Cartilage Tissue Engineering Using Dermis Isolated Adult Stem Cells: The Use of Hypoxia during Expansion versus Chondrogenic Differentiation

Dermis isolated adult stem (DIAS) cells, a subpopulation of dermis cells capable of chondrogenic differentiation in the presence of cartilage extracellular matrix, are a promising source of autologous cells for tissue engineering. Hypoxia, through known mechanisms, has profound effects on in vitro chondrogenesis of mesenchymal stem cells and could be used to improve the expansion and differentiation processes for DIAS cells. The objective of this study was to build upon the mechanistic knowledge of hypoxia and translate it to tissue engineering applications to enhance chondrogenic differentiation of DIAS cells through exposure to hypoxic conditions (5% O₂) during expansion and/or differentiation. DIAS cells were isolated and expanded in hypoxic (5% O₂) or normoxic (20% O₂) conditions, then differentiated for 2 weeks in micromass culture on chondroitin sulfate-coated surfaces in both environments. Monolayer cells were examined for proliferation rate and colony forming efficiency. Micromasses were assessed for cellular, biochemical, and histological properties. Differentiation in hypoxic conditions following normoxic expansion increased per cell production of collagen type II 2.3 fold and glycosaminoglycans 1.2 fold relative to continuous normoxic culture (p<0.0001). Groups expanded in hypoxia produced 51% more collagen and 23% more GAGs than those expanded in normoxia (p<0.0001). Hypoxia also limited cell proliferation in monolayer and in 3D culture. Collectively, these data show hypoxic differentiation following normoxic expansion significantly enhances chondrogenic differentiation of DIAS cells, improving the potential utility of these cells for cartilage engineering.     

Moderate Hypoxia Influences Potassium Outward Currents in Adipose-Derived Stem Cells

Moderate hypoxic preconditioning of adipose-derived stem cells (ASCs) enhances properties such as proliferation and secretion of growth factors, representing a valuable strategy to increase the efficiency of cell-based therapies. In a wide variety of cells potassium (K⁺) channels are key elements involved in the cellular responses to hypoxia, suggesting that ASCs cultured under low oxygen conditions may display altered electrophysiological properties. Here, the effects of moderate hypoxic culture on proliferation, whole-cell currents, and ion channel expression were investigated using human ASCs cultured at 5% and 20% oxygen. Although cell proliferation was greatly enhanced, the dose-dependent growth inhibition by the K⁺ channel blocker tetraethylammonium (TEA) was not significantly affected by hypoxia. Under both normoxic and hypoxic conditions, ASCs displayed outward K⁺ currents composed by Ca²⁺-activated, delayed rectifier, and transient components. Hypoxic culture reduced the slope of the current-voltage curves and caused a negative shift in the voltage activation threshold of the whole-cell currents. However, the TEA-mediated shift of voltage activation threshold was not affected by hypoxia. Semiquantitative real-time RT-PCR revealed that expression of genes encoding for various ion channels subunits related to oxygen sensing and proliferation remained unchanged after hypoxic culture. In conclusion, outward currents are influenced by moderate hypoxia in ASCs through a mechanism that is not likely the result of modulation of TEA-sensitive K⁺ channels.     

The metabolism of human mesenchymal stem cells during proliferation and differentiation

Human mesenchymal stem cells (MSCs) reside under hypoxic conditions in vivo, between 4% and 7% oxygen. Differentiation of MSCs under hypoxic conditions results in inhibited osteogenesis, while chondrogenesis is unaffected. The reasons for these results may be associated with the inherent metabolism of the cells. The present investigation measured the oxygen consumption, glucose consumption and lactate production of MSCs during proliferation and subsequent differentiation towards the osteogenic and chondrogenic lineages. MSCs expanded under normoxia had an oxygen consumption rate of ～98 fmol/cell/h, 75% of which was azide-sensitive, suggesting that these cells derive a significant proportion of ATP from oxidative phosphorylation in addition to glycolysis. By contrast, MSCs differentiated towards the chondrogenic lineage using pellet culture had significantly reduced oxygen consumption after 24 h in culture, falling to ～12 fmol/cell/h after 21 days, indicating a shift towards a predominantly glycolytic metabolism. By comparison, MSCs retained an oxygen consumption rate of ～98 fmol/cell/h over 21 days of osteogenic culture conditions, indicating that these cells had a more oxidative energy metabolism than the chondrogenic cultures. In conclusion, osteogenic and chondrogenic MSC cultures appear to adopt the balance of oxidative phosphorylation and glycolysis reported for the respective mature cell phenotypes. The addition of TGF-β to chondrogenic pellet cultures significantly enhanced glycosaminoglycan accumulation, but caused no significant effect on cellular oxygen consumption. Thus, the differences between the energy metabolism of chondrogenic and osteogenic cultures may be associated with the culture conditions and not necessarily their respective differentiation.     

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.     

HIF1α‐mediated AIMP3 suppression delays stem cell aging via the induction of autophagy

Senescence in stem cells, which occurs as a consequence of chronic responses to the environment, defines the capacity of stem cells for proliferation and differentiation as well as their potential for tissue regeneration and homeostasis maintenance. Although stem cells reside under low oxygen pressure and the availability of oxygen is known to be a crucial determinant in their fate, the key modulators in stem cell aging and the underlying mechanism have yet to be unraveled. Human placenta‐derived mesenchymal stem cells (hpMSCs) were cultured under hypoxia (3% O₂) or normoxia (21% O₂) to investigate the key factors that regulate stem cell senescence under hypoxic conditions. RNA sequencing results suggested that the expression of aminoacyl‐tRNA synthetase‐interacting multifunctional protein 3 (AIMP3, EEF1E1), an aging inducer, in the hpMSCs was dramatically repressed under hypoxia with concurrent suppression of the aging marker p16^INK4a. The hpMSCs that overexpressed AIMP3 under hypoxic conditions displayed significantly decreased proliferation and fewer stem cell characteristics, whereas the downregulation of AIMP3 ameliorated the age‐related senescence of MSCs. Consistent with the results of the hpMSCs, MSCs isolated from the adipose tissue of AIMP3‐overexpressing mice exhibited decreased stem cell functions. Interestingly, AIMP3‐induced senescence is negatively regulated by hypoxia‐inducible factor 1α (HIF1α) and positively regulated by Notch3. Furthermore, we showed that AIMP3 enhanced mitochondrial respiration and suppressed autophagic activity, indicating that the AIMP3‐associated modulation of metabolism and autophagy is a key mechanism in the senescence of stem cells and further suggesting a novel target for interventions against aging.     

Recapitulation of mesenchymal condensation enhances in vitro chondrogenesis of human mesenchymal stem cells

Mesenchymal condensation is a critical transitional stage that precedes cartilage formation during embryonic development. We hypothesized that "priming" hMSCs to recapitulate mesenchymal condensation events prior to inducing differentiation would enhance their subsequent chondrogenic properties. Our prior studies have suggested that exposing hMSCs to hypoxia (2% O(2)) induces condensation-like effects. We therefore assessed the effect of preconditioning for different time periods on the expression of condensation specific genes by growing hMSCs in expansion medium under different normoxic (20% O(2)) and hypoxic conditions for up to 2 weeks, and subsequently induced chondrogenesis of preconditioned hMSCs. The total cultivation time for each group was 4 weeks and the chondrogenic properties were assessed using gene expression, biochemical analysis, and histological staining. Our results demonstrated the benefits of preconditioning were both time- and oxygen-dependent. Condensation specific genes, SOX-9 and NCAM, were significantly up-regulated in hypoxic conditions at the end of 1 week. COL X and MMP13 expression was also lower than the normoxic samples at this time point. However, this group did not exhibit more efficient chondrogenesis after 4 weeks. Instead, hMSCs preconditioned for 1 week and subsequently differentiated, both under 20% O(2), resulted in the most efficient chondrogenesis. Interestingly, while hypoxia appears to positively enhance expression of chondrogenic genes, this did not produce an enhanced matrix accumulation. The results of this study emphasize the significance of considering the timing of specific cues in developing protocols for stem cell-based therapies and underscore the complexity in regulating stem cell differentiation and tissue formation.     

Short-term exposure of multipotent stromal cells to low oxygen increases their expression of CX3CR1 and CXCR4 and their engraftment in vivo

The ability of stem/progenitor cells to migrate and engraft into host tissues is key to their potential use in gene and cell therapy. Among the cells of interest are the adherent cells from bone marrow, referred to as mesenchymal stem cells or multipotent stromal cells (MSC). Since the bone marrow environment is hypoxic, with oxygen tensions ranging from 1% to 7%, we decided to test whether hypoxia can upregulate chemokine receptors and enhance the ability of human MSCs to engraft in vivo. Short-term exposure of MSCs to 1% oxygen increased expression of the chemokine receptors CX3CR1and CXCR4, both as mRNA and as protein. After 1-day exposure to low oxygen, MSCs increased in vitro migration in response to the fractalkine and SDF-1alpha in a dose dependent manner. Blocking antibodies for the chemokine receptors significantly decreased the migration. Xenotypic grafting into early chick embryos demonstrated cells from hypoxic cultures engrafted more efficiently than cells from normoxic cultures and generated a variety of cell types in host tissues. The results suggest that short-term culture of MSCs under hypoxic conditions may provide a general method of enhancing their engraftment in vivo into a variety of tissues.     

Priming adult stem cells by hypoxic pretreatments for applications in regenerative medicine

The efficiency of regenerative medicine can be ameliorated by improving the biological performances of stem cells before their transplantation. Several ex-vivo protocols of non-damaging cell hypoxia have been demonstrated to significantly increase survival, proliferation and post-engraftment differentiation potential of stem cells. The best results for priming cultured stem cells against a following, otherwise lethal, ischemic stress have been obtained with brief intermittent episodes of hypoxia, or anoxia, and reoxygenation in accordance with the extraordinary protection afforded by the conventional maneuver of ischemic preconditioning in severely ischemic organs. These protocols of hypoxic preconditioning can be rather easily reproduced in a laboratory; however, more suitable pharmacological interventions inducing stem cell responses similar to those activated in hypoxia are considered among the most promising solutions for future applications in cell therapy. Here we want to offer an up-to-date review of the molecular mechanisms translating hypoxia into beneficial events for regenerative medicine. To this aim the involvement of epigenetic modifications, microRNAs, and oxidative stress, mainly activated by hypoxia inducible factors, will be discussed. Stem cell adaptation to their natural hypoxic microenvironments (niche) in healthy and neoplastic tissues will be also considered.     

Reconstitution activity of hypoxic cultured human cord blood CD34-positive cells in NOG mice

Hematopoietic stem cells (HSCs) reside in hypoxic areas of the bone marrow. However, the role of hypoxia in the maintenance of HSCs has not been fully characterized. We performed xenotransplantation of human cord blood cells cultured in hypoxic or normoxic conditions into adult NOD/SCID/IL-2Rγ^null (NOG) mice. Hypoxic culture (1% O₂) for 6 days efficiently supported the maintenance of HSCs, although cell proliferation was suppressed compared to the normoxic culture. In contrast, hypoxia did not affect in vitro colony-forming ability. Upregulation of a cell cycle inhibitor, p21, was observed in hypoxic culture. Immunohistochemical analysis of recipient bone marrow revealed that engrafted CD34⁺CD38⁻ cord blood HSCs were hypoxic. Taken together, these results demonstrate the significance of hypoxia in the maintenance of quiescent human cord blood HSCs.     

Characterization and evaluation of mesenchymal stem cells derived from human embryonic stem cells and bone marrow

Embryonic stem cells (ESCs) and mesenchymal stem cells (MSCs) have been studied for years as primary cell sources for regenerative biology and medicine. MSCs have been derived from cell and tissue sources, such as bone marrow (BM), and more recently from ESCs. This study investigated MSCs derived from BM, H1- and H9-ESC lines in terms of morphology, surface marker and growth factor receptor expression, proliferative capability, modulation of immune cell growth and multipotency, in order to evaluate ESC-MSCs as a cell source for potential regenerative applications. The results showed that ESC-MSCs exhibited spindle-shaped morphology similar to BM-MSCs but of various sizes, and flow cytometric immunophenotyping revealed expression of characteristic MSC surface markers on all tested cell lines except H9-derived MSCs. Differences in growth factor receptor expression were also shown between cell lines. In addition, ESC-MSCs showed greater capabilities for cell proliferation, and suppression of leukocyte growth compared to BM-MSCs. Using standard protocols, induction of ESC-MSC differentiation along the adipogenic, osteogenic, or chondrogenic lineages was less effective compared to that of BM-MSCs. By adding bone morphogenetic protein 7 (BMP7) into transforming growth factor beta 1 (TGFβ1)-supplemented induction medium, chondrogenesis of ESC-MSCs was significantly enhanced. Our findings suggest that ESC-MSCs and BM-MSCs show differences in their surface marker profiles and the capacities of proliferation, immunomodulation, and most importantly multi-lineage differentiation. Using modified chondrogenic medium with BMP7 and TGFβ1, H1-MSCs can be effectively induced as BM-MSCs for chondrogenesis.     

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.