Human umbilical cord mesenchymal stem cell-derived exosomes attenuate neuroinflammation and oxidative stress through the NRF2/NF-κB/NLRP3 pathway.

AIMS: We investigated whether human umbilical cord mesenchymal stem cell (hUC-MSC)-derived exosomes bear therapeutic potential against lipopolysaccharide (LPS)-induced neuroinflammation. METHODS: Exosomes were isolated from hUC-MSC supernatant by ultra-high-speed centrifugation and characterized by transmission electron microscopy and western blotting. Inflammatory responses were induced by LPS in BV-2 cells, primary microglial cultures, and C57BL/6J mice. H2 O2 was also used to induce inflammation and oxidative stress in BV-2 cells. The effects of hUC-MSC-derived exosomes on inflammatory cytokine expression, oxidative stress, and microglia polarization were studied by immunofluorescence and western blotting. RESULTS: Treatment with hUC-MSC-derived exosomes significantly decreased the LPS- or H2 O2 -induced oxidative stress and expression of pro-inflammatory cytokines (IL-6 and TNF-α) in vitro, while promoting an anti-inflammatory (classical M2) phenotype in an LPS-treated mouse model. Mechanistically, the exosomes increased the NRF2 levels and inhibited the LPS-induced NF-κB p65 phosphorylation and NLRP3 inflammasome activation. In contrast, the reactive oxygen species scavenger NAC and NF-κB inhibitor BAY 11-7082 also inhibited the LPS-induced NLRP3 inflammasome activation and switched to the classical M2 phenotype. Treatment with the NRF2 inhibitor ML385 abolished the anti-inflammatory and anti-oxidative effects of the exosomes. CONCLUSION: hUC-MSC-derived exosomes ameliorated LPS/H2 O2 -induced neuroinflammation and oxidative stress by inhibiting the microglial NRF2/NF-κB/NLRP3 signaling pathway.

Immunosuppressive therapy and COVID-19 infection in patients with NMOSD.

INTRODUCTION: To evaluate whether treated with immunosuppressants in neuromyelitis optica spectrum disorder (NMOSD) shows an effect on the severity and outcomes of COVID-19 Omicron variant. METHODS: This is a substudy of a single-center clinical trial involving human umbilical cord mesenchymal stem cells (hUC-MSCs) in NMOSD patients. NMOSD patients with hUC-MSCs treatment, NMOSD patients without hUC-MSCs treatment, and matched healthy controls (HC) were included. Demographic information, NMOSD-related clinical features, comorbidities, use of disease-modifying therapy, COVID-19 vaccination status, COVID-19 clinical features, COVID-19 clinical outcomes, and NMOSD-related disease activity were obtained through online questionnaires or phone calls. RESULTS: The majority of NMOSD patients received long-term treatment with mycophenolate mofetil (68.8%) or azathioprine (22.9%), and 50% received oral glucocorticoid. During the epidemic, 97.4% of NMOSD patients infected with COVID-19 had asymptomatic or mild forms, with only two patients (2.6%) requiring hospitalization. None of these patients required tracheal intubation or admission to the intensive care unit. Clinical symptoms were found to be more prevalent in HC groups. Additionally, the HC groups had higher fever-recorded temperatures. NMOSD patients who received hUC-MSCs treatment had shorter disease duration than patients who did not receive hUC-MSCs treatment. DISCUSSION: Immunosuppressant-treated patients with NMOSD have a similar risk of COVID-19 infection as the general population, but the disease duration is shorter and the clinical symptoms are less severe. Among our NMOSD patients who received hUC-MSCs treatment, COVID-19 outcomes were favorable, with no increased risk of severe COVID-19. Prospective studies on immunotherapies are needed to help determine best treatment practices.

Human Umbilical Cord Mesenchymal Stem Cells to Treat Neuromyelitis Optica Spectrum Disorder (hUC-MSC-NMOSD): A Study Protocol for a Prospective, Multicenter, Randomized, Placebo-Controlled Clinical Trial.

Background: Neuromyelitis Optica spectrum disorder (NMOSD) is severe relapsing and disabling autoimmune disease of the central nervous system. Its optimal first-line treatment to reduce relapse rate and ameliorate neurological disability remains unclear. We will conduct a prospective, multicenter, randomized, placebo-controlled clinical trial to study the safety and effectiveness of human umbilical cord mesenchymal stem cells (hUC-MSCs) in treating NMOSD. Methods: The trial is planned to recruit 430 AQP4-IgG seropositive NMOSD patients. It consists of three consecutive stages. The first stage will be carried out in the leading center only and aims to evaluate the safety of hUC-MSCs. Patients will be treated with three different doses of hUC-MSCs: 1, 2, or 5 × 106 MSC/kg·weight for the low-, medium-, and high-dose group, respectively. The second and third stages will be carried out in six centers. The second stage aims to find the optimal dosage. Patients will be 1:1:1:1 randomized into the low-, medium-, high-dose group and the controlled group. The third stage aims to evaluate the effectiveness. Patients will be 1:1 randomized into the optimal dose and the controlled group. The primary endpoint is the first recurrent time and secondary endpoints are the recurrent times, EDSS scores, MRI lesion numbers, OSIS scores, Hauser walking index, and SF-36 scores. Endpoint events and side effects will be evaluated every 3 months for 2 years. Discussion: Although hUC-MSC has shown promising treatment effects of NMOSD in preclinical studies, there is still a lack of well-designed clinical trials to evaluate the safety and effectiveness of hUC-MSC among NMOSD patients. As far as we know, this trial will be the first one to systematically demonstrate the clinical safety and efficacy of hUC-MSC in treating NMOSD and might be able to determine the optimal dose of hUC-MSC for NMOSD patients. Trial registration: The study was registered with the Chinese Clinical Trial Registry (CHICTR.org.cn) on 2 March 2016 (registration No. ChiCTR-INR-16008037), and the revised trial protocol (Protocol version 1.2.1) was released on 16 March 2020.

Melatonin treatment improves human umbilical cord mesenchymal stem cell therapy in a mouse model of type II diabetes mellitus via the PI3K/AKT signaling pathway.

BACKGROUND: Mesenchymal stem cells (MSCs) are promising candidates for tissue regeneration and disease treatment. However, long-term in vitro passaging leads to stemness loss of MSCs, resulting in failure of MSC therapy. This study investigated whether the combination of melatonin and human umbilical cord mesenchymal stem cells (hUC-MSCs) was superior to hUC-MSCs alone in ameliorating high-fat diet and streptozocin (STZ)-induced type II diabetes mellitus (T2DM) in a mouse model. METHODS: Mice were divided into four groups: normal control (NC) group; T2DM group; hUC-MSCs treatment alone (UCMSC) group and pretreatment of hUC-MSCs with melatonin (UCMSC/Mel) group. RESULTS: RNA sequence analysis showed that certain pathways, including the signaling pathway involved in the regulation of cell proliferation signaling pathway, were regulated by melatonin. The blood glucose levels of the mice in the UCMSC and UCMSC/Mel treatment groups were significantly reduced compared with the T2DM group without treatment (P < 0.05). Furthermore, hUC-MSCs enhance the key factor in the activation of the PI3K/Akt pathway in T2DM mouse hepatocytes. CONCLUSION: The pretreatment of hUC-MSCs with melatonin partly boosted cell efficiency and thereby alleviated impaired glycemic control and insulin resistance. This study provides a practical strategy to improve the application of hUC-MSCs in diabetes mellitus and cytotherapy. Overview of the PI3K/AKT signaling pathway. (A) Underlying mechanism of UCMSC/Mel inhibition of hyperglycemia and insulin resistance T2DM mice via regulation of PI3K/AKT pathway. hUC-MSCs stimulates glucose uptake and improves insulin action thus should inhibition the clinical signs of T2DM, through activation of the p-PI3K/Akt signaling pathway and then regulates glucose transport through activating AS160. UCMSC/Mel increases p53-dependent expression of BCL2, and inhibit BAX and Capase3 protein activation. Leading to the decrease in apoptosis. (B) Melatonin modulated PI3K/AKT signaling pathway. Melatonin activated PI3K/AKT response pathway through binding to MT1and MT2 receptor. Leading to the increase in hUC-MSCs proliferation, migration and differentiation. → (Direct stimulatory modification); ┴ ( Direct Inhibitory modification); → ┤ (Multistep inhibitory modification); ↑ (Up regulate); ↓ (Down regulate); PI3K (Phosphoinositide 3-Kinase); AKT ( protein kinase B); PDK1 (Phosphoinositide-dependent protein kinase 1); IR, insulin receptor; GLUT4 ( glucose transporter type 4); ROS (reactive oxygen species); BCL-2 (B-cell lymphoma-2); PDK1 (phosphoinositide-dependent kinase 1) BAX (B-cell lymphoma-2-associated X protein); PCNA (Proliferating cell nuclear antigen); Cell cycle-associated proteins (KI67, cyclin A, cyclin E).

Human umbilical cord mesenchymal stem cell-derived extracellular vesicles enhance the regenerative capability of fibrotic liver / 器官移植

Objective To investigate the role of human umbilical cord mesenchymal stem cell-derived extracellular vesicle (hUC-MSC-EV) in the regeneration of fibrotic liver. Methods C57BL/6 mice were randomly divided into the 70% normal liver resection group (Oil+PHx group), 70% liver fibrosis resection group (CCl4+PHx group) and 70% liver fibrosis resection+mesenchymal stem cell-derived extracellular vesicle (MSC-EV) treatment group (CCl4+PHx+MSC-EV group), with 8 mice in each group. LX-2 cell lines were assigned into the phosphate buffer solution (PBS) group, transforming growth factor (TGF)-β group and TGF-β+MSC-EV group. The serum levels of alanine aminotransferase (ALT), aspartate aminotransferase (AST) and lactate dehydrogenase (LDH) in mice after partial liver resection were detected in each group. The expression levels of liver fibrosis and proliferation-related parameters were analyzed in each group. The messenger RNA (mRNA) expression levels of epidermal growth factor (EGF), fibroblast growth factor (FGF), vascular endothelial growth factor (VEGF) and hepatocyte growth factor (HGF) in LX-2 cells were detected in each group, and their effects on HGF expression in mouse liver were observed. Results Compared with the Oil+PHx group, the serum levels of AST, ALT and LDH were up-regulated, and the degree of fibrosis was more severe, the positive area of Sirius red and α-smooth muscle actin (α-SMA) staining was larger, and the expression level of α-SMA protein was up-regulated in the CCl4+PHx group. Compared with the CCl4+PHx group, the serum levels of AST, ALT and LDH were decreased, the degree of fibrosis was slighter, the positive area of Sirius red and α-SMA staining was decreased, and the expression level of α-SMA protein was down-regulated in the CCl4+PHx+MSC-EV group, and the differences were statistically significant (all P < 0.05). Compared with the Oil+PHx group, the protein expression levels of Ki67 and proliferating cell nuclear antigen (PCNA) were lower in the CCl4+PHx group. Compared with the CCl4+PHx group, the protein expression levels of Ki67 and PCNA were increased in the CCl4+PHx+MSC-EV group, and the differences were statistically significant (all P < 0.05). Compared with the PBS group, the expression level of CollagenⅠ mRNA in LX-2 cells was increased, the expression level of α-SMA protein was up-regulated and the expression level of HGF protein was decreased in the TGF-β group. Compared with the TGF-β group, the expression level of CollagenⅠ mRNA in LX-2 cells was decreased, the expression levels of HGF mRNA and protein were increased, and the expression level of α-SMA protein was decreased in the TGF-β+MSC-EV group, the differences were statistically significant (all P < 0.05). The expression level of HGF protein in the CCl4+PHx group was lower than that in the Oil+PHx group, whereas the difference was not statistically significant (P > 0.05). The expression level of HGF protein in the CCl4+PHx+MSC-EV group was higher than that in the CCl4+PHx group, and the difference was statistically significant (P < 0.05). Conclusions The regenerative capacity of fibrotic liver is weaker than that of normal liver. hUC-MSC-EV may alleviate liver fibrosis and improve liver regeneration by promoting HGF secretion from actived hepatic stellate cells and effectively enhancing the regenerative capacity of fibrotic liver.

Research progress on mesenchymal stem cell in treatment of ischemic-type biliary lesion / 器官移植

At present, surgical and endoscopic interventions are mainly employed to treat ischemic-type biliary lesion (ITBL). Due to the disadvantages of single therapeutic strategy, high difficulty and expensive medical cost, it is urgent to identify a novel treatment option. Mesenchymal stem cell (MSC) has become potential seed cell for tissue and organ repair in regenerative medicine due to its high self-renewal capability, multi-directional differentiation potential, low immunogenicity and immunoregulatory effects, etc. Recent studies have demonstrated that MSC transplantation into ITBL animal models may not only home to the injured area, but also promote the repair of injured biliary tract tissues through anti-apoptotic and pro-angiogenic effect, which indicates that MSC transplantation is expected to become a new strategy for the treatment of ITBL. In this article, the biological characteristics of MSC, the mechanism and clinical application of MSC transplantation for ITBL were reviewed.

Mechanism of human umbilical cord mesenchymal stem cells alleviating ischemia-reperfusion injury of hepatocytes through mitochondrial transfer / 器官移植

Objective To explore the mechanism of human umbilical cord mesenchymal stem cell (HUC-MSC) alleviating ischemia-reperfusion injury (IRI) of liver cells through mitochondrial transfer. Methods Normal human liver cell line L02 was divided into the blank control group, oxygen-glucose deprivation (OGD) group, experimental control group, and L02 and HUC-MSC co-culture group (L02+HUC-MSC group). L02+HUC-MSC group was further divided into 10:1 co-culture subgroup (group A), 4:1 co-culture subgroup (group B), 2:1 co-culture subgroup (group C), 1:1co-culture subgroup (group D) and 1:2 co-culture subgroup (group E) according to different co-culture ratio of L02 and HUC-MSC. The apoptosis rate and relative reactive oxygen species (ROS) level of L02 cells were detected by flow cytometry. The MitoTracker positive rate of L02 cells was detected by flow cytometry. The mitochondrial transfer from HUC-MSC to L02 cells was observed by laser confocal microscope. Results The apoptosis rate and relative ROS level of L02 cells in the OGD group were significantly higher than those in the blank control group (both P < 0.05). Compared with the OGD group, the apoptosis rates of L02 cells in group B, C, D and E were significantly decreased (all P < 0.05), and the relative ROS level of L02 cells in group E was significantly declined (P < 0.05). The MitoTracker positive rate of L02 cells did not significantly differ between group A and experimental control group (P>0.05), whereas the MitoTracker positive rates of L02 cells in group B, C, D and E were significantly higher than that in the experimental control group in a concentration-dependent manner (all P < 0.05). Under the laser confocal microscope, mitochondrial transfer fromHUC-MSC to L02 cells could be observed through tunneling nanotube (TNT). Conclusions HUC-MSC may alleviate cell apoptosis and reduce ROS level of liver cells after IRI via direct mitochondrial transfer between cells.