Differentiation Of Muscle-derived Stem Cells Research Articles

IGF-1-induced Muscle-Derived Stem Cells as a Potential Treatment of Stress Urinary Incontinence in Female Rats.

Stress urinary incontinence (SUI) is prevalent among elderly women. This study aimed to discuss the potential of muscle-derived stem cells (MDSCs)-based therapy in treating SUI by exploring the effect of Insulin-like growth factor-1 (IGF-1) on transplanted MDSC and urethral sphincter function. Bilaterally pudendal nerve-transected (PNT) female rats were divided into four groups: sham, PNT+ phosphate buffered solution (PBS) injection, PNT+IGF-1/MDSCs and PNT+ green fluorescent protein (GFP)/MDSCs. IGF-1 was expressed in MDSCs by lentiviral vector. Viable MDSCs were detected by laser scanning confocal microscopy (LSCM). The expression of Myosin heavy chain (MyHC), vascular endothelial growth factor (VEGF), VEGF receptor 2 (VEGFR-2), microvessel density (MID) and urethral resistance function were assessed. IGF-1 promoted the survival and differentiation of MDSCs. IGF-1-expressing MDSCs facilitated local angiogenesis and muscle fiber regeneration, and alleviated symptoms of SUI. IGF-1-expressing MDSCs may be used as a novel treatment for patients with SUI.

Differentially expressed microRNAs during the differentiation of muscle-derived stem cells into insulin-producing cells, a promoting role of microRNA-708-5p/STK4 axis.

Stem cell therapy is a promising approach for diabetes via promoting the differentiation of insulin-producing cells (IPCs). This study aimed to screen the differentially expressed miRNAs (DEmiRNAs) during the differentiation of muscle-derived stem cells (MDSCs) into IPCs, and uncover the underlying function and mechanism of a specific DEmiRNA, miR-708-5p. MDSCs were successfully isolated from the leg muscle of rats, and were induced for IPCs differentiation through a five-stage protocol. miRNA microarray assay was performed for screening DEmiRNAs during differentiation. The features of MDSCs-derived IPCs were identified by qRT-PCR, flow cytometry, and immunofluorescence staining. The targeting of STK4 by miR-708-5p was examined by luciferase assay. The protein expression of STK4, YAP1, and p-YAP1 was determined by Western blot and immunofluorescence staining. MDSCs were successfully isolated and differentiated into IPCs. A total of 12 common DEmiRNAs were obtained during five-stage differentiation. Among them, miR-708-5p that highly expressed in MDSCs-derived IPCs was selected. Overexpression of miR-708-5p upregulated some key transcription factors (Pdx1, Ngn3, Nkx2.2, Nkx6.1, Gata4, Gata6, Pax4, and Pax6) involving in IPCs differentiation, and increased insulin positive cells. In addition, STK4 was identified as the target gene of miR-708-5p. miR-708-5p overexpression downregulated the expression of STK4 and the downstream phosphorylated YAP1. There were 12 DEmiRNAs involved in the differentiation of MDSCs into IPCs. miR-708-5p promoted MDSCs differentiation into IPCs probably by targeting STK4-mediated Hippo-YAP1 signaling pathway.

Evaluating the Effects of Brain Death Physiology and Immunosuppression on the Muscle-derived Stem Cell Niche in a Large Animal Transplant Model

BACKGROUND: Muscle-derived stem cells (MDSCs) have been shown to be robust mediators of tissue regeneration and mixed chimerism in small animal murine transplant models. The effects of transplant and brain death (BD) physiology on MDSCs are understudied in large animal translational models. Our study aim was to evaluate the viability, quantity, and stem cell differentiation of MDSCs under various conditions in a porcine vascularized composite allografts (VCA) model. We hypothesized that BD negatively affect the MDSC stem cell-niche in VCA. METHODS: MDSCs were harvested from the hind-limbs of pigs under the following conditions: (1) pre-BD donor, (2) post-BD donor, (3) immunosuppressed transplanted donor VCA, and (4) immunosuppressed recipient muscle (contralateral leg). Stem cell markers were evaluated in all groups using flow cytometry with standard stem markers (CFS, APC, PERCP) and stem cell presence further confirmed in the pre-BD group with differentiation assays. Samples were harvested at the time of transplant and 7 days after transplantation. MDSC populations were isolated using a modified collagen-sorting preplate technique. Fluorescent and bright field microscopy was used at various time points (0–10 days) to evaluate cell expansion and growth. Images were analyzed for confluence and differentiation stains using ImageJ, and statistical analysis was performed using Mathematica. RESULTS: Flow-cytometry demonstrated significantly higher population of stem cell markers PERCP (27% versus 7%), CFS (43% versus 14%), and APC (61% versus 12%) in pre-BD versus post-BD groups (P = 0.001). Differentiation assays confirmed stem cell presence in the pre-BD differentiation assays, with significantly increased differentiation with later pre-plates for osteogenesis, chrondrogenesis, and adipogenesis (P < 0.01).Percent confluence at 10 days was greatest in the pre-BD and lowest in post-BD conditions (81% versus 39%; P < 0.01). The immunosuppressed transplant donor VCA showed greater percent confluence compared to the immunosuppressed recipient muscle (71% versus 55%; P < 0.01), although this remained at a lower level than the pre-BD condition (71% versus 81%; P < 0.01). CONCLUSION: BD and immunosuppression each impair MDSC niche expansion and mitosis based on time to confluence. However, our study demonstrates that successful transplantation partially reverses the negative effects of BD. Future investigations will focus on further ameliorating BD effects to optimize the MDSC stem cell niche prior to transplant.

근육유래 줄기세포의 평활근으로 분화유도를 위한 VEGF/Laminin 탑재 맴브레인

근육유래 줄기세포의 평활근으로 분화유도를 위한 VEGF/Laminin 탑재 맴브레인

Progress of Experimental Research on Differentiation of Muscle-Derived Stem Cells into Haematopoietic Lineages in Vitro -Review

Muscle-derived stem cells (MDSC) are a population of multipotent stem cells in the muscular tissue. It provide an excellent prospect of hemopathy treatment due to their superiorities, such as rich sources, convenient material resource and a high survival rate after transplantation and so on. However, there are great differences in sampling, separation, purification, and proliferation when MDSC were cultured in vitro. In addition, the proliferation conditions of the MDSC in vitro are yet unclear. The related regulatory mechanisms, which MDSC transformed into haematopoietic cells, need to be investigated. In this article, the experimental researches on the differentiation of MDSC into haematopoietic lineages are reviewed, the concrete problems discussed in this review are culture of MDSC in vitro, identification of MDSC, proleferation of MDSC, differention of MDSC in to hematopoietic lineages and so on.

Sustained Release of Bone Morphogenetic Protein 2 via Coacervate improves Muscle Derived Stem Cell Mediated Cartilage Regeneration in MIA-induced Osteoarthritis

Objectives:Individuals who participate in sports have an increased risk of osteoarthritis (OA), characterized by articular cartilage degeneration. Currently, there is no cure for OA with treatment aimed at symptom relief and improved function. Muscle-derived stem cells (MDSCs) have been shown to exhibit long-term proliferation, high self-renewal, and multipotent differentiation capabilities in vitro. Previously, we have demonstrated that murine MDSCs retrovirally transduced to express chondrogenic proteins (BMPs) differentiate into chondrocytes and enhance cartilage repair in vivo. Direct injection of therapeutic proteins can promote cartilage healing; however, they have relatively short half-lives requiring muitiple injections of high dosages. This presents a challenge in terms of maintaining adequate local BMP levels and could negatively affect both injured and normal structures and lead to side effects such as osteophyte formation. Gene therapy is a promising approach that addresses this problem; however, its utilization in clinical applications is much further down the road. In order to circumvent viral transduction of cells for cartilage regeneration, we developed a unique growth factor delivery platform comprised of native heparin and a synthetic polycation, poly(ethylene argininylaspartate diglyceride) (PEAD) incorporated with BMP2 (BMP2 coacervate). In this study, we show that sustained delivery of BMP2 via a BMP2 coacervate can induce the differentiation of MDSCs to a chondrocyte lineage for in vivo cartilage regeneration and healing in a Monoiodoacetate (MIA)-induced osteoarthritis model.Methods:mMDSCs were isolated from muscle biopsies via a modified pre-plated technique. The BMP2 coacervates were prepared as previously described. The release profiles of BMP2 coacervate were tested by ELISA. The chondrogenic effects that delivery of BMP2 had on MDSCs were evaluated by RT-PCR. The efficacy of MDSC with BMP2 coacervate were evaluated in vivo in a MIA-induced OA model in C57B6 mice. Mice received an intraarticular injection of 20μl of MIA to induce osteoarthritic lesions. Two weeks after MIA injection, mice were injected with PBS (control), MDSC + free BMP2, or MDSC + BMP2 coacervate. Histologic evaluations of cartilage regeneration were conducted at week 4. Institutional Animal Care and Use Committee approval was obtained prior to all animal studies.Results:Release profiles of BMP2 showed sustained release for more than 28 days demonstrating sustained release. The chondrogenic differentiation of MDSCs following BMP2 delivery was assayed using cell culture. The mRNA expression of Aggrecan and Col2A were significantly higher in each BMP2 group compared to control or vehicle only (P<0.05). Multi-dosage free BMP2 demonstrated significantly higher Aggrecan expression compared to single dose free BMP2 (p<0.05). Col2A and aggrecan expression in the BMP coacervate group was superior to both single and mult-dose free BMP2 delivery (p<0.05) (Fig 1A). Histologic examination demonstrated superior cartilage repair and integration in the BMP2 coacervate group (Fig. 1B).Conclusion:This study demonstrates that sustained growth factor delivery (BMP2) is a potential therapeutic option for muscle-derived stem cell based cartilage regeneration for the treatment of osteoarthritis. Our results demonstrate an effective method for prolonged exposure to BMP2 via a non-gene therapy approach which is preferred and clinically translatable.

In vitro induced differentiation of mouse muscle-derived stem cells into Schwann-like cells

In vitro induced differentiation of mouse muscle-derived stem cells into Schwann-like cells

Engineered Human Muscle Tissue from Skeletal Muscle Derived Stem Cells and Induced Pluripotent Stem Cell Derived Cardiac Cells.

During development, cardiac and skeletal muscle share major transcription factors and sarcomere proteins which were generally regarded as specific to either cardiac or skeletal muscle but not both in terminally differentiated adult cardiac or skeletal muscle. Here, we investigated whether artificial muscle constructed from human skeletal muscle derived stem cells (MDSCs) recapitulates developmental similarities between cardiac and skeletal muscle. We constructed 3-dimensional collagen-based engineered muscle tissue (EMT) using MDSCs (MDSC-EMT) and compared the biochemical and contractile properties with EMT using induced pluripotent stem (iPS) cell-derived cardiac cells (iPS-EMT). Both MDSC-EMT and iPS-EMT expressed cardiac specific troponins, fast skeletal muscle myosin heavy chain, and connexin-43 mimicking developing cardiac or skeletal muscle. At the transcriptional level, MDSC-EMT and iPS-EMT upregulated both cardiac and skeletal muscle-specific genes and expressed Nkx2.5 and Myo-D proteins. MDSC-EMT displayed intracellular calcium ion transients and responses to isoproterenol. Contractile force measurements of MDSC-EMT demonstrated functional properties of immature cardiac and skeletal muscle in both tissues. Results suggest that the EMT from MDSCs mimics developing cardiac and skeletal muscle and can serve as a useful in vitro functioning striated muscle model for investigation of stem cell differentiation and therapeutic options of MDSCs for cardiac repair.

Isolation and osteogenic differentiation of skeletal muscle-derived stem cells for bone tissue engineering

The purpose of this study was to investigate the isolation and culture of muscle‑derived stem cells (MDSCs) and their capability to differentiate into osteoblasts invitro. Skeletal muscle tissue was obtained from double hind limbs of New Zealand white rabbits under sterile conditions and isolated by collagenase digestion. Following passages in basic medium, the primary cells were desmin(+), myosin(+) and CD105(+). Differentiation of MDSCs was induced by osteogenic medium. Using a 3‑(4,5‑dimethylthiazol‑2‑yl)‑2,5‑diphenyl tetrazolium bromide assay, the differentiated cell population was found to proliferate faster than the undifferentiated. Alkaline phosphatase staining and alizarin red staining revealed that the differentiated cells were mineralized invitro. Quantitative polymerase chain reaction assays also showed increased mRNA expression of osteogenic genes in differentiated cells. In conclusion, stem cells were successfully isolated and cultured from rabbit skeletal muscle tissue and were able to differentiate into osteoblasts following induction. These observations may indicate an ideal stem cell source for tissue engineering.

Differentiation of muscle-derived stem cells in the rat penile corpus cavernosum into endothelial cells in vitro

Objective To observe the potential of the rat penile corpus cavernosum muscle-derived stem cells (MDSCs) differentiation into endothelial cells (ECs) in the presence of vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (bFGF) in vitro.Methods MDSCs were purified through density gradient centrifugation and differential attachment.PP1-PP6 cells were obtained,and the expression of stem cell marker Sea-1 and myoblast marker Desmim in PP6 cells was detected by using immunofluorescence.The PP6 cells at third passage were induced to differentiate into endothelial cells within 21 days after VEGF and bFGF stimulation.The differentiating MDSCs were assessed by using immunofluorescence of ECs markers [CD31,fetal liver kinase-1 (Flk-1)],reverse transcriptase-polymerase chain reaction (RT-PCR) of ECs markers [CD31,endothelial nitric oxide synthase (eNOS),Flk-1] and stem cell markers (Oct-4) at 0,5,10,15 and 21 days.Dil-Ac-LDL uptake test was used to test the ability of cells taking up acetylated LDL,and scratch tests was used to observe the scratch space distance after 24 h.Results We observed that PP6 cells expressed MDSCs markers (Desmin,Sca-1) and the differentiating MDSCs expressed ECs markers (CD31,Flk-1) within 21 days after VEGF and bFGF stimulation.As compared with 0 day,the expression of Oct-4 was declined by 0.445 ± 0.025 times,and that of ECs-specific marker mRNA was significantly increased in the differentiated MDSCs at 21 days by 2.541 ± 0.146,2.364 ± 0.175 and 2.538 ± 0.100 times for C D31,eNOS and Flk-1,respectively (P ＜ 0.05 for all).An increased uptake of Dil-Ac-LDL in MDSCs-derived ECs was observed at 21 days compared to undifferentiated MDSCs,and the migration ability of MDSCs-derived ECs was significantly increased.The scratch space distance of differentiated MDSCs was 282.01 ± 3.84 and that of undifferentiated MDSCs was 450.35 ± 1.86 with the difference between statistically significant (P ＜ 0.05).Conclusion The rat penile corpus cavernosum MDSCs can differentiate into ECs in the presence of VEGF and bFGF in vitro and differentiated MDSCs have the typical functions for mature ECs. Key words: Penile corpus cavernosum; Muscle-derived stem cells; Vascular endothelial growth factor; Basic fibroblast growth factor; Differentiation; Endothelial cells

Induction of Neuronal Differentiation of Rat Muscle-Derived Stem Cells in Vitro Using Basic Fibroblast Growth Factor and Ethosuximide

Several studies have demonstrated that basic fibroblast growth factor (bFGF) can induce neural differentiation of mesenchymal stem cells. In this study, we investigated the neural differentiation of muscle-derived stem cells (MDSCs) following treatment with bFGF and ethosuximide, a small molecule used as an anticonvulsant in humans. Stem cells isolated from rat skeletal muscle (rMDSCs) were pre-induced by culturing with 25 ng/mL bFGF for 24 h and then were transferred to a medium supplemented with or without 4 mM ethosuximide. Neuronal differentiation was assessed by immunocytochemical and western blotting analyses of marker expression. Immunocytochemistry of rMDSCs treated with bFGF and ethosuximide identified abundant cells expressing neuronal markers (TuJ1, neuron-specific class III β-tubulin; NeuN, neuronal nuclear antigen; and NF-MH; neurofilament M and H). Olig2 (oligodendrocyte transcription factor 2)-positive cells were also observed, indicating the presence of oligodendrocyte lineage cells. These findings were substantiated by western blotting analysis of marker proteins. In particular, the expression of NeuN and TuJ1 was significantly higher in rMDSCs treated with ethosuximide and bFGF than in cells stimulated with bFGF alone (NeuN, p < 0.05 and TuJ1, p < 0.001). Expression of the astrocyte marker GFAP (glial fibrillary acidic protein) was not detected in this study. Collectively, the results showed that treatment with bFGF and ethosuximide induced effective transdifferentiation of rMDSCs into cells with a neural-like phenotype. Notably, rMDSCs treated with a combination of bFGF plus ethosuximide showed enhanced differentiation compared with cells treated with bFGF alone, implying that ethosuximide may stimulate neuronal differentiation.

Role of TGF-β1 in the process of differentiation of denervated skeletal muscle-derived stem cells in vitro

Objective To investigate the effect on differentiation of denervated skeletal muscle-derived stem cells (MDSCs) induced by TGF-β1 in vitro.Methods MDSCs were obtained from the rat denervated skeletal muscle by preplate technique,with TGF-β1 adding on medium.Cultured cells were divided into two groups.A,control group; B,10 ng/ml TGF-β1 group.Cell growth was observed with phase contrast microscope.lmmunocytochemistry,quantitative RT-PCR and Western blot was used to detect the expression of Sca-1,COL-Ⅰ,COL-Ⅲ,α-SMA and vimentin in denervated MDSCs.Results The synthesis of COL-Ⅰ,COL-Ⅲ,α-SMA and vimentin by denervated MDSCs was extremely low at protein level in vitro,while Sca-1 level was really high.Belong to the treatment with TGF-β1,COL-Ⅰ,COL-Ⅲ,oα-SMA and vimentin in the denervated MDSCs had strong expression,but Sca-1 in which had a weak expression.Under the stimulation of TGF-β1,COL-Ⅰ expression reached peak at the 2nd day (12.5591 ± 0.3389),which was about 3 times as control group.COL-Ⅲ reached highest value at the 5th day (0.8956 ± 0.0438),which was about 23 times as control group.α-SMA topped out to 18 times at the 5th day (1.1090 ± 0.0018).Vimentin expression rose by 8.5 times and peaked at the 5th day (0.1794 ± 0.0019).The expression of Sca-1 began to decline at the 2nd day,with a remarkable reduction at the 5th day (0.0636 ± 0.0015).Conclusion TGF-β1 could induce differentiation of the denervated MDSCs to myofibroblasts in vitro,and promote the synthesis and excretion of extracellular matrix. Key words: TGF-β1; Muscle-derived stem cells; Myofibroblasts; Denervated skeletal muscle

NF-κB Negatively Impacts the Myogenic Potential of Muscle-derived Stem Cells

Inhibition of the inhibitor of kappa B kinase (IKK)/nuclear factor-kappa B (NF-κB) pathway enhances muscle regeneration in injured and diseased skeletal muscle, but it is unclear exactly how this pathway contributes to the regeneration process. In this study, we examined the role of NF-κB in regulating the proliferation and differentiation of muscle-derived stem cells (MDSCs). MDSCs isolated from the skeletal muscles of p65(+/-) mice (haploinsufficient for the p65 subunit of NF-κB) had enhanced proliferation and myogenic differentiation compared to MDSCs isolated from wild-type (wt) littermates. In addition, selective pharmacological inhibition of IKKβ, an upstream activator of NF-κB, enhanced wt MDSC differentiation into myotubes in vitro. The p65(+/-) MDSCs also displayed a higher muscle regeneration index than wt MDSCs following implantation into adult mice with muscular dystrophy. Additionally, using a muscle injury model, we observed that p65(+/-) MDSC engraftments were associated with reduced inflammation and necrosis. These results suggest that inhibition of the IKK/NF-κB pathway represents an effective approach to improve the myogenic regenerative potential of MDSCs and possibly other adult stem cell populations. Moreover, our results suggest that the improved muscle regeneration observed following inhibition of IKK/NF-κB, is mediated, at least in part, through enhanced stem cell proliferation and myogenic potential.

BMP9-Induced Osteogenetic Differentiation and Bone Formation of Muscle-Derived Stem Cells

Efficient osteogenetic differentiation and bone formation from muscle-derived stem cells (MDSCs) should have potential clinical applications in treating nonunion fracture healing or bone defects. Here, we investigate osteogenetic differentiation ability of MDSCs induced by bone morphogenetic protein 9 (BMP9) in vitro and bone formation ability in rabbit radius defects repairing model. Rabbit's MDSCs were extracted by type I collagenase and trypsin methods, and BMP9 was introduced into MDSCs by infection with recombinant adenovirus. Effects of BMP9-induced osteogenetic differentiation of MDSCs were identified with alkaline phosphatase (ALP) activity and expression of later marker. In stem-cell implantation assay, MDSCs have also shown valuable potential bone formation ability induced by BMP9 in rabbit radius defects repairing test. Taken together, our findings suggest that MDSCs are potentiated osteogenetic stem cells which can be induced by BMP9 to treat large segmental bone defects, nonunion fracture, and/or osteoporotic fracture.

Inhibitory effect of Salvia miltiorrhiza on myofibroblastic differentiation of skeletal muscle-derived stem cells induced by transformation growth factor-β1

Objective To investigate the inhibitory effect of Salvia miltiorrhiza on differentiation of skeletal muscle-derived stem cells into myofibroblasts.Methods MDSCs in SD rats were isolated by differential adhesion method.Differentiation of MDSCs was induced by transformation growth factor-β1 ( TGF-β1 )administration.The cells cultivated in vitro was divided into three groups:control group (A),10 μg/LTGF-β1 group ( B),10 μg/L TGF-β1 + 150 mg/L Salvia miltiorrhiza group (C).The mRNA expression levels of α-smooth muscle actin (α-SMA) and Vimentin in each group were detected by quantiteve realtime reverse transcription-polymerase chain reaction (qRT-PCR) and there protein secretions were detected by Western blotting.Results As compared with group A,the mRNA after TGF-31 intervention and protein expression of α-SMA and Vimentin at the 3rd,4nd,6nd,8nd day in groups B and C were prominently increased (P ＜ 0.05 ).As Compared with group B,the mRNA and protein expression levels of α-SMA and Vimentin in group C were obviously down-regulated (P ＜ 0.05 ).Conclusion Salvia miltiorrhiza could inhibit myofibroblastic differentiation and further ameliorate fibrosis of denervated skeletal muscles. Key words: Salvia miltiorrhiza; TGF-β1 ; MDSCs; Myofibroblasts

The role of histone acetyltransferases in cardiomyogenesis

Wnt signaling plays a crucial role in regulating cell proliferation, differentiation and inducing cardiomyogenesis. Skeletal muscle-derived stem cells (MDSCs) have been shown to be multipotent; however, their potential to aid in the healing of the heart after myocardial infarction appears to be due to the paracrine effects they impart on the host environment. The goal of this study was to investigate whether Wnt11 could promote the differentiation of MDSCs into cardiomyocytes and enhance the repair of infarcted myocardium. MDSCs transduced with a lentivirus encoding for Wnt11 increased mRNA and protein expression of the early cardiac markers NK2 transcription factor related 5 (NKx2.5) and Connexin43 (Cx43) and also led to an increased expression of late-stage cardiac markers including: α, β-myosin heavy chain (MHC) and brain natriuretic protein (BNP) at the mRNA level, and MHC and Troponin I (TnI) at the protein level. We also observed that Wnt11 expression significantly enhanced c-jun N-terminal kinase activity in transduced MDSCs, and that some of the cells beat spontaneously but are not fully differentiated cardiomyocytes. Finally, lentivirus-Wnt11-transduced MDSCs showed greater survival and cardiac differentiation after being transplanted into acutely infarct-injured myocardium. These findings could one day lead to strategies that could be utilized in cardiomyoplasty treatments of myocardial infarction.

Inhibition of Fibroblast Differentiation of Muscle-Derived Stem Cells in Cell Implantation Treatment of Stress Urinary Incontinence

The aim of this study was to investigate the effects of transforming growth factor-β1 (TGF-β1) stimulation and the blocking of the TGF-β1/Smad3 signaling pathway by vector-mediated Smad3 shRNA on muscle-derived stem cells (MDSCs) in cell implantation treatment of stress urinary incontinence (SUI) of the rat. MDSCs were infected with the GC-shSmad3 lentivirus vector. Five days after infection, the cells were treated with TGF-β1. The expression levels of desmin (a marker of muscle differentiation) and vimentin (a marker of fibroblast differentiation) were tested by real-time PCR and Western blot. GC-shSmad3 lentivirus-infected MDSCs were injected into the bladder neck and proximal urethra of SUI rats. Urodynamic test was used to measure leak point pressure (LPP) at 2 weeks and 4 weeks after MDSC transplantation. Upregulated expression of vimentin and downregulated expression of desmin were found in MDSCs after culture with TGF-β1 in vitro. GC-shSmad3 lentivirus infection inhibited fibroblast differentiation of MDSCs but allowed muscle differentiation with desmin expression. In vivo experiments showed that GC-shSmad3 lentivirus infection could improve MDSC-mediated repairing of urethra sphincter function. In conclusion, blocking Smad3 expression inhibits the fibroblast differentiation of MDSCs induced by TGF-β1 in vitro and improves the repairing of urethral sphincter function by inhibiting the fibroblast differentiation of MDSCs in a rat model of SUI in vivo.

Lentivirus-mediated Wnt11 Gene Transfer Enhances Cardiomyogenic Differentiation of Skeletal Muscle-derived Stem Cells

Wnt signaling plays a crucial role in regulating cell proliferation, differentiation and inducing cardiomyogenesis. Skeletal muscle-derived stem cells (MDSCs) have been shown to be multipotent; however, their potential to aid in the healing of the heart after myocardial infarction appears to be due to the paracrine effects they impart on the host environment. The goal of this study was to investigate whether Wnt11 could promote the differentiation of MDSCs into cardiomyocytes and enhance the repair of infarcted myocardium. MDSCs transduced with a lentivirus encoding for Wnt11 increased mRNA and protein expression of the early cardiac markers NK2 transcription factor related 5 (NKx2.5) and Connexin43 (Cx43) and also led to an increased expression of late-stage cardiac markers including: α, β-myosin heavy chain (MHC) and brain natriuretic protein (BNP) at the mRNA level, and MHC and Troponin I (TnI) at the protein level. We also observed that Wnt11 expression significantly enhanced c-jun N-terminal kinase activity in transduced MDSCs, and that some of the cells beat spontaneously but are not fully differentiated cardiomyocytes. Finally, lentivirus-Wnt11-transduced MDSCs showed greater survival and cardiac differentiation after being transplanted into acutely infarct-injured myocardium. These findings could one day lead to strategies that could be utilized in cardiomyoplasty treatments of myocardial infarction.

Bioactive Porous Beads as an Injectable Urethral Bulking Agent: TheirIn VitroEvaluation on Smooth Muscle Cell Differentiation

Growth factor (basic fibroblast growth factor or vascular endothelial growth factor)-immobilized polycaprolactone (PCL)/Pluronic F127 porous beads were prepared as an injectable bulking agent for effective treatment of urinary incontinence. The growth factor-immobilized porous beads may stimulate smooth muscle cell (SMC) differentiation of muscle-derived stem cells or defect tissues around urethra to improve the sphincter function (bioactive therapy) as well as to provide a bulking effect (passive therapy). The porous PCL/F127 beads were fabricated by an isolated particle-melting/melt-molding particulate-leaching method. The growth factors were easily immobilized onto the surfaces of the PCL/F127 porous beads via heparin binding and were continuously released for up to 28 days. Both growth factor-immobilized porous beads had a positive effect for the SMC differentiation of muscle-derived stem cells, as were demonstrated by the analyses of quantitative polymerase chain reactions, Western blot using SMC-specific markers, and immunohistochemical staining. In particular, the basic fibroblast growth factor-immobilized porous beads showed desirable SMC differentiation behavior that can be applied as an injectable bulking agent for the treatment of urinary incontinence.

Effect of Phosphatidyl Inositol 3-Kinase, Extracellular Signal-Regulated Kinases 1/2, and p38 Mitogen-Activated Protein Kinase Inhibition on Osteogenic Differentiation of Muscle-Derived Stem Cells

Skeletal muscle-derived stem cells (MDSCs) can undergo osteogenesis when treated with bone morphogenetic proteins (BMPs), making them a potential cell source for bone tissue engineering. The signaling pathways that regulate BMP4-induced osteogenesis in MDSCs are not well understood, although they may provide a means to better regulate differentiation during bone regeneration. The objective of this study was to characterize the signaling pathways involved in the BMP4-induced osteogenesis of MDSCs. Cells were treated with BMP4 and specific inhibitors to the extracellular signal-regulated kinases 1/2 (ERK1/2), p38 mitogen-activated protein kinase (MAPK), and phosphatidyl inositol 3-kinase (PI3K) pathways (PD98059, SB203580, and Ly294002, respectively). Cellular proliferation, expression of osteoblast-related genes, alkaline phosphatase (ALP) activity, and tissue mineralization were measured to determine the role of each pathway in the osteogenic differentiation of MDSCs. Inhibition of the ERK1/2 pathway increased ALP activity and mineralization, whereas inhibition of the p38 MAPK pathway decreased osteogenesis, suggesting opposing roles of these pathways in the BMP4-induced osteogenesis of MDSCs. Inhibition of the PI3K pathway significantly increased mineralization by MDSCs. These findings highlight the involvement of the ERK1/2, p38 MAPK, and PI3K pathways in opposing capacities in MDSC differentiation and warrant further investigation, as it may identify novel therapeutic targets for the development of stem cell-based therapies for bone tissue engineering.