Correction of a CADASIL point mutation using adenine base editors in hiPSCs and blood vessel organoids.

Cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL) is a monogenic small vessel disease caused by mutations in the NOTCH3 gene. However, the pathogenesis of CADASIL remains unclear, and patients have limited treatment options. Here, we use human induced pluripotent stem cells (hiPSCs) generated from the peripheral blood mononuclear cells of a patient with CADASIL carrying a heterozygous NOTCH3 mutation (c.1261C>T, p.R421C) to develop a disease model. The correction efficiency of different adenine base editors (ABEs) is tested using the HEK293T-NOTCH3 reporter cell line. ABEmax is selected based on its higher efficiency and minimization of predicted off-target effects. Vascular smooth muscle cells (VSMCs) differentiated from CADASIL hiPSCs show NOTCH3 deposition and abnormal actin cytoskeleton structure, and the abnormalities are recovered in corrected hiPSC-derived VSMCs. Furthermore, CADASIL blood vessel organoids generated for in vivo modeling show altered expression of genes related to disease phenotypes, including the downregulation of cell adhesion, extracellular matrix organization, and vessel development. The dual adeno-associated virus (AAV) split-ABEmax system is applied to the genome editing of vascular organoids with an average editing efficiency of 8.82%. Collectively, we present potential genetic therapeutic strategies for patients with CADASIL using blood vessel organoids and the dual AAV split-ABEmax system.

Transplantation of Gelatin Microspheres Loaded with Wharton's Jelly Derived Mesenchymal Stem Cells Facilitates Cartilage Repair in Mice.

BACKGROUND: Knee osteoarthritis (KOA) is a prevalent chronic joint disease caused by various factors. Mesenchymal stem cells (MSCs) therapy is an increasingly promising therapeutic option for osteoarthritis. However, the chronic inflammation of knee joint can severely impede the therapeutic effects of transplanted cells. Gelatin microspheres (GMs) are degradable biomaterial that have various porosities for cell adhesion and cell-cell interaction. Excellent elasticity and deformability of GMs make it an excellent injectable vehicle for cell delivery. METHODS: We created Wharton's jelly derived mesenchymal stem cells (WJMSCs)-GMs complexes and assessed the effects of GMs on cell activity, proliferation and chondrogenesis. Then, WJMSCs loaded in GMs were transplanted in the joint of osteoarthritis mice. After four weeks, joint tissue was collected for histological analysis. Overexpressing-luciferase WJMSCs were performed to explore cell retention in mice. RESULTS: In vitro experiments demonstrated that WJMSCs loaded with GMs maintained cell viability and proliferative potential. Moreover, GMs enhanced the chondrogenesis differentiation of WJMSCs while alleviated cell hypertrophy. In KOA mice model, transplantation of WJMSCs-GMs complexes promoted cartilage regeneration and cartilage matrix formation, contributing to the treatment of KOA. Compared with other groups, in WJMSCs+GMs group, there were fewer cartilage defects and with a more integrated tibia structure. Tracking results of stable-overexpressing luciferase WJMSCs demonstrated that GMs significantly extended the retention time of WJMSCs in knee joint cavity. CONCLUSION: Our results indicated that GMs facilitate WJMSCs mediated knee osteoarthritis healing in mice by promoting cartilage regeneration and prolonging cell retention. It might potentially provide an optimal strategy for the biomaterial-stem cell based therapy for knee osteoarthritis.

pH-Responsive Wound Dressing Based on Biodegradable CuP Nanozymes for Treating Infected and Diabetic Wounds.

Nanozymes, emerging nanomaterials for wound healing, exhibit enzyme-like activity to modulate the levels of reactive oxygen species (ROS) at wound sites. Yet, the solo regulation of endogenous ROS by nanozymes often falls short, particularly in chronic refractory wounds with complex and variable pathological microenvironments. In this study, we report the development of a multifunctional wound dressing integrating a conventional alginate (Alg) hydrogel with a newly developed biodegradable copper hydrogen phosphate (CuP) nanozyme, which possesses good near-infrared (NIR) photothermal conversion capabilities, sustained Cu ion release ability, and pH-responsive peroxidase/catalase-mimetic catalytic activity. When examining acute infected wounds characterized by a low pH environment, the engineered Alg/CuP composite hydrogels demonstrated high bacterial eradication efficacy against both planktonic bacteria and biofilms, attributed to the combined action of catalytically generated hydroxyl radicals and the sustained release of Cu ions. In contrast, when applied to chronic diabetic wounds, which typically have a high pH environment, these composite hydrogels exhibit significant angiogenic performance. This is driven by the provision of catalytically generated dissolved oxygen and a beneficial supplement of Cu ions released from the degradable CuP nanozyme. Further, a mild thermal effect induced by NIR irradiation amplifies the catalytic activities and bioactivity of Cu ions, thereby enhancing the healing process of both infected and diabetic wounds. Our study validates that the synergistic integration of photothermal effects, catalytic activity, and released Cu ions can concurrently yield high antibacterial efficiency and tissue regenerative activity, rendering it highly promising for various clinical applications in wound healing.

3D-printed GelMA/CaSiO3 composite hydrogel scaffold for vascularized adipose tissue restoration.

The increased number of mastectomies, combined with rising patient expectations for cosmetic and psychosocial outcomes, has necessitated the use of adipose tissue restoration techniques. However, the therapeutic effect of current clinical strategies is not satisfying due to the high demand of personalized customization and the timely vascularization in the process of adipose regeneration. Here, a composite hydrogel scaffold was prepared by three-dimensional (3D) printing technology, applying gelatin methacrylate anhydride (GelMA) as printing ink and calcium silicate (CS) bioceramic as an active ingredient for breast adipose tissue regeneration. The in vitro experiments showed that the composite hydrogel scaffolds could not only be customized with controllable architectures, but also significantly stimulated both 3T3-L1 preadipocytes and human umbilical vein endothelial cells in multiple cell behaviors, including cell adhesion, proliferation, migration and differentiation. Moreover, the composite scaffold promoted vascularized adipose tissue restoration under the skin of nude mice in vivo. These findings suggest that 3D-printed GelMA/CS composite scaffolds might be a good candidate for adipose tissue engineering.

Zn2 SiO4 Bioceramic Attenuates Cardiac Remodeling after Myocardial Infarction.

In the pursuit of therapeutic strategies for myocardial infarction (MI), a pivotal objective lies in the concurrent restoration of blood perfusion and reduction of cardiomyocyte apoptosis. However, achieving these dual goals simultaneously presents a considerable challenge. In this study, a Zn2 SiO4 bioceramic capable of concurrently sustaining the release of bioactive SiO3 2- and Zn2+ ions, which exhibit a synergistic impact on endothelial cell angiogenesis promotion, cardiomyocyte apoptosis inhibition, and myocardial mitochondrial protection against oxygen-free radical (reactive oxygen species) induced injury is developed. Furthermore, in vivo outcomes from a murine MI model demonstrate that either systemic administration via tail vein injection of Zn2 SiO4 extract or local application through intramyocardial injection of a Zn2 SiO4 composite hydrogel promotes cardiac function and reduces cardiac fibrosis, thus aiding myocardial repair. This research is the first to elucidate the advantageous effects of dual bioactive ions in myocardial protection and may offer a novel therapeutic avenue for ischemic heart disease based on meticulously engineered bioceramics.

Silicate Nanoplatelets Promotes Neuronal Differentiation of Neural Stem Cells and Restoration of Spinal Cord Injury.

Neural stem cell (NSC) transplantation has been suggested as a promising therapeutic strategy to replace lost neurons after spinal cord injury (SCI). However, the low survival rate and neuronal differentiation efficiency of implanted NSCs within the lesion cavity limit the application. Furthermore, it is difficult for transplanted cells to form connections with host cells. Thus, effective and feasible methods to enhance the efficacy of cell transplantation are needed. In this study, the effect of Laponite nanoplatelets, a type of silicate nanoplatelets, on stem cell therapy is explored. Laponite nanoplatelets can induce the neuronal differentiation of NSCs in vitro within five days, and RNA sequencing and protein expression analysis demonstrated that the NF-κB pathway is involved in this process. Moreover, histological results revealed that Laponite nanoplatelets can increase the survival rate of transplanted NSCs and promote NSCs to differentiate into mature neurons. Finally, the formation of connections between transplanted cells and host cells is confirmed by axon tracing. Hence, Laponite nanoplatelets, which drove neuronal differentiation and the maturation of NSCs both in vitro and in vivo, can be considered a convenient and practical biomaterial to promote repair of the injured spinal cord by enhancing the efficacy of NSC transplantation.

Silicate Ions Derived from Calcium Silicate Extract Decelerate Ang II-Induced Cardiac Remodeling.

BACKGROUND: Pathological cardiac hypertrophy is one of the main activators of heart failure. Currently, no drug can completely reverse or inhibit the development of pathological cardiac hypertrophy. To this end, we proposed a silicate ion therapy based on extract derived from calcium silicate (CS) bioceramics for the treatment of angiotensin II (Ang II) induced cardiac hypertrophy. METHODS: In this study, the Ang II induced cardiac hypertrophy mouse model was established, and the silicate ion extract was injected to mice intravenously. The cardiac function was evaluated by using a high-resolution Vevo 3100 small animal ultrasound imaging system. Wheat germ Agglutinin, Fluo4-AM staining and immunofluorescent staining was conducted to assess the cardiac hypertrophy, intracellular calcium and angiogenesis of heart tissue, respectively. RESULTS: The in vitro results showed that silicate ions could inhibit the cell size of cardiomyocytes, reduce cardiac hypertrophic gene expression, including atrial natriuretic peptide (ANP), brain natriuretic peptide (BNP) and ß-myosin heavy chain (ß-MHC), decrease the content of intracellular calcium induced by Ang II. In vivo experiments in mice confirmed that intravenous injection of silicate ions could remarkably inhibit the cardiac hypertrophy and promote the formation of capillaries, further alleviating Ang II-induced cardiac function disorder. CONCLUSION: This study demonstrated that the released silicate ions from CS possessed potential value as a novel therapeutic strategy of pathological cardiac hypertrophy, which provided a new insight for clinical trials.

A biomaterial-based therapy for lower limb ischemia using Sr/Si bioactive hydrogel that inhibits skeletal muscle necrosis and enhances angiogenesis.

Muscle necrosis and angiogenesis are two major challenges in the treatment of lower-limb ischemic diseases. In this study, a triple-functional Sr/Si-containing bioceramic/alginate composite hydrogel with simultaneous bioactivity in enhancing angiogenesis, regulating inflammation, and inhibiting muscle necrosis was designed to treat lower-limb ischemic diseases. In particular, sodium alginate, calcium silicate and strontium carbonate were used to prepare injectable hydrogels, which was gelled within 10 min. More importantly, this composite hydrogel sustainedly releases bioactive Sr2+ and SiO3 2- ions within 28 days. The biological activity of the bioactive ions released from the hydrogels was verified on HUVECs, SMCs, C2C12 and Raw 264.7 cells in vitro, and the therapeutic effect of the hydrogel was confirmed using C57BL/6 mouse model of femoral artery ligation in vivo. The results showed that the composite hydrogel stimulated angiogenesis, developed new collateral capillaries, and re-established the blood supply. In addition, the bioactive hydrogel directly promoted the expression of muscle-regulating factors (MyoG and MyoD) to protect skeletal muscle from necrosis, inhibited M1 polarization, and promoted M2 polarization of macrophages to reduce inflammation, thereby protecting skeletal muscle cells and indirectly promoting vascularization. Our results indicate that these bioceramic/alginate composite bioactive hydrogels are effective biomaterials for treating hindlimb ischemia and suggest that biomaterial-based approaches may have remarkable potential in treating ischemic diseases.

Mild Heat-Assisted Polydopamine/Alginate Hydrogel Containing Low-Dose Nanoselenium for Facilitating Infected Wound Healing.

In clinical practice, it has become urgent to develop multifunctional wound dressings that can combat infection and prompt wound healing simultaneously. In this study, we proposed a polydopamine/alginate/nanoselenium composite hydrogel (Alg-PDA-Se) for the treatment of infected wounds. In particular, polydopamine endows the composite hydrogel with controllable near-infrared photothermal properties, while low-dosage selenium nanoparticles (Se NPs) offer excellent anti-oxidation, anti-inflammatory, pro-proliferative, pro-migration, and pro-angiogenic performances, which are verified by multiple cells, including macrophages, fibroblasts, and endothelial cells. More interestingly, the combination of mild temperature with low-dosage Se NPs produces a synergistic effect on combating both Staphylococcus aureus (S. aureus) and Escherichia coli (E. coli) and promoting the healing of bacteria-infected wounds in vivo. We anticipate that the designed composite hydrogel might be a potential candidate for anti-infection bioactive dressing.

Gelatin Microspheres Loaded with Wharton's Jelly Mesenchymal Stem Cells Promote Acute Full-Thickness Skin Wound Healing and Regeneration in Mice.

Objective: At present, there is an urgent need to develop a novel and practical therapeutic approach to accelerate the healing of acute wounds. Mesenchymal stem cell (MSC)-based therapy is emerging as a promising therapeutic approach for acute skin wounds. However, there are still challenges in clinical application of this strategy, such as low survivability, low retention time, and less engraftment in skin wounds. Approach: Wharton's jelly mesenchymal stem cells (WJMSCs) were seeded into three-dimensional (3D) gelatin microspheres (GMs) to identify the biocompatibility of GMs. WJMSCs were embedded in GMs and then encapsulated with Pluronic F-127 (PF-127) and sodium ascorbyl phosphate (SAP) combination to transplant onto acute full-thickness skin wound in mice. Histology, immunohistochemistry, and immunofluorescence assay were used to investigate the skin wound healing, dermis regeneration, collagen deposition, cell proliferation, and neovascularization. Results: Three-dimensional GM had strong biocompatibility, compared with two-dimensional adherent culturing, GM loading increased the cell viability and proliferation ability of WJMSCs. WJMSCs+GM+PF-127+SAP transplantation increased skin wound healing rate, dermis regeneration, and type III collagen deposition through improving macrophage polarization, cell proliferation, neovascularization, cell retention, and engraftment at skin wound site. Innovation: The effective 3D encapsulation technology for WJMSCs solved the main problems of cell activity and residence time during MSC transplantation. WJMSCs+GM+PF-127+SAP transplantation will be a new and effective MSC biomaterials-based therapeutic strategy for acute skin traumatic wounds. Conclusion: WJMSCs+GM+PF-127+SAP transplantation facilitated acute full-thickness skin wound healing and regeneration and might be a new and effective therapy for acute skin traumatic wounds.

Insufficient HtrA2 causes meiotic defects in aging germinal vesicle oocytes.

BACKGROUND: High-temperature requirement protease A2 (HtrA2/Omi) is a mitochondrial chaperone that is highly conserved from bacteria to humans. It plays an important role in mitochondrial homeostasis and apoptosis. In this study, we investigated the role of HtrA2 in mouse oocyte maturation. METHODS: The role of HtrA2 in mouse oocyte maturation was investigated by employing knockdown (KD) or overexpression (OE) of HtrA2 in young or old germinal vesicle (GV) oocytes. We employed immunoblotting, immunostaining, fluorescent intensity quantification to test the HtrA2 knockdown on the GV oocyte maturation progression, spindle assembly checkpoint, mitochondrial distribution, spindle organization, chromosome alignment, actin polymerization, DNA damage and chromosome numbers and acetylated tubulin levels. RESULTS: We observed a significant reduction in HtrA2 protein levels in aging germinal vesicle (GV) oocytes. Young oocytes with low levels of HtrA2 due to siRNA knockdown were unable to complete meiosis and were partially blocked at metaphase I (MI). They also displayed significantly more BubR1 on kinetochores, indicating that the spindle assembly checkpoint was triggered at MI. Extrusion of the first polar body (Pb1) was significantly less frequent and oocytes with large polar bodies were observed when HtrA2 was depleted. In addition, HtrA2 knockdown induced meiotic spindle/chromosome disorganization, leading to aneuploidy at metaphase II (MII), possibly due to the elevated level of acetylated tubulin. Importantly, overexpression of HtrA2 partially rescued spindle/chromosome disorganization and reduced the rate of aneuploidy in aging GV oocytes. CONCLUSIONS: Collectively, our data suggest that HtrA2 is a key regulator of oocyte maturation, and its deficiency with age appears to contribute to reproduction failure in females.

Transplantation of Wharton's jelly mesenchymal stem cells encapsulated with Hydroactive® Gel promotes diabetic wound antifibrotic healing in type 2 diabetic rats.

Diabetic cutaneous ulcers (DCU) are a complication for diabetes patients, mostly occurring in the foot and causing non-healing diabetic foot ulcers. Mesenchymal stem cell (MSC)-based therapy is currently being investigated as a therapeutic avenue for chronic diabetic ulcers. However, poor engraftment, short retention, and low survival still limit the treatment effectiveness. Hydroactive® Gel is a sterile transparent gel made of natural hydrocolloid, which has been widely used for wound management. Whether transplantation of Wharton's jelly mesenchymal stem cells (WJMSCs) encapsulated with Hydroactive® Gel is helpful to diabetic ulcers wound healing remains to be explored. The biocompatibility experiments showed that WJMSCs embedded in Hydroactive® Gel did not influence the cell viability, survival, proliferation, and apoptosis of WJMSCs in vitro. RNA-seq results also implied that Hydroactive® Gel + WJMSCs transplantation activated the "cytokine-cytokine receptor interaction", "mononuclear cell differentiation", "regulation of cell-cell adhesion", and "chemokine receptor activity" to accelerate the inflammatory reaction and epidermis regeneration in diabetic wounds. Histological analysis results demonstrated that Hydroactive® Gel encapsulated WJMSCs transplantation promoted diabetic wound healing and regeneration, indicating improved dermis regeneration, sebaceous gland formation, and type III collagen fiber deposition. Besides, immunohistochemical analysis results showed that Hydroactive® Gel + WJMSCs transplantation also facilitated the transformation of pro-inflammatory M1 macrophages to anti-inflammatory M2 macrophages, cell proliferation, and neovascularization at the wound site. Hydroactive® Gel encapsulation further prolonged the retention time of WJMSCs at the diabetic wound site. Above all, Hydroactive® Gel accelerates WJMSCs-mediated diabetic wound healing by promoting macrophage transformation, facilitating cell proliferation and angiogenesis, and prolonging cell retention time. Our findings may potentially provide a useful therapeutic strategy based on the combination of WJMSCs and biomedical materials for patients with diabetic cutaneous ulcers.

Putative MicroRNA-mRNA Networks Upon Mdfi Overexpression in C2C12 Cell Differentiation and Muscle Fiber Type Transformation.

Mdfi, an inhibitor of myogenic regulatory factors, is involved in myoblast myogenic development and muscle fiber type transformation. However, the regulatory network of Mdfi regulating myoblasts has not been revealed. In this study, we performed microRNAs (miRNAs)-seq on Mdfi overexpression (Mdfi-OE) and wild-type (WT) C2C12 cells to establish the regulatory networks. Comparative analyses of Mdfi-OE vs. WT identified 66 differentially expressed miRNAs (DEMs). Enrichment analysis of the target genes suggested that DEMs may be involved in myoblast differentiation and muscle fiber type transformation through MAPK, Wnt, PI3K-Akt, mTOR, and calcium signaling pathways. miRNA-mRNA interaction networks were suggested along with ten hub miRNAs and five hub genes. We also identified eight hub miRNAs and eleven hub genes in the networks of muscle fiber type transformation. Hub miRNAs mainly play key regulatory roles in muscle fiber type transformation through the PI3K-Akt, MAPK, cAMP, and calcium signaling pathways. Particularly, the three hub miRNAs (miR-335-3p, miR-494-3p, and miR-709) may be involved in both myogenic differentiation and muscle fiber type transformation. These hub miRNAs and genes might serve as candidate biomarkers for the treatment of muscle- and metabolic-related diseases.

Wharton's jelly mesenchymal stem cells embedded in PF-127 hydrogel plus sodium ascorbyl phosphate combination promote diabetic wound healing in type 2 diabetic rat.

BACKGROUND: Diabetic cutaneous ulcers (DCU) are a complication of diabetes with diabetic foot ulcers being the most common, and the wounds are difficult to heal, increasing the risk of bacterial infection. Cell-based therapy utilizing mesenchymal stem cells (MSCs) is currently being investigated as a therapeutic avenue for both chronic diabetic ulcers and severe burns. Wharton's jelly mesenchymal stem cell (WJMSC) with PF-127 hydrogel and sodium ascorbyl phosphate (SAP) improved skin wound healing in mice. Whether this combination strategy is helpful to diabetic ulcers wound healing remains to be explored. METHODS: Firstly, the WJMSCs embedded in PF-127 and SAP combination were transplanted onto excisional cutaneous wound bed in type 2 diabetic Sprague Dawley (SD) rats. Two weeks after transplantation, the skin tissue was collected for histological and immunohistochemical analysis. Further, overexpressing-EGFP WJMSCs were performed to investigate cell engraftment in the diabetic cutaneous ulcer. The apoptosis of WJMSCs which encapsulated with combination of PF-127 and SAP was detected by TUNEL fluorescence assay and RT-PCR in vitro. And the mitochondrial damage induced by oxidative stress assessed by MitoTracker and CMH2DCFDA fluorescence assay. RESULTS: In diabetic cutaneous wound rat model, PF-127 plus SAP-encapsulated WJMSCs transplantation promoted diabetic wound healing, indicating improving dermis regeneration and collagen deposition. In diabetic wound healing, less pro-inflammatory M1 macrophages, more anti-inflammatory M2 tissue-healing macrophages, and neovascularization were observed in PF-127 + SAP + WJMSCs group compared with other groups. SAP supplementation alleviated the apoptosis ratio of WJMSCs embedded in the PF-127 in vitro and promoted cell survival in vivo. CONCLUSION: PF-127 plus SAP combination facilitates WJMSCs-mediated diabetic wound healing in rat through promoting cell survival, the macrophage transformation, and angiogenesis. Our findings may potentially provide a helpful therapeutic strategy for patients with diabetic cutaneous ulcer.

Mdfi Promotes C2C12 Cell Differentiation and Positively Modulates Fast-to-Slow-Twitch Muscle Fiber Transformation.

Muscle development requires myoblast differentiation and muscle fiber formation. Myod family inhibitor (Mdfi) inhibits myogenic regulatory factors in NIH3T3 cells, but how Mdfi regulates myoblast myogenic development is still unclear. In the present study, we constructed an Mdfi-overexpression (Mdfi-OE) C2C12 cell line by the CRISPR/Cas9 system and performed RNA-seq on Mdfi-OE and wild-type (WT) C2C12 cells. The RNA-seq results showed that the calcium signaling pathway was the most significant. We also established the regulatory networks of Mdfi-OE on C2C12 cell differentiation and muscle fiber type transformation and identified hub genes. Further, both RNA-seq and experimental verification demonstrated that Mdfi promoted C2C12 cell differentiation by upregulating the expression of Myod, Myog, and Myosin. We also found that the positive regulation of Mdfi on fast-to-slow-twitch muscle fiber transformation is mediated by Myod, Camk2b, and its downstream genes, such as Pgc1a, Pdk4, Cs, Cox4, Acadm, Acox1, Cycs, and Atp5a1. In conclusion, our results demonstrated that Mdfi promotes C2C12 cell differentiation and positively modulates fast-to-slow-twitch muscle fiber transformation. These findings further our understanding of the regulatory mechanisms of Mdfi in myogenic development and muscle fiber type transformation. Our results suggest potential therapeutic targets for muscle- and metabolic-related diseases.

PF-127 hydrogel plus sodium ascorbyl phosphate improves Wharton's jelly mesenchymal stem cell-mediated skin wound healing in mice.

BACKGROUND: Factors such as poor engraftment, retention, and survival of the transplanted stem cells are deemed to limit their therapeutic efficacy for wound regeneration. Hence, it is necessary to explore these issues in order to resolve them. In this study, we aim to investigate the role of Pluronic F-127 (PF-127) hydrogel plus antioxidant sodium ascorbyl phosphate (SAP) in enhancing Wharton's jelly mesenchymal stem cell (WJMSC)-mediated effectiveness on full-thickness skin wound healing in mice. METHODS: First, the cytotoxicity of PF-127 and the biological effect of SAP on the survival of WJMSCs were tested in vitro using cell viability and proliferation assays. Next, a cell suspension containing WJMSCs, PF-127, and SAP was topically administered onto an 8-mm diameter excisional full-thickness wound bed. Eight days after transplantation, the mice were sacrificed and the skin tissue was excised for histological and immunohistochemical analysis. Finally, in vivo distribution of transplanted WJMSCs was traced to investigate cell engraftment and the potential therapeutic mechanism. RESULTS: PF-127 was found to be cytotoxic to WJMSCs while SAP significantly improved the survival of PF-127-embedded WJMSCs. When this combination was topically transplanted onto the wound bed, wound healing was facilitated and dermis regeneration was achieved on the 8th day after surgery, as evidenced by an increase in dermal thickness, newly developed hair follicles, and collagen fiber deposition accompanied by a reduction in scar width. Further, immunohistochemical analysis demonstrated a higher number of anti-inflammatory M2 macrophages, proliferating cells, and newly formed blood vessels in the WJMSCs/PF-127/SAP group relative to all other groups. In addition, in vivo tracking results revealed a highly enhanced engraftment of WJMSCs accumulated in the dermis in the WJMSCs/PF-127/SAP group. CONCLUSIONS: SAP significantly improves the survival of WJMSCs in PF-127 encapsulation. Further, PF-127 plus SAP is an effective combination that enhances WJMSC engraftment in the dermis, which then promotes full-thickness wound healing through potential M2 macrophage formation and angiogenesis.

MiR-27b Promotes Muscle Development by Inhibiting MDFI Expression.

BACKGROUND/AIMS: Skeletal muscle plays an essential role in the body movement. However, injuries to the skeletal muscle are common. Lifelong maintenance of skeletal muscle function largely depends on preserving the regenerative capacity of muscle. Muscle satellite cells proliferation, differentiation, and myoblast fusion play an important role in muscle regeneration after injury. Therefore, understanding of the mechanisms associated with muscle development during muscle regeneration is essential for devising the alternative treatments for muscle injury in the future. METHODS: Edu staining, qRT-PCR and western blot were used to evaluate the miR-27b effects on pig muscle satellite cells (PSCs) proliferation and differentiation in vitro. Then, we used bioinformatics analysis and dual-luciferase reporter assay to predict and confirm the miR-27b target gene. Finally, we elucidate the target gene function on muscle development in vitro and in vivo through Edu staining, qRT-PCR, western blot, H&E staining and morphological observation. RESULT: miR-27b inhibits PSCs proliferation and promotes PSCs differentiation. And the miR-27b target gene, MDFI, promotes PSCs proliferation and inhibits PSCs differentiation in vitro. Furthermore, interfering MDFI expression promotes mice muscle regeneration after injury. CONCLUSION: our results conclude that miR-27b promotes PSCs myogenesis by targeting MDFI. These results expand our understanding of muscle development mechanism in which miRNAs and genes work collaboratively in regulating skeletal muscle development. Furthermore, this finding has implications for obtaining the alternative treatments for patients with the muscle injury.

Integrated Analyses Reveal Overexpressed Notch1 Promoting Porcine Satellite Cells' Proliferation through Regulating the Cell Cycle.

Notch signaling as a conserved cell fate regulator is involved in the regulation of cell quiescence, proliferation, differentiation and postnatal tissue regeneration. However, how Notch signaling regulates porcine satellite cells (PSCs) has not been elucidated. We stably transfected Notch1 intracellular domain (N1ICD) into PSCs to analyze the gene expression profile and miRNA-seq. The analysis of the gene expression profile identified 295 differentially-expressed genes (DEGs) in proliferating-N1ICD PSCs (P-N1ICD) and nine DEGs on differentiating-N1ICD PSCs (D-N1ICD), compared with that in control groups (P-Control and D-Control, respectively). Analyzing the underlying function of DEGs showed that most of the upregulated DEGs enriched in P-N1ICD PSCs are related to the cell cycle. Forty-four and 12 known differentially-expressed miRNAs (DEMs) were identified in the P-N1ICD PSCs and D-N1ICD PSCs group, respectively. Furthermore, we constructed the gene-miRNA network of the DEGs and DEMs. In P-N1ICD PSCs, miR-125a, miR-125b, miR-10a-5p, ssc-miR-214, miR-423 and miR-149 are downregulated hub miRNAs, whose corresponding hub genes are marker of proliferation Ki-67 (MKI67) and nuclear receptor binding SET domain protein 2 (WHSC1). By contrast, miR-27a, miR-146a-5p and miR-221-3p are upregulated hub miRNAs, whose hub genes are RUNX1 translocation partner 1 (RUNX1T1) and fibroblast growth factor 2 (FGF2). All the hub miRNAs and genes are associated with cell proliferation. Quantitative RT-PCR results are consistent with the gene expression profile and miRNA-seq results. The results of our study provide valuable information for understanding the molecular mechanisms underlying Notch signaling in PSCs and skeletal muscle development.

MiR-34c represses muscle development by forming a regulatory loop with Notch1.

Since pork accounts for about 40% of global meat consumption, the pig is an important economic animal for meat production. Pig is also a useful medical model for humans due to its similarity in size and physiology. Understanding the mechanism of muscle development has great implication for animal breeding and human health. Previous studies showed porcine muscle satellite cells (PSCs) are important for postnatal skeletal muscle growth, and Notch1 signaling pathway and miRNAs regulate the skeletal muscle development. Notch1 signal pathway regulates the transcription of certain types of miRNAs which further affects target gene expression. However, the specific relationship between Notch1 and miRNAs during muscle development has not been established. We found miR-34c is decreased in PSCs overexpressed N1ICD. Through the overexpression and inhibition of mi-34c, we demonstrated that miR-34c inhibits PSCs proliferation and promotes PSCs differentiation. Using dual-luciferase reporter assay and Chromatin immunoprecipitation, we demonstrate there is a reciprocal regulatory loop between Notch1 and miR-34c. Furthermore, injection of miR-34c lentivirus into mice caused repression of gastrocnemius muscle development. In summary, our data revealed that miR-34c can form a regulatory loop with Notch1 to repress muscle development, and this result expands our understanding of muscle development mechanism.

Novel antiviral effect of lithium chloride on mammalian orthoreoviruses in vitro.

Reovirus not only causes considerable economic loss in the swine industry of the United States and other countries, but also threatens the public health due to its zoonotic potential. According to previous reports, LiCl has antiviral activity against a number of viruses. The inhibitory effects of LiCl on reovirus life cycle in Vero cells were evaluated. The unpaired t-test and one-way ANOVA were used to analyze the differences between experimental groups. We first found that LiCl treatment significantly inhibited reovirus replication in a dose-dependent manner. Furthermore, we found that this antiviral activity of LiCl targets the early stage of viral replication. LiCl could be a potential drug against reovirus infection.