Tendon injuries are common musculoskeletal system disorders in clinical, but the regeneration ability of tendon is limited. Tendon stem cells (TSCs) have shown promising effect on tissue engineering and been used for the treatment of tendon injury. Exosomes that serve as genetic information carriers have been implicated in many diseases and physiological processes, but effect of exosomes from TSCs on tendon injury repair is unclear. The aim of this study is to make clear that the effect of exosomes from TSCs on tendon injury healing. Exosomes were harvested from conditioned culture media of TSCs by a sequential centrifugation process. Rat Achilles tendon tendinopathy model was established by collagenase‐I injection. This was followed by intra‐Achilles‐tendon injection with TSCs or exosomes. Tendon healing and matrix degradation were evaluated by histology analysis and biomechanical test at the post‐injury 5 weeks. In vitro, TSCs treated with interleukin 1 beta were added by conditioned medium including exosomes or not, or by exosomes or not. Tendon matrix related markers and tenogenesis related markers were measured by immunostaining and western blot. We found that TSCs injection and exosomes injection significantly decreased matrix metalloproteinases (MMP)‐3 expression, increased expression of tissue inhibitor of metalloproteinase‐3 (TIMP‐3) and Col‐1a1, and increased biomechanical properties of the ultimate stress and maximum loading. In vitro, conditioned medium with exosomes and exosomes also significantly decreased MMP‐3, and increased expression of tenomodulin, Col‐1a1 and TIMP‐3. Exosomes from TSCs could be an ideal therapeutic strategy in tendon injury healing for its balancing tendon extracellular matrix and promoting the tenogenesis of TSCs.
BackgroundThe stem cell-based therapies for intervertebral disc degeneration have been widely studied. However, the mechanisms of mesenchymal stem cells interacting with intervertebral disc cells, such as nucleus pulposus cells (NPCs), remain unknown. Exosomes as a vital paracrine mechanism in cell–cell communication have been highly focused on. The purpose of this study was to detect the role of exosomes derived from bone marrow mesenchymal stem cells (BM-MSCs) and NPCs in their interaction with corresponding cells.MethodsThe exosomes secreted by BM-MSCs and NPCs were purified by differential centrifugation and identified by transmission electron microscope and immunoblot analysis of exosomal marker proteins. Fluorescence confocal microscopy was used to examine the uptake of exosomes by recipient cells. The effects of NPC exosomes on the migration and differentiation of BM-MSCs were determined by transwell migration assays and quantitative RT-PCR analysis of NPC phenotypic genes. Western blot analysis was performed to examine proteins such as aggrecan, sox-9, collagen II and hif-1α in the induced BM-MSCs. Proliferation and the gene expression profile of NPCs induced by BM-MSC exosomes were measured by Cell Counting Kit-8 and qRT-PCR analysis, respectively.ResultsBoth the NPCs and BM-MSCs secreted exosomes, and these exosomes underwent uptake by the corresponding cells. NPC-derived exosomes promoted BM-MSC migration and induced BM-MSC differentiation to a nucleus pulposus-like phenotype. BM-MSC-derived exosomes promoted NPC proliferation and healthier extracellular matrix production in the degenerate NPCs.ConclusionOur study indicates that the exosomes act as an important vehicle in information exchange between BM-MSCs and NPCs. Given a variety of functions and multiple advantages, exosomes alone or loaded with specific genes and drugs would be an appropriate option in a cell-free therapy strategy for intervertebral disc degeneration.
Mitogen activated protein kinase kinase kinase 18 (MAPKKK18) mediated signaling cascade plays important roles in Arabidopsis drought stress tolerance. However, the post‐translational modulation patterns of MAPKKK18 are not characterized. In this study, we found that the protein level of MAPKKK18 was tightly controlled by the 26S proteasome. Ubiquitin ligases RGLG1 and RGLG2 ubiquitinated MAPKKK18 at lysine residue K32 and K154, and promoted its degradation. Deletion of RGLG1 and RGLG2 stabilized MAPKKK18 and further enhanced the drought stress tolerance of MAPKKK18‐overexpression plants. Our data demonstrate that RGLG1 and RGLG2 negatively regulate MAPKKK18‐mediated drought stress tolerance in Arabidopsis.
Stem cell therapies for intervertebral disc degeneration have been demonstrated as a promising strategy. Previous studies have shown that human nucleus pulposus cell- (NPC-) derived exosomes can induce the differentiation of mesenchymal stem cells (MSCs) into NP-like cells in vitro. However, the mechanism of MSC differentiation into NP-like cells with the induction of NPC exosomes is still unclear. Here, we verified the induction effects of NPC exosomes on the differentiation of MSCs into NP-like cells. In addition, the Notch1 pathway was downregulated in this process. Then, DAPT and soluble Jagged1 (SJAG) were applied to inhibit or enhance the expression of the Notch1 pathway, respectively, resulting in the upregulation or downregulation of collagen II, aggrecan, and Sox9 in MSCs. Knocking down of Notch1 protein facilitated the effects of NPC exosomes on the differentiation of MSCs into NP-like cells. NPC exosomes were more effective than an indirect coculture system in terms of the differentiation of MSCs into NP-like cells. Inhibition of NPC exosome secretion with Rab27a siRNA prevented the induction effects of an indirect coculture system on the differentiation of MSCs into NP-like cells. Transwell migration assays revealed that NPC exosomes could promote the migration of MSCs. Taken together, the Notch1 pathway was negatively associated with the differentiation of MSCs into NP-like cells with the treatments of NPC exosomes. Inhibition of the Notch1 pathway facilitates NPC exosome-induced differentiation of MSCs into NP-like cells in vitro. NPC exosomes play a key role in the differentiation of MSCs into NP-like cells in an indirect coculture system of NPCs and MSCs.
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