Mesenchymal stem cell‐derived extracellular vesicles reduce senescence and extend health span in mouse models of aging

Aging drives progressive loss of the ability of tissues to recover from stress, partly through loss of somatic stem cell function and increased senescent burden. We demonstrate that bone marrow‐derived mesenchymal stem cells (BM‐MSCs) rapidly senescence and become dysfunctional in culture. Injection of BM‐MSCs from young mice prolonged life span and health span, and conditioned media (CM) from young BM‐MSCs rescued the function of aged stem cells and senescent fibroblasts. Extracellular vesicles (EVs) from young BM‐MSC CM extended life span of Ercc1 ^−/− mice similarly to injection of young BM‐MSCs. Finally, treatment with EVs from MSCs generated from human ES cells reduced senescence in culture and in vivo, and improved health span. Thus, MSC EVs represent an effective and safe approach for conferring the therapeutic effects of adult stem cells, avoiding the risks of tumor development and donor cell rejection. These results demonstrate that MSC‐derived EVs are highly effective senotherapeutics, slowing the progression of aging, and diseases driven by cellular senescence.     

microRNA‐19b‐3p‐containing extracellular vesicles derived from macrophages promote the development of atherosclerosis by targeting JAZF1

Atherosclerosis has been regarded as a major contributor to cardiovascular disease. The role of extracellular vesicles (EVs) in the treatment of atherosclerosis has been increasingly reported. In this study, we set out to investigate the effect of macrophages‐derived EVs (M‐EVs) containing miR‐19b‐3p in the progression of atherosclerosis, with the involvement of JAZF1. Following isolation of EVs from macrophages, the M‐EVs were induced with ox‐low density lipoprotein (LDL) (ox‐LDL‐M‐EVs), and co‐cultured with vascular smooth muscle cells (VSMCs). RT‐qPCR and western blot assay were performed to determine the expression of miR‐19b‐3p and JAZF1 in M‐EVs and in VSMCs. Lentiviral infection was used to overexpress or knock down miR‐19b‐3p. EdU staining and scratch test were conducted to examine VSMC proliferation and migration. Dual‐luciferase gene reporter assay was performed to examine the relationship between miR‐19b‐3p and JAZF1. In order to explore the role of ox‐LDL‐M‐EVs carrying miR‐19b‐3p in atherosclerotic lesions in vivo, a mouse model of atherosclerosis was established through high‐fat diet induction. M‐EVs were internalized by VSMCs. VSMC migration and proliferation were promoted by ox‐LDL‐M‐EVs. miR‐19b‐3p displayed upregulation in ox‐LDL‐M‐EVs. miR‐19b‐3p was transferred by M‐EVs into VSMCs, thereby promoting VSMC migration and proliferation. mir‐19b‐3p targeted JAZF1 to decrease its expression in VSMCs. Atherosclerosis lesions were aggravated by ox‐LDL‐M‐EVs carrying miR‐19b‐3p in ApoE^−/− mice. Collectively, this study demonstrates that M‐EVs containing miR‐19b‐3p accelerate migration and promotion of VSMCs through targeting JAZF1, which promotes the development of atherosclerosis.     

Exosomes from microRNA-126 overexpressing mesenchymal stem cells promote angiogenesis by targeting the PIK3R2-mediated PI3K/Akt signalling pathway

microRNA-126 (miR-126), an endothelial-specific miRNA, is associated with vascular homeostasis and angiogenesis. However, the efficiency of miR-126-based treatment is partially compromised due to the low efficiency of miRNA delivery in vivo. Lately, exosomes have emerged as a natural tool for therapeutic molecule delivery. Herein, we investigated whether exosomes derived from bone marrow mesenchymal stem cells (BMMSCs) can be utilized to deliver miR-126 to promote angiogenesis. Exosomes were isolated from BMMSCs overexpressed with miR-126 (Exo-miR-126) by ultracentrifugation. In vitro study, Exo-miR-126 treatment promoted the proliferation, migration and angiogenesis of human umbilical vein endothelial cells (HUVECs). Furthermore, the gene/protein expression of angiogenesis-related vascular endothelial growth factor (VEGF) and angiotensin-1 (Ang-1) were up-regulated after incubation with Exo-miR-126. Additionally, the expression level of phosphoinositol-3 kinase regulatory subunit 2 (PIK3R2) showed an inverse correlation with miR-126 in HUVECs. Particularly, the Exo-miR-126 treatment contributed to enhanced angiogenesis of HUVECs by targeting PIK3R2 to activate the PI3K/Akt signalling pathway. Similarly, Exo-miR-126 administration profoundly increased the number of newly formed capillaries in wound sites and accelerated the wound healing in vivo. The results demonstrate that exosomes derived from BMMSCs combined with miR-126 may be a promising strategy to promote angiogenesis.     

Follicle‐stimulating hormone impairs dental pulp stem cells odontogenic differentiation

In addition to bone, the dentin‐pulp complex is also influenced by menopause, showing a decreased regenerative capacity. High levels of follicle‐stimulating hormone (FSH) during menopause could directly regulate bone metabolism. Here, the role of FSH in the odontogenic differentiation of the dentin‐pulp complex was investigated. Dental pulp stem cells (DPSCs) were isolated. CCK‐8 assays, cell apoptosis assays, Western blotting (WB), real‐time RT‐PCR, alkaline phosphatase activity assays, and Alizarin Red S staining were used to clarify the effects of FSH on the proliferation, apoptosis and odontogenic differentiation of the DPSCs. MAPK pathway‐related factors were explored by WB assays. FSH and its inhibitor were used in OVX rats combined with a direct pulp‐capping model. HE and immunohistochemistry were used to detect reparative dentin formation and related features. The results indicated that FSH significantly decreased the odontogenic differentiation of the DPSCs without affecting cell proliferation and apoptosis. Moreover, FSH significantly activated the JNK signalling pathway, and JNK inhibitor partly rescued the inhibitory effect of FSH on DPSC differentiation. In vivo, FSH treatment attenuated the dentin bridge formation and mineralization‐related protein expression in the OVX rats. Our findings indicated that FSH reduced the odontogenic capacity of the DPSCs and was involved in reparative dentinogenesis during menopause.     

Mesenchymal stromal cell‐derived extracellular vesicles as cell‐free biologics for the ex vivo expansion of hematopoietic stem cells

Hematopoietic stem cell transplantation (HSCT) is the ultimate choice of treatment for patients with hematological diseases and cancer. The success of HSCT is critically dependent on the number and engraftment efficiency of the transplanted donor hematopoietic stem cells (HSCs). Various studies show that bone marrow‐derived mesenchymal stromal cells (MSCs) support hematopoiesis and also promote ex vivo expansion of HSCs. MSCs exert their therapeutic effect through paracrine activity, partially mediated through extracellular vesicles (EVs). Although the physiological function of EVs is not fully understood, inspiring findings indicate that MSC‐derived EVs can reiterate the hematopoiesis, supporting the ability of MSCs by transferring their cargo containing proteins, lipids, and nucleic acids to the HSCs. The activation state of the MSCs or the signaling mechanism that prevails in them also defines the composition of their EVs, thereby influencing the fate of HSCs. Modulating or preconditioning MSCs to achieve a specific composition of the EV cargo for the ex vivo expansion of HSCs is, therefore, a promising strategy that can overcome several challenges associated with the use of naïve/unprimed MSCs. This review aims to speculate upon the potential role of preconditioned/primed MSC‐derived EVs as “cell‐free biologics,” as a novel strategy for expanding HSCs in vitro.     

SHED aggregate exosomes shuttled miR‐26a promote angiogenesis in pulp regeneration via TGF‐β/SMAD2/3 signalling

ObjectivesPulp regeneration brings big challenges for clinicians, and vascularization is considered as its determining factor. We previously accomplished pulp regeneration with autologous stem cells from deciduous teeth (SHED) aggregates implantation in teenager patients, however, the underlying mechanism needs to be clarified for regenerating pulp in adults. Serving as an important effector of mesenchymal stem cells (MSCs), exosomes have been reported to promote angiogenesis and tissue regeneration effectively. Here, we aimed to investigate the role of SHED aggregate‐derived exosomes (SA‐Exo) in the angiogenesis of pulp regeneration.Materials and MethodsWe extracted exosomes from SHED aggregates and utilized them in the pulp regeneration animal model. The pro‐angiogenetic effects of SA‐Exo on SHED and human umbilical vein endothelial cells (HUVECs) were evaluated. The related mechanisms were further investigated.ResultsWe firstly found that SA‐Exo significantly improved pulp tissue regeneration and angiogenesis in vivo. Next, we found that SA‐Exo promoted SHED endothelial differentiation and enhanced the angiogenic ability of HUVECs, as indicated by the in vitro tube formation assay. Mechanistically, miR‐26a, which is enriched in SA‐Exo, improved angiogenesis both in SHED and HUVECs via regulating TGF‐β/SMAD2/3 signalling.ConclusionsIn summary, these data reveal that SA‐Exo shuttled miR‐26a promotes angiogenesis via TGF‐β/SMAD2/3 signalling contributing to SHED aggregate‐based pulp tissue regeneration. These novel insights into SA‐Exo may facilitate the development of new strategies for pulp regeneration.     

Simvastatin treatment promotes proliferation of human dental pulp stem cells via modulating PI3K/AKT/miR-9/KLF5 signalling pathway

Simvastatin serves as an effective therapeutic potential in the treatment of dental disease via alternating proliferation of dental pulp stem cells. First, western-blot and real-time quantitative PCR were used to detect the effect of simvastatin or LY294002 on the expression levels of AKT, miR-9 and KLF5, or determine the effect of miR-9. Simvastatin, KLF5 and AKT significantly enhanced the proliferation of pulp stem cells, whilst this effect induced by simvastatin was suppressed by LY294002, AKT siRNA, KLF5 siRNA and miR-9, and simvastatin dose-dependently upregulated the expression of PI3K. Furthermore, simvastatin upregulated PI3K and p-AKT expression in a concentration-dependent manner. LY294002 abrogated the upregulation of p-AKT expression levels induced by simvastatin, and LY294002 induced the miR-9 expression and simvastatin dose-dependently inhibited the expression of miR-9, by contrast, LY294002 reduced the KLF5 expression and simvastatin dose-dependently promoted the expression of KLF5. And using computational analysis, KLF5 was found to be a candidate target gene of miR-9, and which was further verified using luciferase assay. Finally, the level of KLF5 in cells was much lower following the transfection with miR-9 and KLF5 siRNA, and the level of AKT mRNA in cells was significantly inhibited after transfection with AKT siRNA than control. These findings suggested simvastatin could promote the proliferation of pulp stem cells, possibly by suppressing the expression of miR-9 via activating the PI3K/AKT signalling pathway, and the downregulation of miR-9 upregulated the expression of its target gene, KLF5, which is directly responsible for the enhanced proliferation of pulp stem cells.     

Extracellular vesicle‐enclosed miR‐486‐5p mediates wound healing with adipose‐derived stem cells by promoting angiogenesis

Adipose‐derived stem cells (ASC) are said to have a pivotal role in wound healing. Specifically, ASC‐secreted extracellular vesicles (EV) carry diverse cargos such as microRNAs (miRNAs) to participate in the ASC‐based therapies. Considering its effects, we aimed to investigate the role of ASC‐EVs in the cutaneous wound healing accompanied with the study on the specific cargo‐medicated effects on wound healing. Two full‐thickness excisional skin wounds were created on mouse dorsum, and wound healing was recorded at the indicated time points followed by histological analysis and immunofluorescence staining for CD31 and α‐SMA. Human skin fibroblasts (HSFs) and human microvascular endothelial cells (HMECs) were co‐cultured with EVs isolated from ASC (ASC‐EVs), respectively, followed by the evaluation of their viability and mobility using CCK‐8, scratch test and transwell migration assays. Matrigel‐based angiogenesis assays were performed to evaluate vessel‐like tube formation by HMECs in vitro. ASC‐EVs accelerated the healing of full‐thickness skin wounds, increased re‐epithelialization and reduced scar thickness whilst enhanced collagen synthesis and angiogenesis in murine models. However, miR‐486‐5p antagomir abrogated the ASC‐EVs‐induced effects. Intriguingly, miR‐486‐5p was found to be highly enriched in ASC‐EVs, exhibiting an increase in viability and mobility of HSFs and HMECs and enhanced the angiogenic activities of HMECs. Notably, we also demonstrated that ASC‐EVs‐secreted miR‐486‐5p achieved the aforesaid effects through its target gene Sp5. Hence, our results suggest that miR‐486‐5p released by ASC‐EVs could be a critical mediator to develop an ASC‐based therapeutic strategy for wound healing.     

Distal C‐terminus of Cav1.2 is indispensable for the chondrogenic differentiation of rat dental pulp stem cells

The α1 subunit (Cav1.2) of the L‐type calcium channel (LTCC), which is presently existing in both excitatory cells and non‐excitatory cells, is involved in the differentiation and proliferation of mesenchymal stem cells (MSCs). Dental pulp stem cells (DPSCs), MSCs derived from dental pulp, exhibit multipotent characteristics similar to those of MSCs. The aim of the present study was to examine the contribution of Cav1.2 and its distal C‐terminus (DCT) to the commitment of rat DPSCs (rDPSCs) toward chondrocytes and adipocytes in vitro. The expression of Cav1.2 was obviously elevated in chondrogenic differentiation but did not differ significantly in adipogenic differentiation. The chondrogenic differentiation but not adipogenic of rDPSCs was inhibited by either blocking LTCC using nimodipine or knockdown of Cav1.2 via short hairpin RNA (shRNA). Overexpression of DCT rescued the inhibition by Cav1.2‐shRNA during chondrogenic differentiation, indicating that DCT is essential for the chondrogenic differentiation of rDPSCs. However, the protein level of DCT decreased after chondrogenic differentiation in wild‐type cells, and overexpression of DCT in rDPSCs inhibited the phenotype. These data suggest that DCT is indispensable for chondrogenic differentiation of rDPSCs but that superfluous DCT inhibits this process. Through the analysis of differentially expressed genes using RNA‐seq data, we speculated that the regulation of DCT might be mediated by the mitogen‐activated protein kinase/extracellular‐regulated kinase and c‐Jun N‐terminal kinase signaling pathways, or Chondromodulin‐1.