Umbilical Mesenchymal Stem Cell-derived Exosomes Research Articles

Umbilical mesenchymal stem cell-derived exosomes promote spinal cord functional recovery through the miR-146b/TLR4 -mediated NF-κB p65 signaling pathway in rats

Spinal cord injury (SCI) is an incurable central nervous system impairment that lack of efficient treatment. Exosomes derived from mesenchymal stem cells (MSCs) are widely applied in disease treatment. This work aimed to determine the promising therapeutic effects of MSC-derived exosomal miRNA146b on SCI. A rat spinal cord injury (SCI) model and lipopolysaccharide (LPS)-induced PC12 cell model were established. Exosomes were extracted from human umbilical cord mesenchymal stem cells (hUCMSCs). The identification of exosomes was performed by using transmission electronic microscope (TEM) and nanoparticle tracking analysis (NTA). Hematoxylin and eosin (HE) staining and TUNEL assay were performed to assess tissue damage and apoptosis, respectively. ELISA was performed to detect levels of inflammatory cytokines. Cell viability was checked by cell counting kit 8 (CCK-8). Gene expression and protein levels were detected by qPCR and western blotting assay. The interaction between miR-146 b and Toll-like receptor 4 (TLR4) was assessed by luciferase reporter gene assay. The hUCMSC-derived exosomes could notably alleviate the spinal cord injury and cell apoptosis. The exosomal miR-146 b treatment suppressed the release of IL-1 β, IL-6, and TNFα. The miR-146 b suppressed the expression of TLR4, directly interact with the 3′-untranslated region (3′UTR) of TLR4, and inactivated the nuclear factor κB (NF-κB) signaling. The hUCMSCs-derived exosomal miR-146 b protects neurons from spinal cord injury through targeting the TLR4 and inactivating the NF-κB signaling. Our findings supported the application of hUCMSCs-derived exosomal miR-146 b for the protection of SCI.

Immunomodulatory effects of umbilical mesenchymal stem cell-derived exosomes on CD4+ T cells in patients with primary Sjögren's syndrome

BackgroundPrimary Sjögren's syndrome (pSS) is an autoimmune disease that leads to the destruction of exocrine glands and multisystem lesions. Abnormal proliferation, apoptosis, and differentiation of CD4+ T cells are key factors in the pathogenesis of pSS. Autophagy is one of the important mechanisms to maintain immune homeostasis and function of CD4+ T cells. Human umbilical cord mesenchymal stem cell-derived exosomes (UCMSC-Exos) may simulate the immunoregulation of MSCs while avoiding the risks of MSCs treatment. However, whether UCMSC-Exos can regulate the functions of CD4+ T cells in pSS, and whether the effects via the autophagy pathway remains unclear.MethodsThe study analyzed retrospectively the peripheral blood lymphocyte subsets in pSS patients, and explored the relationship between lymphocyte subsets and disease activity. Next, peripheral blood CD4+ T cells were sorted using immunomagnetic beads. The proliferation, apoptosis, differentiation, and inflammatory factors of CD4+ T cells were determined using flow cytometry. Autophagosomes of CD4+ T cells were detected using transmission electron microscopy, autophagy-related proteins and genes were detected using western blotting or RT-qPCR.ResultsThe study demonstrated that the peripheral blood CD4+ T cells decreased in pSS patients, and negatively correlated with disease activity. UCMSC-Exos inhibited excessive proliferation and apoptosis of CD4+ T cells in pSS patients, blocked them in the G0/G1 phase, inhibited them from entering the S phase, reduced the Th17 cell ratio, elevated the Treg ratio, inhibited IFN-γ, TNF-α, IL-6, IL-17A, and IL-17F secretion, and promoted IL-10 and TGF-β secretion. UCMSC-Exos reduced the elevated autophagy levels in the peripheral blood CD4+ T cells of patients with pSS. Furthermore, UCMSC-Exos regulated CD4+ T cell proliferation and early apoptosis, inhibited Th17 cell differentiation, promoted Treg cell differentiation, and restored the Th17/Treg balance in pSS patients through the autophagy pathway.ConclusionsThe study indicated that UCMSC-Exos exerts an immunomodulatory effect on the CD4+ T cells, and maybe as a new treatment for pSS.

Human umbilical cord mesenchymal stem cell-derived exosomes inhibit ovarian granulosa cells inflammatory response through inhibition of NF-κB signaling in polycystic ovary syndrome

Polycystic ovary syndrome (PCOS) is a common endocrine disorder and the leading cause of anovulatory infertility in women of reproductive age. Chronic low-grade inflammation is considered to be the major contributors to ovarian dysfunction. Human umbilical mesenchymal stem cell-derived exosomes (hUC-MSC-exos) have been proven to exhibit highly effective anti-inflammatory ability. However, its ability to protect against inflammatory response in PCOS remains unclear. Therefore, the aim of the present study was to explore the effect and underlying mechanisms of hUC-MSC-exos on inflammatory response of ovarian granulosa cells (GCs) in PCOS. In this study, we demonstrated that hUC-MSC-exos enhanced the expression of anti-inflammatory factor IL-10 and inhibited the expression of pro-inflammatory factors TNF-α and IFN-γ. Moreover, hUC-MSC-exos had the effects of inhibiting apoptosis and promoting progesterone production. Mechanistically, exposure to hUC-MSC-exos inhibited NF-κB signaling through significantly decreased the levels of the phosphorylation IκB and p65 in GCs. Moreover, the nuclear translocation of p65 was inhibited. Taken together, these results indicate that hUC-MSC-exos ameliorates the inflammation of granulosa cells in patients with PCOS by inhibiting NF-κB signaling pathway.

Umbilical mesenchymal stem cell-derived exosomes facilitate spinal cord functional recovery through the miR-199a-3p/145-5p-mediated NGF/TrkA signaling pathway in rats

BackgroundAlthough exosomes, as byproducts of human umbilical cord mesenchymal stem cells (hUC-MSCs), have been demonstrated to be an effective therapy for traumatic spinal cord injury (SCI), their mechanism of action remains unclear.MethodsWe designed and performed this study to determine whether exosomes attenuate the lesion size of SCI by ameliorating neuronal injury induced by a secondary inflammatory storm and promoting neurite outgrowth. We determined the absolute levels of all exosomal miRNAs and investigated the potential mechanisms of action of miR-199a-3p/145-5p in inducing neurite outgrowth in vivo and in vitro.ResultsmiR-199a-3p/145-5p, which are relatively highly expressed miRNAs in exosomes, promoted PC12 cell differentiation suppressed by lipopolysaccharide (LPS) in vitro through modulation of the NGF/TrkA pathway. We also demonstrated that Cblb was a direct target of miR-199a-3p and that Cbl was a direct target of miR-145-5p. Cblb and Cbl gene knockdown resulted in significantly decreased TrkA ubiquitination levels, subsequently activating the NGF/TrkA downstream pathways Akt and Erk. Conversely, overexpression of Cblb and Cbl was associated with significantly increased TrkA ubiquitination level, subsequently inactivating the NGF/TrkA downstream pathways Akt and Erk. Western blot and coimmunoprecipitation assays confirmed the direct interaction between TrkA and Cblb and TrkA and Cbl. In an in vivo experiment, exosomal miR-199a-3p/145-5p was found to upregulate TrkA expression at the lesion site and also promote locomotor function in SCI rats.ConclusionsIn summary, our study showed that exosomes transferring miR-199a-3p/145-5p into neurons in SCI rats affected TrkA ubiquitination and promoted the NGF/TrkA signaling pathway, indicating that hUC-MSC-derived exosomes may be a promising treatment strategy for SCI.

Human umbilical mesenchymal stem cells-derived exosomes modulate the proliferation, apoptosis and migration of human retinal pigment epithelial cells in hypoxia

Objective: To study the effects of human umbilical mesenchymal stem cells (HUMSCs) exosomes on the proliferation and apoptotic as well as migration of human retinal pigment epithelial cells (HRPE) in hypoxia, and explore its mechanism. Method: Direct adherent culture was adopted to cultivate umbilical cord mesenchymal stem cells and amplified to the fourth generation. Markers on the cell surface were identified by flow cytometry. Culture medium was collect without serum from the 4th generation umbilical cord mesenchymal stem cells. Exosomes were separated and extracted, then the ultrastructure was observed under electron microscope and examined expression of CD63 and CD9 protein by Western blot method with isolated and extracted exosomes. HRPE was cultivated in vitro culture, proliferation was detected at the time point of 0, 1, 2, 3, 4, 5 d with MTT assay under hypoxic condition. Meanwhile, the cell migration was quantified by Wound-Healing Assay under hypoxic condition at 0, 24, 48 and 72 h respectively combined with apoptosis test. The HRPE cells in the growth period were divided into 5 groups: the control group, the hypoxia group and the pretreated exosomes group (100, 200, 300 μg/ml). In all groups, apoptosis was observed by Annexin V/PI dual-dye flow cytometry after 48 h's incubation. Proliferation was observed by MTT assay and the migration was observed with Wound-Healing Assay. Results: Flow cytometry detection of the surface marker of HUMSCs in the 4th generation showed strong positive expression of CD105, CD73, CD90. It was suggested that HUMSCs with isolated culture had MSC specific phenotype with duction of lipids and osteoblasts in vitro. The separated exosomes were observed with spherical membranous structures in different sizes by scanning electron microscopy, and Western blot detected positive expression of CD63 and CD9. In vitro culture of HRPE detected by MTT assay for cell proliferation at the time of hypoxic 0, 1, 2, 3, 4, 5 d, the results showed that, comparing with time point 0 d, other groups had statistically significant OD values. In the first 2 days, the proliferation ability of RPE cells gradually increased as the time of hypoxia prolonged(1.862±0.135, 2.278±0.244). After 3 d, the proliferation ability of RPE cells gradually decreased(1.419±0.124, 1.599±0.156). Wound-Healing Assay results showed that the migration distance gradually increased as[(29.883±4.504), (36.200±1.928) μm] the time of hypoxia increased from 0 to 72 h. The cells were fully covering at the point of 72 h [(1.223±0.194), (0.430±0.299) μm]. Apoptosis test results showed that the number of apoptotic cells was different(3.628%±1.348%, 20.123%±1.183%) with the extension of hypoxia Oxygen before 2 d from 0 to 72 h. At the time of d3, there were more apoptotic cells(42.290%±3.217%). There is a significant difference from pre-2d.RPE cells were divided into 5 groups: the control group, the hypoxia group and the pretreated exosomes group (100, 200, 300 μg/ml).After 48 h hypoxia incubation, MTT assay results showed that, compared with the control group (1.870±0.499), the number of cell proliferation was significantly increased (t=-3.116, P<0.05), while compared with the hypoxia group(2.616±0.307), the proliferation number of exosomes was significantly reduced [(2.041±0.115), (1.931±0.205), (1.929±0.025); t=-4.920, -4.540, -5.286, P<0.01], and there was no significant difference between groups with different doses of the exosomes (F=1.181,P>0.05). Annexin V/PI dual-dye flow cytometry was used to observe the apoptosis results. Compared with the control group 1.180%±0.689%, the number of apoptosis in hypoxia group was significantly increased (19.273%±1.194%, t=-32.141, P<0.01), while compared with the hypoxia group, the number of apoptosis in the exosomes was significantly decreased (12.318%±1.087%, 11.878%±1.348%, 11.090%±1.716%; t=-10.547, -10.057, 9.589, P<0.01). There was no significant difference between the groups with different doses of exosomes (F=1.173, P>0.05). Wound-Healing Assay results showed that, compared with the control group(68.047±2.851) μm, the migration distance of the hypoxia group was significantly increased [(13.470±2.255)μm, t=36.778, P<0.01] while compared with the hypoxia group, the migration distance of the exosomes was reduced (33.110±1.774, 24.650±1.175, 26.440±1.674; t=11.766, 10.770, 11.311, P<0.01), and there was no significant difference between the groups of the exosomes (F=1.179, P>0.05). Conclusion: Human umbilical cord mesenchymal stem cells can effectively inhibit the apoptosis and migration of HRPE cells in hypoxia. It provides a theoretical basis for the research and treatment of RPE related diseases. (Chin J Ophthalmol, 2019, 55: 933-941).