Shi-Cong Tao

Exosomes derived from miR-140-5p-overexpressing human synovial mesenchymal stem cells enhance cartilage tissue regeneration and prevent osteoarthritis of the knee in a rat model.

OBJECTIVES: Osteoarthritis (OA) is the most common joint disease throughout the world. Exosomes derived from miR-140-5p-overexpressing synovial mesenchymal stem cells (SMSC-140s) may be effective in treating OA. We hypothesized that exosomes derived from SMSC-140 (SMSC-140-Exos) would enhance the proliferation and migration abilities of articular chondrocytes (ACs) without harming extracellular matrix (ECM) secretion. METHODS: SMSCs were transfected with or without miR-140-5p. Exosomes derived from SMSCs or SMSC-140s (SMSC-Exos or SMSC-140-Exos) were isolated and identified. Proliferation, migration and ECM secretion were measured in vitro and compared between groups. The mechanism involving alternative Wnt signalling and activation of Yes-associated protein (YAP) was investigated using lentivirus, oligonucleotides or chemical drugs. The preventative effect of exosomes in vivo was measured using Safranin-O and Fast green staining and immunohistochemical staining. RESULTS: Wnt5a and Wnt5b carried by exosomes activated YAP via the alternative Wnt signalling pathway and enhanced proliferation and migration of chondrocytes with the side-effect of significantly decreasing ECM secretion. Highly-expressed miR-140-5p blocked this side-effect via RalA. SMSC-140-Exos enhanced the proliferation and migration of ACs without damaging ECM secretion in vitro, while in vivo, SMSC-140-Exos successfully prevented OA in a rat model. CONCLUSIONS: These findings highlight the promising potential of SMSC-140-Exos in preventing OA. We first found a potential source of exosomes and studied their merits and shortcomings. Based on our understanding of the molecular mechanism, we overcame the shortcomings by modifying the exosomes. Such exosomes derived from modified cells hold potential as future therapeutic strategies.

Advantages of pure platelet-rich plasma compared with leukocyte- and platelet-rich plasma in promoting repair of bone defects

BackgroundHigh levels of pro-inflammatory cytokines in leukocyte- and platelet-rich plasma (L-PRP) may activate the nuclear factor κB (NF-κB) pathway to counter the beneficial effect of the growth factors on bone regeneration. However, to date, no relevant studies have substantiated this.MethodsL-PRP and pure platelet-rich plasma (P-PRP) were isolated. The in vitro effects of L-PRP and P-PRP on the proliferation, viability and migration of human bone marrow-derived mesenchymal stem cells (HBMSCs) and EaHy926, tube formation of EaHy926, and osteogenic differentiation of HBMSCs were assessed by cell counting, flow cytometry, scratch assay, tube formation assay, and real-time quantitative polymerase chain reaction (RT-PCR), western blotting and Alizarin red staining, respectively. The in vitro effects of L-PRP and P-PRP on the nuclear translocation of NF-κB p65, mRNA expression of inducible nitric oxide synthase and cyclooxygenase-2, and production of prostaglandin E2 and nitric oxid were assessed by western blotting, RT-PCR, enzyme-linked immunosorbent assay and Griess reaction, respectively. The in vivo effects of L-PRP or P-PRP preprocessed β-tricalcium phosphate (β-TCP) on the calvarial defects in rats were assessed by histological and immunofluorescence examinations.ResultsP-PRP, which had similar platelet and growth factors concentrations but significantly lower concentrations of leukocytes and pro-inflammatory cytokines compared with L-PRP, promoted the proliferation, viability and migration of HBMSCs and EaHy926, tube formation of EaHy926 and osteogenic differentiation of HBMSCs in vitro, compared with L-PRP. The implantation of P-PRP preprocessed β-TCP also yielded better histological results than the implantation of L-PRP preprocessed β-TCP in vivo. Moreover, L-PRP treatment resulted in the activation of the NF-κB pathway in HBMSCs and EaHy926 in vitro while the postoperative delivery of caffeic acid phenethyl ester, an inhibitor of NF-κB activation, enhanced the histological results of the implantation of L-PRP preprocessed β-TCP in vivo.ConclusionsLeukocytes in L-PRP may activate the NF-κB pathway via the increased pro-inflammatory cytokines to induce the inferior effects on bone regeneration of L-PRP compared with P-PRP. Hence, P-PRP may be more suitable for bone regeneration compared with L-PRP, and the combined use of P-PRP and β-TCP represents a safe, simple, and effective alternative option for autogenous bone graft in the treatment of bone defects.

Exosomes derived from platelet-rich plasma promote the re-epithelization of chronic cutaneous wounds via activation of YAP in a diabetic rat model.

Chronic wounds have become an economic, social, and public health burden and need advanced treatment. Platelet-rich plasma (PRP) has been used extensively in treatment of chronic wounds because it contains an abundance of growth factors secreted by platelets. The exosomes derived from PRP (PRP-Exos) have been proven to encapsulate principal growth factors from platelets. This study is the first to show that these exosomes may exert the function of PRP. PRP-Exos can effectively induce proliferation and migration of endothelial cells and fibroblasts to improve angiogenesis and re-epithelialization in chronic wounds. We regulated YAP to verify the PRP-Exos-dependent effect on fibroblast proliferation and migration through YAP activation. In vivo, we observed the cutaneous healing process in chronic wounds treated with PRP-Exos in a diabetic rat model. We provide evidence of the probable molecular mechanisms underlying the PRP effect on healing of chronic ulcers and describe a promising resource of growth factors from exosomes without species restriction.

Chitosan Wound Dressings Incorporating Exosomes Derived from MicroRNA‐126‐Overexpressing Synovium Mesenchymal Stem Cells Provide Sustained Release of Exosomes and Heal Full‐Thickness Skin Defects in a Diabetic Rat Model

There is a need to find better strategies to promote wound healing, especially of chronic wounds, which remain a challenge. We found that synovium mesenchymal stem cells (SMSCs) have the ability to strongly promote cell proliferation of fibroblasts; however, they are ineffective at promoting angiogenesis. Using gene overexpression technology, we overexpressed microRNA‐126‐3p (miR‐126‐3p) and transferred the angiogenic ability of endothelial progenitor cells to SMSCs, promoting angiogenesis. We tested a therapeutic strategy involving controlled‐release exosomes derived from miR‐126‐3p‐overexpressing SMSCs combined with chitosan. Our in vitro results showed that exosomes derived from miR‐126‐3p‐overexpressing SMSCs (SMSC‐126‐Exos) stimulated the proliferation of human dermal fibroblasts and human dermal microvascular endothelial cells (HMEC‐1) in a dose‐dependent manner. Furthermore, SMSC‐126‐Exos also promoted migration and tube formation of HMEC‐1. Testing this system in a diabetic rat model, we found that this approach resulted in accelerated re‐epithelialization, activated angiogenesis, and promotion of collagen maturity in vivo. These data provide the first evidence of the potential of SMSC‐126‐Exos in treating cutaneous wounds and indicate that modifying the cells—for example, by gene overexpression—and using the exosomes derived from these modified cells provides a potential drug delivery system and could have infinite possibilities for future therapy. Stem Cells Translational Medicine 2017;6:736–747

Exosomes derived from human platelet-rich plasma prevent apoptosis induced by glucocorticoid-associated endoplasmic reticulum stress in rat osteonecrosis of the femoral head via the Akt/Bad/Bcl-2 signal pathway

An excess of glucocorticoids (GCs) is reported to be one of the most common causes of osteonecrosis of the femoral head (ONFH). In addition, GCs can induce bone cell apoptosis through modulating endoplasmic reticulum (ER) stress. Among the three main signal pathways in ER stress, the PERK (protein kinase RNA-like ER kinase)/CHOP (CCAAT-enhancer-binding protein homologous protein) pathway has been considered to be closely associated with apoptosis. Platelet-rich plasma (PRP) has been referred to as a concentration of growth factors and the exosomes derived from PRP (PRP-Exos) have a similar effect to their parent material. The enriched growth factors can be encapsulated into PRP-Exos and activate Akt and Erk pathways to promote angiogenesis. Activation of the Akt pathway may promote the expression of anti-apoptotic proteins like Bcl-2, while CHOP can inhibit B-cell lymphoma 2 (Bcl-2) expression to increase the level of cleaved caspase-3 and lead to cell death. Consequently, we hypothesized that PRP-Exos prevent apoptosis induced by glucocorticoid-associated ER stress in rat ONFH via the Akt/Bad/Bcl-2 signal pathway. To verify this hypothesis, a dexamethasone (DEX)-treated in vitro cell model and methylprednisolone (MPS)-treated in vivo rat model were adopted. Characterization of PRP-Exos, and effects of PRP-Exos on proliferation, apoptosis, angiogenesis, and osteogenesis of cells treated with GCs in vitro and in vivo were examined. Furthermore, the mechanism by which PRP-Exos rescue the GC-induced apoptosis through the Akt/Bad/Bcl-2 pathway was also investigated. The results indicate that PRP-Exos have the capability to prevent GC-induced apoptosis in a rat model of ONFH by promoting Bcl-2 expression via the Akt/Bad/Bcl-2 signal pathway under ER stress.

Exosomes from Human Synovial-Derived Mesenchymal Stem Cells Prevent Glucocorticoid-Induced Osteonecrosis of the Femoral Head in the Rat.

Osteonecrosis of the femoral head (ONFH) represents a debilitating complication following glucocorticoid (GC)-based therapy. Synovial-derived mesenchymal stem cells (SMSCs) can exert protective effect in the animal model of GC-induced ONFH by inducing cell proliferation and preventing cell apoptosis. Recent studies indicate the transplanted cells exert therapeutic effects primarily via a paracrine mechanism and exosomes are an important paracrine factor that can be directly used as therapeutic agents for tissue engineering. Herein, we provided the first demonstration that the early treatment of exosomes secreted by human synovial-derived mesenchymal stem cells (SMSC-Exos) could prevent GC-induced ONFH in the rat model. Using a series of in vitro functional assays, we found that SMSC-Exos could be internalized into bone marrow derived stromal cells (BMSCs) and enhance their proliferation and have anti-apoptotic abilities. Finally, SMSC-Exos may be promising for preventing GC-induced ONFH.

Fabrication of hydroxyapatite/chitosan composite hydrogels loaded with exosomes derived from miR-126-3p overexpressed synovial mesenchymal stem cells for diabetic chronic wound healing

The exploration of an effective diabetic chronic wound healing process still remains a great challenge. Herein, we used gene overexpression technology to obtain synovial mesenchymal stem cells (SMSCs) and the miR-126-3p highly expressed SMSCs (SMSCs-126). The exosomes derived from miR-126-3p overexpressed SMSCs (SMSCs-126-Exos) with a particle size of 85 nm were encapsulated in hydroxyapatite/chitosan (HAP-CS) composite hydrogels (HAP-CS-SMSCs-126-Exos) as wound dressings. The SMSCs-126-Exos, CS and low-crystallinity HAP nanorods with a length of 200 nm and a diameter of 50 nm are uniformly dispersed within the whole composite hydrogel. The HAP-CS-SMSCs-126-Exos possess the controlled release property of SMSCs-126-Exos for at least 6 days. The released SMSCs-126-Exos nanoparticles stimulate the proliferation and migration of human dermal fibroblasts and human dermal microvascular endothelial cells (HMEC-1). At the same time, the migration and capillary-network formation of HMEC-1 are promoted through the activation of MAPK/ERK and PI3K/AKT. In vivo tests demonstrate that the HAP-CS-SMSCs-126-Exos successfully promote wound surface re-epithelialization, accelerate angiogenesis, and expedite collagen maturity due to the presence of HAP, CS and SMSCs-126-Exos. Therefore, the HAP-CS-SMSCs-126-Exos possess great potential application for diabetic chronic wound healing, and especially provide the possibility of using exosomes derived from modified cells as a new approach to bring wonderful functionality and controllability in future chronic wound therapy.

Proton-sensing GPCR-YAP Signalling Promotes Cancer-associated Fibroblast Activation of Mesenchymal Stem Cells

The pHs of extracellular fluids (ECFs) in normal tissues are commonly maintained at 7.35 to 7.45. The acidification of the ECF is one of the major characteristics of tumour microenvironment. In this study, we report that decreased extracellular pH promotes the transformation of mesenchymal stem cells (MSCs) into cancer-associated fibroblasts (CAFs), termed CAF activation. Furthermore, we demonstrate that GPR68, a proton-sensing G-protein-coupled receptor (GPCR), is required for the pH-dependent regulation of the differentiation of MSCs into CAFs. We then identify Yes-associated protein 1 (YAP) as a downstream effector of GPR68 for CAF activation. Finally, we show that knockdown of GPR68 in MSCs can prevent the CAF activation under cancer microenvironment. Systemic transplantation of GPR68-silenced MSCs suppresses in-situ tumour growth and prolong life span after cancer graft.

Proton-sensing GPCR-YAP Signalling Promotes Cell Proliferation and Survival.

The pHs of extracellular fluids (ECFs) in humans are consistently maintained at 7.35 to 7.45 in physiological conditions. Pathological changes, including infarction, tumourigenesis and inflammation, commonly result in decreases in the ECF pH in the affected tissues. In this study, we report that proliferation is promoted and apoptosis is inhibited by decreases in extracellular pH. Furthermore, we demonstrated that proton-sensing G-protein-coupled receptors (GPCRs) are required for the pH-dependent regulation of proliferation and apoptosis through the G protein α subunit 12/13 (G12/13) and Rho GTPases. Next, we identified Yes-associated protein 1 (YAP) as a downstream effector of Rho signalling. Together, the results from our study demonstrate that extracellular pH can modulate cell proliferation and apoptosis by regulating the proton-sensing GPCR-YAP pathway.

Modularized Extracellular Vesicles: The Dawn of Prospective Personalized and Precision Medicine

Abstract Extracellular vesicles (EVs) are ubiquitous nanosized membrane vesicles consisting of a lipid bilayer enclosing proteins and nucleic acids, which are active in intercellular communications. EVs are increasingly seen as a vital component of many biological functions that were once considered to require the direct participation of stem cells. Consequently, transplantation of EVs is gradually becoming considered an alternative to stem cell transplantation due to their significant advantages, including their relatively low probability of neoplastic transformation and abnormal differentiation. However, as research has progressed, it is realized that EVs derived from native‐source cells may have various shortcomings, which can be corrected by modification and optimization. To date, attempts are made to modify or improve almost all the components of EVs, including the lipid bilayer, proteins, and nucleic acids, launching a new era of modularized EV therapy through the “modular design” of EV components. One high‐yield technique, generating EV mimetic nanovesicles, will help to make industrial production of modularized EVs a reality. These modularized EVs have highly customized “modular design” components related to biological function and targeted delivery and are proposed as a promising approach to achieve personalized and precision medicine.