Human Muscle-derived Stem Cells Research Articles

Human MuStem cells are competent to fuse with nonhuman primate myofibers in a clinically relevant transplantation context: A proof-of-concept study.

We previously reported that human muscle-derived stem cells (hMuStem cells) contribute to tissue repair after local administration into injured skeletal muscle or infarcted heart in immunodeficient rodent models. However, extrapolation of these findings to a clinical context is problematic owing to the considerable differences often seen between in vivo findings in humans versus rodents. Therefore, we investigated whether the muscle regenerative behavior of hMuStem cells is maintained in a clinically relevant transplantation context. Human MuStem cells were intramuscularly administered by high-density microinjection matrices into nonhuman primates receiving tacrolimus-based immunosuppression thereby reproducing the protocol that has so far produced the best results in clinical trials of cell therapy in myopathies. Four and 9 weeks after administration, histological analysis of cell injection sites revealed large numbers of hMuStem cell-derived nuclei in all cases. Most graft-derived nuclei were distributed in small myofiber groups in which no signs of a specific immune response were observed. Importantly, hMuStem cells contributed to simian tissue repair by fusing mainly with host myofibers, demonstrating their capacity for myofiber regeneration in this model. Together, these findings obtained in a valid preclinical model provide new insights supporting the potential of hMuStem cells in future cell therapies for muscle diseases.

Force-Mediated Endocytosis of Iron Oxide Nanoparticles for Magnetic Targeting of Stem Cells.

Stem cell therapy represents one of the most promising approaches for tissue repair and regeneration. However, the full potential of stem cell therapy remains to be realized. One major challenge is the insufficient homing and retention of stem cells at the desired sites after in vivo delivery. Here, we provide a proof-of-principle demonstration of magnetic targeting and retention of human muscle-derived stem cells (hMDSCs) in vitro through magnetic force-mediated internalization of magnetic iron oxide nanoparticles (MIONs) and the use of a micropatterned magnet. We found that the magnetic force-mediated cellular uptake of MIONs occurs through an endocytic pathway, and the MIONs were exclusively localized in the lysosomes. The intracellular MIONs had no detrimental effect on the proliferation of hMDSCs or their multilineage differentiation, and no MIONs were translocated to other cells in a coculture system. Using hMDSCs and three other cell types including human umbilical vein endothelial cells (HUVECs), human dermal fibroblasts (HDFs), and HeLa cells, we further discovered that the magnetic force-mediated MION uptake increased with MION size and decreased with cell membrane tension. We found that the cellular uptake rate was initially increased with MION concentration in solution and approached saturation. These findings provide important insight and guidance for magnetic targeting of stem cells in therapeutic applications.

Poster 107: The Use of Coacervate Sustained Release System to Identify the Most Potent BMP for Bone Regeneration

Poster 107: The Use of Coacervate Sustained Release System to Identify the Most Potent BMP for Bone Regeneration

Comparison of Autologous Blood Clots with Fibrin Sealant as Scaffolds for Promoting Human Muscle-Derived Stem Cell-Mediated Bone Regeneration.

Background. Fibrin sealant has been used as a scaffold to deliver genetically modified human muscle-derived stem cells (hMDSCs) for bone regeneration. Alternatively, autologous blood clots are safe, economic scaffolds. This study compared autologous blood clot (BC) with fibrin sealant (FS) as a scaffold to deliver lenti-BMP2/GFP-transduced hMDSCs for bone regeneration. Methods. In vitro osteogenic differentiation was performed using 3D pellet culture and evaluated using microCT and Von Kossa staining. The lenti-GFP transduced cells were then mixed with human blood for evaluation of osteogenic differentiation. Furthermore, a murine critical- sized calvarial defect model was utilized to compare BC and FS scaffolds for lenti-BMP2/GFP-transduced hMDSCs mediated bone regeneration and evaluated with micro-CT and histology. Results. Lenti-BMP2/GFP transduced hMDSCs formed significantly larger mineralized pellets than non-transduced hMDSCs. hMDSCs within the human blood clot migrated out and differentiated into ALP+ osteoblasts. In vivo, BC resulted in significantly less new bone formation within a critical-sized calvarial bone defect than FS scaffold, despite no difference observed for GFP+ donor cells, osteoclasts, and osteoblasts in the newly formed bone. Conclusions. Human lenti-BMP2/GFP-transduced hMDSCs can efficiently undergo osteogenic differentiation in vitro. Unexpectedly, the newly regenerated bone in BC group was significantly less than the FS group. The autologous blood clot scaffold is less efficacious for delivering stem cells for bone regeneration than fibrin sealant.

A comparison of BMP2 delivery by coacervate and gene therapy for promoting human muscle-derived stem cell-mediated articular cartilage repair

BackgroundOsteoarthritis and cartilage injury treatment is an unmet clinical need. Therefore, development of new approaches to treat these diseases is critically needed. Previous work in our laboratory has shown that murine muscle-derived stem cells (MDSCs) can efficiently repair articular cartilage in an osteochondral and osteoarthritis model. However, the cartilage repair capacity of human muscle-derived stem cells has not been studied which prompt this study.MethodIn this study, we tested the in vitro chondrogenesis ability of six populations of human muscle-derived stem cells (hMDSCs), before and after lenti-BMP2/GFP transduction using pellet culture and evaluated chondrogenic differentiation of via histology and Raman spectroscopy. We further compared the in vivo articular cartilage repair of hMDSCs stimulated with BMP2 delivered through coacervate sustain release technology and lenti-viral gene therapy-mediated gene delivery in a monoiodoacetate (MIA)-induced osteoarthritis (OA) model. We used microCT and histology to evaluate the cartilage repair.ResultsWe observed that all hMDSCs were able to undergo chondrogenic differentiation in vitro. As expected, lenti-BMP2/GFP transduction further enhanced the chondrogenic differentiation capacities of hMDSCs, as confirmed by Alcian blue and Col2A1staining as well as Raman spectroscopy analysis. We observed through micro-CT scanning, Col2A1 staining, and histological analyses that delivery of BMP2 with coacervate could achieve a similar articular cartilage repair to that mediated by hMDSC-LBMP2/GFP. We also found that the addition of soluble fms-like tyrosine kinase-1 (sFLT-1) protein further improved the regenerative potential of hMDSCs/BMP2 delivered through the coacervate sustain release technology. Donor cells did not primarily contribute to the repaired articular cartilage since most of the repair cells are host derived as indicated by GFP staining.ConclusionsWe conclude that the delivery of hMDSCs and BMP2 with the coacervate technology can achieve a similar cartilage repair relative to lenti-BMP2/GFP-mediated gene therapy. The use of coacervate technology to deliver BMP2/sFLT1 with hMDSCs for cartilage repair holds promise for possible clinical translation into an effective treatment modality for osteoarthritis and traumatic cartilage injury.

Characterization of the chondrogenic and osteogenic potential of male and female human muscle-derived stem cells: Implication for stem cell therapy.

People of all backgrounds are susceptible to bone and cartilage damage, and these injuries can be debilitating. Current treatments for bone and cartilage injuries are less than optimal, and we are interested in developing new approaches to treat these diseases, specifically using human muscle-derived stem cells (hMDSCs). Our lab previously demonstrated that sex differences exist between male and female murine MDSCs; thus, this paper sought to investigate whether sex differences also exist in hMDSCs. In the present study, we characterized the chondrogenic and osteogenic sex differences of hMDSCs in vitro and in vivo. We performed in vitro osteogenic and chondrogenic differentiation using hMDSC pellet cultures. As demonstrated by microCT, histology, and immunohistochemistry, male hMDSCs were more chondrogenic and osteogenic than their female counterparts in vitro. No differences were observed based on the sex of hMDSCs in osteogenic and chondrogenic gene expression and cell surface markers. For our in vivo study, we transduced hMDSCs with lenti-BMP2/GFP and transplanted these cells into critical-sized calvarial defects in mice. MicroCT results revealed that male hMDSCs regenerated more bone at 2 weeks and demonstrated higher bone density at 4 and 6 weeks than female hMDSCs. Histology demonstrated that both male and female hMDSCs regenerated functional bone. Clinical relevance: These studies reinforce that stem cells isolated from male and female patients differ in function, and we should disclose the sex of cells used in future studies. Considering sex differences of hMDSCs may help to improve cell-based therapies for autologous cell treatment of bone and cartilage damage. © 2019 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 37:1339-1349, 2019.

Muscle Derived Stem Cells and Tissue Engineering for Urological Regeneration

Human multipotent stem cells might be ideal cells for tissue regeneration. Muscle-derived stem cells (MDSC) would be a better choice to do research as it can avoid the ethical issues associated with embryonic stem cells (ESCs). In addition, self-transplantation of MDSC also provides benefit for clinical application. Human MDSC have been successfully isolated using modified preplating technique (MPT). These cells have also been purified using immunoselective method. These purified cells were proven to have the ability of differentiation. Laboratory data showed that by changing the microenvironment of the cells using different proportion of differentiating medium and different kinds of genes and growth factors would successfully differentiate these purified cells into smooth muscle cells, skeletal muscle, adipocytes, osteocytes, chondrocyte and neurogenic cells. These results proved that multipotent stem cells from human skeletal muscle tissue have been successfully isolated. Tissue engineering in Urology may become a more considered approach from idealistic claims and predictions. The approaches of tissue engineering include the management of specific cell such as MDSCs, and management of natural and synthetic biomaterials. In the near future, cell transplantation of tissue reconstructive engineering using human MDSCs (hMDSCs) would offer further benefits to people for bladder reconstitution, treatment of stress urinary incontinence (SUI) and erectile dysfunction (ED).

Novel murine xenograft model for the evaluation of stem cell therapy for profound dysphagia.

Dysphagia is common and costly. Treatments are limited and innovative therapies are required. The tongue is essential for safe, effective swallowing and is a natural target for regenerative therapy. Muscle-derived stem cells (MDSCs) hold potential to restore dynamic function, and their application in the damaged tongue is appealing. We examined the safety and efficacy of human MDSC implantation into a novel mouse tongue model. Animal study. Adult immune-deficient mice were randomized to surgical (hemiglossectomy) and nonsurgical groups. Animals underwent lingual injection of human MDSCs or saline (control). Groups were followed for 12 weeks. The primary outcome was MDSC survival measured by an in vivo imaging system (IVIS). Secondary outcomes included animal survival and weight. Comparisons were made using a Mann-Whitney U test with an α of .05. Human MDSCs survived to the endpoint demonstrating 132% ± 465% and 15% ± 11% bioluminescence by IVIS at 12 weeks in hemiglossectomy and nonsurgical groups, respectively. All but one animal (hemiglossectomy with saline injection) survived to the study endpoint. Mean weight increased from baseline in all groups, with the greatest change observed in hemiglossectomy mice with MDSC injection (baseline 24.5 g ± 3.9 g; delta 5.9 g ± 4.6 g), exceeding the weight gain seen in surgical control mice (baseline 24.9 g ± 4.2 g, delta 2.7 g ± 1.4 g) (P = .04). MDSCs exhibited over 100% survival at 3 months when injected into an immune-deficient hemiglossectomy mouse model. Tongue-injured animals injected with MDSCs exhibited superior weight gain after hemiglossectomy than control animals (P < .05). These data support further investigation into the use of autologous MDSCs as a potential treatment for dysphagia secondary to tongue weakness and fibrosis LEVEL OF EVIDENCE: NA Laryngoscope, 127:E359-E363, 2017.

Capacity of muscle derived stem cells and pericytes to promote tendon graft integration and ligamentization following anterior cruciate ligament reconstruction.

The aim of this study is to examine the capacity of muscle tissue preserved on hamstring tendons forming candy-stripe grafts in order to improve tendon to bone ingrowth and ligamentization. We hypothesized that muscle tissue does possess a stem cell population that could enhance the healing process of the ACL graft when preserved on the tendons. Human samples from gracilis and semitendinosus muscles were collected during ACL surgery from ten patients and from these tissue samples human muscle-derived stem cells and tendon-derived stem cells were isolated and propagated. Both stem cell populations were in-vitro differentiated into osteogenic lineage. Alkaline phosphatase activity was determined at days zero and 14 of the osteogenic induction and von Kossa staining to assess mineralization of the cultures. Total RNA was collected from osteoblast cultures and real time quantitative PCR was performed. Western-blot for osteocalcin and collagen type I followed protein isolation. Immunofluorescence double labeling of pericytes in muscle and tendon tissue was performed. Mesenchymal stem cells from muscle and tendon tissue were isolated and expanded in cell culture. More time was needed to grow the tendon derived culture compared to muscle derived culture. Muscle derived stem cells exhibited more alkaline phosphatase actvity compared to tendon derived stem cells, whereas tendon derived stem cells formed more mineralized nodules after 14days of osteoinduction. Muscle derived stem cells exhibited higher expression levels of bone sialoprotein, and tendon derived stem cells showed higher expression of dental-matrix-protein 1 and osteocalcin. Immunofluorescent staining against pericytes indicated that they are more abundant in muscle tissue. These results indicate that muscle tissue is a better source of stem cells than tendon tissue. Achievement of this study is proof that there is vast innate capacity of muscle tissue for enhancement of bone-tendon integration and ligamentization of ACL hamstring grafts and consequently muscle tissue should not be treated as waste after harvesting.

240. Lenti-BMP2/GFP Gene Therapy Enhance the Chondrogenic and Osteogenic Differentiation of Human Muscle Derived Stem Cells In Vitro

Human muscle derived stem cells (hMDSCs) have been shown to be multipotent in vitro and can repair critical size bone defects as efficiently as human bone marrow stem cells, but require BMPs. In this study, we constructed a new lenti-BMP2/GFP vector and tested its ability to promote in vitro chondrogenesis and osteogenesis of the hMDSCs. Material and methods: 1. Lenti-BMP2/GFP was made by inserting human BMP2 gene up-stream of an eGFP tag gene in a Lenti-viral vector with a CMV promoter. 2. hMDSCs were isolated from skeletal muscle via the preplate technique. All 6 populations were transduced with the Lenti-BMP2/GFP virus, expanded and sorted via FACS based on GFP fluorescence. BMP2 secretion was measured using an R&D ELISA kit. 3. Osteogenesis:3D pellet cultures were used for osteogenic differentiation of non-transduced and Lenti-BMP2/GFP transduced hMDSCs. After 4 weeks, the cell pellets were fixed and scanned using VivaCT40 to detect mineralization. The pellets were embedded in NEG medium and snap frozen in liquid nitrogen, sectioned and von Kossa stained. Osteogenic differentiation was detected by staining for osteocalcin. 4. Chondrogenesis was performed on non-transduced and Lenti-BMP2/GFP transduced hMDSCs using a 3D pellet culture method. Cell pellets were cultured for 24 days in chondrogenic medium, embedded in freezing medium and cryosectioned. Alcian blue staining and Col2A1 immunohistochemistry was also performed. Raman Spectroscopy was used to quantify chondroitin sulphate and collagen. Results: 1. Lenti-BMP2/GFP transduction enhanced osteogenesis of the hMDSCs. BMP2 secretion levels of the sorted lenti-BMP2/GFP transduced cells are showed in Fig. 1AFig. 1A. MicroCT results of the lenti-BMP2/GFP transduced cells showed they had an increase in mineralized pellet volume (Fig. 1B, CFig. 1B, C). Von Kossa staining indicated that the non-transduced hMDSCs had variable brown-black mineralization while the lenti-BMP2/GFP transduced cells all showed stronger mineralization (Fig. 1DFig. 1D). Osteocalcin staining showed lenti-BMP2/GFP transduction enhanced osteogenic differentiation of the hMDSCs (Fig. 1EFig. 1E). 2. Lenti-BMP2/GFP transduction enhanced chondrogenesis of the hMDSCs. Alcian blue staining showed a significantly bluer chondrogenic matrix in the Lenti-BMP2/GFP transduced cell pellets compared to non-transduced hMDSCs (Fig2A and 2BFig2A and 2B). Raman Spectroscopy indicated the Lenti-BMP2/GFP transduced hMDSC exhibited higher amounts of chondroitin sulphate and collagen (Fig. 2C, DFig. 2C, D). Col2A1 staining showed more Col2A1 in the lenti-BMP2/GFP transduced hMDSCs compared to non-transduced hMDSCs (Fig. 2EFig. 2E). Conclusion: We found that all the Lenti-BMP2/GFP transduced hMDSCs showed enhanced osteogenesis and chonrogenesis compared to non-transduced cells. This in vitro study lays the foundation for using Lenti-BMP2/GFP gene transfer to repair bone and cartilage defects.View Large Image | Download PowerPoint SlideView Large Image | Download PowerPoint Slide

446. Gender Differences in the Osteogenic & Chondrogenic Potential of Human Muscle-Derived Stem Cells In Vitro

Introduction: It has been shown that murine muscle-derived stem cells (MDSCs) are capable of multipotent differentiation1xSee all References. These murine cells exhibit sexual dimorphism of osteogenic and chondrogenic potentials1xSee all References. Previous studies have shown that male mouse MDSCs (M-MDSCs) differentiate into chondrocytes more effectively and display better cartilage regeneration potentials than female MDSCs (F-MDSCs)2xSee all References. M-MDSCs have also been shown to be more osteogenic and demonstrate more rapid bone formation compared to F-MDSCs1. In vivo murine studies have also demonstrated that M-MDSCs form more bone than F-MDSCs1xSee all References, 3xSee all References. Adipose derived stem cells also display sexual dimorphism in their multipotent differentiation potential4xSee all References. This study further investigates this sexual dimorphism in human muscle-derived stem cells (hMDSCs). Methods: Two populations of human MDSCs, one male and one female, were used in these experiments. (1) Osteogenesis: M- and F-hMDSCs 3D pellets were cultured in osteogenic medium containing 100ng/ml bone morphogenic protein 2 (BMP-2) for 28d according to literature1xSee all References. Cell pellets were fixed in neutral buffered formalin and subjected to micro CT scanning. After scanning, cell pellets were embedded in NEG50 freezing medium for sectioning. Von Kossa staining was used to detect mineralization. Osteocalcin immunohistochemistry (IHC) was used to detect osteogenic differentiation. (2) Chondrogenesis: M- and F-MDSC 3D pellets were cultured for 24d in chondrogenic medium using Stem Pro complete chondrogenic medium. After culture, all pellets were fixed and sectioned. Alcian blue and Col2A1 were used as indicators of chondrogenesis, and the percentage of each pellet stained with Alcian blue staining was quantified. Results: Osteogenesis: MicroCT analysis indicated that M-hMDSCs had significantly more pellet mineralization compared to F-MDSCs (p<0.001) (Fig. 1A, 1BFig. 1A, 1B). Von Kossa staining showed stronger brown-black mineral deposition in male pellets than female pellets (Fig. 1CFig. 1C). Osteocalcin IHC indicated higher expression in male hMDSCs than female hMDSCs (Fig. 1DFig. 1D). Chondrogenesis: Alcian blue staining demonstrated that male cells have stronger blue cartilage matrices than female cells (Fig. 2AFig. 2A). Quantification of the blue matrix indicated significantly more matrix in male hMDSC pellets than female pellets (Fig. 2BFig. 2B). Col2A1 IHC showed stronger staining in male hMDSCs than female hMDSCs (Fig. 2CFig. 2C). Conclusions: Our results suggest that both gender hMDSCs can undergo osteogenic and chondrogenic differentiation, but male hMDSCs are more osteogenic and chondrogenic than female hMDSCs. Therefore, sex difference is an important factor to consider in the development and application of cell-based therapies for bone and articular cartilage repair.View Large Image | Download PowerPoint SlideView Large Image | Download PowerPoint Slide

Bladder reconstruction using stem cells seeded on multilayered scaffolds in a mucosa preserving partial cystectomy model

Recently, studies have focused more towards using biocompatible scaffolds and stem cells to augment or replace the abnormal bladder. But, due to the lack of biomaterials with appropriate thickness as a suitable scaffold for smooth muscle regeneration, several structural, mechanical, and biocompatibility problems are encountered. Therefore, we aimed to demonstrate whether human muscle-derived stem cells (h-MDSCs) seeded on multilayered polycaprolactone (PCL) nanofiber is an appropriate scaffold for bladder smooth muscle regeneration. h-MDSCs were seeded on a multilayered PCL/collagen nanofiber sheet and implanted in the bladder of a mucosa preserving partial cystectomy rat. From our findings, h-MDSCs seeded on multilayered PCL showed efficient cell seeding and proliferation. In addition, the histological and immunohistochemical analysis showed cell survival in between the multilayered nanofiber sheet, which led to smooth muscle cell regeneration with improved pro-angiogenesis in the regenerated region of the bladder. Therefore, h-MDSCs seeded nanofibers could be a promising tool in treating neurogenic bladder and related diseases.

999 Bladder reconstruction using human muscle derived stem cells seeded multi-layered polycaprolactone in a mucosa preserving partial cystectomy rat model

999 Bladder reconstruction using human muscle derived stem cells seeded multi-layered polycaprolactone in a mucosa preserving partial cystectomy rat model

Combined biophysical and soluble factor modulation induces cardiomyocyte differentiation from human muscle derived stem cells.

Cellular cardiomyoplasty has emerged as a novel therapy to restore contractile function of injured failing myocardium. Human multipotent muscle derived stem cells (MDSC) can be a potential abundant, autologous cell source for cardiac repair. However, robust conditions for cardiomyocyte (CM) differentiation are not well established for this cell type. We have developed a new method for CM differentiation from human MDSC that combines 3-dimensional artificial muscle tissue (AMT) culture with temporally controlled biophysical cell aggregation and delivery of 4 soluble factors (microRNA-206 inhibitor, IWR-1, Lithium Chloride, and BMP-4) (4F-AG-AMT). The 4F-AG-AMT displayed cardiac-like response to β-adrenergic stimulation and contractile properties. 4F-AG-AMT expressed major cardiac (NKX2-5, GATA4, TBX5, MEF2C) transcription factors and structural proteins. They also express cardiac gap-junction protein, connexin-43, similar to CMs and synchronized spontaneous calcium transients. These results highlight the importance of temporal control of biophysical and soluble factors for CM differentiation from MDSCs.

BMP2 is Superior to BMP4 for Promoting Human Muscle-Derived Stem Cell-Mediated Bone Regeneration in a Critical-Sized Calvarial Defect Model

Muscle-derived cells have been successfully isolated using a variety of different methods and have been shown to possess multilineage differentiation capacities, including an ability to differentiate into articular cartilage and bone in vivo; however, the characterization of human muscle-derived stem cells (hMDSCs) and their bone regenerative capacities have not been fully investigated. Genetic modification of these cells may enhance their osteogenic capacity, which could potentially be applied to bone regenerative therapies. We found that hMDSCs, isolated by the preplate technique, consistently expressed the myogenic marker CD56, the pericyte/endothelial cell marker CD146, and the mesenchymal stem cell markers CD73, CD90, CD105, and CD44 but did not express the hematopoietic stem cell marker CD45, and they could undergo osteogenic, chondrogenic, adipogenic, and myogenic differentiation in vitro. In order to investigate the osteoinductive potential of hMDSCs, we constructed a retroviral vector expressing BMP4 and GFP and a lentiviral vector expressing BMP2. The BMP4-expressing hMDSCs were able to undergo osteogenic differentiation in vitro and exhibited enhanced mineralization compared to nontransduced cells; however, when transplanted into a calvarial defect, they failed to regenerate bone. Local administration of BMP4 protein and cell pretreatment with N-acetylcysteine (NAC), which improves cell survival, did not enhance the osteogenic capacity of the retro-BMP4-transduced cells. In contrast, lenti-BMP2-transduced hMDSCs not only exhibited enhanced in vitro osteogenic differentiation but also induced robust bone formation and nearly completely healed a critical-sized calvarial defect in CD-1 nude mice 6 weeks following transplantation. Herovici's staining of the regenerated bone demonstrated that the bone matrix contained a large amount of type I collagen. Our findings indicated that the hMDSCs are likely mesenchymal stem cells of muscle origin and that BMP2 is more efficient than BMP4 in promoting the bone regenerative capacity of the hMDSCs in vivo.

178. Human Muscle Derived Stem Cells Exhibit Bone Regeneration Capacity Similar to Bone Marrow Mesenchymal Stem Cells When Genetically Modified To Express BMP2 Both In Vitro and In Vivo

178. Human Muscle Derived Stem Cells Exhibit Bone Regeneration Capacity Similar to Bone Marrow Mesenchymal Stem Cells When Genetically Modified To Express BMP2 Both In Vitro and In Vivo

Effect of Human Muscle–derived Stem Cells on Cryoinjured Mouse Bladder Contractility

To investigate the effect of human muscle-derived stem cells (hMDSCs) on ameliorating impaired detrusor contractility in a cryoinjured bladder murine model. The hMDSCs were isolated and cultured by modified preplate technique, and only CD34-positive hMDSCs were extracted by Mini-MACS kits. Isolated hMDSCs were prelabeled with PKH26 and injected into the cryoinjured bladder to observe the pattern and characteristics. The nude mice were subdivided into three groups: normal group (N), cryoinjury bladder group with saline injection (C), and hMDSCs injection group after cryoinjury (M). At 2 weeks after injecting hMDSCs, we compared the contractility of bladder muscle strip stimulated by electrical field stimulation (EFS), acetylcholine (Ach.), and adenosine triphosphate (ATP), and the bladder smooth muscle tissue was examined by immunohistochemistry. The contractile powers of bladder muscle strip in the C group were more decreased than the N group after EFS, Ach, and ATP treatment (P < .05). The bladder contractility of the M group was more increased than in the C group (P < .05), but was lower than the N group after EFS and Ach treatment. However, there was no significant difference of contractile power between the C and M groups after ATP stimulation. In immunohistochemical staining, the thickness of the bladder smooth muscle layer in the M group was significantly increased compared with the C group, and PKH26-labeled implanted cells were positive for smooth muscle cell differentiation marker (α-SMA) in the injected region. hMDSCs injection increased cholinergic bladder contractile power but not the purinergic component of bladder contraction after cryoinjury.

Human Muscle-Derived Cell Populations Isolated by Differential Adhesion Rates: Phenotype and Contribution to Skeletal Muscle Regeneration in Mdx/SCID Mice

Muscle-derived stem cells (MDSCs) isolated from murine skeletal tissue by the preplate method have displayed the capability to commit to the myogenic lineage and regenerate more efficiently than myoblasts in skeletal and cardiac muscle in murine Duchenne Muscular Dystrophy mice (mdx). However, until now, these studies have not been translated to human muscle cells. Here, we describe the isolation, by a preplate technique, of candidate human MDSCs, which exhibit myogenic and regenerative characteristics similar to their murine counterparts. Using the preplate isolation method, we compared cells that adhere faster to the flasks, preplate 2 (PP2), and cells that adhere slower, preplate 6 (PP6). The human PP6 cells express several markers of mesenchymal stem cells and are distinct from human PP2 (a myoblast-like population) based on their expression of CD146 and myogenic markers desmin and CD56. After transplantation to the gastrocnemius muscle of mdx/SCID mice, we observe significantly higher levels of PP6 cells participating in muscle regeneration as compared with the transplantation of PP2 cells. This study supports some previous findings related to mouse preplate cells, and also identifies some differences between mouse and human muscle preplate cells.

Biological and morphological characterization of in vitro expanded human muscle-derived stem cells

Stem cells are generally characterised as clonogenic and undifferentiated cells with the capacity of self-renewal and plasticity. Over the past few years, the adult stem cells have been derived from various types of tissues including the skeletal muscle. The main goal of the present study was the isolation, in vitro expansion and characterisation of muscle-derived stem cells (MDSCs). Thereby obtained results showed that MDSCs have a fibroblast-like shape with a large nucleus having one to four nucleoli. The cytoplasm was transparent without any signs of vacuolisation. TEM analysis showed an ultrastructure of cells with high proteosynthetic activity. MDSCs had a large and irregular nucleus with variable number of nucleoli. The cytoplasm contained a richly developed and rough endoplasmic reticulum, prominent Golgi apparatus cisterns as well as transport vesicles containing glycogen granules and variable microvilli and filopodia. They expressed alpha-actin and desmin. Results of the phenotypic characterization showed that the analyzed cells were positive for CD29, CD34, CD44, CD90, CD105 and HLA Class I. They did not express CD14, CD45, CD235a, HLA Class II and human fibroblast surface protein. According to these results it should be emphasised that MDSCs after performing the detailed studies focused on their immunological properties and differentiation potential may be used in the cell therapy of many degenerative diseases.

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.