the addition of CXCR4 selective antagonist AMD3100.

Conclusions

Hypoxic preconditioning may improve the survival and retention of c-kit+ CPCs in the ischemic heart tissue through activating the SDF-1α/CXCR4 axis and the downstream anti-apoptosis pathway. Strategies targeting this aspect may enhance the effectiveness of cell-based cardiac regenerative therapy.     

Hypoxia,a dynamic tool to amplify the gingival mesenchymal stem cells potential for neurotrophic factor secretion

Gingival mesenchymal stem cells (GMSCs) have significant regenerative potential. Their potential applications range from the treatment of inflammatory diseases, wound healing, and oral disorders. Preconditioning these stem cells can optimize their biological properties. Hypoxia preconditioning of MSCs improves stem cell properties like proliferation, survival, and differentiation potential. This research explored the possible impact of hypoxia on the pluripotent stem cell properties that GMSCs possess. We evaluated the morphology, stemness, neurotrophic factors, and stemness-related genes. We compared the protein levels of secreted neurotrophic factors between normoxic and hypoxic GMSC-conditioned media (GMSC-CM). Results revealed that hypoxic cultured GMSC’s had augmented expression of neurotrophic factors BDNF, GDNF, VEGF, and IGF1 and stemness-related gene NANOG. Hypoxic GMSCs showed decreased expression of the OCT4 gene. In hypoxic GMSC-CM, the neurotrophic factors secretions were significantly higher than normoxic GMSC-CM. Our data demonstrate that culturing of GMSCs in hypoxia enhances the secretion of neurotrophic factors that can lead to neuronal lineage differentiation.     

Conditioned medium enhances the fusion capability of rat bone marrow mesenchymal stem cells and cardiomyocytes

Mesenchymal stem cells (MSCs) show accelerated regeneration potential when these cells experience hypoxic stress. This “preconditioning” has shown promising results with respect to cardio-protection as it stimulates endogenous mechanisms resulting in multiple cellular responses. The current study was carried out to analyze the effect of hypoxia on the expression of certain growth factors in rat MSCs and cardiomyocytes (CMs). Both cell types were cultured and assessed separately for their responsiveness to hypoxia by an optimized dose of 2,4,-dinitrophenol (DNP). These cells were allowed to propagate under normal condition for either 2 or 24 h and then analyzed for the expression of growth factors by RT-PCR. Variable patterns of expression were observed which indicate that their expression depends on the time of re-oxygenation and extent of hypoxia. To see whether the growth factors released during hypoxia affect the fusion of MSCs with CMs, we performed co-culture studies in normal and conditioned medium. The conditioned medium is defined as the medium in which CMs were grown for re-oxygenation till the specified time period of either 2 or 24 h after hypoxia induction. The results showed that the fusion efficiency of cells was increased when the conditioned medium was used as compared to that in the normal medium. This may be due to the presence of certain growth factors released by the cells under hypoxic condition that promote cell survival and enhance their fusion or regenerating ability. This study would serve as another attempt in designing a therapeutic strategy in which conditioned MSCs can be used for ischemic diseases and provide more specific therapy for cardiac regeneration.     

Ginkgolides mimic the effects of hypoxic preconditioning to protect C6 cells against ischemic injury by up-regulation of hypoxia-inducible factor-1 alpha and erythropoietin

Hypoxic preconditioning can play a significant neuroprotective role. However, it has not been employed clinically because of safety concerns. To find a safer preconditioning stimulus that is both practical and effective, we investigated whether ginkgolides are capable of preconditioning as hypoxia to protect C6 cells against ischemic injury. We demonstrated that both ginkgolides (37.5microg/mL) and hypoxia (1% O(2) for 16h) can significantly increase cell viabilities and expression of phosphorylated glycogen synthase kinase (p-GSK), phosphorylated extracellular signal-regulated kinase (p-ERK), hypoxia-inducible factor-1 alpha (HIF-1alpha) and erythropoietin (EPO) in ischemic cells. The inhibitors of mitogen-activated protein kinase (MAPK) or phosphatidylinositol 3'-kinase (PI3K) significantly but not completely reduced the enhanced expression of these proteins and cell viabilities induced by ginkgolides and hypoxic preconditioning. These indicated that ginkgolides could mimic hypoxic preconditioning by increasing expression of HIF-1alpha as well as its target protein EPO and that the ginkgolides and hypoxic preconditioning role might be partly mediated by the activation of the p42/p44-mitogen-activated protein kinase and phosphatidylinositol 3'-kinase/AKT/glycogen synthase kinase 3beta pathways. The similar tendency in the changes of protein expression, cell viabilities and responses to MAPK or PI3K inhibitors of the cells treated with ginkgolides and hypoxia suggests that ginkgolides and hypoxic preconditioning might operate by similar mechanisms. The findings also imply that ginkgolides might have the potential for clinical use to prevent injury in high-risk conditions.     

Hypoxic preconditioning increases survival and angiogenic potency of peripheral blood mononuclear cells via oxidative stress resistance

Cell-based angiogenesis is a promising treatment for ischemic diseases; however, the survival of implanted cells is impaired by oxidative stress in the ischemic microenvironment. We tested the hypothesis that hypoxic preconditioning of implanted cells enhances their resistance against oxidative stress, increasing cell survival and angiogenic potency after implantation into ischemic tissue. Mouse peripheral blood mononuclear cells (PBMNCs) were collected and subjected to hypoxic preconditioning by culture for 24 h in 2% O(2) at 33 degrees C. Hypoxic preconditioning of PBMNCs increased the expression of various genes related to antioxidant and survival signals remarkably. Compared with cells cultured under normoxia, the hypoxia-preconditioned PBMNCs showed significantly lower reactive oxygen species (ROS) accumulation and higher cell survival under oxidative stress induced by LY-83583 (a superoxide generator). Three days after intramuscular implantation into the ischemic hindlimbs of mice, survival of the hypoxia-preconditioned PBMNCs was high, whereas that of the normoxia-cultured PBMNCs was relatively low. Furthermore, 28 days after treatment microvessel density and blood flow in the ischemic hindlimbs were significantly better in the mice implanted with hypoxia-preconditioned PBMNCs than in those implanted with normoxia-cultured PBMNCs. Hypoxic preconditioning increased the survival and angiogenic potency of PBMNCs, through oxidative stress resistance mechanisms.     

Increased expression of CXCR4 and integrin αM in hypoxia‐preconditioned cells contributes to improved cell retention and angiogenic potency

Cell‐based angiogenesis is a promising method for the treatment of ischemic diseases, but the poor retention of implanted cells in targeted tissues is a major drawback. We tested whether hypoxic preconditioning increased retention and angiogenic potency of implanted cells in ischemic tissue. Hypoxic preconditioning of mouse peripheral blood mononuclear cells (PBMNCs) was done with 24 h of culture under 2% O₂. Normoxia‐cultured PBMNCs were used as a control. Hypoxic preconditioning increased the adhesion capacity of the PBMNCs. Moreover, the expression of integrin αM and CXCR4 was significantly higher in the hypoxia‐preconditioned PBMNCs than in the normoxia‐cultured PBMNCs. Interestingly, the expression of intercellular adhesion molecule‐1 (ICAM‐1), a ligand of integrin αM, and stromal cell‐derived factor‐1 (SDF‐1), a chemokine for CXCR4, were remarkably increased in the ischemic hindlimbs. The retention of the hypoxia‐preconditioned PBMNCs was significantly higher than that of the normoxia‐cultured PBMNCs, 3 days after their intramuscular implantation into ischemic hindlimbs. We also noted better blood flow in the ischemic hindlimbs implanted with the hypoxia‐preconditioned PBMNCs than in those implanted with the normoxia‐cultured PBMNCs, 14 days after treatment. Furthermore, antibody neutralization of integrin αM and CXCR4 abolished completely the increased cell retention and angiogenic potency of the hypoxia‐preconditioned PBMNCs after implantation into the ischemic hindlimbs. These results indicate that hypoxic preconditioning of implanted cells is a feasible method of enhancing therapeutic angiogenesis by increasing their retention. J. Cell. Physiol. 220: 508–514, 2009. © 2009 Wiley‐Liss, Inc.     

Tissue stem cells: new tools and functional diversity

The detailed understanding of adult tissue stem cells has significance for both regenerative medicine and oncology. This perspective will discuss how major advances in our ability to identify and monitor these cells, which include genetic lineage tracing, FACS purification, and robust in vitro clonogenic assays, have changed our view of their roles in many organs. Label retention and quiescence are no longer considered obligatory stem cell features. Furthermore, some tissues have more than one type of stem cell, each used in only particular situations of regenerative stress. Thus, there is no "one size fits all" adult tissue stem cell paradigm.     

Hypoxic preconditioning-induced mitochondrial protection is not disrupted in a cell model of mtDNA T8993G mutation-induced F1F0-ATP synthase defect: the role of mitochondrial permeability transition

Transient opening of the mitochondrial permeability transition pore plays a crucial role in hypoxic preconditioning-induced protection. Recently, the cyclophilin-D component of the mitochondrial permeability transition pore has been shown to interact with and regulate the F1F0-ATP synthase. However, the precise role of the F1F0-ATP synthase and the interaction between cyclophilin-D and F1F0-ATP synthase in the mitochondrial permeability transition pore and hypoxic preconditioning remain uncertain. Here we found that a 1-h hypoxic preconditioning delayed apoptosis and improved cell survival after stimulation with various apoptotic inducers including H₂O₂, ionomycin, and arachidonic acid in mitochondrial DNA T8993G mutation (NARP) osteosarcoma 143B cybrids, an F1F0-ATP synthase defect cell model. This hypoxic preconditioning protected NARP cybrid cells against focal laser irradiation-induced oxidative stress by suppressing reactive oxygen species formation and preventing the depletion of cardiolipin. Furthermore, the protective functions of transient opening of the mitochondrial permeability transition pore in both NARP cybrids and wild-type 143B cells can be augmented by hypoxic preconditioning. Disruption of the interaction between cyclophilin-D and F1F0-ATP synthase by cyclosporin A attenuated the mitochondrial protection induced by hypoxic preconditioning in both NARP cybrids and wild-type 143B cells. Our results demonstrate that the interaction between cyclophilin-D and F1F0-ATP synthase is important in the hypoxic preconditioning-induced cell protection. This finding improves our understanding of the mechanism of mitochondrial permeability transition pore opening in cells in response to hypoxic preconditioning, and will be helpful in further developing new pharmacological agents targeting hypoxia–reoxygenation injury and mitochondria-mediated cell death     

Advances in cell lineage reprogramming

As a milestone breakthrough of stem cell and regenerative medicine in recent years,somatic cell reprogramming has opened up new applications of regenerative medicine by breaking through the ethical shackles of embryonic stem cells.However,induced pluripotent stem(iPS) cells are prepared with a complicated protocol that results in a low reprogramming rate.To obtain differentiated target cells,iPS cells and embryonic stem cells still need to be induced using step-by-step procedures.The safety of induced target cells from iPS cells is currently a further concerning matter.More broadly conceived is lineage reprogramming that has been investigated since 1987.Adult stem cell plasticity,which triggered interest in stem cell research at the end of the last century,can also be included in the scope of lineage reprogramming.With the promotion of iPS cell research,lineage reprogramming is now considered as one of the most promising fields in regenerative medicine,will hopefully lead to customized,personalized therapeutic options for patients in the future.     

Role of hypoxia preconditioning in therapeutic potential of mesenchymal stem-cell-derived extracellular vesicles

The use of mesenchymal stem-cells (MSC) in cell therapy has received considerable attention because of their properties. These properties include high expansion and differentiation in vitro, low immunogenicity, and modulation of biological processes, such as inflammation, angiogenesis and hematopoiesis. Curiously, the regenerative effect of MSC is partly due to their paracrine activity. This has prompted numerous studies, to investigate the therapeutic potential of their secretome in general, and specifically their extracellular vesicles (EV). The latter contain proteins, lipids, nucleic acids, and other metabolites, which can cause physiological changes when released into recipient cells. Interestingly, contents of EV can be modulated by preconditioning MSC under different culture conditions. Among them, exposure to hypoxia stands out; these cells respond by activating hypoxia-inducible factor (HIF) at low O₂ concentrations. HIF has direct and indirect pleiotropic effects, modulating expression of hundreds of genes involved in processes such as inflammation, migration, proliferation, differentiation, angiogenesis, metabolism, and cell apoptosis. Expression of these genes is reflected in the contents of secreted EV. Interestingly, numerous studies show that MSC-derived EV conditioned under hypoxia have a higher regenerative capacity than those obtained under normoxia. In this review, we show the implications of hypoxia responses in relation to tissue regeneration. In addition, hypoxia preconditioning of MSC is being evaluated as a very attractive strategy for isolation of EV, with a high potential for clinical use in regenerative medicine that can be applied to different pathologies.     

Stem cells, regenerative medicine, and animal models of disease

The field of stem cell biology and regenerative medicine is rapidly moving toward translation to clinical practice, and in doing so has become even more dependent on animal donors and hosts for generating cellular reagents and assaying their potential therapeutic efficacy in models of human disease. Advances in cell culture technologies have revealed a remarkable plasticity of stem cells from embryonic and adult tissues, and transplantation models are now needed to test the ability of these cells to protect at-risk cells and replace cells lost to injury or disease. With such a mandate, issues related to acceptable sources and controversial (e.g., chimeric) models have challenged the field to provide justification of their potential efficacy before the passage of new restrictions that may curb anticipated breakthroughs. Progress from the use of both in vitro and in vivo regenerative medicine models already offers hope both for the facilitation of stem cell phenotyping in recursive gene expression profile models and for the use of stem cells as powerful new therapeutic reagents for cancer, stroke, Parkinson's, and other challenging human diseases that result in movement disorders. This article describes research in support of the following three objectives: (1) To discover the best stem or progenitor cell in vitro protocols for isolating, expanding, and priming these cells to facilitate their massive propagation into just the right type of neuronal precursor cell for protection or replacement protocols for brain injury or disease, including those that affect movement such as Parkinson's disease and stroke; (2) To discover biogenic factors--compounds that affect stem/progenitor cells (e.g., from high-throughput screening and other bioassay approaches)--that will encourage reactive cell genesis, survival, selected differentiation, and restoration of connectivity in central nervous system movement and other disorders; and (3) To establish the best animal models of human disease and injury, using both small and large animals, for testing new regenerative medicine therapeutics.     

Hypoxic stress activates chaperone-mediated autophagy and modulates neuronal cell survival

Autophagy is a conserved mechanism responsible for the continuous clearance of unnecessary organelles or misfolded proteins in lysosomes. Three types of autophagy have been reported in the difference of substrate delivery to lysosome: macroautophagy, microautophagy, and chaperone-mediated autophagy (CMA). Among these types, CMA is a unique autophagy system that selectively degrades substrates detected by heat shock cognate protein 70 (HSC70). Recently, autophagic cell death has been reported to be involved in neuronal death following brain ischemia; however, the contribution of CMA to neuronal death/survival after ischemic stress has not been addressed. In the present study, we determined whether quantitative alterations in LAMP-2A, which is the key molecule in CMA, would modulate neuronal cell survival under hypoxic conditions. Incubation of Neuro2A cells in a hypoxic chamber (1% O(2), 5% CO(2)) increased the level of LAMP-2A and induced accumulation of LAMP-2A-positive lysosomes in the perinuclear area, which is a hallmark of CMA activation. The activation of CMA in response to hypoxia was also confirmed by the GAPDH-HaloTag CMA indicator system at the single cell level. Next, we asked whether CMA was involved in cell survival during hypoxia. Blocking LAMP-2A expression with siRNA increased the level of cleaved caspase-3 and the number of propidium iodide-positive cells after hypoxic stress regardless of whether macroautophagy could occur, whereas the administration of mycophenolic acid, a potent CMA activator, rescued hypoxia-mediated cell death. Finally, we asked whether CMA was activated in the neurons after middle cerebral artery occlusion in vivo. The expression of LAMP-2A was significantly increased in the ischemic hemisphere seven days after brain ischemia. These results indicate that CMA is activated during hypoxia and contributes to the survival of cells under these conditions.     

缺氧预处理对神经干细胞间隙连接蛋白的表达及半通道功能的影响

目的:检测缺氧预处理对神经干细胞间隙连接蛋白的表达情况及半通道功能的影响,为探究缺氧预处理后神经干细胞间细胞通讯提供理论依据。方法:采用免疫组织化学方法检测对照组及缺氧预处理1小时组、缺氧预处理4小时组、缺氧预处理8小时组、缺氧预处理12小时组神经干细胞中cx43、cx36、cx47的表达情况。通过激光共聚焦激光扫描显微镜测量钙黄绿素滤过情况以评估半通道功能的变化。结果:与对照组(1.03±0.14%)比较,在0.5%氧气条件下暴露8小时(3.05±0.40%)和12小时(5.51±0.31%)的神经干细胞坏死率较显著升高(P0.05)。与对照组相比,缺氧预处理4、8、12小时组神经干细胞中cx43、cx36、cx47的表达显著增加,且随着缺氧预处理时间的延长而显著增加(P0.05)。与对照组及加入半通道阻滞剂组相比,缺氧预处理组半通道滤过功能显著增强(P0.05)。结论:缺氧预处理可以以时间依赖性方式提高神经干细胞间隙连接蛋白的表达和半通道滤过功能。神经干细胞暴露于低氧环境而不引起坏死显著增加的最佳时间为4小时。     

Deferoxamine Preconditioning of Neural-Like Cells Derived from Human Wharton’s Jelly Mesenchymal Stem Cells as a Strategy to Promote Their Tolerance and Therapeutic Potential: An In Vitro Study

Transplantation of neural-like cells is considered as a promising therapeutic strategy developed for neurodegenerative disease in particular for ischemic stroke. Since cell survival is a major concern following cell implantation, a number of studies have underlined the protective effects of preconditioning with hypoxia or hypoxia mimetic pharmacological agents such as deferoxamine (DFO), induced by activation of hypoxia inducible factor-1 (HIF-1) and its target genes. The present study has investigated the effects of DFO preconditioning on some factors involved in cell survival, angiogenesis, and neurogenesis of neural-like cells derived from human Wharton’s jelly mesenchymal stem cells (HWJ-MSCs) in presence of hydrogen peroxide (H₂O₂). HWJ-MSCs were differentiated toward neural-like cells for 14 days and neural cell markers were identified using immunocytochemistry. HWJ-MSC-derived neural-like cells were then treated with 100 µM DFO, as a known hypoxia mimetic agent for 48 h. mRNA and protein expression of HIF-1 target genes including brain-derived neurotrophic factors (BDNF) and vascular endothelial growth factor (VEGF) significantly increased using RT-PCR and Western blotting which were reversed by HIF-1α inhibitor, while, gene expression of Akt-1, Bcl-2, and Bax did not change significantly but pAkt-1 was up-regulated as compared to poor DFO group. However, addition of H₂O₂ to DFO-treated cells resulted in higher resistance to H₂O₂-induced cell death. Western blotting analysis also showed significant up-regulation of HIF-1α, BDNF, VEGF, and pAkt-1, and decrease of Bax/Bcl-2 ratio as compared to poor DFO. These results may suggest that DFO preconditioning of HWJ-MSC-derived neural-like cells improves their tolerance and therapeutic potential and might be considered as a valuable strategy to improve cell therapy.     

Hypoxic preconditioning improves spatial cognitive ability in mice

Although it has been reported in a lot of studies that hypoxic preconditioning could protect the brain from hypoxic/ischemic injury, it is not clear whether hypoxic preconditioning could affect brain functions such as cognitive ability. This work aims at investigating the effect of hypoxic preconditioning on spatial cognitive ability in mice after acute and repeated hypoxic exposures. The mice were randomly divided into 3 groups: a control group in which mice were not exposed to hypoxia (H0) and experimental groups in which mice encountered hypoxia either once (H1) or 4 times (H4). Neural cell adhesion molecule (NCAM) expression, long-term potentiation (LTP) recording and Morris water maze test were used to measure the animals' cognitive ability. The tolerance time was progressively prolonged as exposure went on. The expression of both NCAM mRNA and NCAM protein as well as the LTP induction rate decreased in group H1, but recovered to control level in group H4. The performance of mice in the maze test was improved in H4 in comparison with that in both H1 and H0. These findings may indicate that spatial cognitive ability is improved in adult mice by their hypoxic preconditioning.     

The Effect of Hypoxic Preconditioning on Induced Schwann Cells under Hypoxic Conditions

Object

Our objective was to explore the protective effects of hypoxic preconditioning on induced Schwann cells exposed to an environment with low concentrations of oxygen. It has been observed that hypoxic preconditioning of induced Schwann cells can promote axonal regeneration under low oxygen conditions.

Method

Rat bone marrow mesenchymal stem cells (MSCs) were differentiated into Schwann cells and divided into a normal oxygen control group, a hypoxia-preconditioning group and a hypoxia group. The ultrastructure of each of these groups of cells was observed by electron microscopy. In addition, flow cytometry was used to measure changes in mitochondrial membrane potential. Annexin V-FITC/PI staining was used to detect apoptosis, and Western blots were used to detect the expression of Bcl-2/Bax. Fluorescence microscopic observations of axonal growth in NG-108 cells under hypoxic conditions were also performed.

Results

The hypoxia-preconditioning group maintained mitochondrial cell membrane and crista integrity, and these cells exhibited less edema than the hypoxia group. In addition, the cells in the hypoxia-preconditioning group were found to be in early stages of apoptosis, whereas cells from the hypoxia group were in the later stages of apoptosis. The hypoxia-preconditioning group also had higher levels of Bcl-2/Bax expression and longer NG-108 cell axons than were observed in the hypoxia group.

Conclusion

Hypoxic preconditioning can improve the physiological state of Schwann cells in a severe hypoxia environment and improve the ability to promote neurite outgrowth.     

Umbilical cord blood: a unique source of pluripotent stem cells for regenerative medicine

It is estimated that almost 1 in 3 individuals in the United States might benefit from regenerative medicine therapy. Unfortunately, embryonic stem (ES) cell therapies are currently limited by ethical, political, biological and regulatory hurdles. Thus, for the foreseeable future, the march of regenerative medicine to the clinic will depend upon the development of non-ES cell therapies. Current sources of non-ES cells easily available in large numbers can be found in the bone marrow, adipose tissue and umbilical cord blood. Each of these types of stem cells has already begun to be utilized to treat a variety of diseases. This review will show that cord blood (CB) contains multiple populations of ES-like and other pluripotential stem cells, capable of giving rise to hematopoietic, epithelial, endothelial, and neural tissues both in vitro and in vivo. Cumulatively, the identification and isolation of these populations of pluripotent stem cells within cord blood represents a scientific breakthrough that could potentially impact every field of medicine, via their use in regenerative medicine. Thus, CB stem cells are amenable to treatment of a wide variety of diseases including cardiovascular, hepatic, ophthalmic, orthopaedic, neurological and endocrine diseases.