Hypoxic Culture Conditions Research Articles

Hypoxic culture enhances the antimicrobial activity of amnion-derived mesenchymal stem cells, thereby reducing bacterial load and promoting wound healing in diabetic mice

BackgroundConditioned medium from amnion-derived mesenchymal stem cells (AMSCs) enhances wound healing, a process that is further improved under hypoxic culture conditions. Diabetic foot ulcers are difficult to treat and are frequently complicated by a high rate of bacterial infections, mainly Staphylococcus aureus, which can lead to limb amputation and death. Here, we topically applied conditioned medium from AMSCs cultured under hypoxic conditions to S. aureus-infected wounds in diabetic mice to investigate its effect on bacterial counts and wound healing. MethodsWe prepared conditioned medium by culturing AMSCs under 21% or 1% O2 and investigated its effects on S. aureus. We infected skin wounds of diabetic mice with S. aureus and treated these with hydrogels containing the conditioned medium to examine its effect on bacterial inhibition and wound healing. ResultsConditioned medium from AMSCs cultured under 1% O2 contained higher levels of the antimicrobial peptide LL-37. It significantly inhibited S. aureus growth in vitro, reduced bacterial counts in infected wounds, and facilitated wound closure in diabetic mice. ConclusionsHydrogels containing conditioned medium from hypoxically cultured AMSCs inhibited the growth of S. aureus and promoted wound healing in a mouse model of diabetic wounds.

Synovial Membrane Is a Major Producer of Extracellular Inorganic Pyrophosphate in Response to Hypoxia.

Calcium pyrophosphate dehydrate (CPPD) crystals are found in the synovial fluid of patients with articular chondrocalcinosis or sometimes with osteoarthritis. In inflammatory conditions, the synovial membrane (SM) is subjected to transient hypoxia, especially during movement. CPPD formation is supported by an increase in extracellular inorganic pyrophosphate (ePPi) levels, which are mainly controlled by the transporter Ank and ectonucleotide pyrophosphatase/phosphodiesterase 1 (ENPP1). We demonstrated previously that transforming growth factor (TGF)-β1 increased ePPi production by inducing Ank and Enpp1 expression in chondrocytes. As the TGF-β1 level raises in synovial fluid under hypoxic conditions, we investigated whether hypoxia may transform SM as a major source of ePPi production. Synovial fibroblasts and SM explants were exposed to 10 ng/mL of TGF-β1 in normoxic or hypoxic (5% O2) culture conditions. Ank and Enpp1 expression were assessed by quantitative PCR, Western blot and immunohistochemistry. ePPi was quantified in culture supernatants. RNA silencing was used to define the respective roles of Ank and Enpp1 in TGF-β1-induced ePPi generation. The molecular mechanisms involved in hypoxia were investigated using an Ank promoter reporter plasmid for transactivation studies, as well as gene overexpression and RNA silencing, the respective role of hypoxia-induced factor (HIF)-1 and HIF-2. Our results showed that TGF-β1 increased Ank, Enpp1, and therefore ePPi production in synovial fibroblasts and SM explants. Ank was the major contributor in ePPi production compared to ENPP1. Hypoxia increased ePPi levels on its own and enhanced the stimulating effect of TGF-β1. Hypoxic conditions enhanced Ank promoter transactivation in an HIF-1-dependent/HIF-2-independent fashion. We demonstrated that under hypoxia, SM is an important contributor to ePPi production in the joint through the induction of Enpp1 and Ank. These findings are of interest as a rationale for the beneficial effect of anti-inflammatory drugs on SM in crystal depositions.

Analysis on promotive effect of rocking culture on keratinocyte differentiation in 3-dimensional reconstitution human epidermis.

A 3-dimensional culture system of keratinocytes achieves cornification as a terminal differentiation that can mimic the formation of stratified epidermis. At the onset of keratinocyte differentiation, air-exposure treatment is essential for promotion. We have previously reported that the stimulation of differentiation is accompanied by downregulation of the transcriptional activity of the hypoxia-inducible factor (HIF) and also found that rocking treatment of cultured keratinocytes in the submerged condition restored their differentiation. A comparative study of cultured keratinocytes with and without rocking was then carried out to investigate the characteristics of the recovered differentiation by morphological and biochemical analyses. In addition, transcriptome analysis revealed the expected similar pattern between air-exposed and rocking cultures, including HIF-regulating transcripts. Furthermore, the promotive effect of rocking treatment was impaired under hypoxic culture conditions (1% O2). We showed that the restored promotion of differentiation by rocking culture is mainly due to the abrogation of transcriptional events by hypoxia.

Thrombopoietin exerts a neuroprotective effect by inhibiting the suppression of neuronal proliferation and axonal outgrowth in intrauterine growth restriction rats

Chronic hypoxia in utero causes intrauterine growth restriction (IUGR) of the fetus. IUGR infants are known to be at higher risk for neurodevelopmental disorders, but the mechanism is unclear. In this study, we analyzed the structure of the cerebral cortex using IUGR model rats generated through a reduced uterine perfusion pressure operation. IUGR rats exhibited thinner cerebral white matter and enlarged lateral ventricles compared with control rats. Expression of neuron cell markers, Satb2, microtubule-associated protein (MAP)-2, α-tubulin, and nestin was reduced in IUGR rats, indicating that neurons were diminished at various developmental stages in IUGR rats, from neural stem cells to mature neurons. However, there was no increase in apoptosis in IUGR rats. Cells positive for Ki67, a marker of cell proliferation, were reduced in neurons and all glial cells of IUGR rats. In primary neuron cultures, axonal elongation was impaired under hypoxic culture conditions mimicking the intrauterine environment of IUGR infants. Thus, in IUGR rats, chronic hypoxia in utero suppresses the proliferation of neurons and glial cells as well as axonal elongation, resulting in cortical thinning and enlarged lateral ventricles. Thrombopoietin (TPO), a platelet growth factor, inhibited the decrease in neuron number and promoted axon elongation in primary neurons under hypoxic conditions. Intraperitoneal administration of TPO to IUGR rats resulted in increases in the number of NeuN-positive cells and the area coverage of Satb2. In conclusion, suppression of neuronal proliferation and axonal outgrowth in IUGR rats resulted in cortical thinning and enlargement of lateral ventricles. TPO administration might be a novel therapeutic strategy for treating brain dysmaturation in IUGR infants.

Abstract 6005: Generation of new oncology cell models through long-term acclimation under hypoxic and hyperbaric culture conditions

Abstract Traditional cancer cell lines used as surrogate models in vitro and in vivo have made significant contributions to cancer research and drug discovery. While cell culture and animal studies are critical steps in determining the efficacy, pharmacodynamics, and mechanism of action of novel anti-cancer drugs, it is well-known that cells grown in 2D culture systems have modified growth characteristics and different responses to chemotherapeutic drugs compared to tumors in vivo as a result of differing structural and environmental conditions that fail to replicate in vivo tumor microenvironments. AVATAR bioreactors (Xcell Biosciences, San Francisco, CA) were invented to address these limitations by providing discrete control over oxygen and hyperbaric pressure levels to better mimic the in vivo environment and allow cultured cells to be grown in conditions which mimic the human body. To better serve preclinical research needs, we generated new tumor cell models by long-term acclimating (LTA) existing cancer cells in the AVATAR incubator system using 1% O2 and 2 PSI conditions similar to the conditions expected in the tumor microenvironment (TME). Six different cancer cell lines of diverse origins including pancreas (MIA PaCa-2), breast (BT-474), colon (HCT116), prostate (LNCaP, PC-3) and melanoma (A-375) were adapted for 2-3 months with phenotypic changes recorded. Key marker changes were commonly observed in LTA cells compared to parental lines. For example, in HCT116 colorectal cancer cells, TME culture conditions induced a higher expression level of E-cadherin in both protein and mRNA levels (2.2-fold change), indicating a mesenchymal-epithelial transition of the cell after adaptation. This coincides with the observation that LTA adapted HCT116 cell bodies had a more angular cell morphology as compared to parental cells grown under conventional normoxic culture conditions. In-depth gene-expression analysis using a Nanostring tumor signaling pathway panel identified several common and tissue-specific gene and pathway changes. These included VEGFA, EGF receptor signaling pathway (EGFR, E-Cadherin, GRB7, c-Fos), TGF pathway (IGFBP3, TGFB1, TGFB2, SMAD7), and extracellular matrix signaling (LAMB3, ITGA5, ITGB3, ITGB8, PDGFA, CCND2). Current studies are focusing on drug response testing in 2D and 3D cultures based on above-mentioned gene and signaling pathway changes. Cell proliferation and migration capacity of xenograft LTA cells will be evaluated for the purposes of creating novel murine models for drug screening applications. Citation Format: Yewei Xing, Abriel Czachorowski, James Lim, Scott Wise. Generation of new oncology cell models through long-term acclimation under hypoxic and hyperbaric culture conditions [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6005.

Abstract 5271: Pseudohypoxia by HIF-PHD inhibitors activates tumor immune response for MSS colorectal cancer

Abstract Purpose: Microsatellite-stable (MSS) colorectal cancer is resistant for immune therapy, necessitating the exploration of novel modalities for its conquest. Recent revelations have revealed the genetic upregulation of HIF-1α as a potential activator of CD8+ T-cells, enhancing an anti-tumor effect. Pseudohypoxia, characterized by normoxic upregulation of Hypoxia-Inducible Factor (HIF-1α), is known to be induced by iron chelators, including HIF-PHD inhibitors. In this study, we aimed to examine whether pseudohypoxia by HIF-PHD inhibitor can enhance immune response and elucidate the underlying mechanism. Method: Colon 26, SW480 and HT29 were used as murine and human MSS colorectal cancer model. Human and murine CD8+ T-cells were represented by Jurkat cells and isolated splenic CD8+ T-cells, respectively. HIF-PHD inhibitors Roxadustat and Vadadustat were used in the study, and a subcutaneous Colon 26 tumor model was established in BALB/c WT and nude mice. Hypoxic culture conditions were maintained for 48 hours at 2% O2. Results: Roxadustat and Vadadustat did not suppress the proliferation of cancer cells under conditions marked by HIF-1α upregulation. Roxadustat and Vadadustat suppress the colon 26 subcutaneous tumor growth in WT mice, the effect was cancelled in the nude mice. The treatment regimen was associated with an augmentation in the total number and effector-like subtypes of CD8+ T-cells (Tex) infiltrating the tumor. Combination therapy involving Roxadustat and PD-1 antibody synergistically inhibited tumor growth and was accompanied by an increase in PD-1lo, Ly108-, TIM-3+ CD8+ T-cells within the tumor microenvironment. Immunohistochemistry revealed that Interleukin-2 (IL-2) expression of murine spleen was stronger in Roxadustat than control group. Interestingly, HIF-PHD inhibitors induced the secretion of IL-2 from isolated CD3+ T-cells of murine spleens in ELISA, a phenomenon uniquely observed under pseudo-hypoxic conditions created by HIF-PH inhibitors, rather than actual hypoxia. This result indicates that pseudo-hypoxia may induce more robust hypoxic response than real hypoxia. Conclusion: The induction of pseudohypoxia by HIF-PHD inhibitors activates the tumor immune response for MSS colorectal cancer via induction of splenic IL-2. Citation Format: Toshiaki Ohara, Yuehua Chen, Yusuke Hamada, Seitaro Nishimura, Hotaka Kawai, Kazuhiro Noma, Hiroshi Tazawa, Toshiyoshi Fujiwara, Akihiro Matsukawa. Pseudohypoxia by HIF-PHD inhibitors activates tumor immune response for MSS colorectal cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5271.

Abstract 1240: Enhancing NK cell function in the ‘cold’ tumor microenvironment of prostate cancer with a novel tri-specific killer engager

Abstract Introduction: Natural killer (NK) cells are associated with good prognosis in patients with metastatic castration-resistant prostate cancer (mCRPC). However, NK cell function is hindered in the immunosuppressive and hypoxic tumor microenvironment (TME) of mCRPC. To improve NK cell-mediated responses against mCRPC, we designed a novel Tri-specific Killer Engager (TriKE®) that engages with NK cell activating receptor CD16, binds to prostate-specific membrane antigen (PSMA) that is highly and specifically expressed on mCRPC, and has an interleukin (IL)-15 moiety that is essential for NK cell survival, proliferation, and priming. Methods: PSMA TriKE was produced using the mammalian expression system and tested using healthy donor and prostate cancer patient-derived NK cells. Flow cytometry-based functional and dye dilution proliferation assays were used to compare activation and proliferation of NK cells treated with PSMA TriKE or IL-15. NK cell cytolytic capacity against C4-2 cells, a PSMA-expressing prostate cancer line, was measured using IncuCyte live cell imaging. In various assays, hypoxic (1% oxygen) culture condition and cytokine-induced myeloid-derived suppressor cells (MDSC) were incorporated to better examine PSMA TriKE function in the physiological setting of mCRPC. In vivo testing of the PSMA TriKE was performed using C4-2 xenograft model and expanded NK cells in NSG mice. Results: PSMA TriKE significantly enhanced expansion of peripheral blood NK cells derived from healthy donors up to 8-fold. Additionally, PSMA TriKE induced significantly higher NK cell degranulation and intracellular IFNγ and TNFα buildup, when compared to IL-15 treatment, after incubation with C4-2 cells. Responses of NK cells derived from prostate cancer patients were equivalent to healthy controls. Although NK cell activation was not observed against PSMA knockout (KO) C4-2, indicating specificity of PSMA TriKE treatment, bystander killing of PSMA-KO C4-2 was achieved with PSMA TriKE treatment when wildtype C4-2 cells were cocultured. This suggests potential PSMA TriKE benefit in controlling tumor antigen escape, through natural cytotoxicity once primed. In settings that mimic the TME of mCRPC, NK cells treated with PSMA TriKE in prolonged exposure to hypoxia showed retention of cytotoxicity against C4-2 while IL-15 treated NK cells showed greatly impaired cytotoxicity. Similarly, MDSC suppressed NK cell cytotoxicity in the presence of IL-15, but PSMA TriKE treatment abrogated MDSC-induced suppression. Finally, PSMA TriKE enhanced tumor control and improved survival of mice as compared to IL-15 and no treatment groups in vivo. Conclusion: PSMA TriKE demonstrates potential in overcoming suppression of NK cells in the TME of mCRPC and is a promising candidate for advanced prostate cancer therapy. Citation Format: Shee Kwan Phung, Yvette Soignier, Nicholas Zorko, Rhett L. Waller, Josh Walker, Trygve Nelson, Carly Selleck, Laura Bendzick, Philippa Kennedy, Jeffrey S. Miller, Martin Felices. Enhancing NK cell function in the ‘cold’ tumor microenvironment of prostate cancer with a novel tri-specific killer engager [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1240.

Hypoxia potentiated pro-angiogenic and anti-fibrotic effect of nanovesicles derived from human mesenchymal stem cells

Skin acts as a frontline defense that protects against external invasion of pathogens and harmful substances. When the skin if injured, wound healing to reconstruct the skin is mainly supervised by fibroblasts. For optimal wound healing, proper neovascularization and reduced scar formation are essential factors that contribute to skin regeneration. In this context, various studies have examined the application of stem cells or stem cell-derived materials such as nanovesicles (NVs) in wound healing. In the present study, a hypoxic cell culture condition was used to engineer mesenchymal stem cells (MSCs) to generate NVs with enhanced angiogenic and anti-fibrotic efficacy. Hypoxic culture condition (2 % O2, 48 h) MSCs-derived NVs (H-NVs) showed more angiogenic and less pro-inflammatory factor expression compared to NVs derived from mesenchymal stem cell under normoxic culture condition (N-NVs). The H-NVs treatment showed upregulated angiogenesis with increased migration and proliferation of skin fibroblasts. Moreover, H-NVs treatment downregulated myofibroblast differentiation, which is critical for scar formation. In summary, this study confirmed that the use of a hypoxic cell culture condition can be a key factor enhancing the therapeutic efficacy of NVs in terms of effective angiogenesis and anti-fibrosis.

MESENCHYMAL STEM CELLS: EXOSOMES AND CONDITIONED MEDIA AS INNOVATIVE STRATEGIES IN THE TREATMENT OF PATIENTS WITH AUTOIMMUNE DISEASES

Introduction. Autoimmune diseases are a class of immunopathological conditions heterogeneous in clinical manifestations, characterized by immune disorders that cause the loss of the body's autoimmune tolerance and, as a result, abnormal reactivity of B-cells and T-cells, which leads to damage to own tissues. Today, about 10% of the population suffers from diseases of this class, which are clinically manifested in the form of more than 80 forms of autoimmune diseases.
 The aim of the study. Summarize current ideas about the therapeutic potential of conditioned media and exosomes of MSCs in the treatment of patients with autoimmune diseases based on data from open sources of information.
 Materials and methods. Publications were selected based on PubMed, Clinical Key Elsevier, Cochrane Library, eBook Business Collection and Google Scholar databases, which covered information on the use of conditioned media and MSC exosomes in the treatment of diseases of premature newborns using the
 Keywords:
 mesenchymal stem cells, conditioned media, secretion, autoimmune diseases.
 Results. The technical complexity and high costs associated with the production and regulatory approval procedures of MSC therapy create barriers to their clinical use. Studies have shown that the cell-free secretome of MSCs, which consists of a wide range of growth factors, cytokines, chemokines and extracellular vesicles, exhibits a pluripotent effect. Today, extracellular vesicles are classified according to their diameter into apoptotic bodies (>1000 nm), microvesicles (100–1000 nm) and exosomes (30–150 nm). Exosome activity can be easily manipulated by preconditioning MSCs, by simply adding cytokines or chemicals to the culture medium, by introducing gene modifications, or by using hypoxic culture conditions. A number of studies have demonstrated the comparable effectiveness of conditioned media and MSC exosomes in the treatment of patients with autoimmune diseases.
 Conclusions. Exosomes and conditioned media with MSCs have the potential to replace cell therapy or serve as a comparable clinical strategy to biological therapy in neonatology. MSC preconditioning will allow modulating the therapeutic effects of exosomes and will become the basis for establishing recommendations and standards for effective and safe cell-free therapy.

Sarcoma protein kinase inhibition alleviates liver fibrosis by promoting hepatic stellate cells ferroptosis

Abstract Liver fibrosis is a type of chronic pathological liver damage involving liver tissue hypoxia and abnormal extracellular matrix deposits. Hepatic stellate cells (HSCs) activation is critical for liver fibrosis. Currently, inhibiting HSCs activation or inducing HSCs ferroptosis is considered an effective strategy for the treatment of liver fibrosis. Sarcoma protein kinase (Src) is an important member of the tyrosine protein kinase family. Hypoxia causes Src phosphorylation at tyrosine 416 (Tyr 416), and inhibiting Src activation can alleviate liver fibrosis. There is currently little research on the relationship between Src activation and ferroptosis in liver fibrosis. 1-(1,1-Dimethylethyl)-1-(4-methylphenyl)-1H-pyrazolo[3,4-d]pyrimidin-4-amine (PP1) is an inhibitor of Src activation at Tyr 416. Therefore, in this study we treated HSC-T6 cells with PP1 under normoxic and hypoxic culture conditions; moreover, PP1 was also used to treat a carbon tetrachloride-induced mouse liver fibrosis model. We explored whether inhibiting Src activation could alleviate liver fibrosis by promoting HSCs ferroptosis in vitro and in vivo. In vitro experiments showed that inhibiting Src activation in HSC-T6 cells significantly reduced hypoxia-inducible factor-1α (HIF-1α) expression and HSC-T6 cells activation, and ferroptosis was significantly increased. In vivo experiments revealed that inhibiting Src activation in fibrotic livers reduced HIF-1α expression; meanwhile, ferroptosis was promoted, and liver fibrosis was alleviated. Therefore, inhibiting Src activation, which increases HSCs ferroptosis, can alleviate liver fibrosis.

A Microfluidic Platform for the Time-Resolved Interrogation of Polarized Retinal Pigment Epithelial Cells.

Cells grown in milliliter volume devices have difficulty measuring low-abundance secreted factors due to low resulting concentrations. Using microfluidic devices increases concentration; however, the constrained geometry makes phenotypic characterization with transepithelial electrical resistance more difficult and less reliable. Our device resolves this problem. We designed and built a novel microfluidic "Puck" assembly using laser-cut pieces from preformed sheets of silicone and commercial off-the-shelf parts. Transwell membranes containing polarized retinal pigment epithelial (RPE) cells were reversibly sealed within the Puck and used to study polarized protein secretion. Protein secretion from the apical and basal surfaces in response to hypoxic conditions was quantified using an immunoassay method. Computational fluid modeling was performed on the chamber design. Under hypoxic culture conditions (7% O2), basal vascular endothelial growth factor (VEGF) secretion by polarized RPE cells increased significantly from 1.40 to 1.68 ng/mL over the first 2 hours (P < 0.0013) and remained stably elevated through 4 hours. Conversely, VEGF secretion from the apical side remained constant under the same hypoxic conditions. The Puck can be used to measure spatiotemporal protein secretion by polarized cells into apical and basal microniches in response to environmental conditions. Computational model results support the absence of biologically significant shear stress to the cells caused by the device. The Puck can be used validate the mature phenotypic health of autologous induced pluripotent stem cells (iPSC)-derived RPE cells prior to transplantation.

EGCG inhibits the inflammation and senescence inducing properties of MDA-MB-231 triple-negative breast cancer (TNBC) cells-derived extracellular vesicles in human adipose-derived mesenchymal stem cells

BackgroundTriple-negative breast cancer (TNBC) cells’ secretome can induce a pro-inflammatory phenotype in human adipose-derived mesenchymal stem cells (hADMSC). This can be prevented by the green tea polyphenol epigallocatechin-3-gallate (EGCG). The impact of EGCG on the paracrine regulation that the extracellular vesicles (EVs) specifically exert within the TNBC secretome remains unknown.MethodsEVs were obtained from a TNBC-derived serum-starved MDA-MB-231 cell model treated or not with EGCG under normoxic or hypoxic (< 1% O2) culture conditions. RNA-Seq analysis was used to assess the EVs’ genetic content. The modulation of inflammatory and senescence markers in hADMSC was evaluated by RT-qPCR using cDNA arrays and validated by immunoblotting. A protein profiler phospho-kinase array was used to explore signaling pathways.ResultsWhile hypoxic culture conditions did not significantly alter the genetic content of MDA-MB-231-secreted EVs, the addition of EGCG significantly modified EVs genetic material at low oxygen tension. Gene expression of cancer-associated adipocyte pro-inflammatory markers CXCL8, CCL2 and IL-1β was increased in hADMSC treated with EVs. Concomitantly, EVs isolated from MDA-MB-231 treated with EGCG (EGCG-EVs) downregulated CCL2 and IL-1β, while inducing higher expression of CXCL8 and IL-6 levels. EVs activated CHK-2, c-Jun, AKT and GSK-3β signaling pathways in hADMSC, whereas EGCG-EVs specifically reduced the latter two as well as the serum starvation-induced senescence markers p21 and β-galactosidase. Finally, the mitochondrial content within the TNBC cells-derived EVs was found reduced upon EGCG treatment.ConclusionThis proof of concept study demonstrates that the chemopreventive properties of diet-derived polyphenols may efficiently target the paracrine regulation that TNBC cells could exert upon their surrounding adipose tissue microenvironment.

Genetic and Functional Modifications Associated with Ovarian Cancer Cell Aggregation and Limited Culture Conditions.

The aggregation of cancer cells provides a survival signal for disseminating cancer cells; however, the underlying molecular mechanisms have yet to be elucidated. Using qPCR gene arrays, this study investigated the changes in cancer-specific genes as well as genes regulating mitochondrial quality control, metabolism, and oxidative stress in response to aggregation and hypoxia in our progressive ovarian cancer models representing slow- and fast-developing ovarian cancer. Aggregation increased the expression of anti-apoptotic, stemness, epithelial-mesenchymal transition (EMT), angiogenic, mitophagic, and reactive oxygen species (ROS) scavenging genes and functions, and decreased proliferation, apoptosis, metabolism, and mitochondrial content genes and functions. The incorporation of stromal vascular cells (SVF) from obese mice into the spheroids increased DNA repair and telomere regulatory genes that may represent a link between obesity and ovarian cancer risk. While glucose had no effect, glutamine was essential for aggregation and supported proliferation of the spheroid. In contrast, low glucose and hypoxic culture conditions delayed adhesion and outgrowth capacity of the spheroids independent of their phenotype, decreased mitochondrial mass and polarity, and induced a shift of mitochondrial dynamics towards mitophagy. However, these conditions did not reduce the appearance of polarized mitochondria at adhesion sites, suggesting that adhesion signals that either reversed mitochondrial fragmentation or induced mitobiogenesis can override the impact of low glucose and oxygen levels. Thus, the plasticity of the spheroids' phenotype supports viability during dissemination, allows for the adaptation to changing conditions such as oxygen and nutrient availability. This may be critical for the development of an aggressive cancer phenotype and, therefore, could represent druggable targets for clinical interventions.

Characteristics of Human Nasal Turbinate Stem Cells under Hypoxic Conditions.

This study investigated the influence of hypoxic culture conditions on human nasal inferior turbinate-derived stem cells (hNTSCs), a subtype of mesenchymal stem cells (MSCs). It aimed to discern how hypoxia affected hNTSC characteristics, proliferation, and differentiation potential compared to hNTSCs cultured under normal oxygen levels. After obtaining hNTSCs from five patients, the samples were divided into hypoxic and normoxic groups. The investigation utilized fluorescence-activated cell sorting (FACS) for surface marker analysis, cell counting kit-8 assays for proliferation assessment, and multiplex immunoassays for cytokine secretion study. Differentiation potential-osteogenic, chondrogenic, and adipogenic-was evaluated via histological examination and gene expression analysis. Results indicated that hNTSCs under hypoxic conditions preserved their characteristic MSC phenotype, as confirmed by FACS analysis demonstrating the absence of hematopoietic markers and presence of MSC markers. Proliferation of hNTSCs remained unaffected by hypoxia. Cytokine expression showed similarity between hypoxic and normoxic groups throughout cultivation. Nevertheless, hypoxic conditions reduced the osteogenic and promoted adipogenic differentiation potential, while chondrogenic differentiation was relatively unchanged. These insights contribute to understanding hNTSC behavior in hypoxic environments, advancing the development of protocols for stem cell therapies and tissue engineering.

M6A RNA MODIfiCATION AND A DRIVER OF CELLULAR PLASTICITY AND INTRA-TUMOURAL HETEROGENEITY IN GLIOBLASTOMA

Abstract AIMS The immense cellular plasticity of glioblastoma is a barrier to successful therapy. N6-methyladenosine (m6A) methylation of RNA is increasingly recognised as a key driver of cellular differentiation. Crucially, it is a reversible and dynamic modification that alters cellular phenotype via the regulation of mRNA metabolism. We hypothesise that m6A methylation mediates glioblastoma plasticity, regulating transcriptional networks and thus influencing cellular adaptation under different microenvironmental conditions. Our aims are to assess whether total m6A levels and m6A-regulatory gene expression are responsive to change in oxygenation levels and cellular confluency. Furthermore, we aim to characterise the spatial distribution of m6A in 3D cell culture models. METHOD We have investigated m6A levels in patient-derived cell lines from the invasive region of adult glioblastoma. Mass spectrometry and anti-m6A immunofluorescence confocal microscopy was used to quantify total m6A levels. We have compared total m6A levels in normoxic versus hypoxic culture conditions and in low versus high confluency 2D cell culture, including cell migration during a scratch assay. We have measured expression levels of key m6A-regulatory genes using rtPCR. RESULTS Total m6A levels and regulatory gene expression were not significantly different in normoxic versus hypoxic conditions. However, m6A levels were decreased in high confluency versus low confluency conditions. We will further characterise the spatial distribution of m6A in 3D cell culture models using immunofluorescence confocal microscopy. CONCLUSIONS We demonstrate a degree of variability in m6A levels in response to micro-environmental conditions, such as cellular confluency. These findings indicate a role for m6A in tumoral heterogeneity and cellular adaptation.

Promotion of NAD+ recycling by the hypoxia-induced shift in the lactate dehydrogenase isozyme profile reduces the senescence of human bone marrow-derived endothelial progenitor cells

Expansion of bone marrow-derived endothelial progenitor cells (EPCs) in vitro to obtain required cell numbers for therapeutic applications faces the challenge of growing cell senescence under the traditional normoxic culture condition. We previously found that 1% O2 hypoxic culture condition is favorable for reducing senescence of EPCs, but the mechanisms underlying the favorability are still unclear. Here, we found that, compared with normoxia, hypoxia induced a shift in lactate dehydrogenase (LDH) isozyme profile, which manifested as decreased LDH2 and LDH1 and increased LDH5, LDH4 and total LDHs. Moreover, under hypoxia, EPCs presented higher LDH activity, which could promote the conversion of pyruvate to lactate, as well as a higher level of NAD+, Bcl2 interacting protein 3 (BNIP3) expression and mitophagy. Additionally, under hypoxia, knock-down of the LDHA subunit increased the LDH2 and LDH1 levels and knock-down of the LDHB subunit increased the LDH5 level, while the simultaneous knock-down of LDHA and LDHB reduced total LDHs and NAD+ level. Inhibition of NAD+ recycling reduced BNIP3 expression and mitophagy and promoted cell senescence. Taken together, these data demonstrated that 1% O2 hypoxia induces a shift in the LDH isozyme profile, promotes NAD+ recycling, increases BNIP3 expression and mitophagy, and reduces EPC senescence. Our findings contribute to a better understanding of the connection between hypoxic culture conditions and the senescence of bone marrow-derived EPCs and provide a novel strategy to improve in vitro expansion of EPCs.

Adipose-Derived Stem Cells (ADSCs) in preconditioning of hypoxic cultures compared to normoxic cultures to prevent the formation of fibrous tissue: a review article

Background: Stem cells are a key element in regenerative medicine for their ability to differentiate into various cell phenotypes. According to the literature review, one process that might improve the functionality of ADSCs is hypoxic culture conditions. Hypoxic oxygen culture conditions at 5% concentration are said to increase stem cell proliferation and viability after transplantation. Methods: A comprehensive literature search was conducted by the author to obtain relevant studies from the PubMed, MEDLINE, Embase, PreMEDLINE, Embase, PsycINFO, Scopus, and Cochrane databases for the last twenty years (January 2002 – July 2022). The author used a search strategy with the following keywords: Adipose-Derived Stem Cells, hypoxic culture, normoxic culture, or pre-conditioned stem cells. Discussion: Hypoxic culture preconditioning of ADSCs with an oxygen concentration of 5% can improve proliferation capabilities and stem cell viability post-transplantation. Hypoxic preconditioning will enhance the expression of HIF-1

Targeting the monocarboxylate transporter MCT2 and lactate dehydrogenase A LDHA in cancer cells with FX-11 and AR-C155858 inhibitors.

In 1930, Otto Warburg reported that "aerobic glycolysis" is the intrinsic property of all tumor cells' fermentation of glucose to L-Lactate by lactate dehydrogenase A (LDHA) activity. This only produces per mole of glucose two moles of adenosine triphosphate (ATP), compared with 32 moles of ATP in a normal cell. Thus, tumor cells have to uptake 30 folds more glucose, the resulting accumulated lactate are then transported by a monocarboxylate transporter (MCT) with the participation of a CD147 molecule. Inhibition of MCT1 by RNA interference (RNAi) disrupted the unique metabolism of the tumor and caused tumor cell death. However, the effectiveness of the strategies depends on the targeted delivery of the therapeutics. In this study, a synergistic approach was used to target LDHA and MCT1 with small molecule inhibitors FX11 and AR-C155858, respectively. Cell cytotoxicity assays (AlamarBlue assay), and Mitochondria Membrane Potential (JC-1) dye assays were performed on human breast cancer cells MCF-7 and colorectal cancer cells HCT116. To achieve this aim, the following objectives were proposed: the effect of metabolic inhibitors on tumor glycolytic metabolite environment, and the efficacy of metabolite inhibitors on human breast and colorectal cancer cells in vitro. Then, gene expression analysis was performed using Qiagen RT2 Profiler PCR array for apoptosis. All these assays were performed on human breast cancer cells MCF-7 and colorectal cancer cells HCT116. Normal human fibroblasts were used as control cells under normal and hypoxic culture conditions. In this study, the use of FX-11 inhibitors under normoxia or hypoxia in two or more cancer and normal cell lines has a direct effect on LDHA, whereby it inhibits its production, and this reduces the growth and cell proliferation of tumors. One of the more significant findings to emerge from this study is that using AR-C155858 inhibitor alone has increased the cell proliferation and showed no significant changes compared with the control. The other major finding was that combination of the two inhibitors, FX-11 and AR-C155858, under normoxia or hypoxia in two different cell lines MCF-7 and HCT-116 measured a decrease in the cells proliferative and red/green ratio. We successfully demonstrated that a combination of MCT1 inhibitor and LDHA inhibitor led to better outcomes. Indeed, this makes LDHA an ideal metabolic therapeutic target.

The optimum oxygen level in hypoxic culture conditions of ligament derived stem cells: experimental research.

This study aimed to investigate the optimum oxygen level for rabbit cruciate ligament fibroblast cells culture and Ligament Derived Conditioned Medium/LD-CM (Secretome) preparation in vitro. Fibroblasts were isolated from the cruciate ligament of the rabbit's knee. Cultured of rabbit cruciate ligament Fibroblast Cells (fifth passage) were assigned to the slight (5% O2), middle (3% O2), and severe hypoxia (1% O2) groups and the normoxia (21% O2) group. Measurement of growth factors: TGF-β1, PDGF, FGF, and VEGF in LD-CM (Secretome) used an enzyme-linked immunosorbent assay. The highest number of cultured cells were in the 5% O2 group compared to the normoxia, 1 and 3% groups. The hypoxia 5% group also had increased productions of PDGF, FGF, and VEGF proteins in LD-CM (secretome) compared to the 1, 3%, and normoxia groups. TGF-β1 production was slightly higher in the 3 group than the 5% group. The hypoxic precondition of 5% oxygen was the optimum condition for ligament culture and ligament derived conditioned medium (secretome) preparation in vitro.

H2O2 transit through the mitochondrial intermembrane space promotes tumor cell growth in vitro and in vivo

Cancer cells experience increased levels of oxidant stress as a consequence of oncogene activation, nucleotide biosynthesis, and growth factor receptor signaling. Mitochondria contribute to this redox stress by generating reactive oxygen species (ROS) along the electron transport chain, which are released to the matrix and the intermembrane space (IMS). Assessing the contribution of mitochondrial ROS in cancer cells is technically difficult, as electron transport chain inhibitors can increase or decrease ROS generation, while they also block oxidative phosphorylation and ATP synthesis. Mitochondria-targeted antioxidant compounds can scavenge ROS in the matrix compartment but do not act on ROS released to the IMS. We assessed the importance of mitochondrial ROS for tumor cell proliferation, survival, and for tumor xenograft growth by stably expressing a hydrogen peroxide (H2O2) scavenger, peroxiredoxin-5, in the mitochondrial IMS (IMS-Prdx5) in 143B osteosarcoma and HCT116 colorectal cancer cell lines. IMS-Prdx5 attenuates hypoxia-induced ROS signaling as assessed independently in cytosol and IMS, HIF-1α stabilization and activity, and cellular proliferation under normoxic and hypoxic culture conditions. It also suppressed tumor growth invivo. Stable expression of nondegradable HIF-1α only partially rescued proliferation in IMS-Prdx5-expressing cells, indicating that mitochondrial H2O2 signaling contributes to tumor cell proliferation and survival through HIF-dependent and HIF-independent mechanisms.