Medium Of Mesenchymal Stem Cells Research Articles

High-Risk Keratoplasty Using Conditioned Media of Mesenchymal Stem Cells in an Experiment

High-Risk Keratoplasty Using Conditioned Media of Mesenchymal Stem Cells in an Experiment

Metabolomics to investigate the effect of preconditioned mesenchymal stem cells with crocin on pulmonary epithelial cells exposed to 2-chloroethyl ethyl sulfide

Metabolomics significantly impacts drug discovery and precise disease management. This study meticulously assesses the metabolite profiles of cells treated with Crocin, Dexamethasone, and mesenchymal stem cells (MSCs) under oxidative stress induced by 2-chloroethyl ethyl sulfide (CEES). Gas chromatography/mass spectrometry (GC/MS) analysis unequivocally identified substantial changes in 37 metabolites across the treated groups. Notably, pronounced alterations were observed in pathways associated with aminoacyl-tRNA biosynthesis and the metabolism of aspartate, serine, proline, and glutamate. These findings demonstrate the potent capacity of the analyzed treatments to effectively reduce inflammation, mitigate reactive oxygen species production, and enhance cell survival rates. Significance•Crocin, Dexamethasone, and the metabolites of the conditioned media of mesenchymal stem cells to decline the injury caused by CEES.•Metabolites can acquaint treatment groups in diminishing inflammation and ROS production and expanding the percentage of cell survival.•Aminoacyl-tRNA biosynthesis, nitrogen metabolism, glyoxylate and dicarboxylate metabolism, and propanoate metabolism were significant pathways involved among groups.

Extracellular vesicles derived from mesenchymal stem cells ameliorate cognitive impairment caused by neuroinflammation in young but not aged mice

Aim: The aim of this work was to study the effects of extracellular vesicles (EVs) derived from mesenchymal stem cells (MSCs) on inflammation-impaired cognitive functions and the brain of mice. Methods: Young mice (~3-month-old) and aged mice (~18-month-old) were injected with bacterial lipopolysaccharide (LPS) and obtained intravenously donor 106 human umbilical cord MSCs, EVs isolated from a similar amount of MSCs or conditioned medium (CM) of MSCs. Subsequently, the mice were examined in behavioral tests and the mouse brains were analyzed for the levels of pro-inflammatory cytokines, α7 nicotinic acetylcholine receptors (α7 nAChRs) and amyloid beta 1-42 (Aβ1-42). Results: EVs prevented LPS-induced memory impairment in mice, whereas CM provided a weaker and temporal effect. Both EVs and MSCs injected once after regular injections of LPS stably improved memory of young mice. In contrast, both cells and EVs provided only transient effect in aged mice injected with LPS. The brains of aged LPS-treated mice contained elevated amounts of IL-1β and IL-6; both MSCs and EVs decreased them significantly. The brains of non-treated aged mice contained decreased levels of α7 nAChRs and increased levels of Aβ1-42 and α7-bound Aβ1-42 compared to the brains of young mice. LPS treatment decreased α7 nAChRs in both young and aged mice, while both MSCs and EVs restored them up to the control level. In young mice, LPS treatment increased the level of Aβ1-42 and α7-bound Aβ1-42, whereas MSCs and EVs decreased it. In contrast, neither LPS nor MSCs/EVs influenced the elevated level of Aβ1-42 but increased α7-bound Aβ1-42 in the brains of aged mice. Conclusions: Regenerative potential of MSCs and MSC-derived EVs is sufficient to support cognitive functions of LPS-treated young mice but is quite poor for aged animals, possibly, due to decreased levels of α7 nAChRs and accumulated Aβ1-42 in their brains.

Mesenchymal Stem Cells-Conditioned Medium; An Effective Cell-Free Therapeutic Option for in vitro Maturation of Oocytes.

Infertility is a major reproductive health issue worldwide. One of the main problems in infertile women is the failure to generate or release a mature egg. Therefore, the development of new technologies for in vitro generation or induction of mature oocytes can improve various ART procedures. Recently, stem cell-based therapy has opened a new window for several pathological complications. Mesenchymal stem cells (MSCs) are multipotent stem cells with the capacity to self-renew and differentiate into the mesodermal lineage. MSCs contain various bioactive molecules which are involved in the regulation of key biological processes. They can secret multiple paracrine factors, such as VEGF, IGF, HGF, EGF, and FGF to stimulate egg maturation. Although MSCs represent a promising source for cell therapy, the potential risk of tumor development reduces their clinical applications. Recent studies have suggested that the supernatant or conditioned medium of MSCs also contains similar components and regulates the oocyte behavior. The MSC-conditioned medium can eliminate the safety concerns associated with MSC transplantation and avoid rejection problems. Although MSC and MSC-CM could improve oocyte quality, ovarian function, and fertility, these improvements have not yet been demonstrated in clinical trials in humans. Hereby, we summarized recent research findings of MSCs-derived conditioned medium in in vitro development of immature oocytes.

ISOLATION OF BONE MARROW MESENCHYMAL STEM CELLS EMBEDDED IN NATIVE TISSUE STROMA YIELDS ENRICHED HARVEST, IMPROVED ADHERENCE AND PROLIFERATION, AND UNIQUE SECRETOME

Background: The field of orthobiologics traditionally utilizes cellular products, including bone-marrow aspirate concentrate (BMAC), micronized adipose tissue, and platelet preparations to address pain from degenerative processes, orthopedic injuries and medical conditions characterized by chronic inflammation and tissue degradation. For BMAC, maximizing the concentration of mesenchymal stem cells (MSCs) in a reduced volume is thought to allow for the therapeutic delivery of the cellular concentrate, secretome, and extracellular vesicles to a site of orthopedic injury or surgical repair. The extracellular matrix (ECM) within the bone marrow stroma contains collagens and proteoglycans known to regulate cell proliferation, migration, differentiation, and cell-cell communication among resident bone marrow cells. This study aimed to evaluate the cellular effects on MSC health and function when harvested to retain their native tissue stroma. Methods: We evaluated a novel and unique processing method and device (BMAX™) to mechanically generate a purified MSC product derived from bone marrow in a nonenzymatic manner. BMAX™ products, including cells and stroma, were plated in MSC culture media and incubated for 3–14 days (P0-P1) before evaluation with flow cytometry for cell phenotyping and immunoassays for secretome profiling. Results: The orthobiologic product containing three-dimensional stromal components can be produced in minutes using an automated bedside device requiring minimal benchtop space. We found increased MSC adherence, improved proliferative density in culture, and significantly elevated enrichment of stromal-derived MSCs versus traditional BMAC centrifugation-based preparations. Further, we demonstrate a unique secretome profile in BMAX™ versus traditional BMAC centrifugation-based preparations. Conclusions: These qualities provide a novel and unique platform for autologous and allogeneic bone-marrow-derived therapy to better address inflammatory and destructive processes that may improve bone-marrow-derived cell therapies’ efficacy.

Exploration of altered miRNA expression and function in MSC-derived extracellular vesicles in response to hydatid antigen stimulation.

Hydatid disease is caused by Echinococcus parasites and can affect various tissues and organs in the body. The disease is characterized by the presence of hydatid cysts, which contain specific antigens that interact with the host's immune system. Mesenchymal stem cells (MSCs) are pluripotent stem cells that can regulate immunity through the secretion of extracellular vesicles (EVs) containing microRNAs (miRNAs). In this study, hydatid antigens were isolated from sheep livers and mice peritoneal cavities. MSCs derived from mouse bone marrow were treated with different hydatid antigens, and EVs were isolated and characterized from the conditioned medium of MSCs. Small RNA library construction, miRNA target prediction, and differential expression analysis were conducted to identify differentially expressed miRNAs. Functional enrichment and network construction were performed to explore the biological functions of the target genes. Real-time PCR and Western blotting were used for miRNA and gene expression verification, while ELISA assays quantified TNF, IL-1, IL-6, IL-4, and IL-10 levels in cell supernatants. The study successfully isolated hydatid antigens and characterized MSC-derived EVs, demonstrating the impact of antigen concentration on MSC viability. Key differentially expressed miRNAs, such as miR-146a and miR-9-5p, were identified, with functional analyses revealing significant pathways like Endocytosis and MAPK signaling associated with these miRNAs' target genes. The miRNA-HUB gene regulatory network identified crucial miRNAs and HUB genes, such as Traf1 and Tnf, indicating roles in immune modulation and osteogenic differentiation. Protein-protein interaction (PPI) network analysis highlighted central HUB genes like Akt1 and Bcl2. ALP activity assays confirmed the influence of antigens on osteogenic differentiation, with reduced ALP activity observed. Expression analysis validated altered miRNA and chemokine expression post-antigen stimulation, with ELISA analysis showing a significant reduction in CXCL1 expression in response to antigen exposure. This study provides insights into the role of MSC-derived EVs in regulating parasite immunity. The findings suggest that hydatid antigens can modulate the expression of miRNAs in MSC-derived EVs, leading to changes in chemokine expression and osteogenic capacity. These findings contribute to a better understanding of the immunomodulatory mechanisms involved in hydatid disease and provide potential therapeutic targets for the development of new treatment strategies.

Induction of mesenchymal stem cell (MSC) differentiation from iPS cells using MSC medium.

Mesenchymal stem cells (MSCs) and induced pluripotent stem (iPS) cells have great potential as cell sources for tissue engineering and regenerative medicine. This study aimed to investigate whether iPS cells can be differentiated into MSCs using MSCGM, a commercially available MSC culture system. The cells were characterized by flow cytometry, immunostaining, and gene expression analyses. We also examined their potential to differentiate into osteoblasts and chondrocytes. Our results showed that iPS cells cultured in MSCGM (iPS-MSCGM) exhibited a fibroblast-like morphology and expressed CD73 and CD90 genes, as well as positive markers for CD73, CD90, and CD105. Moreover, iPS-MSCGM cells demonstrated the ability to differentiate into osteoblasts and chondrocytes in vitro. This study demonstrates a new and simple method for inducing the differentiation of iPS cells to MSCs using MSCGM.

Thermosensitive Capturer Coupled with the CD63 Aptamer for Highly Efficient Isolation of Exosomes.

Exosomes are bioactive substances secreted by various cells that play a crucial role in cell communication. Due to their nanoscale size and interference from nonexosome proteins, the rapid capture and nondestructive release of exosomes remain a technical challenge which significantly hinders their biomedical application. To overcome this obstacle, we have designed a CD63 aptamer-based thermosensitive copolymer for the effective isolation of exosomes from mesenchymal stem cells (MSCs). A thermal-responsive copolymer, poly(N-isopropylacrylamide-co-butyl methacrylate-co-N-hydroxysuccinimide methacrylate) P(NIPAM-co-BMA-co-NHSMA, PNB), was prepared, which could realize reversible hydrophilic/hydrophobic phase transition by varying temperature. Then, CD63 aptamers were further modified to the copolymer to form the PNB-aptamer, where the aptamer units, acting as a "lock and key", specifically bind exosomes. Under the low critical solution temperature (LCST) of the PNB-aptamer, it can maintain a hydrophilic state, capturing exosomes from the cell culture medium. Subsequently, exosome-carrying PNB-aptamers can endure from hydrophilic to hydrophobic phase transition by increasing the temperature above its LCST, and then they can be collected after centrifugation. By introducing the complementary sequence of the CD63 aptamer, the stronger binding affinity between the complementary sequence and the aptamers facilitates the release of exosomes from the PNB-aptamer. The yield of exosome samples captured from a MSC culture medium by the PNB-aptamer system (around 62%) is considerably higher than that obtained by the current "gold standard" ultrafiltration (UC) approach (around 42%). Thus, the PNB-aptamer capturer provides a potential strategy for highly efficient exosome isolation.

MSC-derived exosomes enhance the anticancer activity of drugs in 3D spheroid of breast cancer cells

The unique properties of the MSC-derived exosome (MSC-Exos) make it an interesting drug-delivery tool for tumor cells. Drug delivery via exosomes helps overcome challenges to access the core of solid tumors. Here, we investigated exosomal chemotherapy drug formulations on the 3D multicellular spheroid models of breast cancer cell lines. The multicellular 3D spheroid of BT-474 and MDA-MB-231 breast cancer cell lines were made by liquid overlay technique. MSC-Exos were isolated from adipose-derived mesenchymal stem cells (ADMSCs) culture medium and characterized by their biological characteristics. Then paclitaxel (PTX) and cisplatin (CIS) were loaded into MSC-Exos. The cellular uptake and cytotoxic effect of these nanocarriers were evaluated by MTT and flow cytometry. MSC-Exos could efficiently facilitate cellular uptake in multicellular spheroids of breast cancer cell lines. EXO-PTX significantly inhibited the growth of spheroid in both MDA-MB-231 and BT-474 multicellular spheroids (P < 0.001). Moreover, apoptosis was more prominently inducted by EXO-PTX compared to the free PTX (P < 0.03). For cisplatin, although the exosomal formulation contained lower drug concentration compared to the free drug, the potency was increased. Based on our findings, the exosomal formulation of drugs induced higher toxicity and apoptosis in the 3D multicellular spheroids model with considerably lower drug concentrations. Drug-loaded MSC-Exos could be a potentially suitable alternative for cancer chemotherapy practices.

Clinical evaluation of intralesional umbilical cord-derived mesenchymal stem cells, conditioned medium and triamcinolone acetonide injection for keloid treatment: A pilot study.

Topical keloid therapy is performed with triamcinolone acetonide (TA) intralesional injection. However, the recurrence rate is high with various side effects. Mesenchymal stem cells (MSCs) have high proliferative abilities and reduce the activity and proliferation of fibroblast cells in keloids. To overcome the costs and limitations, conditioned medium (CM) is used. This study aims to evaluate feasibility of intralesional injection of umbilical cord MSC (UC-MSC) and conditioned medium (UC-CM) compared to TA for keloid therapy. Twenty-four patients with keloids who met the inclusion criteria were included, randomized into three treatment groups and then got assessed for the sociodemographic data, keloid volume, histopathology (type 1:3 collagen ratio), interleukin-10 (IL-10) levels and Patient and Observer Scar Assessment Scale (POSAS) score during visits. Largest volume regression occurred in the UC-MSC group, followed by UC-CM and then the TA group (UC-MSC: 45.32% ± 2.61%; UC-CM: 43.61% ± 3.67%; TA: 28.34% ± 3.81%; p = 0.003). Similar pattern was also observed in increase in IL-10 levels, the decrease in POSAS scores and the reduction of type 1:3 collagen ratio. Hence, UC-MSC and UC-CM are promisingly more effective than TA for keloid therapy, showcasing their superiority in reducing keloid volume, symptoms and type 1:3 collagen ratio, as well as increasing the levels of IL-10.

Effect assessment of a type of xeno-free and serum-free human adipose-derived mesenchymal stem cells culture medium by proliferation and differentiation capacities.

Human mesenchymal stem cells (hMSCs) possess broad prospects in pre-clinical research. In vitro amplification of hMSCs requires appropriate medium to reach the number of seed cells with clinical significance. However, the uncertainty of the heterologous components of the traditional fetal bovine serum (FBS) culture medium has great safety risks. Moreover, existing commercial hMSCs medium is very expensive, therefore a safer and more optimal hMSCs medium is urgently needed. Accordingly, we developed five components adipose-derived hMSCs (hADMSCs) medium without xenogenic components, named E5 SFM. which is mainly composed of knockout serum replacement (KSR), and additionally four components such as fibroblast growth factor and transferrin. Here, we mainly compared the E5 SFM with traditional FBS-containing medium and a commercial medium by surface markers testing, proliferation assay as well as osteogenic, adipogenicand chondrogenic differentiation assessment. We demonstrated that hADMSCs cultured in the E5 SFM showed similar morphological characteristics and immunophenotypes to those in other media. Notably, cell proliferative capability was similar to that in the commercial medium, but higher than that in the FBS-containing medium and other media. Additionally, their capabilities of adipogenic and osteogenic differentiation were significantly higher than those of other media. Consequently, we concluded that the E5 SFM medium can not only effectively promote cell proliferation of hMSCs, but also has optimal differentiative capacity and clear and simple ingredients.

Myricetin alleviates H2O2-induced senescence and apoptosis in rat nucleus pulposus-derived mesenchymal stem cells.

Transplantation of mesenchymal stem cells (MSCs) has been reported to be a novel promising target for the regeneration of degenerated intervertebral discs (IVDs). However, the culture and survival limitations of MSCs remain challenging for MSC-based biological therapy. Myricetin, a common natural flavonoid, has been suggested to possess antiaging and antioxidant abilities. Therefore, we investigated the biological function of myricetin, and its related mechanisms involving cell senescence in intervertebral disc degeneration (IDD). The nucleus pulposus-derived mesenchymal stem cells (NPMSCs) were isolated from 4-month-old Sprague-Dawley (SD) rats and identified by examining surface markers and multipotent differentiation. Rat NPMSCs were cultured in an MSC culture medium or culture medium with different concentrations of H2O2. Myricetin or the combination of myricetin and EX527 were added to the culture medium to investigate the effects of myricetin. Cell viability was evaluated by cell counting kit-8 assays (CCK-8). The apoptosis rate was determined using Annexin V/PI dual staining. The mitochondrial membrane potential (MMP) was analyzed by a fluorescence microscope after JC-1 staining. The cell senescence was determined by SA-β-Gal staining. MitoSOX green was used to selectively estimate mitochondrial reactive oxygen species (ROS) Apoptosis-associated proteins (Bax, Bcl2, and cleaved caspase-3), senescence markers (p16, p21, and p53), and SIRT1/PGC-1α signaling pathway-related proteins (SIRT1 and PGC-1α) were evaluated by western blotting. The cells isolated from nucleus pulposus (NP) tissues met the criteria for MSCs. Myricetin showed no cytotoxicity up to a concentration of 100 μM in rat NPMSCs cultured for 24 h. Myricetin pretreatment exhibited protective effects against H₂O₂-induced apoptosis. Myricetin could also alleviate H₂O₂-induced mitochondrial dysfunctions of increased mitochondrial ROS production and reduced MMP. Moreover, myricetin pretreatment delayed rat NPMSC senescence, as evidenced by decreased exppression of senescence indicators. Pretreatment of NPMSCs with 10 μM EX527, a selective inhibitor of SIRT1, prior to exposure to 100 μM H2O2, reversed the inhibitory effects of myricetin on cell apoptosis. Myricetin could affect the SIRT1/PGC-1α pathway to protect mitochondrial functions and alleviate cell senescence in H₂O₂-treated NPMSCs.

Mesenchymal stem cells transplantation combined with IronQ attenuates ICH-induced inflammation response via Mincle/syk signaling pathway

BackgroundIntracerebral hemorrhage (ICH) is a severe brain-injured disease accompanied by cerebral edema, inflammation, and subsequent neurological deficits. Mesenchymal stem cells (MSCs) transplantation has been used as a neuroprotective therapy in nervous system diseases because of its anti-inflammatory effect. Nevertheless, the biological characteristics of transplanted MSCs, including the survival rate, viability, and effectiveness, are restricted because of the severe inflammatory response after ICH. Therefore, improving the survival and viability of MSCs will provide a hopeful therapeutic efficacy for ICH. Notably, the biomedical applications of coordination chemistry-mediated metal-quercetin complex have been verified positively and studied extensively, including growth-promoting and imaging probes. Previous studies have shown that the iron-quercetin complex (IronQ) possesses extraordinary dual capabilities with a stimulating agent for cell growth and an imaging probe by magnetic resonance imaging (MRI). Therefore, we hypothesized that IronQ could improve the survival and viability of MSCs, displaying the anti-inflammation function in the treatment of ICH while also labeling MSCs for their tracking by MRI. This study aimed to explore the effects of MSCs with IronQ in regulating inflammation and further clarify their potential mechanisms.MethodsC57BL/6 male mice were utilized in this research. A collagenase I-induced ICH mice model was established and randomly separated into the model group (Model), quercetin gavage group (Quercetin), MSCs transplantation group (MSCs), and MSCs transplantation combined with IronQ group (MSCs + IronQ) after 24 h. Then, the neurological deficits score, brain water content (BWC), and protein expression, such as TNF-α, IL-6, NeuN, MBP, as well as GFAP, were investigated. We further measured the protein expression of Mincle and its downstream targets. Furthermore, the lipopolysaccharide (LPS)-induced BV2 cells were utilized to investigate the neuroprotection of conditioned medium of MSCs co-cultured with IronQ in vitro.ResultsWe found that the combined treatment of MSCs with IronQ improved the inflammation-induced neurological deficits and BWC in vivo by inhibiting the Mincle/syk signaling pathway. Conditioned medium derived from MSCs co-cultured with IronQ decreased inflammation, Mincle, and its downstream targets in the LPS-induced BV2 cell line.ConclusionsThese data suggested that the combined treatment exerts a collaborative effect in alleviating ICH-induced inflammatory response through the downregulation of the Mincle/syk signaling pathway following ICH, further improving the neurologic deficits and brain edema.

Potent antitumor effects of the conditioned medium of bone marrow-derived mesenchymal stem cells via IGFBP-4.

Cell transfer therapy using mesenchymal stem cells (MSCs) has pronounced therapeutic potential, but concerns remain about immune rejection, emboli formation, and promotion of tumor progression. Because the mode of action of MSCs highly relies on their paracrine effects through secretion of bioactive molecules, cell-free therapy using the conditioned medium (CM) of MSCs is an attractive option. However, the effects of MSC-CM on tumor progression have not been fully elucidated. Herein, we addressed this issue and investigated the possible underlying molecular mechanisms. The CM of MSCs derived from human bone marrow greatly inhibited the in vitro growth of several human tumor cell lines and the in vivo growth of the SCCVII murine squamous cell carcinoma cell line with reduced neovascularization. Exosomes in the MSC-CM were only partially involved in the inhibitory effects. The CM contained a variety of cytokines including insulin-like growth factor binding proteins (IGFBPs). Among them, IGFBP-4 greatly inhibited the in vitro growth of these tumors and angiogenesis, and immunodepletion of IGFBP-4 from the CM significantly reversed these effects. Of note, the CM greatly reduced the phosphorylation of AKT, ERK, IGF-1 receptor beta, and p38 MAPK in a partly IGFBP4-dependent manner, possibly through its binding to IGF-1/2 and blocking the signaling. The CM depleted of IGFBP-4 also reversed the inhibitory effects on in vivo tumor growth and neovascularization. Thus, MSC-CM has potent inhibitory effects on tumor growth and neovascularization in an IGFBP4-dependent manner, suggesting that cell-free therapy using MSC-CM could be a safer promising alternative for even cancer patients.

Brain-Derived Neurotrophic Factor Levels in Mesenchymal Stem Cell Culture Fluid Under Different Culture Conditions.

Mesenchymal stem cells are used in various fields, such as cellular therapy, regeneration, or tissue engineering. It has been shown that they exhibit many protective factors and also work as a modulating chief within the region in which they are administered. There are studies on both the therapeutic and neuroprotective effects of brain-derived neurotrophic factor. Also, there are many studies on the improvement of culture conditions for in vitro reproduction of mesenchymal stem cells, which can be obtained from many sources in various bodies, such as adipose tissue and Wharton's jelly. Improving and standardizing these culture conditions will increase the effectiveness and reliability of stem cell therapies. Studies evaluating many culture conditions, such as O2 level, type of medium, monolayer culture, and the transition from in vitro 3D models, are ongoing. In our study, groups were formed by using stem cells originating from adipose tissue and Wharton's jelly. Stem cell cultures were made using Hillex-II and Pronectin-F microcarriers. Cell culture O2 level was adjusted as 1% and 5% for each group separately. Enzyme-linked immunosorbent assay was used to analyze brain-derived neurotrophic factor levels in stem cell culture fluid. The highest brain-derived neurotrophic factor level in mesenchymal stem cells culture medium was observed in an adipose-derived stem cell culture with an in vitro fertilization (non-treated) dish, using a Hillex microcarrier in a 1% O2 microenvironment. As a result of our observations, we think that cells could exhibit greater therapeutic potential in a dynamic adhesion environment.

Anti-apoptotic and autophagic effect: Using conditioned medium from human bone marrow mesenchymal stem cells to treat human trabecular meshwork cells.

Glaucoma is a vision-threatening disease associated with accelerated aging of trabecular meshwork (TM) which results in elevated intraocular pressure (IOP). Increased oxidative stress in TM plays an important role in cellular molecular damage which leads to senescence. Autophagy is an intracellular lysosomal degradation process which is activated when cells are under stressful condition, and emerging studies have demonstrated increased expression of modulators of apoptosis and expression of autophagic cascade in ex-vivo TM specimens or cultured TM cells under oxidative stress. Recently, studies have shown neuroprotective and IOP-lowering effects after transplanting mesenchymal stem cells (MSCs) or injecting condition medium (CM) of MSCs into ocular hypertension animal models. However, knowledge of the underlying mechanism accounting for these effects is limited. Using condition medium (CM) from human bone marrow-derived mesenchymal stem cells (BM-MSCs), we investigated the effects of the CM derived from BM-MSCs on TM autophagy and apoptosis. H2O2 was added to culture medium of human TM cells to mimic oxidative damage in glaucomatous eyes, and the autophagic and anti-apoptotic effects of BM-MSCs-derived CM was explored on the oxidatively damaged cells. Mitochondrial ROS production was examined by MitoSOX™, apoptosis was evaluated using terminal deoxynucleotidyl transferase (dUTP) nick end labeling (TUNEL) staining, and the expression of proteins involved in autophagy as well as extracellular matrix was investigated via Western blot. There were no significant differences in TM cell viability when the cells were treated with different concentrations of CM in the absence of oxidative stress. Cell viability was significantly higher in oxidatively damaged TM cells treated with 1X or 5X CM compared to untreated TM cells under oxidative stress. The mitochondrial ROS level significantly increased with oxidative stress, which was mitigated in the CM treatment groups. DNA fragmentation significantly decreased in oxidatively stressed TM cells after treatment with CM. LCB3 II/LCB3 I was significantly elevated in the oxidative stress group compared to the control group and was significantly decreased in the CM treatment groups. Expression of fibronectin was not significantly different among the groups. The CM derived from human BM-MSCs has the capacity to rescue oxidatively damaged human TM cells associated with decreased autophagy and apoptosis. The BM-MSCs CM has potential for slowing down age- and disease-related degeneration of TM in patients with glaucoma, facilitating success in the control of IOP.

Protective effects of conditioned media of immortalized stem cells from human exfoliated deciduous teeth on pressure ulcer formation.

Pressure ulcers (PUs) are increasing with aging worldwide, but there is no effective causal therapy. Although mesenchymal stem cells (MSCs) promote cutaneous wound healing, the effects of the conditioned medium (CM) of MSCs on cutaneous PU formation induced by ischemia-reperfusion injury have been poorly investigated. To address this issue, herein, we first established an immortalized stem cell line from human exfoliated deciduous teeth (SHED). This cell line was revealed to have superior characteristics in that it grows infinitely and vigorously, and stably and consistently secretes a variety of cytokines. Using the CM obtained from the immortalized SHED cell line, we investigated the therapeutic potential on a cutaneous ischemia-reperfusion mouse model for PU formation using two magnetic plates. This is the first study to show that CM from immortalized SHEDs exerts therapeutic effects on PU formation by promoting angiogenesis and oxidative stress resistance through vascular endothelial growth factor and hepatocyte growth factor. Thus, the CM of MSCs has potent therapeutic effects, whereas these therapies have not been implemented in human medicine. To try to meet the regulatory requirements for manufacturing and quality control as much as possible, it is necessary to produce CM that is consistently safe and effective. The immortalization of stem cells could be one of the breakthroughs to meet the regulatory requirements and consequently open up a novel avenue to create a novel type of cell-free regenerative medicine, although further investigation into the quality control is warranted.

Study of the Cellular Senescence Process in Human Umbilical Cord Wharton's Jelly-derived Mesenchymal Stem Cells

Introduction: Embryonic stem cells are candidates for the treatment of regenerative medicine, but their use is faced with limitations due to ethical issues. The human umbilical cord-derived mesenchymal stem cells (MSCs) are appropriate options because the cells have no ethical difficulties and have self-renewal. Senescence is known as a gradual functional loss that heterogeneously occurs in multiple organ systems. Objective: The objective of this study was to investigate the cellular senescence process in human umbilical cord Wharton's jelly-derived mesenchymal cells. Materials and Methods: Umbilical cord was obtained from healthy newborns at the General Hospital of Tabriz. Under sterile conditions, Wharton's jelly was removed from the blood vessels and minced into small pieces of about 0.5 mm. These were cultured in Dulbecco's Modified Eagle's Medium (MSC medium). Real-time polymerase chain reaction for p16INK4a and senescence-associated β-galactosidase (SA-β-gal) staining was performed to investigate the cellular senescence process. Results: The results showed the different expressions in the different passages, but it was significantly increased from the fifth passage compared to the first passage. SA-β-gal staining also showed increased colour intensity in the fifth passage compared to the first passage. Conclusion: SA-β-gal is not a specific marker for senescence, while p16INK4a is a specific marker. Further studies are required for the investigation of the senescence mechanism, such as the evaluation of genes involved in the senescence.

Overcoming the resistance of hepatocellular carcinoma to PD-1/PD-L1 inhibitor and the resultant immunosuppression by CD38 siRNA-loaded extracellular vesicles

ABSTRACT Extracellular vesicles (EVs) are promising tools for drug delivery across different biological barriers. Here, we evaluated the potential of EVs-mediated delivery of CD38 siRNA on the immunosuppression of hepatocellular carcinoma (HCC). EVs were isolated from bone marrow mesenchymal stem cell culture medium and loaded with CD38 siRNA to prepare EVs/siCD38. Loss-of-function assays were conducted to investigate the biological functions of EVs/siCD38 in HCC cells. Xenograft mouse models were performed for further validation. High CD38 expression was found in HCC. EVs/siCD38 inhibited CD38 enzyme activity, decreased adenosine production, and promoted macrophage repolarization to M1 type, thus inhibiting HCC cell growth and metastasis in vitro as well as tumor growth in mice. Mechanistically, CD38 was upregulated in mice resistant to PD-1/PD-L1 inhibitor and EVs/siCD38 reversed the resistance of tumor to PD-1/PD-L1 inhibitor in vivo. Our results provide functional evidence for the use of EV-mediated delivery of CD38 siRNA to prevent immunosuppression feature of HCC.

Conditioned medium of mesenchymal stem cells pretreated with H2O2 promotes intestinal mucosal repair in acute experimental colitis

Mesenchymal stem cells (MSCs) are a new therapeutic strategy for inflammatory bowel disease (IBD), and their efficacy has been widely recognized. However, there are still some challenges in cell therapy, including stable cell passage, laboratory conditions for cell culture, high-cost burden, and poor transplantation. The conditioned medium (CM) of MSCs is considered be an excellent alternative to cell transplantation, but the paracrine group in MSC-CM is limited in variety and low in concentration, which cannot meet the therapeutic needs of injured tissues and needs to be optimized. Pretreatment with low concentration of hydrogen peroxide (H2O2) can not only protect cells from oxidative damage, but also play a role similar to growth factors and regulate the physiological function of stem cells, to obtain an improved conditioned medium. To determine the optimal protocol for pretreatment of MSCs with H2O2, and to study the efficacy and potential mechanism of MSC-CM pretreated with H2O2 on Dextran Sulfate Sodium (DSS)-induced acute experimental colitis. MSCs were exposed to different concentrations of H2O2, and the optimal H2O2 pretreatment conditions were determined by evaluating their critical cell functional properties. H2O2-pretreated MSC-CM was transplanted into experimental mouse colitis by enema at 2, 4, and 6 days in modeling, and the changes of colonic tissue structure, the levels of inflammation and oxidative stress, the molecular changes of Nrf2/Keap1/ARE axis, and the related indicators of apoptosis in colonic epithelial cells were observed in each group. In vitro, Pretreated MSCs with 25 μM H2O2 significantly enhanced cell proliferation, migration, and survival, but had no effect on apoptosis. In vivo, MSC-CM treatment decreased apoptosis and extracellular matrix deposition, and maintained the mechanical barrier and permeability of colonic epithelial cells in experimental mouse colitis. Mechanistically, H2O2-pretreated MSC-CM against reactive oxygen species (ROS) production and MDA generation, accompanied by increases in components of the enzymatic antioxidant system includes SOD, CAT, GSH-PX, and T-AOC, which is through the up-regulation of the Nrf2, HO-1, and NQO-1 antioxidant genes. Our data confirmed that 25 μM H2O2 pretreated MSC-CM treatment could effectively improve intestinal mucosal repair in experimental colitis, which may be achieved by activating Nrf2/Keap1/ARE pathway.