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ABSTRACT: BACKGROUND: Epigenetics is the study of changes in gene expression or cellular phenotype caused by mechanisms other than changes in the underlying DNA sequence. It is widely accepted that cancer has genetic and epigenetic origins. The idea of epigenetic reprogramming of cancer cells by an embryonic microenvironment possesses potential interest from the prospect of both basic science and potential therapeutic strategies. Chick embryo extract (CEE) has been used for the successful expansion of many specific stem cells and has demonstrated the ability to facilitate DNA demethylation. QUESTIONS/PURPOSES: The current study was conducted to compare the status of DNA methylation in highly metastatic and less metastatic osteosarcoma cells and to investigate whether CEE may affect the epigenetic regulation of tumor suppressor genes and thus change the metastatic phenotypes of highly metastatic osteosarcoma cells. METHODS: K7M2 murine OS cells were treated with CEE to determine its potential effect on DNA methylation, cell apoptosis, and invasion capacity. RESULTS: Our current results suggest that the methylation status of tumor suppressor genes (p16, p53, and E-cadherin) is significantly greater in highly metastatic mouse ostoesarcoma K7M2 cells in comparison with less metastatic mouse osteosarcoma K12 cells. CEE treatment of K7M2 cells caused demethylation of p16, p53, and E-cadherin genes, upregulated their expression, and resulted in the reversion of metastatic phenotypes in highly metastatic osteosarcoma cells. CONCLUSIONS: CEE may promote the reversion of metastatic phenotypes of osteosarcoma cells and can be a helpful tool to study osteosarcoma tumor reversion by epigenetic reprogramming. CLINICAL RELEVANCE: Demethylation of tumor suppressor genes in osteosarcoma may represent a novel strategy to diminish the metastatic potential of this neoplasm. Further studies, both in vitro and in vivo, are warranted to evaluate the clinical feasibility of this approach as an adjuvant to current therapy.
Clinical Orthopaedics and Related Research 06/2013; · 2.53 Impact Factor
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ABSTRACT: OBJECTIVE: We previously reported that mechanical stimulation increased the effectiveness of muscle-derived stem cells (MDSCs) for tissue repair. The objective of this study was to determine the importance of vascular endothelial growth factor (VEGF) on mechanically stimulated MDSCs in a murine model of muscle regeneration. APPROACH AND RESULTS: MDSCs were transduced with retroviral vectors encoding the LacZ reporter gene (lacZ-MDSCs), the soluble VEGF receptor Flt1 (sFlt1-MDSCs), or a short hairpin RNA (shRNA) targeting messenger RNA of VEGF (shRNA_VEGF MDSCs). Cells were subjected to 24 hours of mechanical cyclic strain and immediately transplanted into the gastrocnemius muscles of mdx/scid mice. Two weeks after transplantation, angiogenesis, fibrosis, and regeneration were analyzed. There was an increase in angiogenesis in the muscles transplanted with mechanically stimulated lacZ-MDSCs compared with nonstimulated lacZ-MDSCs, sFlt1-MDSCs, and shRNA _VEGF MDSCs. Dystrophin-positive myofiber regeneration was significantly lower in the shRNA_VEGF-MDSC group compared with the lacZ-MDSC and sFlt1-MDSC groups. In vitro proliferation of MDSCs was not decreased by inhibition of VEGF; however, differentiation into myotubes and adhesion to collagen were significantly lower in the shRNA_VEGF-MDSC group compared with the lacZ-MDSC and sFlt1-MDSC groups. CONCLUSIONS: The beneficial effects of mechanical stimulation on MDSC-mediated muscle repair are lost by inhibiting VEGF.
Arteriosclerosis Thrombosis and Vascular Biology 05/2013; · 6.37 Impact Factor
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ABSTRACT: Heterotopic ossification (HO) and fatty infiltration (FI) often occur in diseased skeletal muscle and have been previously described in various animal models of Duchenne muscular dystrophy (DMD); however, the pathological mechanisms remain largely unknown. Dystrophin-deficient mdx mice and dystrophin/utrophin double-knockout (dKO) mice are mouse models of DMD; however, mdx mice display a strong muscle regeneration capacity, while dKO mice exhibit a much more severe phenotype, which is similar to patients with DMD. Our results revealed that more extensive HO, but not FI, occurred in the skeletal muscle of dKO mice versus mdx mice, and RhoA activation specifically occurred at the sites of HO. Moreover, the gene expression of RhoA, BMPs, and several inflammatory factors were significantly up-regulated in muscle stem cells isolated from dKO mice; while inactivation of RhoA in the cells with RhoA/ROCK inhibitor Y-27632 led to reduced osteogenic potential and improved myogenic potential. Finally, inactivation of RhoA signaling in the dKO mice with Y-27632 improved muscle regeneration and reduced the expression of BMPs, inflammation, HO, and intramyocellular lipid accumulation in both skeletal and cardiac muscle. Our results revealed that RhoA represents a major molecular switch in the regulation of HO and muscle regeneration in dystrophic skeletal muscle of mice.-Mu, X., Usas, A., Tang, Y., Lu, A., Wang, B., Weiss, K., Huard, J. RhoA mediates defective stem cell function and heterotopic ossification in dystrophic muscle of mice.
The FASEB Journal 05/2013; · 5.71 Impact Factor
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ABSTRACT: As a new strategy for enhancing recovery after anterior cruciate ligament (ACL) reconstruction, we developed a cell sheet wrapped graft using ACL-derived CD34+ cells. Our results from an ex vivo cell migration assay demonstrated gradual cell migration into the graft from the cell sheet, and an enzyme-linked immunosorbent assay indicated that the cell sheet cultures secreted a significantly greater amount of VEGF than typical monolayer cultures. In the in vivo study, the rats received one of three possible ACL reconstruction treatments; 1) a tendon graft wrapped in an ACL-derived CD34+ cell sheet, 2) a tendon graft and ACL-derived CD34+ cell injection, or 3) the control group that received just the tendon graft. The in vivo studies revealed that the ACL-derived CD34+ cell sheet wrapped grafts demonstrated a greater number of the cells derived from the cell sheets incorporated within the bone tunnel site and grafted tendon. We believe the incorporated CD34+ cells enhanced the healing of both the bone-tendon junction and the grafted tendon in the reconstructed rat ACLs by increasing proprioceptive recovery, graft maturation, and biomechanical strength. ACL-derived CD34+ cell sheet wrapped grafts could become a promising strategy to revitalize tendon autografts and recovery after ACL reconstruction resulting in superior and quicker recovery in patients requiring ACL reconstruction.
Biomaterials 04/2013; · 7.40 Impact Factor
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Bo Zheng,
Guangheng Li,
William C W Chen,
Bridget M Deasy,
Jonathan B Pollett,
Bin Sun,
Lauren Drowley,
Burhan Gharaibeh,
Arvydas Usas,
Bruno Péault, Johnny Huard
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ABSTRACT: We have previously reported the high regenerative potential of murine muscle-derived stem cells (mMDSCs) that are capable of differentiating into multiple mesodermal cell lineages, including myogenic, endothelial, chondrocytic, and osteoblastic cells. Recently, we described a putative human counterpart of mMDSCs, the myogenic endothelial cells (MECs), in adult human skeletal muscle, which efficiently repair/regenerate the injured and dystrophic skeletal muscle as well as the ischemic heart in animal disease models. Nevertheless it remained unclear whether human MECs, at the clonal level, preserve mMDSC-like chondrogenic and osteogenic potentials and classic stem cell characteristics including high proliferation and resistance to stress. Herein, we demonstrated that MECs, sorted from fresh postnatal human skeletal muscle biopsies, can be grown clonally and exhibit robust resistance to oxidative stress with no tumorigeneity. MEC clones were capable of differentiating into chondrocytes and osteoblasts under inductive conditions in vitro and participated in cartilage and bone formation in vivo. Additionally, adipogenic and angiogenic potentials of clonal MECs (cMECs) were observed. Overall, our study showed that cMECs not only display typical properties of adult stem cells but also exhibit chondrogenic and osteogenic capacities in vitro and in vivo, suggesting their potential applications in articular cartilage and bone repair/regeneration. © 2013 Orthopaedic Research Society Published by Wiley Periodicals, Inc. J Orthop Res.
Journal of Orthopaedic Research 04/2013; · 2.81 Impact Factor
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ABSTRACT: INTRODUCTION: Loss of adult stem cell function during aging contributes to impaired tissue regeneration. Here, we tested the aging-related decline in regeneration potential of adult stem cells residing in the skeletal muscle. METHODS: We isolated muscle-derived stem/progenitor cells (MDSPCs) from progeroid Zmpste24-deficient mice (Zmpste24/) with accelerated aging phenotypes to investigate whether mutation in lamin A has an adverse effect on muscle stem/progenitor cell function. RESULTS: Our results indicate that MDSPCs isolated from Zmpste24/ mice show reduced proliferation and myogenic differentiation. In addition, Zmpste24/ MDSPCs showed impaired muscle regeneration, with a limited engraftment potential when transplanted into dystrophic muscle, compared with wild-type (WT) MDSPCs. Exposure of progeroid Zmpste24/ MDSPCs to WT MDSPCs rescued the myogenic differentiation defect in vitro. CONCLUSIONS: These results demonstrate that adult stem/progenitor cell dysfunction contributes to impairment of tissue regeneration and suggest that factors secreted by functional cells are indeed important for the therapeutic effect of adult stem cells.
Stem Cell Research & Therapy 03/2013; 4(2):33. · 3.21 Impact Factor
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ABSTRACT: Direct intracardiac cell injection for heart repair is hindered by numerous limitations including: cell death, poor spreading of the injected cells, arrhythmia, needle injury, etc. Tissue-engineered cell sheet implantation has the potential to overcome some of these limitations. We evaluated whether the transplantation of a muscle-derived stem cell (MDSC) sheet could improve the regenerative capacity of MDSCs in a chronic model of myocardial infarction. MDSC sheet-implanted mice displayed a reduction in left ventricle (LV) dilation and sustained LV contraction compared with the other groups. The MDSC sheet formed aligned myotubes and produced a significant increase in capillary density and a reduction of myocardial fibrosis compared with the other groups. Hearts transplanted with the MDSC sheets did not display any significant arrhythmias and the donor MDSC survival rate was higher than the direct myocardial MDSC injection group. MDSC sheet implantation yielded better functional recovery of chronic infarcted myocardium without any significant arrhythmic events compared with direct MDSC injection, suggesting this cell sheet delivery system could significantly improve the myocardial regenerative potential of the MDSCs.Molecular Therapy (2013); doi:10.1038/mt.2012.266.
Molecular Therapy 01/2013; · 6.87 Impact Factor
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ABSTRACT: BACKGROUND:As a result of recent studies describing the double-bundle anterior cruciate ligament (ACL), selected ACL augmentation procedures, either anteromedial (AM) or posterolateral (PL), have been introduced as the treatment of choice for partial ACL ruptures. The preserved mechanoreceptor and vascularity of the remnant ACL are considered to provide additional biological benefits. Although enhanced knee joint proprioception in ACL augmented patients has been previously reported, there is no study assessing biological healing advantages of the graft after the ACL augmentation procedure. HYPOTHESIS:Selected ACL augmentation for partial tears can accelerate the healing process of the grafted tendon, which promotes better biomechanical recovery of the tendon, compared with conventional ACL reconstruction of complete tears. STUDY DESIGN: Controlled laboratory study. METHODS:Two rat models were established in this study: an ACL augmentation partial tear model and conventional ACL reconstruction for a complete tear. Biological assessments of cellularity and angiogenesis were measured by hematoxylin and eosin staining and immunostaining, respectively. Additionally, rat-specific type III collagen and α-smooth muscle actin were evaluated by immunohistochemical staining to analyze the healing process, whereas anti-rat neurofilament antigen was assessed to examine proprioceptive recovery. Biological assessments of the augmented and reconstructed grafts were conducted postoperatively at week 2, whereas biomechanical testing was performed postoperatively at week 8. RESULTS:An increase in cellularity and angiogenesis was observed in the augmented grafts compared with the conventionally reconstructed grafts. Also, increased amounts of rat-specific type III collagen, α-smooth muscle actin, and anti-rat neurofilament antigen were expressed in the augmented grafts. Biomechanical testing showed that failure to load was significantly higher in the augmentation group compared with the conventional reconstruction group (augmentation, 15.9 ± 1.0; reconstruction, 7.0 ± 1.3; P < .01). CLINICAL RELEVANCE:Selected ACL augmentation could be a good choice for the repair of partial ACL injury by preserving the uninjured portion of the ACL, which in turn could maintain the anatomic position of the ligament and its biomechanical function.
The American journal of sports medicine 01/2013; · 3.61 Impact Factor
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ABSTRACT: Our lab developed and optimized a method, known as the modified pre-plate technique, to isolate stem/progenitor cells from skeletal muscle. This method separates different populations of myogenic cells based on their propensity to adhere to a collagen I-coated surface. Based on their surface markers and stem-like properties, including self-renewal, multi-lineage differentiation, and ability to promote tissue regeneration, the last cell fraction or slowest to adhere to the collagen-coated surface (pre-plate 6; pp6) appears to be early, quiescent progenitor cells termed muscle-derived stem/progenitor cells (MDSPCs). The cell fractions preceding pp6 (pp1-5) are likely populations of more committed (differentiated) cells, including fibroblast- and myoblast-like cells. This technique may be used to isolate MDSPCs from skeletal muscle of humans or mice regardless of age, sex or disease state, although the yield of MDSPCs varies with age and health. MDSPCs can be used for regeneration of a variety of tissues including bone, articular cartilage, skeletal and cardiac muscle, and nerve. MDSPCs are currently being tested in clinical trials for treatment of urinary incontinence and myocardial infarction. MDSPCs from young mice have also been demonstrated to extend life span and healthspan in mouse models of accelerated aging through an apparent paracrine/endocrine mechanism. Here we detail methods for isolation and characterization of MDSPCs.
Methods in molecular biology (Clifton, N.J.) 01/2013; 976:53-65.
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ABSTRACT: Osteosarcoma (OS) is the most common primary malignancy of bone. Mortality is determined by the presence of metastatic disease, but little is known regarding the biochemical events that drive metastases. Two murine OS cell lines, K7M2 and K12, are related but differ significantly in their metastatic potentials: K7M2 is highly metastatic whereas K12 displays much less metastatic potential. Using this experimental system, the mammalian target of rapamycin (mTOR) pathway has been implicated in OS metastasis. We also discovered that aldehyde dehydrogenase (ALDH, a stem cell marker) activity is higher in K7M2 cells than K12 cells. Rapamycin treatment reduces the expression and enzymatic activity of ALDH in K7M2 cells. ALDH inhibition renders these cells more susceptible to apoptotic death when exposed to oxidative stress. Furthermore, rapamycin treatment reduces bone morphogenetic protein-2 (BMP2) and vascular endothelial growth factor (VEGF) gene expression and inhibits K7M2 proliferation, migration, and invasion . Inhibition of ALDH with disulfiram correlated with decreased mTOR expression and activity. In conclusion, we provide evidence for interaction between mTOR activity, ALDH activity, and metastatic potential in murine OS cells. Our work suggests that mTOR and ALDH are therapeutic targets for the treatment and prevention of OS metastasis.
Sarcoma 01/2013; 2013:480713.
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ABSTRACT: Cellular therapy is a potential approach to improve the regenerative capacity of damaged or diseased skeletal muscle. However, its clinical use has often been limited by impaired donor cell survival, proliferation and differentiation following transplantation. Additionally, functional improvements after transplantation are all-too-often negligible. Because the host microenvironment plays an important role in the fate of transplanted cells, methods to modulate the microenvironment and guide donor cell behavior are warranted. The purpose of this study was to investigate whether the use of neuromuscular electrical stimulation (NMES) for 1 or 4 weeks following muscle-derived stem cell (MDSC) transplantation into dystrophic skeletal muscle can modulate the fate of donor cells and enhance their contribution to muscle regeneration and functional improvements. Animals submitted to 4 weeks of NMES after transplantation demonstrated a 2-fold increase in the number of dystrophin+ myofibers as compared to control transplanted muscles. These findings were concomitant with an increased vascularity in the MDSC+NMES group when compared to non-stimulated counterparts. Additionally, animals subjected to NMES (with or without MDSC transplantation) presented an increased maximal specific tetanic force when compared to controls. Although cell transplantation and/or the use of NMES resulted in no changes in fatigue resistance, the combination of both MDSC transplantation and NMES resulted in a faster recovery from fatigue, when compared to non-injected and non-stimulated counterparts. We conclude that NMES is a viable method to improve MDSC engraftment, enhance dystrophic muscle strength, and, in combination with MDSC transplantation, improve recovery from fatigue. These findings suggest that NMES may be a clinically-relevant adjunct approach for cell transplantation into skeletal muscle.
PLoS ONE 01/2013; 8(3):e54922. · 4.09 Impact Factor
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ABSTRACT: Human muscle-derived progenitor cells (hMDPCs) offer great promise for muscle cell-based regenerative medicine; however, prolonged ex-vivo expansion using animal sera is necessary to acquire sufficient cells for transplantation. Due to the risks associated with the use of animal sera, the development of a strategy for the ex vivo expansion of hMDPCs is required. The purpose of this study was to investigate the efficacy of using platelet-rich plasma (PRP) for the ex-vivo expansion of hMDPCs. Pre-plated MDPCs, myoendothelial cells, and pericytes are three populations of hMDPCs that we isolated by the modified pre-plate technique and Fluorescence Activated Cell Sorting (FACS), respectively. Pooled allogeneic human PRP was obtained from a local blood bank, and the effect that thrombin-activated PRP-releasate supplemented media had on the ex-vivo expansion of the hMDPCs was tested against FBS supplemented media, both in vitro and in vivo. PRP significantly enhanced short and long-term cell proliferation, with or without FBS supplementation. Antibody-neutralization of PDGF significantly blocked the mitogenic/proliferative effects that PRP had on the hMDPCs. A more stable and sustained expression of markers associated with stemness, and a decreased expression of lineage specific markers was observed in the PRP-expanded cells when compared with the FBS-expanded cells. The in vitro osteogenic, chondrogenic, and myogenic differentiation capacities of the hMDPCs were not altered when expanded in media supplemented with PRP. All populations of hMDPCs that were expanded in PRP supplemented media retained their ability to regenerate myofibers in vivo. Our data demonstrated that PRP promoted the proliferation and maintained the multi-differentiation capacities of the hMDPCs during ex-vivo expansion by maintaining the cells in an undifferentiated state. Moreover, PDGF appears to be a key contributing factor to the beneficial effect that PRP has on the proliferation of hMDPCs.
PLoS ONE 01/2013; 8(6):e64923. · 4.09 Impact Factor
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ABSTRACT: PURPOSE: Surgical repairs of tears in the vascular region of the meniscus usually heal better than repairs performed in the avascular region, thus we hypothesized that this region might possess a richer supply of vascular-derived stem cells than the avascular region. METHODS: Six menisci harvested from aborted human fetuses and twelve human lateral menisci harvested from adult human subjects undergoing total knee arthroplasty were analyzed. Menisci were immunostained for CD34 (a stem cell marker), and CD146 (a pericyte marker) in situ while other menisci were dissected into 2 regions (peripheral and inner) and used to isolate meniscusderived cells by flow-cytometry. Cell populations expressing CD34 and CD146 were tested for their multilineage differentiation potentials including chondrogenic, osteogenic, and adipogenic lineages. Fetal peripheral meniscus cells were transplanted by intracapsular injection into the knee joints of an athymic rat meniscal tear model. Rat menisci were harvested and histologically evaluated after 4 weeks post-transplantation. RESULTS: Immunohistochemistry and flow cytometric analyses demonstrated that a higher number of CD34 and CD146 positive cells were found in the peripheral region compared to the inner region. The CD34 and CD146 positive cells isolated from the vascular region of both fetal and adult menisci demonstrated multilineage differentiation capacities and were more potent than cells isolated from the inner (avascular) region. Fetal CD34 and CD146 positive cells transplanted into the athymic rat knee joint were recruited into the meniscal tear sites and contributed to meniscus repair. CONCLUSIONS: The vascularized region of the meniscus contains more stem cells than the avascular region. These meniscal-derived stem cells were multipotent and contributed to meniscal regeneration.
Medicine and science in sports and exercise 12/2012; · 3.71 Impact Factor
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ABSTRACT: Skeletal muscle injuries are among the most common and frequently disabling injuries sustained by athletes.Repair of injured skeletal muscle is an area that continues to present a challenge for sports medicine clinicians and researchers due, in part, to complete muscle recovery being compromised by development of fibrosis leading to loss of function and susceptibility to re-injury.Injured skeletal muscle goes through a series of coordinated and interrelated phases of healing including degeneration, inflammation, regeneration and fibrosis. Muscle regeneration initiated shortly after injury can be limited by fibrosis which affects the degree of recovery and predisposes the muscle to reinjury. It has been demonstrated in animal studies that antifibrotic agents that inactivate transforming growth factor (TGF)-β1 have been effective at decreasing scar tissue formation. Several studies have also shown that vascular endothelial growth factor (VEGF) can increase the efficiency of skeletal muscle repair by increasing angiogenesis and, at the same time, reducing the accumulation of fibrosis. We have isolated and thoroughly characterised a population of skeletal muscle-derived stem cells (MDSCs) that enhance repair of damaged skeletal muscle fibres by directly differentiating into myofibres and secreting paracrine factors that promote tissue repair. Indeed, we have found that MDSCs transplanted into skeletal and cardiac muscles have been successful at repair probably because of their ability to secrete VEGF that works in a paracrine fashion. The application of these techniques to the study of sport-related muscle injuries awaits investigation. Other useful strategies to enhance skeletal muscle repair through increased vascularisation may include gene therapy, exercise, neuromuscular electrical stimulation and, potentially, massage therapy. Based on recent studies showing an accelerated recovery of muscle function from intense eccentric exercise through massage-based therapies, we believe that this treatment modality offers a practical and non-invasive form of therapy for skeletal muscle injuries. However, the biological mechanism(s) behind the beneficial effect of massage are still unclear and require further investigation using animal models and potentially randomised, human clinical studies.
British journal of sports medicine 11/2012; · 2.55 Impact Factor
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Chien-Wen Chen,
Masaho Okada,
Jonathan D Proto,
Xueqin Gao,
Naosumi Sekiya,
Sarah A Beckman,
Mirko Corselli,
Mihaela Crisan,
Arman Saparov,
Kimimasa Tobita,
Bruno Péault, Johnny Huard
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ABSTRACT: Human microvascular pericytes (CD146(+) /34(-) /45(-) /56(-) ) contain multipotent precursors and repair/regenerate defective tissues, notably skeletal muscle. However, their ability to repair the ischemic heart remains unknown. We investigated the therapeutic potential of human pericytes, purified from skeletal muscle, for treating ischemic heart disease and mediating associated repair mechanisms in mice. Echocardiography revealed that pericyte transplantation attenuated left ventricular dilatation and significantly improved cardiac contractility, superior to CD56+ myogenic progenitor transplantation, in acutely infarcted mouse hearts. Pericyte treatment substantially reduced myocardial fibrosis and significantly diminished infiltration of host inflammatory cells at the infarct site. Hypoxic pericyte-conditioned medium suppressed murine fibroblast proliferation and inhibited macrophage proliferation in vitro. High expression by pericytes of immunoregulatory molecules, including IL-6, LIF, COX-2 and HMOX-1, was sustained under hypoxia, except for MCP-1. Host angiogenesis was significantly increased. Pericytes supported microvascular structures in vivo and formed capillary-like networks with/without endothelial cells in three-dimensional co-cultures. Under hypoxia, pericytes dramatically increased expression of VEGF-A, PDGF-β, TGF-β1 and corresponding receptors while expression of bFGF, HGF, EGF, and Ang-1 was repressed. The capacity of pericytes to differentiate into and/or fuse with cardiac cells was revealed by GFP-labeling, though to a minor extent. In conclusion, intramyocardial transplantation of purified human pericytes promotes functional and structural recovery, attributable to multiple mechanisms involving paracrine effects and cellular interactions.
Stem Cells 11/2012; · 7.78 Impact Factor
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ABSTRACT: Losartan (LOS) is an FDA approved antihypertensive medication that has a well-tolerated side-effect profile. We have demonstrated that treatment with LOS immediately after injury was effective at promoting muscle healing and inducing an antifibrotic effect in a murine model of skeletal muscle injury. We initially investigated the minimum effective dose of LOS administration immediately after injury and subsequently determined whether the timing of administering a clinically relevant dose of LOS would influence its effectiveness at improving muscle healing after muscle injury. In the first part of this study, mice were administered 3, 10, 30 or 300mg/kg/day of LOS immediately after injury and the healing process was evaluated histologically and physiologically 4 weeks after injury. In the second study, the clinically relevant dose of 10mg/kg/day was administered immediately or started at 3 or 7 days post-injury. The administration of 300mg/kg/day immediately following injury led to a significant increase in muscle regeneration, a significant decrease in fibrosis and an improvement in muscle function. Moreover, we observed a significant decrease in fibrosis and a significant increase in muscle regeneration at 4 weeks post-injury, when the clinically relevant dose of 10mg/kg/day was administered at 3 or 7 days post-injury. Functional evaluation also demonstrated a significant improvement compared to the injured untreated control when LOS treatment was initiated 3 days after injury. Our study revealed accelerated muscle healing when the 300mg/kg/day of LOS was administered immediately after injury and a clinically relevant dose of 10mg/kg/day of LOS was administered at 3 or 7 days post-injury.
Journal of Applied Physiology 11/2012; · 3.75 Impact Factor
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ABSTRACT: 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 for bone regenerative therapies. We found that hMDSCs, isolated by the preplate technique, consistently expressed the myogenic marker CD56, pericyte/endothelial cell marker CD146, mesenchymal stem cell markers CD73, CD90, CD105, 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 non-transduced cells; however, when transplanted into a calvarial defect they failed to regenerate bone. Local administration of BMP4 protein and cell pre-treatment with N-acetyl-cysteine (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 size 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 for promoting the bone regenerative capacity of the hMDSCs in vivo.
Cell Transplantation 11/2012; · 5.13 Impact Factor
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Christian Isaac,
Adam Wright,
Arvydas Usas,
Hongshuai Li,
Ying Tang,
Xiaodong Mu,
Nicholas Greco,
Qing Dong,
Nam Vo,
James Kang,
Bing Wang, Johnny Huard
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ABSTRACT: Duchenne muscular dystrophy (DMD) is a degenerative muscle disorder characterized by the lack of dystrophin expression at the sarcolemma of muscle fibers. In addition, DMD patients acquire osteopenia, fragility fractures, and scoliosis indicating that a deficiency in skeletal homeostasis coexists but little is known about the effects of DMD on bone and other connective tissues within the musculoskeletal system. Recent evidence has emerged implicating adult stem cell dysfunction in DMD myopathogenesis. Given the common mesenchymal origin of muscle and bone, we sought to investigate bone and other musculoskeletal tissues in a DMD mouse model. Here, we report that dystrophin-utrophin double knockout (dko) mice exhibit a spectrum of degenerative changes, outside skeletal muscle, in bone, articular cartilage, and intervertebral discs, in addition to reduced lifespan, muscle degeneration, spinal deformity, and cardiomyopathy previously reported. We also report these mice to have a reduced capacity for bone healing and exhibit spontaneous heterotopic ossification in the hind limb muscles. Therefore, we propose the dko mouse as a model for premature musculoskeletal aging and posit that a similar phenomenon may occur in patients with DMD. © 2012 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res.
Journal of Orthopaedic Research 10/2012; · 2.81 Impact Factor
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Y Mifune,
T Matsumoto,
K Takayama,
S Ota,
H Li,
L B Meszaros,
A Usas,
K Nagamune,
B Gharaibeh,
F H Fu, J Huard
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ABSTRACT: OBJECTIVE: Platelet-rich plasma (PRP) is reported to promote collagen synthesis and cell proliferation as well as enhance cartilage repair. Our previous study revealed that the intracapsular injection of muscle derived stem cells (MDSCs) expressing bone morphogenetic protein 4 (BMP-4) combined with soluble Flt-1 (sFlt-1) was effective for repairing articular cartilage (AC) after osteoarthritis (OA) induction. The current study was undertaken to investigate whether PRP could further enhance the therapeutic effect of MDSC therapy for the OA treatment. METHODS: MDSCs expressing BMP-4 and sFlt-1 were mixed with PRP and injected into the knees of immunodeficient rats with chemically induced OA. Histological assessments were performed 4 and 12 weeks after cell transplantation. Moreover, to elucidate the repair mechanisms, we performed in vitro assays to assess cell proliferation, adhesion, migration and mixed pellet co-culture of MDSCs and OA chondrocytes. RESULTS: The addition of PRP to MDSCs expressing BMP-4 and sFlt-1 significantly improved AC repair histologically at week 4 compared to MDSCs expressing BMP-4 and sFlt-1 alone. Higher numbers of cells producing type II collagen and lower levels of chondrocyte apoptosis were observed by MDSCs expressing BMP-4 and sFlt-1 and mixed with PRP. In the in vitro experiments, the addition of PRP promoted proliferation, adhesion and migration of the MDSCs. During chondrogenic pellet culture, PRP tended to increase the number of type II collagen producing cells and in contrast to the in vivo data, it increased cell apoptosis. CONCLUSIONS: Our findings indicate that PRP can promote the therapeutic potential of MDSCs expressing BMP-4 and sFlt-1 for AC repair (4 weeks post-treatment) by promoting collagen synthesis, suppressing chondrocyte apoptosis and finally by enhancing the integration of the transplanted cells in the repair process.
Osteoarthritis and Cartilage 10/2012; · 3.90 Impact Factor
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ABSTRACT: Muscle-derived stem cells (MDSCs) are known to exhibit sexual dimorphism, by donor sex, of osteogenic, chondrogenic, and myogenic differentiation potential in vitro. Moreover, host sex differences in the myogenic capacity of MDSCs in vivo are also observed. This study investigated the role of host sex and host sex hormones in MDSC-mediated bone formation and healing. Using unaltered male, castrated male, unaltered female, and ovariectomized female mice, both MDSC-mediated ectopic bone formation and cranial defect healing were examined. Male hosts, whether unaltered or castrated, form significantly larger volumes of MDSC-mediated ectopic bone than female hosts (either unaltered or ovariectomized), and no differences in ectopic bone volume were found between hosts of the same sex. In a cranial defect healing model, similar results were found-unaltered and castrated male hosts display larger volumes of bone formed when compared with unaltered and ovariectomized female hosts. However, in this healing model, some volume differences were found between hosts of the same sex. In both models, these differences were attributed to varying rates of endochondral bone formation in male and female hosts.
Tissue Engineering Part A 06/2012; 18(17-18):1751-9. · 4.64 Impact Factor
