ASC-expressing pyroptotic extracellular vesicles alleviate sepsis by protecting B cells.

Pyroptosis is an inflammatory programmed cell death process characterized by membrane rupture. Interestingly, pyroptotic cells can generate plenty of nanosized vesicles. Non-inflammatory apoptotic cell death-derived apoptotic vesicles (apoVs) were systemically characterized and displayed multiple physiological functions and therapeutic potentials. However, the characteristics of pyroptotic cell-generated extracellular vesicles (EVs) are largely unknown. Here, we identified a group of pyroptotic EVs (pyroEVs) from in vitro cultured pyroptotic mesenchymal stem cells (MSCs), as well as from septic mouse blood. Compared with apoVs, pyroEVs express similar levels of annexin V, calreticulin, and common EV markers, but express a decreased level of apoptotic marker cleave caspase-3. PyroEVs, but not apoVs and exosomes, specifically express pyroptotic maker apoptosis-associated speck-like protein containing CARD (ASC). More importantly, MSC-derived pyroEVs protect B cells in the spleen and bone marrow to relieve inflammatory responses and enhance the survival rate of the septic mice. Mechanistically, pyroEV membrane-expressed ASC binds to B cells to repress cell death by repressing Toll-like receptor 4. This study uncovered the characteristics of pyroEVs and their therapeutic role in sepsis and B cell-mediated immune response.

Encapsulation of Nano-Bortezomib in Apoptotic Stem Cell-Derived Vesicles for the Treatment of Multiple Myeloma.

Extracellular vesicles (EVs) are lipid bilayer nanovesicles released from living or apoptotic cells that can transport DNA, RNA, protein, and lipid cargo. EVs play critical roles in cell-cell communication and tissue homeostasis, and have numerous therapeutic uses including serving as carriers for nanodrug delivery. There are multiple ways to load EVs with nanodrugs, such as electroporation, extrusion, and ultrasound. However, these approaches may have limited drug-loading rates, poor EV membrane stability, and high cost for large-scale production. Here, it is shown that apoptotic mesenchymal stem cells (MSCs) can encapsulate exogenously added nanoparticles into apoptotic vesicles (apoVs) with a high loading efficiency. When nano-bortezomib is incorporated into apoVs in culture-expanded apoptotic MSCs, nano-bortezomib-apoVs show a synergistic combination effect of bortezomib and apoVs to ameliorate multiple myeloma (MM) in a mouse model, along with significantly reduced side effects of nano-bortezomib. Moreover, it is shown that Rab7 regulates the nanoparticle encapsulation efficiency in apoptotic MSCs and that activation of Rab7 can increase nanoparticle-apoV production. In this study, a previously unknown mechanism to naturally synthesize nano-bortezomib-apoVs to improve MM therapy is revealed.

ARHGEF19 interacts with BRAF to activate MAPK signaling during the tumorigenesis of non-small cell lung cancer.

Rho guanine nucleotide exchange factors (RhoGEFs) are proteins that activate Rho GTPases in response to extracellular stimuli and regulate various biologic processes. ARHGEF19, one of RhoGEFs, was reported to activate RhoA in the Wnt-PCP pathway controlling convergent extension in Xenopus gastrulation. The goal of our study was to identify the role and molecular mechanisms of ARHGEF19 in the tumorigenesis of non-small cell lung cancer (NSCLC). ARHGEF19 expression was significantly elevated in NSCLC tissues, and ARHGEF19 levels were significantly associated with lymph node status, distant metastasis and TNM stage; Patients with high ARHGEF19 levels had poor overall survival (OS) and progression-free survival (PFS). Our investigations revealed that ARHGEF19 overexpression promoted the cell proliferation, invasion and metastasis of lung cancer cells, whereas knockdown of this gene inhibited these processes. Mechanistically, ARHGEF19 activated the mitogen-activated protein kinase (MAPK) pathway in a RhoA-independent manner: ARHGEF19 interacted with BRAF and facilitated the phosphorylation of its downstream kinase MEK1/2; both the Dbl homology (DH) and Pleckstrin homology (PH) domains of ARHGEF19 were indispensable for the phosphorylation of MEK1/2. Furthermore, downregulation of miR-29b was likely responsible for the increased expression of ARHGEF19 in lung cancer tissues and, consequently, the abnormal activation of MAPK signaling. These findings suggest that ARHGEF19 upregulation, due to the low expression of miR-29 in NSCLC tissues, may play a crucial role in NSCLC tumorigenesis by activating MAPK signaling. ARHGEF19 could serve as a negative prognostic marker as well as a therapeutic target for NSCLC patients.

Olfactomedin 1 negatively regulates NF-κB signalling and suppresses the growth and metastasis of colorectal cancer cells.

Uncontrolled growth and distant metastasis are hallmarks of colorectal cancer (CRC), but the mechanisms are poorly understood. Olfactomedin 1 (OLFM1), a member of the olfactomedin domain-containing protein family, plays an important role in the development of neurogenic tissues. Recently, OLFM1 deregulation was frequently observed in several cancers, and it was induced in colon cell lines after treatment with the demethylating agent 5-aza-2'-deoxycytidine. However, the function of OLFM1 in CRC remains unknown. In this study, we reanalysed published microarray data and found that OLFM1 was significantly down-regulated in primary CRC samples compared to adjacent non-cancerous tissues. The results of immunohistochemistry indicated that decreased OLFM1 expression was significantly associated with lymph node status (p = 0.023), distant metastasis (p < 0.001), and AJCC/TNM stage (p = 0.013), and CRC patients with low OLFM1 expression had consistently poor overall survival (OS; p < 0.001) and progression-free survival (PFS; p < 0.001). Further analysis demonstrated that OLFM1 was epigenetically silenced in CRC tissues and cell lines via promoter hypermethylation. Overexpression and knockdown of OLFM1 attenuated and increased, respectively, CRC cells' proliferation, migration, and invasion in vitro and metastasis to the lung and liver in vivo. Mechanistically, the promotion of growth and metastasis of CRC cells by silencing of OLFM1 was associated with the activation of the non-canonical NF-κB signalling pathway. OLFM1 interacted with NF-κB-inducing kinase (NIK; MAP3K14) and repressed the phosphorylation of its downstream substrate Ikappa B kinase alpha (IKKα). OLFM1 expression was negatively correlated with the phosphorylation level of IKKα in CRC tissue samples. Knockdown of NIK impaired the ability of OLFM1 to repress NF-κB signalling, cell growth or migration. Thus, OLFM1 may be a valuable biomarker and therapeutic target for CRC patients. Copyright © 2016 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Apoptotic vesicles rescue impaired mesenchymal stem cells and their therapeutic capacity for osteoporosis by restoring miR-145a-5p deficiency.

Apoptotic vesicles (apoVs) play a vital role in various pathological conditions; however, we have yet to fully understand their precise biological effects in rescuing impaired mesenchymal stem cells (MSCs) and regulating tissue homeostasis. Here, we proved that systemic infusion of bone marrow MSCs derived from wild-type (WT) mice effectively improved the osteopenia phenotype and hyperimmune state in ovariectomized (OVX) mice. Importantly, the WT MSCs rescued the impairment of OVX MSCs both in vivo and in vitro, whereas OVX MSCs did not show the same efficacy. Interestingly, treatment with apoVs derived from WT MSCs (WT apoVs) restored the impaired biological function of OVX MSCs and their ability to improve osteoporosis. This effect was not observed with OVX MSCs-derived apoVs (OVX apoVs) treatment. Mechanistically, the reduced miR-145a-5p expression hindered the osteogenic differentiation and immunomodulatory capacity of OVX MSCs by affecting the TGF-ß/Smad 2/3-Wnt/ß-catenin signaling axis, resulting in the development of osteoporosis. WT apoVs directly transferred miR-145a-5p to OVX MSCs, which were then reused to restore their impaired biological functions. Conversely, treatment with OVX apoVs did not produce significant effects due to their limited expression of miR-145a-5p. Overall, our findings unveil the remarkable potential of apoVs in rescuing the biological function and therapeutic capability of MSCs derived from individuals with diseases. This discovery offers a new avenue for exploring apoVs-based MSC engineering and expands the application scope of stem cell therapy, contributing to the maintenance of bone homeostasis through a previously unrecognized mechanism.

Mesenchymal stem cell-derived apoptotic vesicles ameliorate impaired ovarian folliculogenesis in polycystic ovary syndrome and ovarian aging by targeting WNT signaling.

Rationale: It has been emergingly recognized that apoptosis generates plenty of heterogeneous apoptotic vesicles (apoVs), which play a pivotal role in the maintenance of organ and tissue homeostasis. However, it is unknown whether apoVs influence postnatal ovarian folliculogenesis. Methods: Apoptotic pathway deficient mice including Fas mutant (Fasmut ) and Fas ligand mutant (FasLmut ) mice were used with apoV replenishment to evaluate the biological function of apoVs during ovarian folliculogenesis. Ovarian function was characterized by morphological analysis, biochemical examination and cellular assays. Mechanistical studies were assessed by combinations of transcriptomic and proteomic analysis as well as molecular assays. CYP17A1-Cre; Axin1fl /fl mice was established to verify the role of WNT signaling during ovarian folliculogenesis. Polycystic ovarian syndrome (PCOS) mice and 15-month-old mice were used with apoV replenishment to further validate the therapeutic effects of apoVs based on WNT signaling regulation. Results: We show that systemic administration of mesenchymal stem cell (MSC)-derived apoptotic vesicles (MSC-apoVs) can ameliorate impaired ovarian folliculogenesis, PCOS phenotype, and reduced birth rate in Fasmut and FasLmut mice. Mechanistically, transcriptome analysis results revealed that MSC-apoVs downregulated a number of aberrant gene expression in Fasmut mice, which were enriched by kyoto encyclopedia of genes and genomes (KEGG) pathway analysis in WNT signaling and sex hormone biosynthesis. Furthermore, we found that apoptotic deficiency resulted in aberrant WNT/ß-catenin activation in theca and mural granulosa cells, leading to responsive action of dickkopf1 (DKK1) in the cumulus cell and oocyte zone, which downregulated WNT/ß-catenin expression in oocytes and, therefore, impaired ovarian folliculogenesis via NPPC/cGMP/PDE3A/cAMP cascade. When WNT/ß-catenin was specially activated in theca cells of CYP17A1-Cre; Axin1fl /fl mice, the same ovarian impairment phenotypes observed in apoptosis-deficient mice were established, confirming that aberrant activation of WNT/ß-catenin in theca cells caused the impairment of ovarian folliculogenesis. We firstly revealed that apoVs delivered WNT membrane receptor inhibitor protein RNF43 to ovarian theca cells to balance follicle homeostasis through vesicle-cell membrane integration. Systemically infused RNF43-apoVs down-regulated aberrantly activated WNT/ß-catenin signaling in theca cells, contributing to ovarian functional maintenance. Since aging mice have down-regulated expression of WNT/ß-catenin in oocytes, we used MSC-apoVs to treat 15-month-old mice and found that MSC-apoVs effectively ameliorated the ovarian function and fertility capacity of these aging mice through rescuing WNT/ß-catenin expression in oocytes. Conclusion: Our studies reveal a previously unknown association between apoVs and ovarian folliculogenesis and suggest an apoV-based therapeutic approach to improve oocyte function and birth rates in PCOS and aging.

Apoptotic vesicles are required to repair DNA damage and suppress premature cellular senescence.

It is well known that DNA damage can cause apoptosis. However, whether apoptosis and its metabolites contribute to DNA repair is largely unknown. In this study, we found that apoptosis-deficient Fasmut and Bim- /- mice show significantly elevated DNA damage and premature cellular senescence, along with a significantly reduced number of 16,000 g apoptotic vesicles (apoVs). Intravenous infusion of mesenchymal stromal cell (MSC)-derived 16,000 g apoVs rescued the DNA damage and premature senescence in Fasmut and Bim-/- mice. Moreover, a sublethal dose of radiation exposure caused more severe DNA damage, reduced survival rate, and loss of body weight in Fasmut mice than in wild-type mice, which can be recovered by the infusion of MSC-apoVs. Mechanistically, we showed that apoptosis can assemble multiple nuclear DNA repair enzymes, such as the full-length PARP1, into 16,000 g apoVs. These DNA repair components are directly transferred by 16,000 g apoVs to recipient cells, leading to the rescue of DNA damage and elimination of senescent cells. Finally, we showed that embryonic stem cell-derived 16,000 g apoVs have superior DNA repair capacity due to containing a high level of nuclear DNA repair enzymes to rescue lethal dose-irradiated mice. This study uncovers a previously unknown role of 16,000 g apoVs in safeguarding tissues from DNA damage and demonstrates a strategy for using stem cell-derived apoVs to ameliorate irradiation-induced DNA damage.

Apoptosis releases hydrogen sulfide to inhibit Th17 cell differentiation.

Over 50 billion cells undergo apoptosis each day in an adult human to maintain immune homeostasis. Hydrogen sulfide (H2S) is also required to safeguard the function of immune response. However, it is unknown whether apoptosis regulates H2S production. Here, we show that apoptosis-deficient MRL/lpr (B6.MRL-Faslpr/J) and Bim-/- (B6.129S1-Bcl2l11tm1.1Ast/J) mice exhibit significantly reduced H2S levels along with aberrant differentiation of Th17 cells, which can be rescued by the additional H2S. Moreover, apoptotic cells and vesicles (apoVs) express key H2S-generating enzymes and generate a significant amount of H2S, indicating that apoptotic metabolism is an important source of H2S. Mechanistically, H2S sulfhydrates selenoprotein F (Sep15) to promote signal transducer and activator of transcription 1 (STAT1) phosphorylation and suppress STAT3 phosphorylation, leading to the inhibition of Th17 cell differentiation. Taken together, this study reveals a previously unknown role of apoptosis in maintaining H2S homeostasis and the unique role of H2S in regulating Th17 cell differentiation via sulfhydration of Sep15C38.

Extracellular Vesicles for Dental Pulp and Periodontal Regeneration.

Extracellular vesicles (EVs) are lipid bound particles derived from their original cells, which play critical roles in intercellular communication through their cargoes, including protein, lipids, and nucleic acids. According to their biogenesis and release pathway, EVs can be divided into three categories: apoptotic vesicles (ApoVs), microvesicles (MVs), and small EVs (sEVs). Recently, the role of EVs in oral disease has received close attention. In this review, the main characteristics of EVs are described, including their classification, biogenesis, biomarkers, and components. Moreover, the therapeutic mechanism of EVs in tissue regeneration is discussed. We further summarize the current status of EVs in pulp/periodontal tissue regeneration and discuss the potential mechanisms. The therapeutic potential of EVs in pulp and periodontal regeneration might involve the promotion of tissue regeneration and immunomodulatory capabilities. Furthermore, we highlight the current challenges in the translational use of EVs. This review would provide valuable insights into the potential therapeutic strategies of EVs in dental pulp and periodontal regeneration.

Apoptotic vesicles resist oxidative damage in noise-induced hearing loss through activation of FOXO3a-SOD2 pathway.

BACKGROUND: Mesenchymal stem cell (MSC) transplantation is a promising therapeutic approach for noise-induced hearing loss (NIHL). As the indispensable role of apoptosis in MSC transplantation was raised, the benefits of MSC-derived apoptotic vesicles (apoVs) in several disease models have been proved. However, whether apoVs benefit in NIHL have not been studied yet. METHODS: Female CBA/J mice and HEI-OC1 cells were used in this study. Flow cytometry, nanoparticle tracking analysis (NTA) and transmission electron microscopy (TEM) were used to characterize apoVs. Proteomic analysis was used to identify function proteins in apoVs. Immunofluorescence was used to reveal distribution pattern. Auditory brainstem response (ABR) test was used to measure the effect of apoVs treatment. DCFH-DA staining and MitoSOX staining were used to indicate oxidative damage. Western-blot and qRT-PCR were used to study the signaling pathways. RESULTS: We found that apoVs can be endocytosed by hair cells through systemic administration. Importantly, apoVs administration effectively attenuated NIHL and reduced hair cell loss by resisting oxidative damage in vivo. Further, apoVs application activated forkhead box o3 (FOXO3a)-mitochondrial superoxide dismutase 2(SOD2) pathway, which may relate to signal transduction and activators of transcription 3 (STAT3) in apoVs. CONCLUSIONS: These findings uncovered the role of apoVs in preventing NIHL and resisting oxidative damage, indicating that apoVs is a promising way for inner ear delivery and a prospective cell-free therapy for NIHL.

Inducing the "re-development state" of periodontal ligament cells via tuning macrophage mediated immune microenvironment.

INTRODUCTION: Periodontal regeneration, specifically the restoration of the cementum-periodontal ligament (PDL)-alveolar bone complex, remains a formidable challenge in the field of regenerative dentistry. In light of periodontal development, harnessing the multi-tissue developmental capabilities of periodontal ligament cells (PDLCs) and reinitiating the periodontal developmental process hold great promise as an effective strategy to foster the regeneration of the periodontal complex. OBJECTIVES: This study aims to delve into the potential effects of the macrophage-mediated immune microenvironment on the "developmental engineering" regeneration strategy and its underlying molecular mechanisms. METHODS: In this study, we conducted a comprehensive examination of the periodontium developmental process in the rat mandibular first molar using histological staining. Through the induction of diverse immune microenvironments in macrophages, we evaluated their potential effects on periodontal re-development events using a cytokine array. Additionally, we investigated PDLC-mediated periodontal re-development events under these distinct immune microenvironments through transcriptome sequencing and relevant functional assays. Furthermore, the underlying molecular mechanism was also performed. RESULTS: The activation of development-related functions in PDLCs proved challenging due to their declined activity. However, our findings suggest that modulating the macrophage immune response can effectively regulate PDLCs-mediated periodontium development-related events. The M1 type macrophage immune microenvironment was found to promote PDLC activities associated with epithelial-mesenchymal transition, fiber degradation, osteoclastogenesis, and inflammation through the Wnt, IL-17, and TNF signaling pathways. Conversely, the M2 type macrophage immune microenvironment demonstrated superiority in inducing epithelium induction, fibers formation, and mineralization performance of PDLCs by upregulating the TGFß and PI3K-Akt signaling pathway. CONCLUSION: The results of this study could provide some favorable theoretical bases for applying periodontal development engineering strategy in resolving the difficulties in periodontal multi-tissue regeneration.

Amelioration of Lung Fibrosis by Total Flavonoids of Astragalus via Inflammatory Modulation and Epithelium Regeneration.

Idiopathic Pulmonary Fibrosis (IPF) is identifiable by the excessive increase of mesenchyme paired with the loss of epithelium. Total flavonoids of Astragalus (TFA), the main biologically active ingredient of the traditional Chinese medicine, Astragalus membranaceus (Huangqi), shows outstanding effects on treating pulmonary disorders, including COVID-19-associated pulmonary dysfunctions. This study was designed to evaluate the efficacy of TFA on treating pulmonary fibrosis and the possible mechanisms behind these effects. A549 cells were treated with TGF-[Formula: see text]1 and TFA to observe the potential effects of TFA on regulating alveolar epithelial cell proliferation, TGF-[Formula: see text]1-induced EMT, and the underlying mechanisms in vitro. Then, mouse pulmonary fibrosis was induced with a single intra-tracheal injection of bleomycin, and TFA was administrated by i.p. injection. Lung fibrosis was evaluated through histological and molecular analyses, and the possible mechanisms were explored using immunological methods. The results demonstrated that TFA could promote cell proliferation but inhibit TGF-[Formula: see text]1-induced EMT on A549 cells. TFA attenuated BLM-induced pulmonary fibrosis in mice by modulating inflammatory infiltration and M2 macrophage polarization; it furthermore modulated EMT through regulating the TGF-[Formula: see text]1/Smad pathway. In addition, TFA augmented the expression of the Wnt7b protein, which plays an important role in alveolar epithelium reparation. In conclusion, TFA alleviated bleomycin-induced mouse lung fibrosis by preventing the fibrotic response and increasing epithelium regeneration.

Apoptotic extracellular vesicles are metabolized regulators nurturing the skin and hair.

Over 300 billion of cells die every day in the human body, producing a large number of endogenous apoptotic extracellular vesicles (apoEVs). Also, allogenic stem cell transplantation, a commonly used therapeutic approach in current clinical practice, generates exogenous apoEVs. It is well known that phagocytic cells engulf and digest apoEVs to maintain the body's homeostasis. In this study, we show that a fraction of exogenous apoEVs is metabolized in the integumentary skin and hair follicles. Mechanistically, apoEVs activate the Wnt/ß-catenin pathway to facilitate their metabolism in a wave-like pattern. The migration of apoEVs is enhanced by treadmill exercise and inhibited by tail suspension, which is associated with the mechanical force-regulated expression of DKK1 in circulation. Furthermore, we show that exogenous apoEVs promote wound healing and hair growth via activation of Wnt/ß-catenin pathway in skin and hair follicle mesenchymal stem cells. This study reveals a previously unrecognized metabolic pathway of apoEVs and opens a new avenue for exploring apoEV-based therapy for skin and hair disorders.

An Appearance Data-Driven Model Visualizes Cell State and Predicts Mesenchymal Stem Cell Regenerative Capacity.

Mesenchymal stem cells (MSCs) are widely used in treating various diseases. However, lack of a reliable evaluation approach to characterize the potency of MSCs has dampened their clinical applications. Here, a function-oriented mathematical model is established to evaluate and predict the regenerative capacity (RC) of MSCs. Processed by exhaustive testing, the model excavates four optimal fitted indices, including nucleus roundness, nucleus/cytoplasm ratio, side-scatter height, and ERK1/2 from the given index combinations. Notably, three of them except ERK1/2 are cell appearance-associated features. The predictive power of the model is validated via screening experiments of these indices by predicting the RC of newly enrolled and chemical inhibitor-treated MSCs. Further RNA-sequencing analysis reveals that cell appearance-based indices may serve as major indicators to visualize the results of integration-weighted signals in and out of cells and reflect MSC stemness. In general, this study proposes an appearance data-driven predictive model for the RC and stemness of MSCs.

Exocrine pancreas regeneration modifies original pancreas to alleviate diabetes in mouse models.

Diabetes is a major public health issue because of its widely epidemic nature and lack of cure. Here, we show that pancreas-derived mesenchymal stem cells (PMSCs) are capable of regenerating exocrine pancreas when implanted into the kidney capsule of mice with streptozotocin (STZ)-induced diabetes. Mechanistically, we found that the regenerated exocrine pancreas elevated interleukin-6 (IL-6) in PMSC implants, which transiently activated tumor necrosis factor-α (TNF-α) and interferon-γ (IFN-γ) to inhibit IL-17, thereby rescuing damaged exocrine pancreas and islet ß cells. In addition, we used knockout mouse models to show that global lack of IL-6, TNF-α, or IFN-γ resulted in increased severity of STZ-induced diabetes and resistance to PMSC implantation therapy, confirming the roles of these factors in safeguarding pancreatic ß cells. Furthermore, removal of the kidney capsule PMSC implants at 28 days after implantation did not affect the PMSC-initiated therapeutic effect on diabetic mice. This study reveals a previously unknown role of exocrine pancreas regeneration in safeguarding ß cells and demonstrates a "soil-rescues-seed" strategy for type 1 diabetes therapy.

CD146 controls the quality of clinical grade mesenchymal stem cells from human dental pulp.

BACKGROUND: Human mesenchymal stem cells from dental pulp (hMSC-DP), including dental pulp stem cells from permanent teeth and exfoliated deciduous teeth, possess unique MSC characteristics such as expression of specific surface molecules and a high proliferation rate. Since hMSC-DP have been applied in numerous clinical studies, it is necessary to establish criteria to evaluate their potency for cell-based therapies. METHODS: We compared stem cell properties of hMSC-DP at passages 5, 10 and 20 under serum (SE) and serum-free (SF) culture conditions. Cell morphology, proliferation capacity, chromosomal stability, surface phenotypic profiles, differentiation and immunoregulation ability were evaluated. In addition, we assessed surface molecule that regulates hMSC-DP proliferation and immunomodulation. RESULTS: hMSC-DP exhibited a decrease in proliferation rate and differentiation potential, as well as a reduced expression of CD146 when cultured under continuous passage conditions. SF culture conditions failed to alter surface marker expression, chromosome stability or proliferation rate when compared to SE culture. SF-cultured hMSC-DP were able to differentiate into osteogenic, adipogenic and neural cells, and displayed the capacity to regulate immune responses. Notably, the expression level of CD146 showed a positive correlation with proliferation, differentiation, and immunomodulation, suggesting that CD146 can serve as a surface molecule to evaluate the potency of hMSC-DP. Mechanistically, we found that CD146 regulates proliferation and immunomodulation of hMSC-DP through the ERK/p-ERK pathway. CONCLUSION: This study indicates that SF-cultured hMSC-DP are appropriate for producing clinical-grade cells. CD146 is a functional surface molecule to assess the potency of hMSC-DP.

Dephosphorylation of Caveolin-1 Controls C-X-C Motif Chemokine Ligand 10 Secretion in Mesenchymal Stem Cells to Regulate the Process of Wound Healing.

Mesenchymal stem cells (MSCs) secrete cytokines in a paracrine or autocrine manner to regulate immune response and tissue regeneration. Our previous research revealed that MSCs use the complex of Fas/Fas-associated phosphatase-1 (Fap-1)/caveolin-1 (Cav-1) mediated exocytotic process to regulate cytokine and small extracellular vesicles (EVs) secretion, which contributes to accelerated wound healing. However, the detailed underlying mechanism of cytokine secretion controlled by Cav-1 remains to be explored. We show that Gingiva-derived MSCs (GMSCs) could secrete more C-X-C motif chemokine ligand 10 (CXCL10) but showed lower phospho-Cav-1 (p-Cav-1) expression than skin-derived MSCs (SMSCs). Moreover, dephosphorylation of Cav-1 by a Src kinase inhibitor PP2 significantly enhances CXCL10 secretion, while activating phosphorylation of Cav-1 by H2O2 restraints CXCL10 secretion in GMSCs. We also found that Fas and Fap-1 contribute to the dephosphorylation of Cav-1 to elevate CXCL10 secretion. Tumor necrosis factor-α serves as an activator to up-regulate Fas, Fap-1, and down-regulate p-Cav-1 expression to promote CXCL10 release. Furthermore, local applying p-Cav-1 inhibitor PP2 could accelerate wound healing, reduce the expression of α-smooth muscle actin and increase cleaved-caspase 3 expression. These results indicated that dephosphorylation of Cav-1 could inhibit fibrosis during wound healing. The present study establishes a previously unknown role of p-Cav-1 in controlling cytokine release of MSC and may present a potential therapeutic approach for promoting scarless wound healing.

Autophagy controls mesenchymal stem cell therapy in psychological stress colitis mice.

Mesenchymal stem cell transplantation (MSCT) has been applied to treat a variety of autoimmune and inflammatory diseases. Psychosocial stress can aggravate disease progression in chronic inflammatory patients. Whether psychological stress affects MSCT is largely unknown. In this study we show that psychological stress attenuates therapeutic effects of MSCT in a DSS-induced colitis mouse model by elevating the levels of exosomal Mir7k/mmu-let-7 k (microRNA 7 k) in circulation. Mechanistically, Mir7k inhibits STAT3 pathway in donor MSCs, leading to upregulated expression of BECN1 (beclin 1, autophagy related) and, thus, activation of macroautophagy/autophagy. Inhibition of autophagy by blocking Mir7k or activating STAT3 signaling can restore MSCT-mediated therapy in psychologically stressed colitis mice. Our study identifies a previously unknown role of autophagy in regulating MSCT therapy via exosomal miRNA Mir7k.Abbreviations: BafA1: bafilomycin A1; BECN1: beclin 1, autophagy related; DAI: disease activity index; DAPI: 4',6-diamidino-2-phenylindole; DSS: dextran sulfate sodium; GFP: green fluorescent protein; HAI: histological activity index; IFNG/IFN-γ: interferon gamma; IL10: interleukin 10; IL1RN/IL-1Rra: interleukin 1 receptor antagonist; KD: knockdown; miRNA: microRNA; MSCs: mesenchymal stem cells; MSCT: mesenchymal stem cell transplantation; NTA: nanoparticle tracking analysis; PGE2: prostaglandin E2; SD: standard deviation; siRNA: small-interfering RNA; STAT3: signal transducer and activator of transcription 3; TEM: transmission electron microscopy; TGFB1/TGF-ß1: transforming growth factor, beta 1; Th17 cell: T helper cell 17; TNF/TNF-α: tumor necrosis factor; TNFAIP6/TSG6: tumor necrosis factor alpha induced protein 6; Tregs: regulatory T cells.

Apoptotic Extracellular Vesicles Ameliorate Multiple Myeloma by Restoring Fas-Mediated Apoptosis.

Apoptosis is critical for maintaining bodily homeostasis and produces a large number of apoptotic extracellular vesicles (apoEVs). Several types of cancer cells display reduced expression of Fas on the cell surface and are thus capable of escaping Fas ligand-induced apoptosis. However, it is unknown whether normal cell-derived apoEVs can regulate tumor growth. In this study, we show that apoEVs can induce multiple myeloma (MM) cell apoptosis and inhibit MM cell growth. Systemic infusion of mesenchymal stem cell (MSC)-derived apoEVs significantly prolongs the lifespan of MM mice. Mechanistically, apoEVs directly contact MM cells to facilitate Fas trafficking from the cytoplasm to the cell membrane by evoking Ca2+ influx and elevation of cytosolic Ca2+. Subsequently, apoEVs use their Fas ligand to activate the Fas pathway in MM cells, leading to the initiation of apoptosis. This study identifies the role of apoEVs in inducing MM apoptosis and suggests a potential for apoEVs to treat MM.

Amphiphilic triblock copolymers of methoxy-poly(ethylene glycol)-b-poly(L-lactide)-b-poly(L-lysine) for enhancement of osteoblast attachment and growth.

Amphiphilic triblock copolymers of methoxy-poly(ethylene glycol)-poly(L-lactide)-poly(L-lysine) (MPEG-b-PLLA-b-PLL) (Mn=8540-22 240) were synthesized through the ring-opening polymerization of Nepsilon-(Z)-lysine-N-carboxyanhydrides (N(epsilon)-(Z)-Lys-NCA) using MPEG-b-PLLA-NH2 as a macroinitiator. The triblock copolymers and diblock precursors were characterized by 1H NMR, ATR-FTIR, and GPC. The chain lengths of each block could be controlled by varying the feed ratios of the monomers. The surface properties of films of PLLA modified by blending with the triblock copolymers were investigated by XPS and AFM and demonstrated an enrichment of PLL blocks on the surface of the PLLA film. No cytotoxicity was detected on a range of modified PLLA films arising from the incorporation of the triblock copolymers. The triblock copolymers MPEG-b-PLLA-b-PLL showed better surface properties in promoting osteoblast adhesion and proliferation compared with pure PLLA and PLLA modified with MPEG-b-PLLA diblock copolymers. This study demonstrated that the triblock copolymers containing free amino groups, which self-segregate on the surface of biodegradable polyesters, have potential for applications in cell delivery and tissue engineering.