Mesenchymal Stem Cells Supernatant Research Articles

Exosome-shuttled miR-150-5p from LPS-preconditioned mesenchymal stem cells down-regulate PI3K/Akt/mTOR pathway via Irs1 to enhance M2 macrophage polarization and confer protection against sepsis.

Sepsis is a life-threatening organ dysfunction and lack of effective measures in the current. Exosomes from mesenchymal stem cells (MSCs) reported to alleviate inflammation during sepsis, and the preconditioning of MSCs could enhance their paracrine potential. Therefore, this study investigated whether exosomes secreted by lipopolysaccharide (LPS)-pretreated MSCs exert superior antiseptic effects, and explored the underlying molecular mechanisms. Exosomes were isolated and characterized from the supernatants of MSCs. The therapeutic efficacy of normal exosomes (Exo) and LPS-pretreated exosomes (LPS-Exo) were evaluated in terms of survival rates, inflammatory response, and organ damage in an LPS-induced sepsis model. Macrophages were stimulated with LPS and treated with Exo or LPS-Exo to confirm the results of the in vivo studies, and to explain the potential mechanisms. LPS-Exo were shown to inhibit aberrant pro-inflammatory cytokines, prevent organ damages, and improve survival rates of the septic mice to a greater extent than Exo. In vitro, LPS-Exo significantly promoted the M2 polarization of macrophages exposed to inflammation. miRNA sequencing and qRT-PCR analysis identified the remarkable expression of miR-150-5p in LPS-Exo compared to that in Exo, and exosomal miR-150-5p was transferred into recipient macrophages and mediated macrophage polarization. Further investigation demonstrated that miR-150-5p targets Irs1 in recipient macrophages and subsequently modulates macrophage plasticity by down-regulating the PI3K/Akt/mTOR pathway. The current findings highly suggest that exosomes derived from LPS pre-conditioned MSCs represent a promising cell-free therapeutic method and highlight miR-150-5p as a novel molecular target for regulating immune hyperactivation during sepsis.

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.

Microvesicles from Mesenchymal Stem CellsOverexpressing MiR-34a Ameliorate Renal Fibrosis In Vivo.

We recently discovered that microvesicles (MVs) derived from mesenchymal stem cells (MSCs) overexpressing miRNA-34a can alleviate experimental kidney injury in mice. In this study, we further explored the effects of miR34a-MV on renal fibrosis in the unilateral ureteral obstruction (UUO) models. Methods. Bone marrow MSCs were modified by lentiviruses overexpressing miR-34a, and MVs were collected from the supernatants of MSCs. C57BL6/J mice were divided into control, unilateral ureteral obstruction (UUO), UUO + MV, UUO + miR-34aMV and UUO + miR-34a-inhibitor-MV groups. MVs were injected to mice after surgery. The mice were then euthanized on day 7 and 14 of modeling, and renal tissues were collected for further analyses by Hematoxylin and eosin, Masson's trichrome, and Immunohistochemical (IHC) staining. Results. The UUO + MV group exhibited a significantly reduced degree of renal interstitial fibrosis with inflammatory cell infiltration, tubular epithelial cell atrophy, and vacuole degeneration compared with the UUO group. Surprisingly, overexpressing miR-34a enhanced these effects of MSC-MV on the UUO mice. Conclusion. Our study demonstrates that miR34a further enhances the effects of MSC-MV on renal fibrosis in mice through the regulation of epithelial-to-mesenchymal transition (EMT) and Notch pathway. miR-34a may be a candidate molecular therapeutic target for the treatment of renal fibrosis. DOI: 10.52547/ijkd.7673.

The Protective Effect of a Human Umbilical Cord Mesenchymal Stem Cell Supernatant on UVB-Induced Skin Photodamage.

The skin is constantly exposed to a range of environmental stressors, including ultraviolet (UV) radiation, which can cause damage to the skin. Repairing UV-damaged skin has been a major focus of research in recent years. The therapeutic potential of human umbilical cord mesenchymal stem cells (HUCMSCs) exhibits anti-photoaging properties. In this study, we developed a strategy for concentrating an HUCMSC supernatant, and examined the protective effects of CHS on UVB exposure in vitro and in vivo. Our results demonstrate that CHS repairs UVB exposure by promoting cell viability and migration and reducing senescent and apoptosis cells. We further found that the photoprotective effect of CHS is due to autophagy activation. Moreover, CHS reduces wrinkles and senescent cells, increases collagen expression, and improves immune function in UVB exposure-induced skin damage. In summary, our study provides a new approach for repairing cell damage, and suggests that CHS might be a potential candidate for preventing UVB-induced skin photodamage.

Intravitreal application of stem cells and their products does not ameliorate experimental autoimmune uveitis

Aims/Purpose: Stem cell therapy has demonstrated potential therapeutic benefits in various medical disciplines. This study evaluated therapeutic effects of mesenchymal stem cells (MSCs) or their by‐products on intraocular inflammation in experimental autoimmune uveitis (EAU) model.Methods: EAU was induced in C57BL/6J mouse strain through administration of IRBP, Freund's adjuvant and pertussis toxin. Conditioned medium was procured through cultivation of MSCs in serum‐free medium, while MSC supernatant was obtained via MSC centrifugation and membrane removal. Either conditioned medium or MSC supernatant was intravitreally administered unilaterally, typically on day 14 post‐EAU induction. Ocular fundus of mice was documented regularly. Experiments were generally concluded on day 28, with eyes processed for FACS and PCR analysis. Infiltration of retina by inflammatory cells (F4/80, CD11c, CD45, CD80, CD86, CD206) and gene expression of pro‐inflammatory factors (Gfap, Iba‐1, IL‐1, iNOS) were quantified.Results: Across all experiments, clinical grading, FACS, and PCR analysis indicated exacerbation of intraocular inflammation. Post‐intravitreal administration of MSC by‐products into eyes with EAU, increased infiltration of retina by inflammatory cells and upregulation in gene expression of pro‐inflammatory factors were observed. These findings were corroborated in vitro, where co‐culturing of retina with EAU in presence of MSCs or their supernatant did not significantly reduce gene expression of pro‐inflammatory factors. Upsurge in pro‐inflammatory factors was also noted in control experiments on healthy eyes post‐intravitreal injection of no substance, serum‐free medium, PBS, or conditioned medium.Conclusions: No improvement in EAU was observed following application of MSC products, both in vivo and in vitro. Instead, inflammation was more severe. Trauma induced by intravitreal application itself significantly influenced outcome.Financial support: GA UK 309621, SVV UK 260631.

Evaluation of the Anti-Oxidative Effects of Monocyte Cells Treated with Bone Marrow Mesenchymal Stem Cells Supernatant on Experimental Colitis in BALB/c Mice

Background: Alternate activation of monocytes could induce anti-inflammatory impacts. This study aimed to investigate whether monocyte cells treated with bone marrow mesenchymal stem cells supernatant (MSC-Sp) could improve anti-inflammatory responses as a cell transfer therapy for colitis. Materials and Methods: The induction of experimental colitis was done by acetic acid in four groups of male BALB/c mice, including the control colitis, treated-monocytes, non-treated-monocytes, and mesalazine groups. Following MSCs culture, the supernatant was harvested, and then 50% conditioned media, or negative control media was added to the monocytes for 24 h. After ten days, peritoneal injection of treated or non-treated-monocytes (105 cells/100µL) was performed in animals' relevant groups of colitis. Ten days later, the oxidative stress profile and histopathological evaluation of colon tissue were assessed. Results: Treated monocytes showed a significant improvement in the oxidative stress profile, namely myeloperoxidase (0.126±0.008), nitric oxide (0.153±0.01), and malondialdehyde (0.148±0.014) compared to the control colitis group (P<0.05). Also, histopathological results revealed that the rate of damage in the treated-monocytes group was less than in normal mice. Conclusion: Our study indicated that the treated monocytes had anti-oxidative potential in colitis mice and were usable as a complementary therapy. [GMJ.2021;10:e2131]

Mesenchymal stem cell-derived exosomes can alleviate GVHD and preserve the GVL effect in allogeneic stem cell transplantation animal models.

Mesenchymal stem cells (MSCs) can alleviate graft-versus-host disease (GVHD) in hematopoietic stem cell transplantation (HSCT). MSCs-derived exosomes (MEXs) can mirror the biological function of their parent cells. Whether MEXs can alleviate GVHD like their parent cells or not is unclear. In this study, we investigate the effects of MEXs on GVHD and graft-versus-leukemia (GVL) effect in vitro and in HSCT animal models. MSCs were produced using bone marrow mononuclear cells (MNCs), and MEXs were separated from the supernatants of MSCs. Electron microscopy, western blot, and nanoparticle tracking analysis (NTA) were used to determine the characteristics of MEXs. The immunomodulatory function of MEXs and their effects on GVHD and GVL were examined in vitro and in vivo. Like other cell-type derived exosomes, our data revealed that MEXs were also disc-shaped vesicles with a diameter of 100-200 nm under electron microscopy and were positive for the exosomal hallmark proteins. MEXs can notably inhibit the expression of costimulatory molecules and functional cytokine secretion of dendritic cells (DCs). Meanwhile, MEXs can exert suppressive effects on T lymphocyte proliferation and activation. Moreover, MEXs can also encourage the polarization of macrophages toward the M2 type. In animal HSCT models, MEXs can promote the differentiation of Treg cells in spleens, decrease the GVHD score, increase the survival rate of mice, and preserve the cytotoxic antileukemia effects of CD8+ T lymphocytes from recipient mice. These findings showed that MEXs exert their effects by inhibiting the immunomodulatory function of DCs, macrophages, and T lymphocytes. In the animal model, MEXs ameliorate the clinical symptoms of GVHD, while maintaining the antitumor effects of CD8+ T lymphocytes. Therefore, it can be inferred that MEXs can separate GVHD from GVL in HSCT. Our study suggests that MEXs have broad clinical application potential in the prevention and treatment of GVHD in HSCT in the near future.

Mesenchymal stem cells prevent H7N9 virus infection via rejuvenating immune environment to inhibit immune-overactivity

BackgroundInfluenza is a clinically important infectious disease with a high fatality rate, which always results in severe pneumonia. Mesenchymal stem cells (MSCs) exhibit promising therapeutic effects on severe viral pneumonia, but whether MSCs prevent virus infection and contribute to the prevention of influenza remains unknown. MethodsICR mice were pretreated with human umbilical cord (hUC) MSCs and then infected with the influenza H7N9 virus. Weight, survival days, and lung index of mice were recorded. Serum antibody against influenza H7N9 virus was detected according to the hemagglutination inhibition method. Before and after virus infection, T cell and B cell subtypes in the peripheral blood of mice were evaluated by flow cytometry. Cytokines in the supernatants of MSCs, innate immune cells, and mouse broncho alveolar lavage fluid (BALF) were determined by enzyme-linked immunosorbent assay (ELISA) or Luminex Assay. ResultsPretreatment with MSCs protected mice against influenza H7N9 virus infection. Weight loss, survival rate, and structural and functional damage to the lungs of infected mice were significantly improved. Mechanistically, MSCs modulated T lymphocyte response in virus-infected mice and inhibited the cGAS/STING pathway. Importantly, the protective effect of MSCs was mediated by cell-to-cell communications and attenuation of cytokine storm caused by immune overactivation.

Experimental application results of mesenchymal stem cell microvesicles in the mouse model of acute renal failure

An important role in restoration of damaged organs and tissues is played by mesenchymal stem cells (MSCs) and microvesicular particles (MV) produced by them. They can be a source of cytokines, anti- apoptotic and growth stimulating factors. In addition, MVs carry out transport of mRNA, miRNA, and signal proteins into damaged tissues. This increases the ability of cells to regenerate and to inhibit apoptosis, promote to angiogenesis and stimulate cell proliferation. The aim of our research was to study the immunoregulatory and pro-regenerative properties of mesenchymal stem cell microvesicles (MSC-MV) in a model of glycerol- induced acute renal failure (ARF) in mice. The experiments were carried out on CBA mice aged 3-4 months. AKI was induced by a single intramuscular injection of 50% glycerol. MSCs were obtained from the bone marrow of healthy animals and cultivated under standard conditions. Microvesicles were obtained by centrifugation at 12000g of MSC supernatant after induction of their apoptosis by culturing under oxygen deprivation conditions and in serum-free medium. MSC-MV was injected intravenously into the retroorbital sinus one day after induction of ARF. The MV dose was calculated as equivalent to (derived from) 1 million MSCs, which was 100 mL per mouse. Animals were taken out of the experiment on days 4 and 11 after MSC-MV injection. Blood plasma was taken to determine the level of creatinine, urine – for albumin analysis, kidneys – for histological examination. It has been shown that MVs induced by MSCs dose-dependently stimulated splenocyte proliferation in both spontaneous and Con-A induced tests. The addition of MV caused a decrease in doxorubicin-induced apoptosis of splenic lymphocytes in mice. Probably, in this case, MV produced by MSCs had an immunostimulatory and antiapoptotic effect. Also, MVs had a positive impact on the restoration of structure and function kidneys in a model of ARF in mice. The use of MSC-MV in treatment of acute renal failure induced by a single injection of 50% glycerol contributed to decrease albumin level urine and restoration of creatinine level in blood serum of animals. Morphological studies have shown decrease in the height cell and collecting duct diameter in the medulla and a decrease in the largest transverse diameter of superficial glomeruli in the renal cortex of sick mice. Thus, the obtained results indicate significant therapeutic and pro-regenerative properties of MSC-MV, which require further study.

In Vitro FVIII Encoding Transgenic Mesenchymal Stem Cells Maintain A Successful Coagulation in FVIII- Deficient Plasma Mimicking Hemophilia A.

Hemophilia A is an X-linked recessive bleeding disorder caused by deficiency of plasma coagulation factor VIII (FVIII), and accounts for about 80-85% of patients with hemophilia. Plasma-derived therapies or recombinant factor VIII concentrates and also FVIII mimicking antibodies are used to prevent and treat the bleeding symptoms. Recently, EMA granted a conditional marketing approval for the first gene therapy of Hemophilia A. Aim of this study was to determine the effectiveness of coagulation in correcting FVIII deficiency with FVIII secreting transgenic mesenchymal stem cells (MSC). A lentiviral vector encoding B-domain-deleted Factor VIII cDNA sequence with CD45 R0 truncated (CD45R0t) surface marker was designed to develop a transgenic FVIII expressing primary cell line by transducing MSC. Efficacy and functionality of secreted FVIII from MSCs was assessed with anti- FVIII ELISA, CD45R0t flow cytometry, FVIII Western Blot and Mixing Test analysis in vitro. Results of this study showed that the transgenic MSCs maintain a persistent FVIII secretion. There was no significant difference in FVIII secretion over time, suggesting stable FVIII expression from the MSCs. Functionality of FVIII protein secreted in MSC supernatant was demonstrated by using Mixing Test in coagulation analysis. In the Mixing Test analysis, FVIIIdeficient human plasma products were mixed either with saline control or FVIII-secreted MSC supernatant. Mean FVIII levels of saline control group were 0.41±0.03 IU/dL whereas mean FVIII levels were 25.41±33.38 IU/dL (p<0.01) in FVIII-secreting MSC supernatant mixed group. Mean activated partial thromboplastin time (aPTT) of saline control group was 92.69±11.38 seconds whereas in FVIII-secreting MSC supernatant mixed group, mean aPTT levels were decreased to 38.60±13.38 seconds (p<0.001). Findings of this in vitro study shows a promising new method applicable to treat Hemophilia A. Accordingly, a FVIII secreting transgenic mesenchymal stem cell study will be initiated in FVIII knock-out animal model.

In Vitro FVIII-Encoding Transgenic Mesenchymal Stem Cells Maintain Successful Coagulation in FVIII-Deficient Plasma Mimicking Hemophilia A

Hemophilia A is an X-linked recessive bleeding disorder caused by a deficiency of plasma coagulation factor VIII (FVIII), and it accounts for about 80%-85% of all cases of hemophilia. Plasma-derived therapies or recombinant FVIII concentrates are used to prevent and treat the bleeding symptoms along with FVIII-mimicking antibodies. Recently, the European Medicines Agency granted conditional marketing approval for the first gene therapy for hemophilia A. The aim of this study was to determine the effectiveness of coagulation in correcting FVIII deficiency with FVIII-secreting transgenic mesenchymal stem cells (MSCs). A lentiviral vector encoding a B domain-deleted FVIII cDNA sequence with CD45R0 truncated (CD45R0t) surface marker was designed to develop a transgenic FVIII-expressing primary cell line by transducing MSCs. The efficacy and functionality of the FVIII secreted from the MSCs was assessed with anti-FVIII ELISA, CD45R0t flow cytometry, FVIII western blot, and mixing test analysis in vitro. The findings of this study showed that the transgenic MSCs maintained persistent FVIII secretion. There was no significant difference in FVIII secretion over time, suggesting stable FVIII expression from the MSCs. The functionality of the FVIII protein secreted in the MSC supernatant was demonstrated by applying a mixing test in coagulation analysis. In the mixing test analysis, FVIII-deficient human plasma products were mixed with either a saline control or FVIII-secreted MSC supernatant. The mean FVIII level of the saline control group was 0.41±0.03 IU/dL, whereas the mean level was 25.41±33.38 IU/dL in the FVIII-secreting MSC supernatant mixed group (p<0.01). The mean activated partial thromboplastin time (aPTT) of the saline control group was 92.69±11.38 s, while in the FVIII-secreting MSC supernatant mixed group, the mean aPTT level decreased to 38.60±13.38 s (p<0.001). The findings of this in vitro study suggest that the new method presented here is promising as a possible treatment for hemophilia A. Accordingly, a study of FVIII-secreting transgenic MSCs will next be initiated in a FVIII-knockout animal model.

Analysis of EMT induction in a non-invasive breast cancer cell line by mesenchymal stem cell supernatant: Study of 2D and 3D microfluidic based aggregate formation and migration ability, and cytoskeleton remodeling.

The process of Epithelial-to-mesenchymal transition (EMT) as a phenotypic invasive shift and the factors affecting it, are under extensive research. Application of supernatants of human adipose-derived mesenchymal stem cells (hADMSCs) on non-invasive cancer cells is a well known method of in vitro induction of EMT like process. While previous researches have focused on the effects of hADMSCs supernatant on the biochemical signaling pathways of the cells through expression of different proteins and genes, we investigated pro-carcinogic alterations of physico-mechanical cues in terms of changes in cell motility and aggregated formation in 3D microenvironments, and cytoskeletal actin-myosin content and fiber arrangement. MCF-7 cancer cells were treated by the supernatant from 48hour-starved hADMSCs, and their vimentin/E-cadherin expressions were evaluated. The invasive potential of treated and non-treated cells was measured and compared through aggregate formation and migration capability. Furthermore, alterations in cell and nucleus morphologies were studied, and F-actin and myosin-II alterations in terms of content and arrangement were investigated. Results indicated that application of hADMSCs supernatant enhanced vimentin expression as the biomarker of EMT, and induced pro-carcinogenic effects on non-invasive cancer cells through increased invasive potential by higher cell motility and reduced aggregate formation, rearrangement of actin structure and generation of more stress fibers, together with increased myosin II that lead to enhanced cell motility and traction force. Our results indicated that in vitro induction of EMT through mesenchymal supernatant influenced biophysical features of cancer cells through cytoskeletal remodeling that emphasizes the interconnection of chemical and physical signaling pathways during cancer progress and invasion. Results give a better insight to EMT as a biological process and the synergy between biochemical and biophysical parameters that contribute to this process, and eventually assist in improving cancer treatment strategies.

JAM-A Overexpression in Human Umbilical Cord-Derived Mesenchymal Stem Cells Accelerated the Angiogenesis of Diabetic Wound By Enhancing Both Paracrine Function and Survival of Mesenchymal Stem Cells.

Mesenchymal stem cells (MSCs) is promising in promoting wound healing mainly due to their paracrine function. Nonetheless, the transplanted MSCs presented poor survival with cell dysfunction and paracrine problem in diabetic environment, thus limiting their therapeutic efficacy and clinical application. JAM-A, an adhesion molecule, has been reported to play multi-functional roles in diverse cells. We therefore investigated the potential effect of JAM-A on MSCs under diabetic environment and explored the underlying mechanism. Indeed, high-glucose condition inhibited MSCs viability and JAM-A expression. However, JAM-A abnormality was rescued by lentivirus transfection and JAM-A overexpression promoted MSCs proliferation, migration and adhesion under hyperglycemia. Moreover, JAM-A overexpression attenuated high-glucose-induced ROS production and MSCs apoptosis. The bio-effects of JAM-A on MSCs under hyperglycemia were confirmed by RNA-seq with enrichment analyses. Moreover, Luminex chip results showed JAM-A overexpression dramatically upregulated PDGF-BB and VEGF in the supernatant of MSCs, which was verified by RT-qPCR and western blotting. The supernatant was further found to facilitate HUVECs proliferation, migration and angiogenesis under hyperglycemia. In vivo experiments revealed JAM-A overexpression significantly enhanced MSCs survival, promoted wound angiogenesis, and thus accelerated diabetic wound closure, partially by enhancing PDGF-BB and VEGF expression. This study firstly demonstrated that JAM-A expression of MSCs was inhibited upon high-glucose stimulation. JAM-A overexpression alleviated high-glucose-induced MSCs dysfunction, enhanced their anti-oxidative capability, protected MSCs from hyperglycemia-induced apoptosis and improved their survival, thus strengthening MSCs paracrine function to promote angiogenesis and significantly accelerating diabetic wound healing, which offers a promising strategy to maximize MSCs-based therapy in diabetic wound.

The effect of mesenchymal stem cell-derived supernatant nasal administration on lung inflammation and immune response in BCG-vaccinated BALB/c mice

Mesenchymal stem cells (MSCs) are among the known cells that can control and modulate immune responses in different circumstances, including autoimmune diseases. Also, various studies have shown that they can prevent and reduces the pulmonary inflammation caused by infectious agents. In the case of tuberculosis and inflammation caused by BCG, the granuloma has destructive effects and improper orientation of the immune response. Therefore, it is possible to prevent airway damage by preventing harmful inflammatory responses and guiding the immune system responses. This study investigates the role of nasal administration of MSCs supernatant by designing an inflammatory model in the BALB/c mice lung with BCG. MSCs are isolated from mice adipose tissue in this study and evaluated for their phenotypic and differentiation properties. After the third passage, these cells' condition medium (CM) was collected. 20 mice were divided into four groups. Group 1 receive BCG (107 CFU in 5 ml volume for 15 min) nasal administration. Group 2 treated with CM, and group 3 initially were treated with CM (in 5 ml volume for 15 min) and, after 24 h, treated with BCG nasal administration. CM treatment was continued every five days for one month. The fourth group of mice was treated with PBS nasal administration of CM and BCG. One week after the last administration, the lung tissue of mice in each group was pathologically examined. In addition, secretion of IL1-β, IL-6, TNF-α, TGF-β, and IL-10 in the alveolar fluid and secretion of IL-4 and IFN-γ cytokines in the supernatant of splenocytes was evaluated by ELISA. The TNF-α/IL-10 ratio in the alveolar lung fluid of the BCG received group is 2/9 and decreased to 0.58 after successive CM treatment. Therefore, it can be concluded that inflammatory responses to BCG infection in the presence of CM are balanced and pave the way for the induction of effective immune responses by reducing lung tissue damage.

Evaluation of immunomodulatory effects of co-culture or supernatant of dexamethasone or IFN-γ-treated adipose-derived mesenchymal stem cells on spleen mononuclear cells

Although mesenchymal stem cells (MSCs) have exhibited promising immunomodulatory potential in preclinical studies, clinical studies have revealed variable results. These results often depend on environmental cues. Pre-conditioning MSCs with cytokines is one of the methods used to enhance their immunomodulatory effects. In this study, we harvested adipose-derived MSCs from mice and cultured them with different doses of the cytokine, IFN-γ, and the corticosteroid drug, dexamethasone, in order to investigate their effects on MSC immunosuppressive function. We found the co-culture or supernatant of MSCs, pre-conditioned with IFN-γ, together with spleen mononuclear cells resulted in a significant reduction of mononuclear cell proliferation. Although the supernatant of MSCs, pre-conditioned with dexamethasone, showed similar results, dexamethasone pre-conditioning of co-cultured MSCs increased mononuclear cell proliferation. The results further our understanding of immune-related effects of MSCs which may provide a basis for further in vivo studies to achieve better clinical results. We propose that pre-conditioning with cytokines might be an effective method to boost the immunomodulatory effects of MSCs.

Cytoplasm or Supernatant: Where Is the Treasury of the Bioactive Antiaging Factor from Mesenchymal Stem Cells?

Cell-free compounds of mesenchymal stem cells (MSCs) could be a safer and cheaper substitution for MSC transplantation and have gained substantial research interest for antiaging skin treatments. However, whether those bioactive components should be obtained from the cytoplasm or supernatant is yet to be determined. In this study, we examined the ingredients of the MSC cytoplasm extract (MSC-ex) and MSC supernatant (MSC-s) and evaluated their effect in a photoaging model. Although MSC-ex has a richer protein composition than MSC-s, the latter has a proteome associated with wound healing and blood vessel development. Over 85% of the proteins in MSC-s were also found in MSC-ex, including extracellular matrix protein and various growth factors. The results of real-time PCR and western blot also demonstrate that both MSC-s and MSC-ex can upregulate collagen, transforming growth factor β (TGF-β), and vascular endothelial growth factor (VEGF) and downregulate IL-1β and matrix metalloproteinase-1 (MMP-1), which were considered critical for antiphotoaging. This supports our observations in the Hematoxylin and Eosin (HE) and Masson staining assay that they have a comparable effect as MSCs in terms of enhancing dermal thickness, and stimulating collagen regeneration. Although MSC-s and MSC-ex showed a weaker immunosuppression effect than MSCs, moisture measurement showed that they repair damage more rapidly than MSCs. Furthermore, the histological results showed that MSC-s maintains a super effect on immunosuppression, epidermal repair, and angiogenesis. That may be associated with the higher content of laminin, TGF-β, and VEGF in MSC-s, as well as its super cytokine transcriptional regulation ability. Thus, both MSC-s and MSC-ex can safely and effectively promote the repair of skin light injury, similar to MSCs. Our findings can broaden the range of active factors available in cell-free treatment, determine the difference between MSC-s and MSC-ex, and provide a reference for the development of similar products in regenerative medicine.

Mesenchymal stem cells pretreated with proinflammatory cytokines accelerate skin wound healing by promoting macrophages migration and M2 polarization.

IntroductionNumerous studies have shown that mesenchymal stem cells (MSCs) promote cutaneous wound healing via paracrine signaling. Our previous study found that the secretome of MSCs was significantly amplified by treatment with IFN-γ and TNF-α (IT). It has been known that macrophages are involved in the initiation and termination of inflammation, secretion of growth factors, phagocytosis, cell proliferation and collagen deposition in wound, which is the key factor during wound healing. In the present study, we used a unique supernatant of MSCs from human umbilical cord-derived MSCs (UC-MSCs) pretreated with IT, designated S-IT MSCs, to explore whether S-IT MSCs have a better effect on improving wound healing by improving the biological function of macrophages than the control supernatant of MSCs (S-MSCs).MethodsIn the present study, we used a unique supernatant of MSCs pretreated with IT subcutaneously injected into a mice total skin excision. We evaluated the effect of S-IT MSCs on wound healing and the quality of wound repair via promoting macrophages migration and M2 polarization in vivo. In addition, the effect of S-IT MSCs on macrophages migration, converting toward M2 phenotype and phagocytosis were also investigated in vitro.ResultsIndeed, S-IT MSCs were found to be more potent in promoting macrophage migration, M2 polarization, phagocytosis, and promoting wound closure than S-MSCs during the wound repair. High levels of CCL2 and IL-6 were found in S-IT MSCs, which indicated that the optimization of macrophage function by S-IT MSCs may be achieved through their high expression of CCL2 and IL-6.ConclusionsOur results suggest that the beneficial paracrine effect of MSCs on wound healing can be amplified by pretreatment with IT, which may represent a new strategy for optimizing the therapeutic effect of MSCs on wound healing.

Therapeutic Effects of Human Pluripotent Stem Cell-Derived Mesenchymal Stem Cells on a Murine Model of Acute Type-2-Dominated Airway Inflammation

Allergic rhinitis and allergic asthma are the most common type-2 inflammatory diseases, which are hardly curable and cause heavy burden to general well-being. Mesenchymal stem cells (MSCs) are multipotent nonhematopoietic cells with potential immunomodulatory effects that have been showning to have a therapeutic effect on allergic diseases. Here, we investigated the effects of human induced pluripotent stem cell (iPSC)-derived MSCs on airway hyperresponsiveness and acute type-2-dominated inflammation throughout the upper and lower airways. In this study, human MSCs, MSC cell culture supernatant, and culture medium (control) was injected into the acute airway inflammatory model via the tail vein. Mouse behavioristics were recorded immediately and mouse lung function was measured 24hours after the last ovalbumin (OVA) challenge. Histological staining, Luminex, Elisa and flow cytometry were employed to evaluate the effects on the production of total/OVA-specific IgG1 and IgE, cytokines expression in lung tissues, and inflammatory cells infiltration in the lung and spleen of the experimental mice. Expressions of eotaxin, IL-4, IL-5, IL-13, IL-33 in nasal and lung lavage were evaluated by Luminex and Elisa. We found that for this acute inflammatory mouse model, human MSC transplantation significantly mitigated the decreased motoring time and the increased lung function Rrs caused by OVA challenge. Serum OVA-IgG1, OVA-IgE, and eosinophil percentages in the splenocytes were significantly decreased. Injection of the MSC supernatant also showed the same trend, but not significantly changed. After treatment, IL-4 and IL-13 were significantly decreased in the lung tissue, and IL-5 and IL-13 were significantly decreased in lung lavage. In conclusion, both human MSC culture supernatant and cell transplantation could alleviate AHR and inflammation in acute inflammatory experimental animals, which demonstrated their potential for clinical therapeutics. Human iPSC-MSCs, MSC cell culture supernatant, or culture medium (control) was injected into the OVA-induced acute airway inflammatory model via the tail vein. Behavioral changes, AHR, serum OVA-specific IgG1 and IgE concentrations, and type-2 inflammations were alleviated.

Hypoxia-induced mesenchymal stem cells inhibit corneal fibroblast proliferation by regulating the WWP2/Notch1 axis.

Aim: This study aimed to explore the role of hypoxic mesenchymal stem cells (MSCs) in corneal alkali burns and the underlying mechanism. Materials & methods: Rat corneal fibroblasts were incubated with IL-6, followed by treatment with hypoxic MSC supernatant. A rat corneal alkali burn model was implemented and processed with hypoxic MSCs. The associated factors were detected by corresponding methods. Results: Hypoxic MSCs reduced the Notch1 level and the proliferation of rat corneal fibroblasts. Hypoxic MSCs or WWP2 overexpression in MSCs enhanced ubiquitination of Notch1. WWP2 interacted with Notch1, and WWP2 silencing reversed the effects of the hypoxic MSCs. Hypoxic MSC treatment in vivo decreased the corneal neovascularization scores and opacity scores. Conclusion: Hypoxic MSCs inhibited inflammation and alleviated corneal injury in alkali burns via the WWP2/Notch1 axis.

Research advances on mesenchymal stem cell-derived extracellular vesicles in promoting angiogenesis of diabetic ulcers

Extracellular vesicles are nanoparticles secreted by most eukaryotic cells and play important roles in material transport and information transmission between cells, involved in inflammation, angiogenesis, antigen presentation, cell apoptosis, cell differentiation, and other biological processes. The culture supernatant of mesenchymal stem cells is rich in extracellular vesicles, and the extracellular vesicles can regulate the formation of new blood vessels, a key step in wound healing and tissue repair. The persistence of diabetic ulcers is closely related to the blocked formation of wound vascular network. This article reviews the role of extracellular vesicles derived from mesenchymal stem cells in promoting angiogenesis of diabetic ulcers, in order to provide a new idea for the treatment of diabetic ulcers.