Oxygen levels and the regulation of cell adhesion in the nervous system: A control point for morphogenesis in development,disease and evolution?

In this article, I discuss the hallmarks of hypoxia in vitro and in vivo and review work showing that many types of stem cell proliferate more robustly in lowered oxygen. I then discuss recent studies showing that alterations in the levels and the types of cell and substrate adhesion molecules are a notable response to reduced O₂ levels in both cultured primary neural stem cells and brain tissues in response to hypoxia in vivo. The ability of O₂ levels to regulate adhesion molecule expression is linked to the Wnt signaling pathway, which can control and be controlled by adhesion events. The ability of O₂ levels to influence cell adhesion also has far-reaching implications for development, ischemic trauma and neural regeneration, as well as for cancer and other diseases. Finally I discuss the possibility that the fluctuations in O₂ levels known to have occurred over evolutionary time could, by influencing adhesion systems, have contributed to early symbiotic events in unicellular organisms and to the emergence of multicellularity. It is not my intention to be exhaustive in these domains, which are far from my own field of study. Rather this article is meant to provoke and stimulate thinking about molecular evolution involving O₂ sensing and signaling during eras of geologic and atmospheric change that might inform modern studies on development and disease.     

Interleukin 6 mediated recruitment of mesenchymal stem cells to the hypoxic tumor milieu

Mesenchymal stem cells (MSCs) are a heterogeneous population of non-hematopoietic precursor cells predominantly found in the bone marrow. They have been recently reported to home towards the hypoxic tumor microenvironment in vivo. Interleukin-6 is a multifunctional cytokine normally involved in the regulation of the immune and inflammatory response. In addition to its normal function, IL-6 signaling has been implicated in tumorigenesis. Solid tumors develop hypoxia as a result of inadequate O₂ supply. Interestingly, tumor types with increased levels of hypoxia are known to have increased resistance to chemotherapy as well as increased metastatic potential. Here, we present evidence that under hypoxic conditions (1.5% O₂) breast cancer cells secrete high levels of IL-6, which serve to activate and attract MSCs. We now report that secreted IL-6 acts in a paracrine fashion on MSCs stimulating the activation of both Stat3 and MAPK signaling pathways to enhance migratory potential and cell survival. Inhibition of IL-6 signaling utilizing neutralizing antibodies leads to attenuation of MSC migration. Specifically, increased migration is dependent on IL-6 signaling through the IL-6 receptor. Collectively, our data demonstrate that hypoxic tumor cells specifically recruit MSCs, which through activation of signaling and survival pathways facilitate tumor progression.     

Hypoxia or in situ normoxia: The stem cell paradigm

Although O₂ concentrations are considerably lowered in vivo, depending on the tissue and cell population in question (some cells need almost anoxic environment for their maintenance) the cell and tissue cultures are usually performed at atmospheric O₂ concentration (20–21%). As an instructive example, the relationship between stem cells and micro‐environmental/culture oxygenation has been recapitulated. The basic principle of stem cell biology, “the generation‐age hypothesis,” and hypoxic metabolic properties of stem cells are considered in the context of the oxygen‐dependent evolution of life and its transposition to ontogenesis and development. A hypothesis relating the self‐renewal with the anaerobic and hypoxic metabolic properties of stem cells and the actual O₂ availability is elaborated (“oxygen stem cell paradigm”). Many examples demonstrated that the cellular response is substantially different at atmospheric O₂ concentration when compared to lower O₂ concentrations which better approximate the physiologic situation. These lower O₂ concentrations, traditionally called “hypoxia” represent, in fact, an in situ normoxia, and should be used in experimentation to get an insight of the real cell/cytokine physiology. The revision of our knowledge on cell/cytokine physiology, which has been acquired ex vivo at non physiological atmospheric (20–21%) O₂ concentrations representing a hyperoxic state for most primate cells, has thus become imperious. J. Cell. Physiol. 219: 271–275, 2009. © 2009 Wiley‐Liss, Inc.     

Design of a stirred multiwell bioreactor for expansion of CD34+ umbilical cord blood cells in hypoxic conditions

Besides having a metabolic role, oxygen is recognized as an important signaling stimulus for stem cells. In hematopoiesis, hypoxia seems to favor stem cell self‐renewal. In fact, long‐term repopulating hematopoietic stem cells reside in bone marrow at concentrations as low as 1% oxygen. However, O₂ concentration is difficult to control in vitro. Thermodynamically, we found significant differences between O₂ solubility in different media, and in presence of serum. Furthermore, we verified that medium equilibration with a hypoxic atmosphere requires several hours. Thus, in a static culture, the effective O₂ concentration in the cell immediate microenvironment is difficult to control and subject to concentration gradients. Stirred systems improve homogeneity within the culture volume. In this work, we developed a stirred bioreactor to investigate hypoxia effect on the expression of stem cell markers in CD34⁺ cells from umbilical cord blood. The stirring system was designed on top of a standard six‐well plate to favor continuity with conventional static conditions and transfer of culture protocols. The bioreactor volume (10 mL/well) is suitable for cell expansion and multiparametric flow cytometry analyses. First, it was tested at 21% O₂ for biocompatibility and other possible effects on the cells compared to static conditions. Then, it was used to study c‐kit expression of CD34⁺ cells at 5% O₂, using 21%‐O₂ cultures as a control. In hypoxia we found that CD34⁺ cells maintained a higher expression of c‐kit. Further investigation is needed to explore the dynamics of interaction between oxygen‐ and c‐kit‐dependent pathways at the molecular level. © 2011 American Institute of Chemical Engineers Biotechnol. Prog., 2011     

Effects of hypoxia on the proliferation and differentiation of NSCs

Oxygen is vital to nearly all forms of life on Earth via its role in energy homeostasis and other cell functions. Until recently, the effects of oxygen on the proliferation and differentiation of neural stem cells (NSCs) have been largely ignored. Some studies have been carried out on the basis of the fact that NSCs exists within a “physiological hypoxic” environment at 1 to 5% O₂ in both embryonic and adult brains. The results showed that hypoxia could promote the growth of NSCs and maintain its survival in vitro. In vivo studies also showed that ischemia/hypoxia increased the number of endogenous NSCs in the subventricular zone and dentate gyrus. In addition, hypoxia could influence the differentiation of NSCs. More neurons, especially more doparminergic neurons, were produced under hypoxic condition. The effects of hypoxia on the other kind of stem cell were briefly introduced as additional evidence. The mechanism of these responses might be primarily involved in the hypoxic inducible factor-1 (HIF-1) signal pathway. The present review summarizes recent works on the role of hypoxia in the proliferation and differentiation of NSCs both in vitro and in vivo, and the mechanism involved in HIF-1 signaling pathway behind this response was also discussed.     

Combined pre‐conditioning with salidroside and hypoxia improves proliferation,migration and stress tolerance of adipose‐derived stem cells

Oxidative stress after ischaemia impairs the function of transplanted stem cells. Increasing evidence has suggested that either salidroside (SAL) or hypoxia regulates growth of stem cells. However, the role of SAL in regulating function of hypoxia‐pre–conditioned stem cells remains elusive. Thus, this study aimed to determine the effect of SAL and hypoxia pre‐conditionings on the proliferation, migration and tolerance against oxidative stress in rat adipose‐derived stem cells (rASCs). rASCs treated with SAL under normoxia (20% O₂) or hypoxia (5% O₂) were analysed for the cell viability, proliferation, migration and resistance against H₂O₂‐induced oxidative stress. In addition, the activation of Akt, Erk1/2, LC3, NF‐κB and apoptosis‐associated pathways was assayed by Western blot. The results showed that SAL and hypoxia treatments synergistically enhanced the viability (fold) and proliferation of rASCs under non‐stressed conditions in association with increased autophagic flux and activation of Akt, Erk1/2 and LC3. H₂O₂‐induced oxidative stress, cytotoxicity, apoptosis, autophagic cell death and NF‐κB activation were inhibited by SAL or hypoxia, and further attenuated by the combined SAL and hypoxia pre‐treatment. The SAL and hypoxia pre‐treatment also enhanced the proliferation and migration of rASCs under oxidative stress in association with Akt and Erk1/2 activation; however, the combined pre‐treatment exhibited a more profound enhancement in the migration than proliferation. Our data suggest that SAL combined with hypoxia pre‐conditioning may enhance the therapeutic capacity of ASCs in post‐ischaemic repair.     

Serotonergic sensory‐motor neurons mediate a behavioral response to hypoxia in pond snail embryos

Oxygen (O₂) is one of the most important environmental factors that affects both physiological processes and development of aerobic animals, yet little is known about the neural mechanism of O₂ sensing and adaptive responses to low O₂ (hypoxia) during development. In the pond snail, Helisoma trivolvis, the first embryonic neurons (ENC1s) to develop are a pair of serotonergic sensory‐motor cells that regulate a cilia‐driven rotational behavior. Here, we report that the ENC1‐ciliary cell circuit mediates an adaptive behavioral response to hypoxia. Exposure of egg masses to hypoxia elicited a dose‐dependent and reversible acceleration of embryonic rotation that mixed capsular fluid, thereby facilitating O₂ diffusion to the embryo. The O₂ partial pressures (Po₂) for threshold, half‐maximal, and maximal rotational response were 60, 28, and 13 mm Hg, respectively. During hypoxia, embryos relocated to the periphery of the egg masses where higher Po₂ levels occurred. Furthermore, intermittent hypoxia treatments induced a sensitization of the rotational response. In isolated ciliary cells, ciliary beating was unaffected by hypoxia, suggesting that in the embryo, O₂ sensing occurs upstream of the motile cilia. The rotational response of embryos to hypoxia was attenuated by application of the serotonin receptor antagonist, mianserin, correlated to the development of ENC1‐ciliary cell circuit, and abolished by laser‐ablation of ENC1s. Together, these data suggest that ENC1s are unique oxygen sensors that may provide a good single cell model for the examination of mechanistic, developmental, and evolutionary aspects of O₂ sensing. © 2002 Wiley Periodicals, Inc. J Neurobiol 52: 73–83, 2002     

The role of oxygen in regulating neural stem cells in development and disease

Oxygen (O₂) is a substrate for energy production in the cell and is a rapid regulator of cellular metabolism. Recent studies have also implicated O₂ and its signal transduction pathways in controlling cell proliferation, fate, and morphogenesis during the development of many tissues, including the nervous system. O₂ tensions in the intact brain are much lower than in room air, and there is evidence that dynamic control of O₂ availability may be a component of the in vivo neural stem cell (NSC) niche. At lower O₂ tensions, hypoxia‐inducible factor 1α (HIF1α) facilitates signal transduction pathways that promote self‐renewal (e.g., Notch) and inhibits pathways that promote NSC differentiation or apoptosis (e.g., bone morphogenetic proteins). Increasing O₂ tension degrades HIF1α, thus promoting differentiation or apoptosis of NSCs and progenitors. These dynamic changes in O₂ tension can be mimicked to optimize ex vivo production methods for cell replacement therapies. Conversely, disrupted O₂ availability may play a critical role in disease states such as stroke or brain tumor progression. Hypoxia during stroke activates precursor proliferation in vivo, while glioblastoma stem cells proliferate maximally in a more hypoxic environment than normal stem cells, which may make them resistant to certain anti‐neoplastic therapies. These findings suggest that O₂ response is central to the normal architecture and dynamics of NSC regulation and in the etiology and treatment of brain diseases. J. Cell. Physiol. 220: 562–568, 2009. © 2009 Wiley‐Liss, Inc.     

Low oxygen tension potentiates proliferation and stemness but not multilineage differentiation of caprine male germline stem cells

The milieu of male germline stem cells (mGSCs) is characterized as a low-oxygen (O₂) environment, whereas, their in-vitro expansion is typically performed under normoxia (20–21% O₂). The comparative information about the effects of low and normal O₂ levels on the growth and differentiation of caprine mGSCs (cmGSCs) is lacking. Thus, we aimed to investigate the functional and multilineage differentiation characteristics of enriched cmGSCs, when grown under hypoxia and normoxia. After enrichment of cmGSCs through multiple methods (differential platting and Percoll-density gradient centrifugation), the growth characteristics of cells [population-doubling time (PDT), viability, proliferation, and senescence], and expression of key-markers of adhesion (β-integrin and E-Cadherin) and stemness (OCT-4, THY-1 and UCHL-1) were evaluated under hypoxia (5% O₂) and normoxia (21% O₂). Furthermore, the extent of multilineage differentiation (neurogenic, adipogenic, and chondrogenic differentiation) under different culture conditions was assessed. The survival, viability, and proliferation were significantly (p?<?0.05) improved, thus, yielding a significantly (p?<?0.05) higher number of viable cells with larger colonies under hypoxia. Furthermore, the expression of stemness and adhesion markers were distinctly upregulated under lowered O₂ conditions. Conversely, the differentiated regions and expression of differentiation-specific genes [C/EBPα (adipogenic), nestin and β-tubulin (neurogenic), and COL2A1 (chondrogenic)] were significantly (p?<?0.05) reduced under hypoxia. Overall, the results demonstrate that culturing cmGSCs under hypoxia augments the growth characteristics and stemness but not the multilineage differentiation of cmGSCs, as compared with normoxia. These data are important to develop robust methodologies for ex-vivo expansion and lineage-committed differentiation of cmGSCs for clinical applications.

     

ROS and cGMP signaling modulate persistent escape from hypoxia in Caenorhabditis elegans

The ability to detect and respond to acute oxygen (O₂) shortages is indispensable to aerobic life. The molecular mechanisms and circuits underlying this capacity are poorly understood. Here, we characterize the behavioral responses of feeding Caenorhabditis elegans to approximately 1% O₂. Acute hypoxia triggers a bout of turning maneuvers followed by a persistent switch to rapid forward movement as animals seek to avoid and escape hypoxia. While the behavioral responses to 1% O₂ closely resemble those evoked by 21% O₂, they have distinct molecular and circuit underpinnings. Disrupting phosphodiesterases (PDEs), specific G proteins, or BBSome function inhibits escape from 1% O₂ due to increased cGMP signaling. A primary source of cGMP is GCY-28, the ortholog of the atrial natriuretic peptide (ANP) receptor. cGMP activates the protein kinase G EGL-4 and enhances neuroendocrine secretion to inhibit acute responses to 1% O₂. Triggering a rise in cGMP optogenetically in multiple neurons, including AIA interneurons, rapidly and reversibly inhibits escape from 1% O₂. Ca²⁺ imaging reveals that a 7% to 1% O₂ stimulus evokes a Ca²⁺ decrease in several neurons. Defects in mitochondrial complex I (MCI) and mitochondrial complex I (MCIII), which lead to persistently high reactive oxygen species (ROS), abrogate acute hypoxia responses. In particular, repressing the expression of isp-1, which encodes the iron sulfur protein of MCIII, inhibits escape from 1% O₂ without affecting responses to 21% O₂. Both genetic and pharmacological up-regulation of mitochondrial ROS increase cGMP levels, which contribute to the reduced hypoxia responses. Our results implicate ROS and precise regulation of intracellular cGMP in the modulation of acute responses to hypoxia by C. elegans.

The ability to detect and respond to acute oxygen shortages is indispensable to aerobic life, but the molecular mechanisms underlying this capacity are poorly understood. This study reveals that high levels of cGMP and reactive oxygen species (ROS) prevent the nematode Caenorhabditis elegans from escaping hypoxia.     

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.     

Autocrine fibroblast growth factor 2‐mediated interactions between human mesenchymal stem cells and the extracellular matrix under varying oxygen tension

Human mesenchymal stromal or stem cells (hMSCs) are being investigated for cell therapy in a wide range of diseases. MSCs are a potent source of trophic factors and actively remodel their immediate microenvironment through the secretion of bioactive factors in response to external stimuli such as oxygen tension. In this study, we examined the hypothesis that hypoxia influences hMSC properties in part through the regulation of extracellular milieu characterized by the extracellular matrix (ECM) matrices and the associated fibroblast growth factor‐2 (FGF‐2). The decellularized ECM matrices derived from hMSC culture under both hypoxic (e.g., 2% O₂) and the standard culture (e.g., 20% O₂) conditions have different binding capacities to the cell‐secreted and exogenenous FGF‐2. The reduced hMSC proliferation in the presence of FGF‐2 inhibitor and the differential capacity of the decellularized ECM matrices in regulating hMSC osteogeneic and adipogenic differentiation suggest an important role of the endogenous FGF‐2 in sustaining hMSC proliferation and regulating hMSC fate. Additionally, the combination of the ECM adhesion and hypoxic culture preserved hMSC viability under serum withdrawal. Together, the results suggest the synergistic effect of hypoxia and the ECM matrices in sustaining hMSC ex vivo expansion and preserving their multi‐potentiality and viability under nutrient depletion. The results have important implication in optimizing hMSC expansion and delivery strategies to obtain hMSCs in sufficient quantity with required potency and to enhance survival and function upon transplantation. J. Cell. Biochem. 114: 716–727, 2013. © 2012 Wiley Periodicals, Inc.     

Time-course of the polycythemic response in normoxic and hypoxic mice with high blood oxygen affinity induced by cyanate administration

The Hb-O₂ affinity and the erythropoietic response as a function of time were studied in mice treated with sodium cyanate for up to 2 months. Cyanate increased the Hb-O₂ affinity in normoxic mice more than in chronically hypoxic mice. The hemoglobin concentration rose as a function of time both in normoxic and hypoxic conditions but reached higher levels in hypoxia. After 42 days of study (21 days of hypoxia) hemoglobin reached maximum levels and thereafter showed a plateau in both cyanate and control animals. It is concluded that a chronic left-shifted oxygen dissociation curve does not avoid the development of hypoxic polycythemia in mice. Moreover, prolonged cyanate administration potentiates the crythropoietic response to chronic hypoxia. Since polycythemia is an index of tissue hypoxia, the results show that the high hemoglobin affinity did not prevent tissue hypoxia in low PO₂ conditions. Results showing beneficial effects of high hemoglobin oxygen affinity induced by cyanate based on acute hypoxic expositions should be cautiously interpreted with regard to their adaptive value in animals chronically exposed to natural or simulated hypoxia.Abbreviations Hb hemoglobin - NaOCN sodium cyanate - ODC oxygen dissociation curve - P ₅₀ PO₂ at which hemoglobin is half saturated with O₂     

Participation of autophagy in renal ischemia/reperfusion injury

Renal ischemia-reperfusion (I/R) injury is inevitable in transplantation, and it results in renal tubular epithelial cells undergoing cell death. We observed an increase in autophagosomes in the tubular epithelial cells of I/R-injured mouse models, and in biopsy specimens from human transplanted kidney. However, it remains unclear whether autophagy functions as a protective pathway, or contributes to I/R-induced cell death. Here, we employed the human renal proximal tubular epithelial cell line HK-2 in order to explore the role of autophagy under hypoxia (1% O₂) or activation of reactive oxygen species (500 μM H₂O₂). When compared to normoxic conditions, 48 h of hypoxia slightly increased LC3-labeled autophagic vacuoles and markedly increased LAMP2-labeled lysosomes. We observed similar changes in the mouse IR-injury model. We then assessed autophagic generation and degradation by inhibiting the downstream lysosomal degradation of autophagic vacuoles using lysosomal protease inhibitor. We found that autophagosomes increased markedly under hypoxia in the presence of lysosomal protease inhibitors, thus suggesting that hypoxia induces high turnover of autophagic generation and degradation. Furthermore, inhibition of autophagy significantly inhibited H₂O₂-induced cell death. In conclusion, high turnover of autophagy may lead to autophagic cell death during I/R injury.     

Hypoxic expression of NLRP3 and VEGF in cultured retinal pigment epithelial cells: contribution of P2Y<Subscript>2</Subscript> receptor signaling

Retinal hypoxia is a major condition of the chronic inflammatory disease age-related macular degeneration. Extracellular ATP is a danger signal which is known to activate the NLRP3 inflammasome in various cell systems. We investigated in cultured human retinal pigment epithelial (RPE) cells whether hypoxia alters the expression of inflammasome-associated genes and whether purinergic receptor signaling contributes to the hypoxic expression of key inflammatory (NLRP3) and angiogenic factor (VEGF) genes. Hypoxia and chemical hypoxia were induced by a 0.2%-O₂ atmosphere and addition of CoCl₂, respectively. Gene expression was determined with real-time RT-PCR. Cytosolic NLRP3 and (pro-) IL-1β levels, and the extracellular VEGF level, were evaluated with Western blot and ELISA analyses. Cell culture in 0.2% O₂ induced expression of NLRP3 and pro-IL-1β genes but not of the pro-IL-18 gene. Hypoxia also increased the cytosolic levels of NLRP3 and (pro-) IL-1β proteins. Inflammasome activation by lysosomal destabilization decreased the cell viability under hypoxic, but not control conditions. In addition to activation of IL-1 receptors, purinergic receptor signaling mediated by a pannexin-dependent release of ATP and a release of adenosine, and activation of P2Y₂ and adenosine A₁ receptors, was required for the full hypoxic expression of the NLRP3 gene. P2Y₂ (but not A₁) receptor signaling also contributed to the hypoxic expression and secretion of VEGF. The data indicate that hypoxia induces priming and activation of the NLRP3 inflammasome in cultured RPE cells. The hypoxic NLRP3 and VEGF gene expression and the secretion of VEGF are in part mediated by P2Y₂ receptor signaling.     

Bone mesenchymal stem cell‐conditioned medium attenuates the effect of oxidative stress injury on NSCs by inhibiting the Notch1 signaling pathway

Numerous studies have demonstrated the therapeutic effect of bone mesenchymal stem cells on spinal cord injury (SCI), especially on neural stem cells (NSCs). However, the predominant mechanisms of bone mesenchymal stem cells (BMSCs) are unclear. Recently, some researchers have found that paracrine signaling plays a key role in the therapeutic capacity of BMSCs and emphasized that the protective effect of BMSCs may be due to paracrine factors. In this study, we aimed to investigate the potential mechanisms of BMSCs to protect NSCs. NSCs were identified by immunocytochemistry. The oxidative stress environment was simulated by H₂O₂ (50, 100, 200 μM) for 2 h. The apoptotic rate of the NSCs was detected via flow cytometry. Lactate dehydrogenase (LDH), malondialdehyde (MDA), and superoxide dismutase (SOD) activity were evaluated via corresponding assay kits. Western blot was used to detect the expressions of Notch1, HES1, caspase‐3, cleave caspase‐3, Bax, and Bcl‐2. We found that H₂O₂ could significantly induce the apoptosis of NSCs, increase LDH, MDA levels, and decrease SOD activity by activating the Notch1 signaling pathway. DAPT (the specific blocker of Notch1) and BMSC‐conditioned medium (BMSC‐CM) could significantly prevent the apoptotic effect and oxidative stress injury on NSCs that were treated with H₂O₂. We also revealed that BMSC‐CM could decrease the expression of Notch1, Hes1, cleave caspase‐3, Bax, and increases the expression of Bcl‐2 in NSCs, which was induced by H₂O₂. These results have revealed that BMSC‐CM can neutralize the effect against oxidative stress injury on the apoptosis of NSCs by inhibiting the Notch1 signaling pathway.     

Hypoxia Promotes Dopaminergic Differentiation of Mesenchymal Stem Cells and Shows Benefits for Transplantation in a Rat Model of Parkinson’s Disease

Mesenchymal stem cells (MSCs) are multipotent cells capable of differentiating into dopaminergic (DAergic) neurons, which is one of the major cell types damaged in Parkinson’s disease (PD). For this reason, MSCs are considered a potential cell source for PD therapy. It has been proved that hypoxia is involved in the proliferation and differentiation of stem cells. In this study, we investigated the effect of hypoxia on MSC proliferation and DAergic neuronal differentiation. Our results demonstrate that 3% O₂ treatment can enhance rat MSC proliferation by upregulation of phosphorylated p38 MAPK and subsequent nuclear translocation of hypoxia inducible factor (HIF)-1α. During neural differentiation, 3% O₂ treatment increases the expression of HIF-1α, phosphorylated ERK and p38 MAPK. These changes are followed by promotion of neurosphere formation and further DAergic neuronal differentiation. Furthermore, we explored the physiological function of hypoxia-induced DAergic neurons from human fetal MSCs by transplanting them into parkinsonian rats. Grafts induced with hypoxia display more survival of DAergic neurons and greater amelioration of behavioral impairments. Altogether, these results suggest that hypoxia can promote MSC proliferation and DAergic neuronal differentiation, and benefit for intrastriatal transplantation. Therefore, this study may provide new perspectives in application of MSCs to clinical PD therapy.     

Ex vivo expansion of human mesenchymal stem cells: A more effective cell proliferation kinetics and metabolism under hypoxia

The low bone marrow (BM) MSC titers demand a fast ex vivo expansion process to meet the clinically relevant cell dosage. Attending to the low oxygen tension of BM in vivo, we studied the influence of hypoxia on human BM MSC proliferation kinetics and metabolism. Human BM MSC cultured under 2% (hypoxia) and 20% O₂ (normoxia) were characterized in terms of proliferation, cell division kinetics and metabolic patterns. BM MSC cultures under hypoxia displayed an early start of the exponential growth phase, and cell numbers obtained at each time point throughout culture were consistently higher under low O₂, resulting in a higher fold increase after 12 days under hypoxia (40 ± 10 vs. 30 ± 6). Cell labeling with PKH26 allowed us to determine that after 2 days of culture, a significant higher cell number was already actively dividing under 2% compared to 20% O₂ and BM MSC expanded under low oxygen tension displayed consistently higher percentages of cells in the latest generations (generations 4–6) until the 5th day of culture. Cells under low O₂ presented higher specific consumption of nutrients, especially early in culture, but with lower specific production of inhibitory metabolites. Moreover, 2% O₂ favored CFU‐F expansion, while maintaining BM MSC characteristic immunophenotype and differentiative potential. Our results demonstrated a more efficient BM MSC expansion at 2% O₂, compared to normoxic conditions, associated to an earlier start of cellular division and supported by an increase in cellular metabolism efficiency towards the maximization of cell yield for application in clinical settings. J. Cell. Physiol. 223: 27–35, 2010. © 2009 Wiley‐Liss, Inc.