Human Wharton's jelly mesenchymal stem cells derived-exosomes enriched by miR-124 promote an anti-fibrotic response in an experimental model of liver fibrosis.

BACKGROUND: Liver fibrosis is a significant challenge to global health that results in organ failure through inflammation and the release of fibrotic biomarkers. Due to the lack of effective treatments for liver fibrosis, anti-fibrotic and anti-inflammatory therapies are being developed. Since there has been an association between aberrant expression of miR-124 and liver disease progression, we investigated whether delivery of miR-124 through human Wharton's jelly mesenchymal stem cells derived-exosomes (hWJMSC-Exo) can improve liver fibrosis. METHODS: We established a 6-week carbon tetrachloride (CCl4)-induced mouse model of liver fibrosis, then we administered hWJMSC-Exo and miR-124-3p-enriched exosomes (ExomiR-124) for three weeks. The extent of fibrosis and inflammation was assessed by histology, biochemistry, Real-time PCR, immunohistochemistry, and Enzyme-linked immunoassays (ELISA). The inflammatory status of the spleen was also investigated using flow cytometry. RESULTS: Based on the gene and protein expression measurement of IL-6, IL-17, TGF-ß, STAT3, α-SMA, and COL1, In vivo administration of Exo and ExomiR-124 effectively reduce collagen accumulation and inhibition of inflammation. Regarding histopathology findings, the therapeutic effect of ExomiR-124 against liver fibrosis was significantly greater than hWJMSC-Exo. In addition, we found that Exo and ExomiR-124 was capable of phenotype switching of splenic monocytes from inflammatory Ly6Chi to restorative Ly6Clo. CONCLUSIONS: MSC-derived exosomes demonstrated anti-inflammatory effect via different aspects. Aside from the therapeutic approach, enrichment of exosomes as a nanocarrier by miR-124 revealed the down-regulation of STAT3, which plays a crucial role in liver fibrosis. The anti-inflammatory and anti-fibrotic properties of ExomiR-124 could be a promising option in liver fibrosis combination therapies.

Umbilical Cord Wharton's Jelly-Derived Mesenchymal Stem Cells Inhibit the TGF-ß1 Pathway in Hepatic Fibrosis Rats Through a Paracrine Regulation Process.

BACKGROUND: The aim is to verify the therapeutic effect and possible mechanism of human umbilical cord Wharton's jelly-derived transplantation of mesenchymal stem cells (UMSCs) on CCl4-induced hepatic fibrosis rats through in vivo studies and to explore the regulatory mechanism of UMSCs on fibrosis of hepatic stellate cells (HSCs) through in vitro experiments. METHODS: In vivo experiment: Rats were randomly divided into blank control group and hepatic fibrosis group. During the entire trial, the blank control group received subcutaneous injection of normal saline, while in the hepatic fibrosis group received injections of 50% CCl4-olive oil subcutaneously for 10 weeks to establish the rat model of liver fibrosis. Hepatic fibrosis rats were then randomly and evenly divided into umbilical cord mesenchymal stem cell (UMSC) group, bone marrow mesenchymal stem cell (BMSC) group, UMSC-culture medium (CM) group, and control group. Rats in each group were infused with the following substances through the caudal vein as follows: 1 mL UMSCs (2 × 106/mL) in UMSC group, 1 mL BMSCs (2 × 106/mL) in BMSC group, 1 mL UMSCs-CM in CM group, and 1 mL saline in control group. Rats of each group were closely observed (weight, hair condition, activity, appetite, diarrhea, etc.), venous blood samples were collected, the number of white blood cells and lymphocytes were measured, and liver function indicators (ALT, AST, TBIL, ALB) were determined. Three weeks later, rat liver specimens were taken, HE stained, pathological changes were examined and quantified. In vitro experiments: HSCs were seeded in 6-well plates at 1.0 × 105/mL, with a serum-free medium for 24 hours. Then, 2 mL of UMSCs-CM was added in the study group, while an equal amount of complete medium was added to the control group. RT-PCR was used to detect TGF-ß1, Collagen-I, TIMP-2 mRNA expression in HSCs, and western blot was used to detect TGF-ß1 protein expression in HSCs. RESULTS: In vivo experiment: Compared with the control group, after the transplantation, the activity status (weight, spirit, appetite, movement, hair, diarrhea, etc.) of rats in the UMSC group, BMSC group, and CM group were improved. The liver function indexes of these groups, such as alanine aminotransferase (ALT), aspartate aminotransferase (AST), and total bilirubin (TBIL) were significantly decreased (p < 0.05), while albumin (ALB) levels were mildly but not significantly increased (p > 0.05). The Knodell score (reflecting the degree of liver inflammation) and Chevallier score (reflecting the degree of liver fibrosis) of liver specimens in pathological examination were also significantly reduced, and the difference in the quantitative scores of those indexes was statistically significant (p < 0.05). There was no statistically significant difference in the number of venous white blood cells and lymphocytes, liver function indexes (ALT, AST, TBIL, ALB), Knodell score, and Chevallier score of liver samples among the UMSC group, BMSC group, and CM group. In vitro experiments: After treatment with UMSCs-CM, the expression of TGF-ß1, Collagen-I, and TIMP-2 mRNA in HSCs was significantly down-regulated compared with that of the control group (treated with complete medium), and it gradually decreased with the extension of the treatment time. Compared with the control group, the expression of TGF-ß1 protein in the HSCs of the experimental group was down-regulated, and this effect was time-dependent, specifically, the control group (2.49 ± 0.43) > the experimental group at 48 hours (1.98 ± 0.26) > the experimental group at 72 hours (1.62 ± 0.20) (F = 7.796, p < 0.05). CONCLUSIONS: In rats with liver fibrosis, transplantation of UMSCs can improve liver function and reduce the inflammatory activity and fibrosis of the liver, possibly through the paracrine mechanism. UMSCs inhibit HSCs fibrosis through a paracrine mechanism, which is time-dependent, possibly by targeting TGF-ß1 and its downstream gene products.

Dysregulation of Caveolin-1 Phosphorylation and Nuclear Translocation Is Associated with Senescence Onset.

We recently reported that the inability of osteoarthritic (OA) chondrocytes to repair oxidative stress (OS) induced DNA damage is linked to Cav-1 overexpression/improper localization. We speculated that the senescent status of OA cells was responsible for this Cav-1 dysregulation. Here, to further investigate this hypothesis, we used Wharton Jelly derived mesenchymal stem cells (WJ-MSCs) and investigated Cav-1 function as cells reached replicative senescence or upon stress induced senescence (SIPS). We showed that Cav-1 is upregulated, phosphorylated and translocated to the nucleus in young WJ-MSCs upon acute exogenous OS, and that it returns back to basal/nonphosphorylated levels and exports the nucleus in the recovery phase. However, as cells reach senescence, this regulation is lost. OS did not induce any Cav-1-mediated response, which is concomitant with the inability of older cells to restore DNA damage. Furthermore, downregulation of Cav-1 resulted in persistent OS-induced DNA damage and subsequent onset of senescence. We also report that the establishment of senescence is mediated by autophagy stimulation, since downregulation of autophagy key molecule Atg5, simultaneously with Cav-1 downregulation, was found to inhibit SIPS. Basically, we propose that Cav-1 involvement in DNA damage response can lead to senescence, either because the damage is extensive or because Cav-1 is absent/unable to perform its homeostatic role.

The effect of vascular complications of diabetes mellitus on human umbilical cord tissue and the number of Wharton Jelly's mesenchymal stem cells.

The current study investigated the change in umbilical cord tissue and the number of markers of Wharton's jelly mesenchymal stem cells (WJ-MSC) in pregnant women with gestational diabetes (GDM), with chronic diabetes who developed nephropathy as vascular complication (VC-PGDM), and healthy pregnant women as the control. The umbilical cords (UC) were investigated by the histomorphological method and the number of WJ-MSC were detected by flow-cytometry using the CD90, CD44, CD105, and CD73 markers in Wharton's jelly (WJ) isolated from fresh umbilical cords. The number of positive cells for CD 90, CD44, CD105, and CD73 were found to be elevated in the GDM group, whereas it was significantly diminished in the VC-PGDM group (p = 0.001, p = 0.001, p = 0.001, and p = 0.001). The only histopathological sign in the GDM group were an increased number of pores in the Wharton jelly. Artery wall thickness/cord diamater ratio was increased, which indicates an increase of the artery wall thickness in the VC- PGDM group (p = 0.039 and p = 0.048). The increase in umbilical cord diameter and number of Wharton jelly mesenchymal stem cells in babies of gestational diabetic mothers was considered as an effect of macrosomia seen in babies of mothers with gestational diabetes. Vasculopathy, a long-term complication of diabetes, is known to affect all tissues by causing marked lower perfusion and hypoxia, as well as a decrease in the MSC number in our study.

Wharton's Jelly-derived mesenchymal stem cells suppress apoptosis of nucleus pulposus cells in intervertebral disc degeneration via Wnt pathway.

OBJECTIVE: Aberrant apoptosis of nucleus pulposus cells (NPCs) is one of the most remarkable pathological changes in intervertebral disc degeneration (IDD) development. Albeit the advances in the application of stem cell-based therapy in IDD treatment, the molecular mechanisms underlying the anti-apoptotic actions of mesenchymal stem cell (MSC) remain poorly elucidated. PATIENTS AND METHODS: The expression patterns of apoptosis-related proteins and Wnt/ß-catenin-related genes in NP samples isolated from patients with mild or severe IDD were compared by performing immunoblot assay and quantitative real-time polymerase chain reaction (qRT-PCR), respectively. NPCs were in vitro treated with compression to induce apoptosis and then co-cultured with Wharton's Jelly-derived MSCs without direct interaction. After that, flow cytometry was carried out to detect the apoptosis rate of NPCs and the activity of Wnt/ß-catenin pathway was determined. DKK-1 was used to inhibit Wnt signaling, in prior to evaluation of the effects of WJ-MSCs on apoptosis within the co-cultured NPCs. RESULTS: Compared to the mild IDD group, there was a significant increase in the expression of Caspase-3 and Bax in the NP tissues from severe IDD patients, whereas Bcl-2 displayed an opposite result. In addition, the expression of Wnt 3a, Wnt 5a, Wnt 10a, GSK-3ß, cyclinD1 and ß-catenin was notably augmented in parallel with IDD progression. After compression treatment, the proportion of apoptotic NPCs was increased, which was then dramatically reversed by WJ-MSCs co-culture. Likewise, WJ-MSCs suppressed compression-induced Wnt-related gene expression and blocking Wnt/ß-catenin pathway using DKK-1 enhanced the anti-apoptotic impacts of WJ-MSCs. In the presence of DKK-1, there was no significant difference between NPCs co-cultured with WJ-MSCs and those cells cultured alone. CONCLUSIONS: WJ-MSCs attenuate the compression-induced apoptosis in NPCs and inhibit the activation of Wnt/ß-catenin signaling. Blocking Wnt/ß-catenin pathway further facilitates the actions of WJ-MSCs in anti-apoptosis, indicating that Wnt/ß-catenin signaling plays a crucial role in this process and may function as a potential therapeutic target for IDD treatment.

A novel extracellular matrix-based leukemia model supports leukemia cells with stem cell-like characteristics.

Acute myeloid leukemia (AML) relapse results from the survival of chemotherapy-resistant and quiescent leukemia stem cells (LSC). These LSCs reside in the bone marrow microenvironment, comprised of other cells and extracellular matrix (ECM), which facilitates LSC quiescence through expression of cell adhesion molecules. We used decellularized Wharton's jelly matrix (DWJM), the gelatinous material in the umbilical cord, as a scaffolding material to culture leukemia cells, because it contains many components of the bone marrow extracellular matrix, including collagen, fibronectin, lumican, and hyaluronic acid (HA). Leukemia cells cultured in DWJM demonstrated decreased proliferation without undergoing significant differentiation. After culture in DWJM, these cells also exhibited changes in morphology, acquiring a spindle-shaped appearance, and an increase in the ALDH+ cell population. When treated with a high-dose of doxorubicin, leukemia cells in DWJM demonstrated less apoptosis compared with cells in suspension. Serial colony forming unit (CFU) assays indicated that leukemia cells cultured in DWJM showed increased colony-forming ability after both primary and secondary plating. Leukemia cell culture in DWJM was associated with increased N-cadherin expression by flow cytometry. Our data suggest that DWJM could serve as an ECM-based model to study AML stem cell-like cell behavior and chemotherapy sensitivity.

Evaluation of nerve growth factor (NGF) treated mesenchymal stem cells for recovery in neurotmesis model of peripheral nerve injury.

BACKGROUND: Peripheral nerve damages are a relatively common type of the nervous system injuries. Although peripheral nerves show some capacity of regeneration after injury, the extent of regeneration is not remarkable. The present study aimed to evaluate the effect of NGF treated mesenchymal stem cells on regeneration of transected sciatic nerve. MATERIALS AND METHODS: In this experimental study, forty-two male Wistar.rats (180-200 g) were randomly divided into 6 groups (n = 7) including control, Membrane + Cell (Mem + Cell), NGF group, NGF + Cell group, NGF + Mem group and NGF + Mem + Cell group. Regeneration of sciatic nerve was evaluated using behavioral analysis, electrophysiological assessment and histological examination. RESULTS: The rats in the NGF + Mem + Cell group showed significant decrease in sciatic functional index (SFI) and hot water paw immersion test during the 2nd to 8th weeks after surgery. (p < 0.001). At 8 weeks after surgery, electrophysiological findings showed that amplitude increased and latency decreased significantly in NGF + Mem + Cell group (p < 0.001). Measured histological parameters showed that number of nerve fibers, number of vessels and percent of vessel area also increased significantly in NGF + Mem + Cell group (p < 0.05). CONCLUSION: The present study showed that NGF in accompany with mesenchymal stem cells improved electrophysiological and histological indices.

Comparison of Advanced Platelet Rich Fibrin (A-PRF) and Culture Media Conditioned Warton's Jelly (CMCWJ) on Fibroblast Cells Proliferation

Objective: To compare the potency of fibroblast cells proliferation in 12.5% and 25% Culture Media Conditioned Warton's Jelly (CMCWJ) and Advanced Platelet Rich Fibrin (A-PRF) cultured medium. Material and Methods: Fibroblast cells were divided into five groups: Group I (Control Group): serum-starved fibroblast without any treatment as a negative control; Group II: fibrolast that supplemented in 12.5% CMCWJ medium; Group III: fibrolast that supplemented 12.5% A-PRF medium; Group IV: fibrolast that supplemented 25% CMCWJ medium, and Group V: fibrolast that supplemented 25% A-PRF medium. The fibroblasts proliferation was counted by an automated cell counter. Statistical analysis was performed using One-way ANOVA and Post hoc Tamhane test was conducted to analyze the potential fibroblast proliferation differences in different concentration of CMCWJ and A-PRF group. Results: There were no significant differences in the fibroblast cell proliferation between GI and GIV, GII and GIV, GII and GIII, GII and GV, also GIV and GV. There were significant differences between GI and GII, GI and GIII, GI and GV, also GIII and GIV. Conclusion: The 12.5% CMCWJ group, 12.5% A-PRF group and 25% A-PRF group has excellent potential ability of fibroblast cells proliferation, meanwhile 25% CMCWJ group has the lowest mean potency of fibroblast cells proliferation compared to other groups. The 12.5% A-PRF Group has the highest mean of fibroblast cell proliferation amongst other groups.

Effects of maternal obesity on Wharton's Jelly mesenchymal stromal cells.

We investigated whether maternal metabolic environment affects mesenchymal stromal/stem cells (MSCs) from umbilical cord's Wharton's Jelly (WJ) on a molecular level, and potentially render them unsuitable for clinical use in multiple recipients. In this pilot study on umbilical cords post partum from healthy non-obese (BMI = 19-25; n = 7) and obese (BMI ≥ 30; n = 7) donors undergoing elective Cesarean section, we found that WJ MSC from obese donors showed slower population doubling and a stronger immunosuppressive activity. Genome-wide DNA methylation of triple positive (CD73+CD90+CD105+) WJ MSCs found 67 genes with at least one CpG site where the methylation difference was ≥0.2 in four or more obese donors. Only one gene, PNPLA7, demonstrated significant difference on methylome, transcriptome and protein level. Although the number of analysed donors is limited, our data suggest that the altered metabolic environment related to excessive body weight might bear consequences on the WJ MSCs.

The effect of transplanted human Wharton's jelly mesenchymal stem cells treated with IFN-γ on experimental autoimmune encephalomyelitis mice.

Interferon gamma (IFN-γ) increases the immunosuppressive property of human Wharton's jelly mesenchymal stem cells (hWJ-MSCs). In this study, we evaluated the therapeutic effects of IFN-γ primed WJ-MSCs in EAE mice. IFN-γ primed WJ-MSCs were injected on days 3 and 11 after EAE induction. 21 days after EAE induction, splenocytes and cervical lymph node cells were isolated and cell proliferation, secretion of inflammatory cytokines and frequency of regulatory T-cells was measured. On day 50 of the study, cell infiltration and gene expression of inflammatory cytokines in brain of mice were studied. Leukocyte infiltration and symptoms were significantly reduced in IFN-γ primed WJ-MSCs treated group compared to other groups. These cells showed significantly reduced proliferation and increased Treg cells as well as decreased secretion and gene expression of inflammatory cytokines in EAE mice. Our data suggest that IFN-γ may be used to stimulate the immunomodulatory property of WJ-MSCs in clinical situations.

Human Wharton's jelly mesenchymal stem cell secretome display antiproliferative effect on leukemia cell line and produce additive cytotoxic effect in combination with doxorubicin.

Mesenchymal stem cell (MSC) therapy moves toward clinic progressively. Recent evidences establish anticancer effect of mesenchymal stem cells. However multiple factors including type of cancer, MSC source, study design, and animal model play role in final outcome. Wharton's jelly - a newly approved source of MSCs - possesses superiorities to bone marrow as the conventional source; therefore investigation of its medical effects can produce beneficial results. In this survey we examined cytotoxic and proapoptotic effect of human Wharton's jelly MSC secretome on K562 human leukemia cells. MSCs were isolated from human Wharton's jelly of umbilical cord by explant culture method, then characterized according to ISCT criteria (morphology and plastic adherence, surface antigenicity and differentiation potential). MSC secretome was collected and its cytotoxic and proapoptotic effects on K562 cells in combination with doxorubicin were evaluated using BrdU cell proliferation assay and Annexin V-PI staining. Our results showed antiproliferative effect of mesenchymal stem cell secretome on K562 cancer cells, the effect was also added to cytotoxic effect of doxorubicin without induction of drug resistance. Human Wharton's jelly derived mesenchymal stem cells exerted cytotoxic effect on leukemia cells. Addition of that effect to anticancer effect of chemotherapeutic agents can leads to cytotoxic drug dose reduction and diminished side effects.

Preeclampsia enhances neuroglial marker expression in umbilical cord Wharton's jelly-derived mesenchymal stem cells.

OBJECTIVE: The aim of the study was to compare the neuroglial phenotype of Wharton's jelly-derived mesenchymal stem cells (WJ-MSC) from pregnancies complicated with preeclampsia and gestational age (GA)-matched controls. METHODS: WJ-MSC were isolated from umbilical cords from both groups and analyzed for the cell surface expression of MSC markers and the gene and protein expression of neuroglial markers. RESULTS: All WJ cells were highly positive for the MSC markers CD105, CD90 and CD73, but negative for markers specific for hematopoietic (CD34) and immunological cells (CD45, CD14, CD19 and HLA-DR). WJ-MSC from both groups expressed neuroglial markers (MAP-2, GFAP, MBP, Musashi-1 and Nestin) at the mRNA and protein level. The protein expressions of neuronal (MAP-2) and oligodendrocytic (MBP) markers were significantly increased in WJ-MSC from preeclampsia versus GA-matched controls. CONCLUSIONS: WJ-MSC from preeclamptic patients are possibly more committed to neuroglial differentiation through the activation of pathways involved both in the pathophysiology of the disease and in neurogenesis.