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Adipose-derived mesenchymal stem cells (ADSCs) are promising candidate for regenerative medicine to repair non-healing bone defects due to their high and easy availability. However, the limited osteogenic differentiation potential greatly hinders the clinical application of ADSCs in bone repair. Accumulating evidences demonstrate that circular RNAs (circRNAs) are involved in stem/progenitor cell fate determination, but their specific role in stem/progenitor cell osteogenesis, remains mostly undescribed. Here, we show that circRNA-vgll3 originating from the vgll3 locus markedly enhances osteogenic differentiation of ADSCs; nevertheless, silencing of circRNA-vgll3 dramatically attenuates ADSC osteogenesis. Furthermore, we validate that circRNA-vgll3 functions in ADSC osteogenesis through a circRNA-vgll3/miR-326-5p/integrin α5 (Itga5) pathway. Itga5 promotes ADSC osteogenic differentiation and miR-326-5p suppresses Itga5 translation. CircRNA-vgll3 directly sequesters miR-326-5p in the cytoplasm and inhibits its activity to promote osteogenic differentiation. Moreover, the therapeutic potential of circRNA-vgll3-modified ADSCs with calcium phosphate cement (CPC) scaffolds was systematically evaluated in a critical-sized defect model in rats. Our results demonstrate that circRNA-vgll3 markedly enhances new bone formation with upregulated bone mineral density, bone volume/tissue volume, trabeculae number, and increased new bone generation. This study reveals the important role of circRNA-vgll3 during new bone biogenesis. Thus, circRNA-vgll3 engineered ADSCs may be effective potential therapeutic targets for bone regenerative medicine.

Retinal progenitor cells (RPCs) hold great potential for the treatment of retinal degenerative diseases. However, their proliferation capacity and differentiation potential towards specific retinal neurons are limited, which limit their future clinical applications. Thus, it is important to improve the RPCs’ ability to proliferate and differentiate. Currently, epidermal growth factor (EGF) is commonly used to stimulate RPC growth in vitro. In this study, we find that betacellulin (BTC), a member of the EGF family, plays important roles in the proliferation and differentiation of RPCs. Our results showed that BTC can significantly promote the proliferation of RPCs more efficiently than EGF. EGF stimulated RPC proliferation through the EGFR/ErbB2‐Erk pathway, while BTC stimulated RPC proliferation more powerfully through the EGFR/ErbB2/ErbB4‐Akt/Erk pathway. Meanwhile, under differentiated conditions, the BTC‐pre‐treated RPCs were preferentially differentiated into retinal neurons, including photoreceptors, one of the most important types of cells for retinal cell replacement therapy, compared to the EGF‐pre‐treated RPCs. In addition, knockdown of endogenous BTC expression can also obviously promote RPC differentiation into retinal neuronal cells. This data demonstrate that BTC plays important roles in promoting RPC proliferation and differentiation into retinal neurons. This study may provide new insights into the study of RPC proliferation and differentiation and make a step towards the application of RPCs in the treatment of retinal degenerative diseases.

MicroRNAs (miRNAs) are endogenous small noncoding ~22-nt RNAs, which have been reported to play a crucial role in maintaining bone development and metabolism. Osteogenesis originates from mesenchymal stem cells (MSCs) differentiating into mature osteoblasts and each period of bone formation is inseparable from the delicate regulation of various miRNAs. Of note, apprehending the sophisticated circuit between miRNAs and osteogenic homeostasis is of great value for artificial skeletal regeneration for severe bone defects. In this review, we highlight how different miRNAs interact with diverse osteo-related genes and endeavor to sketch the contours of potential manipulations of miRNA-modulated bone repair.

Context T helper (Th)17 cells are correlated with many human autoimmune disorders, including Graves disease, and may play key roles in the pathogenesis of Graves orbitopathy (GO). Objective To study the phenotype of Th17 cells in patients with GO and healthy subjects, investigate the fibrosis and adipogenesis in orbital fibroblasts (OFs) modulated by interleukin (IL)-17A, and determine the interaction between Th17 cells and OFs. Design/Setting/Participants Blood samples and orbital tissues from GO patients and healthy controls were collected. Main Outcome Measures We conducted multicolor flow cytometry, immunohistochemical and immunofluorescent stainings, Western blotting, a PathScan intracellular signaling assay, Luminex and enzyme-linked immunosorbent assays, and protein mass spectrum. Results Interferon-γ– and IL-22–expressing Th17 cells are increased in GO patients, which are positively related to clinical activity score. Costimulatory molecules are highly expressed in GO orbits and most GO OFs are CD90+. IL-17A promotes TGF-β–induced fibrosis in CD90+ OFs but impedes 15-deoxy-Δ12,14-prostaglandin J2–induced adipogenesis in CD90− OFs. Th17 cells promote proinflammatory cytokine secretion in both CD90+ and CD90− OFs. Meanwhile, both CD90+ and CD90− OFs contribute to Th17 cell differentiation through prostaglandin E2 production, which can be attenuated by indomethacin. Furthermore, Th17 cells upregulate costimulatory molecule expression on OFs. Conclusion Our findings unravel the pathogenicity of IL-17A in the initiation and progression of GO. In-depth interpretation of the molecular basis of OFs delineated by CD90 and Th17–OF interaction will help to afford a novel approach to better therapeutic strategies for GO.

Increasing evidence has indicated that bone morphogenetic protein 2 (BMP2) coordinates with microRNAs (miRNAs) to form intracellular networks regulating mesenchymal stem cells (MSCs) osteogenesis. This study aimed to identify specific miRNAs in rat adipose-derived mesenchymal stem cells (ADSCs) during BMP2-induced osteogenesis, we selected the most significantly down-regulated miRNA, miR-146a, to systematically investigate its role in regulating osteogenesis and bone regeneration. Overexpressing miR-146a notably repressed ADSC osteogenesis, whereas knocking down miR-146a greatly promoted this process. Drosophila mothers against decapentaplegic protein 4 (SMAD4), an important co-activator in the BMP signaling pathway, was miR-146a’s direct target and miR-146a exerted its repressive effect on SMAD4 through interacting with 3′-untranslated region (3′-UTR) of SMAD4 mRNA. Furthermore, knocking down SMAD4 attenuated the ability of miR-146a inhibitor to promote ADSC osteogenesis. Next, transduced ADSCs were incorporated with poly(sebacoyl diglyceride) (PSeD) porous scaffolds for repairing critical-sized cranial defect, the treatment of miR-146a inhibitor greatly enhanced ADSC-mediated bone regeneration with higher expression levels of SMAD4, Runt-related transcription factor 2 (Runx2) and Osterix in newly formed bone. In summary, our study showed that miR-146a negatively regulates the osteogenesis and bone regeneration from ADSCs both in vitro and in vivo.

Background aims Retinal progenitor cells (RPCs) are a promising cell therapy treatment for retinal degenerative diseases. However, problems with limited proliferation ability and differentiation preference toward glia rather than neurons restrict the clinical application of these RPCs. The extracellular matrix (ECM) has been recognized to provide an appropriate microenvironment to support stem cell adhesion and direct cell behaviors, such as self-renewal and differentiation. Methods In this study, decellularized matrix of adipose-derived mesenchymal stromal cells (DMA) was manufactured using a chemical agent method (0.5% ammonium hydroxide Triton + 20 mmol/L NH4OH) in combination with a biological agent method (DNase solution), and the resulting DMA were evaluated by scanning electron microscopy (SEM) and immunocytochemistry. The effect of DMA on RPC proliferation and differentiation was evaluated by quantitative polymerase chain reaction, Western blot and immunocytochemistry analysis. Results DMA was successfully fabricated, as demonstrated by SEM and immunocytochemistry. Compared with tissue culture plates, DMA may effectively enhance the proliferation of RPCs by activating Akt and Erk phosphorylation; when the two pathways were blocked, the promoting effect was reversed. Moreover, DMA promoted the differentiation of RPCs toward retinal neurons, especially rhodopsin- and recoverin-positive photoreceptors, which is the most interesting class of cells for retinal degeneration treatment. Conclusions These results indicate that DMA has important roles in governing RPC proliferation and differentiation and may contribute to the application of RPCs in treating retinal degenerative diseases.

Elucidating the molecular mechanisms responsible for osteogenesis of human adipose-derived mesenchymal stem cells (hADSCs) will provide deeper insights into the regulatory mechanisms of this process and help develop more efficient methods for cell-based therapies. In this study, we analysed the role of miR-26a in the regulation of hADSC osteogenesis. The endogenous expression of miR-26a increased during the osteogenic differentiation. The overexpression of miR-26a promoted hADSC osteogenesis, whereas osteogenesis was repressed by miR-26a knockdown. Additionally, miR-26a directly targeted the 3′UTR of the GSK3β, suppressing the expression of GSK3β protein. Similar to the effect of overexpressing miR-26a, the knockdown of GSK3β promoted osteogenic differentiation, whereas GSK3β overexpression inhibited this process, suggesting that GSK3β acted as a negative regulator of hADSC osteogenesis. Furthermore, GSK3β influences Wnt signalling pathway by regulating β-catenin and subsequently altered the expression of its downstream target C/EBPα. In turn, C/EBPα transcriptionally regulated the expression of miR-26a by physically binding to the CTDSPL promoter region. Taken together, our data identified a novel feedback regulatory circuitry composed of miR-26a, GSK3β and C/EBPα, the function of which might contribute to the regulation of hADSC osteogenesis. Our findings provided new insights into the function of miR-26a and the mechanisms underlying osteogenesis of hADSCs.

Biocompatible polymer scaffolds are promising as potential carriers for the delivery of retinal progenitor cells (RPCs) in cell replacement therapy for the repair of damaged or diseased retinas. The primary goal of the present study was to investigate the effects of blended electrospun nanofibrous membranes of silk fibroin (SF) and poly(L-lactic acid-co-ε-caprolactone) (PLCL), a novel scaffold, on the biological behaviour of RPCs in vitro. To assess the cell-scaffold interaction, RPCs were cultured on SF/PLCL scaffolds for indicated durations. Our data revealed that all the SF/PLCL scaffolds were thoroughly cytocompatible and the SF:PLCL (1:1) scaffolds yielded the best RPC growth. The in vitro proliferation assays showed that RPCs proliferated more quickly on the SF:PLCL (1:1) than on the other scaffolds and the control. Quantitative polymerase chain reaction (qPCR) and immunocytochemistry analyses demonstrated that RPCs grown on the SF:PLCL (1:1) scaffolds preferentially differentiated toward retinal neurons, including, most interestingly, photoreceptors. In summary, we demonstrated that the SF:PLCL (1:1) scaffolds can not only markedly promote RPC proliferation with cytocompatibility for RPC growth but also robustly enhance RPCs’ differentiation toward specific retinal neurons of interest in vitro, suggesting that SF:PLCL (1:1) scaffolds may have potential applications in retinal cell replacement therapy in the future.

Mesenchymal stem cells (MSCs) have been extensively applied in the field of tissue regeneration. MSCs derived from various tissues exhibit different characteristics. In this study, a cluster of cells were isolated from human ethmoid sinus mucosa membrane and termed as hESMSCs. hESMSCs was demonstrated to have MSC-specific characteristics of self-renewal and tri-lineage differentiation. In particular, hESMSCs displayed strong osteogenic differentiation potential, and also remarkably promoted the proliferation and osteogenesis of rat bone marrow mesenchymal stem cells (rBMSCs) in vitro. Next, hESMSCs were prepared into a cell sheet and combined with a PSeD scaffold seeded with rBMSCs to repair critical-sized calvarial defects in rats, which showed excellent reparative effects. Additionally, ELISA assays revealed that secreted cytokines, such as BMP-2, BMP-4 and bFGF, were higher in the hESMSCs conditioned medium, and immunohistochemistry validated that hESMSCs cell sheet promoted the expression of BMP signaling downstream genes in newly formed bone. In conclusion, hESMSCs were demonstrated to be a class of mesenchymal stem cells that possessed high self-renewal capacity along with strong osteogenic potential, and the cell sheet of hESMSCs could remarkably promote new bone regeneration, indicating that hESMSCs cell sheet could serve as a novel and promising alternative strategy in the management of bone regeneration.

MicroRNAs manifest significant functions in brain neural stem cell (NSC) self-renewal and differentiation through the post-transcriptional regulation of neurogenesis genes. Let-7b is expressed in the mammalian brain and regulates NSC proliferation and differentiation by targeting the nuclear receptor TLX, which is an essential regulator of NSC self-renewal. Whether let-7b and TLX act as important regulators in retinal progenitor cell (RPC) proliferation and differentiation remains unknown. Here, our data show that let-7b and TLX play important roles in controlling RPC fate determination in vitro. Let-7b suppresses TLX expression to negatively regulate RPC proliferation and accelerate the neuronal and glial differentiation of RPCs. The overexpression of let-7b downregulates TLX levels in RPCs, leading to reduced RPC proliferation and increased neuronal and glial differentiation, whereas antisense knockdown of let-7b produces robust TLX expression,enhanced RPC proliferation and decreased differentiation. Moreover, the inhibition of endogenous TLX by small interfering RNA suppresses RPC proliferation and promotes RPC differentiation. Furthermore, overexpression of TLX rescues let-7b-induced proliferation deficiency and weakens the RPC differentiation enhancement caused by let-7b alone. These results suggest that let-7b, by forming a negative feedback loop with TLX, provides a novel model to regulate the proliferation and differentiation of retinal progenitors in vitro.

Zi Wang,1,* Ming Lin,1,* Qing Xie,1 Hao Sun,1 Yazhuo Huang,1 DanDan Zhang,1 Zhang Yu,1 Xiaoping Bi,1 Junzhao Chen,1 Jing Wang,2 Wodong Shi,1 Ping Gu,1 Xianqun Fan1 1Department of Ophthalmology, Ninth People’s Hospital, Shanghai Jiao Tong University School of Medicine, 2Biomaterials and Tissue Engineering Laboratory, College of Chemistry & Chemical Engineering and Biotechnology, Donghua University, Shanghai, People’s Republic of China *These authors contributed equally tothis work Background: Tissue engineering has become a promising therapeutic approach for bone regeneration. Nanofibrous scaffolds have attracted great interest mainly due to their structural similarity to natural extracellular matrix (ECM). Poly(lactide-co-ε-caprolactone) (PLCL) has been successfully used in bone regeneration, but PLCL polymers are inert and lack natural cell recognition sites, and the surface of PLCL scaffold is hydrophobic. Silk fibroin (SF) is a kind of natural polymer with inherent bioactivity, and supports mesenchymal stem cell attachment, osteogenesis, and ECM deposition. Therefore, we fabricated hybrid nanofibrous scaffolds by adding different weight ratios of SF to PLCL in order to find a scaffold with improved properties for bone regeneration.Methods: Hybrid nanofibrous scaffolds were fabricated by blending different weight ratios of SF with PLCL. Human adipose-derived stem cells (hADSCs) were seeded on SF/PLCL nanofibrous scaffolds of various ratios for a systematic evaluation of cell adhesion, proliferation, cytotoxicity, and osteogenic differentiation; the efficacy of the composite of hADSCs and scaffolds in repairing critical-sized calvarial defects in rats was investigated.Results: The SF/PLCL (50/50) scaffold exhibited favorable tensile strength, surface roughness, and hydrophilicity, which facilitated cell adhesion and proliferation. Moreover, the SF/PLCL (50/50) scaffold promoted the osteogenic differentiation of hADSCs by elevating the expression levels of osteogenic marker genes such as BSP, Ocn, Col1A1, and OPN and enhanced ECM mineralization. In vivo assays showed that SF/PLCL (50/50) scaffold improved the repair of the critical-sized calvarial defect in rats, resulting in increased bone volume, higher trabecular number, enhanced bone mineral density, and increased new bone areas, compared with the pure PLCL scaffold.Conclusion: The SF/PLCL (50/50) nanofibrous scaffold facilitated hADSC proliferation and osteogenic differentiation in vitro and further promoted new bone formation in vivo, suggesting that the SF/PLCL (50/50) nanofibrous scaffold holds great potential in bone tissue regeneration. Keywords: human adipose-derived mesenchymal stem cells, silk fibrin, poly(lactide-co-ε-caprolactone), proliferation, osteogenesis

Mesenchymal stem cells (MSCs) are attractive candidates for cell therapy and regenerative medicine because of their potential for proliferation and multilineage differentiation. MSCs secrete various cytokines, acting as trophic mediators to regulate neighboring cells. Osteoblasts are the cells directly responsible for forming new bone, and they are the final target of many osteogenic regulators. However, the induction effect of MSCs on osteoblasts is still unknown. In this study, we isolated osteoblasts from rat calvariae and investigated their proliferation and differentiation under induction of varied concentrations of MSC-conditioned medium (MSC-CM). Cells in the MSC-CM groups showed a reduction in cell proliferation at 3–6 days, and a decrease in the expression of osteocalcin and osteopontin at 3 days, with low levels of alkaline phosphatase activity. The expression of osteogenic markers went back to normal at 7 days. In order to evaluate the molecular mechanisms underlying this suppression, levels of two osteoblastic transcription factors, runt-related transcription factor 2 (Runx2) and osterix (Osx), were detected at both mRNA and protein levels. The results indicated that MSC-CM significantly inhibited Runx2 expression in a concentration-dependent manner. However, the effect was not due to the inhibition of Osx, for Osx was not significantly altered. This work demonstrates that MSCs may suppress osteoblast proliferation and transiently retard osteoblast differentiation by downregulating Runx2. These results highlight the need to take into account the paracrine effect of MSCs when using them in regenerative medicine for the repair of bone defects.