Search

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 in vitro. 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.

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.

Chronic inflammation, promoted by an upregulated NF-kappa B (NF-κB) pathway, has a key role in Duchenne muscular dystrophy (DMD) patients' pathogenesis. Blocking the NF-κB pathway has been shown to be a viable approach to diminish chronic inflammation and necrosis in the dystrophin-defective mdx mouse, a murine DMD model. In this study, we used the recombinant adeno-associated virus serotype 9 (AAV9) carrying an short hairpin RNA (shRNA) specifically targeting the messenger RNA of NF-κB/p65 (p65-shRNA), the major subunit of NF-κB associated with chronic inflammation in mdx mice. We examined whether i.m. AAV9-mediated delivery of p65-shRNA could decrease NF-κB activation, allowing for amelioration of muscle pathologies in 1- and 4-month-old mdx mice. At 1 month after treatment, NF-κB/p65 levels were significantly decreased by AAV gene transfer of p65-shRNA in the two ages of treatment groups, with necrosis significantly decreased compared with controls. Quantitative analysis revealed that central nucleation (CN) of the myofibers of p65-shRNA-treated 1-month-old mdx muscles was reduced from 67 to 34%, but the level of CN was not significantly decreased in treated 4-month-old mdx mice. Moreover, delivery of the p65-shRNA enhanced the capacity of myofiber regeneration in old mdx mice treated at 4 months of age when the dystrophic myofibers were most exhausted; however, such p65 silencing diminished the myofiber regeneration in young mdx mice treated at 1 month of age. Taken together, these findings demonstrate that the AAV-mediated delivery of p65-shRNA has the capacity to ameliorate muscle pathologies in mdx mice by selectively reducing NF-κB/p65 activity.

During liver regeneration, quiescent hepatocytes re-enter the cell cycle to proliferate and compensate for lost tissue. Multiple signals including hepatocyte growth factor, epidermal growth factor, tumor necrosis factor α, interleukin-6, insulin and transforming growth factor β orchestrate these responses and are integrated during the G(1) phase of the cell cycle. To investigate how these inputs influence DNA synthesis as a measure for proliferation, we established a large-scale integrated logical model connecting multiple signaling pathways and the cell cycle. We constructed our model based upon established literature knowledge, and successively improved and validated its structure using hepatocyte-specific literature as well as experimental DNA synthesis data. Model analyses showed that activation of the mitogen-activated protein kinase and phosphatidylinositol 3-kinase pathways was sufficient and necessary for triggering DNA synthesis. In addition, we identified key species in these pathways that mediate DNA replication. Our model predicted oncogenic mutations that were compared with the COSMIC database, and proposed intervention targets to block hepatocyte growth factor-induced DNA synthesis, which we validated experimentally. Our integrative approach demonstrates that, despite the complexity and size of the underlying interlaced network, logical modeling enables an integrative understanding of signaling-controlled proliferation at the cellular level, and thus can provide intervention strategies for distinct perturbation scenarios at various regulatory levels., (© 2012 The Authors Journal compilation © 2012 FEBS.)

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.

Introduction: It has been reported that suramin treatment can improve muscle healing; however, details about optimizing the dosing requirements remain unclear. The purpose of this study was to determine the optimal timing of suramin administration and investigate the effects it had on the expression of myostatin, follistatin, and muscle vascularity after muscle injury., Methods: Contusion injured muscles of mice were treated with suramin at 1, 2, or 3 weeks post-injury and evaluated histologically and physiologically at 1, 2, and 10 days after injection., Results: Suramin treatment initiated at 2 weeks post-injury was observed to promote muscle regeneration and muscle strength, and to decrease fibrosis. Suramin reduced myostatin expression and increased follistatin expression and vascularity in injured skeletal muscle., Conclusions: Suramin's positive effect on muscle regeneration is thought to be due to its enhancement of follistatin expression which increases neoangiogenesis and inhibits myostatin's promotion of fibrosis., (Copyright © 2012 Wiley Periodicals, Inc.)

The accumulation of cellular damage, including DNA damage, is thought to contribute to aging-related degenerative changes, but how damage drives aging is unknown. XFE progeroid syndrome is a disease of accelerated aging caused by a defect in DNA repair. NF-κB, a transcription factor activated by cellular damage and stress, has increased activity with aging and aging-related chronic diseases. To determine whether NF-κB drives aging in response to the accumulation of spontaneous, endogenous DNA damage, we measured the activation of NF-κB in WT and progeroid model mice. As both WT and progeroid mice aged, NF-κB was activated stochastically in a variety of cell types. Genetic depletion of one allele of the p65 subunit of NF-κB or treatment with a pharmacological inhibitor of the NF-κB-activating kinase, IKK, delayed the age-related symptoms and pathologies of progeroid mice. Additionally, inhibition of NF-κB reduced oxidative DNA damage and stress and delayed cellular senescence. These results indicate that the mechanism by which DNA damage drives aging is due in part to NF-κB activation. IKK/NF-κB inhibitors are sufficient to attenuate this damage and could provide clinical benefit for degenerative changes associated with accelerated aging disorders and normal aging.

Stem cell therapy for tissue repair is a rapidly evolving field and the factors that dictate the physiological responsiveness of stem cells remain under intense investigation. In this study we hypothesized that the mechanical loading history of muscle-derived stem cells (MDSCs) would significantly impact MDSC survival, host tissue angiogenesis, and myocardial function after MDSC transplantation into acutely infarcted myocardium. Mice with acute myocardial infarction by permanent left coronary artery ligation were injected with either nonstimulated (NS) or mechanically stimulated (MS) MDSCs. Mechanical stimulation consisted of stretching the cells with equibiaxial stretch with a magnitude of 10% and frequency of 0.5 Hz. MS cell-transplanted hearts showed improved cardiac contractility, increased numbers of host CD31+ cells, and decreased fibrosis, in the peri-infarct region, compared to the hearts treated with NS MDSCs. MS MDSCs displayed higher vascular endothelial growth factor expression than NS cells in vitro. These findings highlight an important role for cyclic mechanical loading preconditioning of donor MDSCs in optimizing MDSC transplantation for myocardial repair.

The anterior cruciate ligament (ACL) usually fails to heal after rupture mainly due to the inability of the cells within the ACL tissue to establish an adequate healing process, making graft reconstruction surgery a necessity. However, some reports have shown that there is a healing potential of ACL with primary suture repair. Although some reports showed the existence of mesenchymal stem cell-like cells in human ACL tissues, their origin still remains unclear. Recently, blood vessels have been reported to represent a rich supply of stem/progenitor cells with a characteristic expression of CD34 and CD146. In this study, we attempted to validate the hypothesis that CD34- and CD146-expressing vascular cells exist in hACL tissues, have a potential for multi-lineage differentiation, and are recruited to the rupture site to participate in the intrinsic healing of injured ACL. Immunohistochemistry and flow cytometry analysis of hACL tissues demonstrated that it contains significantly more CD34 and CD146-positive cells in the ACL ruptured site compared with the noninjured midsubstance. CD34+CD45- cells isolated from ACL ruptured site showed higher expansionary potentials than CD146+CD45- and CD34-CD146-CD45- cells, and displayed higher differentiation potentials into osteogenic, adipogenic, and angiogenic lineages than the other cell populations. Immunohistochemistry of fetal and adult hACL tissues demonstrated a higher number of CD34 and CD146-positive cells in the ACL septum region compared with the midsubstance. In conclusion, our findings suggest that the ACL septum region contains a population of vascular-derived stem cells that may contribute to ligament regeneration and repair at the site of rupture., (© Mary Ann Liebert, Inc.)

We performed this study to investigate the therapeutic role of vascular endothelial growth factor (VEGF) in medial collateral ligament (MCL) healing. Murine muscle derived stem cells (MDSCs) obtained via the preplate technique were retrovirally transduced to express: (1) VEGF and nLacZ (MDSC-VEGF), (2) soluble fms-like tyrosine kinase-1 (sFLT1, a VEGF-specific antagonist) and nLacZ (MDSC-sFLT1), and (3) nLacZ (MDSC-nLacZ). After transecting the MCL of immunodeficient rats, 5 × 10(5)  cells of each of the transduction groups list above were transplanted into the MCL injury site. A control group was injected with phosphate-buffered saline (PBS) only. Immunohistochemical staining demonstrated that there were more Isolectin B4 and β-galactosidase double positive cells in the rats transplanted with MDSC-VEGF transduced cells than the other groups at week 1. Capillary density was significantly higher in the MDSC-VEGF group than the other groups at week 2; however, there were no significant differences in the biomechanical assessment between the MDSC-VEGF and MDSC-nLacZ groups. On the other hand, the MDSC-sFLT1 group revealed a lower capillary density than the other two groups and the functional ligament healing of the MDSC-sFLT1 group was significantly decreased compared to the other groups when assessed biomechanically. The findings of the present study suggest that angiogenesis plays a critical role in the healing process of injured MCL., (Copyright © 2011 Orthopaedic Research Society.)

The relationship between the two coexpressed differentiation markers, profilaggrin and loricrin, is not clear right now. In this study, we explored the interaction of profilaggrin N-terminal domain (PND) with loricrin in keratinocytes and epidermis. Confocal immunofluorescence microscopic analysis of human epidermis showed that PND colocalized with loricrin. Loricrin nucleofected into HaCaT cells colocalized with PND in the nucleus and cytoplasm. The PND localizes to both the nucleus and cytoplasm of epidermal granular layer cells. Nucleofected PND also colocalized with keratin 10 (K10) in the nucleus and cytoplasm. Immunoelectron microscopic analysis of human epidermis confirmed the findings in nucleofected keratinocytes. Yeast two-hybrid assays showed that the B domain of human and mouse PND interacted with loricrin. The glutathione S-transferase (GST) pull-down analysis using recombinant GST-PND revealed that PND interacted with loricrin and K10. Knockdown of PND in an organotypic skin culture model caused loss of filaggrin expression and a reduction in both the size and number of keratohyalin granules, as well as markedly reduced expression of loricrin. Considering that expression of PND is closely linked to keratinocyte terminal differentiation, we conclude that PND interacts with loricrin and K10 in vivo and that these interactions are likely to be relevant for cornified envelope assembly and subsequent epidermal barrier formation.

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.

Skeletal muscle injury and repair are complex processes, including well-coordinated steps of degeneration, inflammation, regeneration, and fibrosis. We have reviewed the recent literature including studies by our group that describe how to modulate the processes of skeletal muscle repair and regeneration. Antiinflammatory drugs that target cyclooxygenase-2 were found to hamper the skeletal muscle repair process. Muscle regeneration phase can be aided by growth factors, including insulin-like growth factor-1 and nerve growth factor, but these factors are typically short-lived, and thus more effective methods of delivery are needed. Skeletal muscle damage caused by traumatic injury or genetic diseases can benefit from cell therapy; however, the majority of transplanted muscle cells (myoblasts) are unable to survive the immune response and hypoxic conditions. Our group has isolated neonatal skeletal muscle derived stem cells (MDSCs) that appear to repair muscle tissue in a more effective manner than myoblasts, most likely due to their better resistance to oxidative stress. Enhancing antioxidant levels of MDSCs led to improved regenerative potential. It is becoming increasingly clear that stem cells tissue repair by direct differentiation and paracrine effects leading to neovascularization of injured site and chemoattraction of host cells. The factors invoked in paracrine action are still under investigation. Our group has found that angiotensin II receptor blocker (losartan) significantly reduces fibrotic tissue formation and improves repair of murine injured muscle. Based on these data, we have conducted a case study on two hamstring injury patients and found that losartan treatment was well tolerated and possibly improved recovery time. We believe this medication holds great promise to optimize muscle repair in humans., (Copyright © 2012 Wiley Periodicals, Inc.)

Background: Despite the advances in surgical procedures to repair the rotator cuff, there is a high incidence of failure. Biologic approaches, such as growth factor delivery and stem cell and gene therapy, are potential targets for optimization to improve the outcome of rotator cuff therapies and reduce rates of reinjury. This article outlines the current evidence for growth factor and stem cell therapy in tendon healing and the augmentation of rotator cuff repair., Methods: Literature on the PubMed-National Center for Biotechnology Information database was searched using the keywords growth factor, factor, gene therapy, stem cell, mesenchymal, or bone marrow in combination with rotator cuff, supraspinatus, or infraspinatus. Articles that studied growth factors or stem cells alone in rotator cuff repair were selected. Only 3 records showed use of stem cells in rotator cuff repair; thus, we expanded our search to include selected studies on stem cells and Achilles or patellar tendon repairs. Bibliographies and proceedings of meetings were searched to include additional applicable studies. We also included hitherto unpublished data by our group on the use of stem cell transplantation for rotator cuff therapy., Results: More than 70 articles are summarized, with focus on recent original research papers and significant reviews that summarized earlier records., Conclusions: Use of growth factors, stem cell therapy, and other tissue-engineering means serve to augment classical surgical rotator cuff repair procedures. The combination of stem cells and growth factors resulted in enhanced repair that emulated uninjured tissue, but the literature search reflected paucity of research in this field. Preclinical evidence from gene therapy and stem cell studies can be used as a start to move therapy from the experimental phase to clinical translation in patients., (Copyright © 2012 Journal of Shoulder and Elbow Surgery Board of Trustees. All rights reserved.)

With ageing, there is a loss of adult stem cell function. However, there is no direct evidence that this has a causal role in ageing-related decline. We tested this using muscle-derived stem/progenitor cells (MDSPCs) in a murine progeria model. Here we show that MDSPCs from old and progeroid mice are defective in proliferation and multilineage differentiation. Intraperitoneal administration of MDSPCs, isolated from young wild-type mice, to progeroid mice confer significant lifespan and healthspan extension. The transplanted MDSPCs improve degenerative changes and vascularization in tissues where donor cells are not detected, suggesting that their therapeutic effect may be mediated by secreted factor(s). Indeed, young wild-type-MDSPCs rescue proliferation and differentiation defects of aged MDSPCs when co-cultured. These results establish that adult stem/progenitor cell dysfunction contributes to ageing-related degeneration and suggests a therapeutic potential of post-natal stem cells to extend health.

Skeletal muscle derived stem cells (MDSCs) transplanted into injured myocardium can differentiate into fast skeletal muscle specific myosin heavy chain (sk-fMHC) and cardiac specific troponin-I (cTn-I) positive cells sustaining recipient myocardial function. We have recently found that MDSCs differentiate into a cardiomyocyte phenotype within a three-dimensional gel bioreactor. It is generally accepted that terminally differentiated myocardium or skeletal muscle only express cTn-I or sk-fMHC, respectively. Studies have shown the presence of non-cardiac muscle proteins in the developing myocardium or cardiac proteins in pathological skeletal muscle. In the current study, we tested the hypothesis that normal developing myocardium and skeletal muscle transiently share both sk-fMHC and cTn-I proteins. Immunohistochemistry, western blot, and RT-PCR analyses were carried out in embryonic day 13 (ED13) and 20 (ED20), neonatal day 0 (ND0) and 4 (ND4), postnatal day 10 (PND10), and 8 week-old adult female Lewis rat ventricular myocardium and gastrocnemius muscle. Confocal laser microscopy revealed that sk-fMHC was expressed as a typical striated muscle pattern within ED13 ventricular myocardium, and the striated sk-fMHC expression was lost by ND4 and became negative in adult myocardium. cTn-I was not expressed as a typical striated muscle pattern throughout the myocardium until PND10. Western blot and RT-PCR analyses revealed that gene and protein expression patterns of cardiac and skeletal muscle transcription factors and sk-fMHC within ventricular myocardium and skeletal muscle were similar at ED20, and the expression patterns became cardiac or skeletal muscle specific during postnatal development. These findings provide new insight into cardiac muscle development and highlight previously unknown common developmental features of cardiac and skeletal muscle.

We demonstrate that subpopulations of adult human skeletal muscle-derived stem cells, myogenic endothelial cells (MECs), and perivascular stem cells (PSCs) can be simultaneously purified by fluorescence-activated cell sorting (FACS) from cryopreserved human primary skeletal muscle cell cultures (cryo-hPSMCs). For FACS isolation, we utilized a combination of cell lineage markers: the myogenic cell marker CD56, the endothelial cell marker UEA-1 receptor (UEA-1R), and the perivascular cell marker CD146. MECs expressing all three cell lineage markers (CD56(+)UEA-1R(+)CD146(+)/CD45(-)) and PSCs expressing only CD146 (CD146(+)/CD45(-)CD56(-)UEA-1R(-)) were isolated by FACS. To evaluate their myogenic capacities, the sorted cells, with and without expansion in culture, were transplanted into the cardiotoxin-injured skeletal muscles of immunodeficient mice. The purified MECs exhibited the highest regenerative capacity in the injured mouse muscles among all cell fractions tested, while PSCs remained superior to myoblasts and the unpurified primary skeletal muscle cells. Our findings show that both MECs and PSCs retain their high myogenic potentials after in vitro expansion, cryopreservation, and FACS sorting. The current study demonstrates that myogenic stem cells are prospectively isolatable from long-term cryopreserved primary skeletal muscle cell cultures. We emphasize the potential application of this new approach to extract therapeutic stem cells from human muscle cells cryogenically banked for clinical purposes.

Multipotent stem/progenitor cells with similar developmental potentials have been independently identified from diverse human tissue/organ cultures. The increasing recognition of the vascular/perivascular origin of mesenchymal precursors suggested blood vessels being a systemic source of adult stem/progenitor cells. Our group and other laboratories recently isolated multiple stem/progenitor cell subsets from blood vessels of adult human tissues. Each of the three structural layers of blood vessels: intima, media, and adventitia has been found to include at least one precursor population, that is, myogenic endothelial cells (MECs), pericytes, and adventitial cells (ACs), respectively. MECs and pericytes efficiently regenerate myofibers in injured and dystrophic skeletal muscles as well as improve cardiac function after myocardial infarction. The applications of ACs in vascular remodeling and angiogenesis/vasculogenesis have been examined. Our recent finding that MECs and pericytes can be purified from cryogenically banked human primary muscle cell culture further indicates their potential applications in personalized regenerative medicine.

We recently reported that the ruptured regions of the human anterior cruciate ligament (ACL) contained vascular-derived stem cells, which showed the potential for high expansion and multilineage differentiation. In this study, we performed experiments to test the hypothesis that ACL-derived CD34(+) cells could contribute to tendon-bone healing. ACL-derived cells were isolated from the rupture site of human ACL by fluorescence-activated cell sorting. Following ACL reconstruction, immunodeficient rats received intracapsular administration of either ACL-derived CD34(+) cells, nonsorted (NS) cells, CD34(+) cells, or phosphate-buffered saline (PBS). We also performed in vitro cell proliferation assays and enzyme-linked immunosorbent assays for vascular endothelial growth factor (VEGF) secretion. We confirmed the recruitment of the transplanted cells into the perigraft site after intracapuslar injection by immunohistochemical staining at week 1. Histological evaluation showed a greater area of collagen fiber formation and more collagen type II expression in the CD34(+) group than the other groups at the week 2 time point. Immunostaining with isolectin B4 and rat osteocalcin demonstrated enhanced angiogenesis and osteogenesis in the CD34(+) group at week 2. Moreover, double immunohistochemical staining for human-specific endothelial cell (EC) and osteoblast (OB) markers at week 2 demonstrated a greater ability of differentiation into ECs and OBs in the CD34(+) group. Microcomputerized tomography showed the greatest healing of perigraft bone at week 4 in the CD34(+) cell group, and the failure load of tensile test at week 8 demonstrated the greatest biomechanical strength in the CD34(+) group. Furthermore, the in vitro studies indicated that the CD34(+) group was superior to the other groups in their cell proliferation and VEGF secretion capacities. We demonstrated that ACL-derived CD34(+) cells contributed to the tendon-bone healing after ACL reconstruction via the enhancement of angiogenesis and osteogenesis, which also contributed to an increase in biomechanical strength.

Identification of cells that are endowed with maximum potency could be critical for the clinical success of cell-based therapies. We investigated whether cells with an enhanced efficacy for cardiac cell therapy could be enriched from adult human skeletal muscle on the basis of their adhesion properties to tissue culture flasks following tissue dissociation. Cells that adhered slowly displayed greater myogenic purity and more readily differentiated into myotubes in vitro than rapidly adhering cells (RACs). The slowly adhering cell (SAC) population also survived better than the RAC population in kinetic in vitro assays that simulate conditions of oxidative and inflammatory stress. When evaluated for the treatment of a myocardial infarction (MI), intramyocardial injection of the SACs more effectively improved echocardiographic indexes of left ventricular (LV) remodeling and contractility than the transplantation of the RACs. Immunohistological analysis revealed that hearts injected with SACs displayed a reduction in myocardial fibrosis and an increase in infarct vascularization, donor cell proliferation, and endogenous cardiomyocyte survival and proliferation in comparison with the RAC-treated hearts. In conclusion, these results suggest that adult human skeletal muscle-derived cells are inherently heterogeneous with regard to their efficacy for enhancing cardiac function after cardiac implantation, with SACs outperforming RACs.

Intramuscular injection of bone morphogenetic proteins (BMPs) has been shown to induce ectopic bone formation. A chondrogenic phase is typically observed in this process, which suggests that there may exist a chondrogenic subpopulation of cells residing in skeletal muscle. Two prospective cell populations were isolated from rat skeletal muscle: fascia-derived cells (FDCs), extracted from gluteus maximus muscle fascia (epimysium) and muscle-derived cells (MDCs) isolated from the muscle body. Both populations were investigated for their cell surface marker profiles (flowcytometry analysis), proliferation rates as well as their myogenic and chondrogenic potentials. The majority of FDCs expressed mesenchymal stromal cell markers but not endothelial cell markers. FDCs underwent chondrogenic differentiation after BMP4 treatment in vitro, but not myogenic differentiation. Although MDCs showed chondrogenic potential, they expressed the myogenic cell marker desmin and readily underwent myogenic differentiation in vitro; however, the chondrogenic potential of the MDCs is confounded by the presence of FDC-like cells residing in the muscle perimysium and endomysium. To clarify the role of the muscle-derived myogenic cells in chondrogenesis, mixed pellets with varying ratios of FDCs and L6 myoblasts were formed and studied for chondrogenic potential. Our results indicated that the chondrogenic potential of the mixed pellets decreased with the increased ratio of myogenic cells to FDCs supporting the role of FDCs in chondrogenesis. Taken together, our results suggest that non-myogenic cells residing in the fascia of skeletal muscle have a strong chondrogenic potential and may represent a novel donor cell source for cartilage regeneration and repair.

Background: Muscle contusions are common muscle injuries. Although these injuries are capable of healing, incomplete functional recovery often occurs. Muscle-derived stem cells (MDSCs) are likely derived from blood vessel cells and have a multilineage differentiation potential., Purpose: The aims of this study are (1) to find optimal timing of MDSC transplantation to enhance muscle healing by stimulating muscle regeneration and preventing scar tissue (fibrosis) formation after skeletal muscle contusion injury, and (2) to investigate the role of angiogenesis in the muscle-healing process after MDSC transplantation., Study Design: Controlled laboratory study., Methods: Muscle-derived stem cells were injected directly into injured tibialis anterior muscles of mice at various time points (1, 4, and 7 days) after the muscle contusion injury. Muscle regeneration, angiogenesis, and fibrosis formation were evaluated by histology and real-time polymerase chain reaction analysis, and functional recovery was measured by physiologic testing., Results: Transplantation of MDSCs at 4 days after injury significantly promoted angiogenesis, which was induced by high levels of vascular endothelial growth factor expression at week 1, and significantly increased muscle regeneration and muscle strength by week 2, when compared with the other groups. A decrease in fibrosis formation was observed at week 4, when compared with the other groups, after the transplantation of MDSCs at 4 and 7 days after injury., Conclusion: Intramuscular injection of MDSCs at 4 days after injury improved and accelerated skeletal muscle healing by increasing angiogenesis and decreasing scar tissue formation., Clinical Relevance: These findings could contribute to the development of biologic treatments to aid in muscle healing after muscle injury.

Enhancing the maturity of the newly formed blood vessels is critical for the success of therapeutic angiogenesis. The maturation of vasculature relies on active participation of mural cells to stabilize endothelium and a basal level of relevant growth factors. We set out to design and successfully achieved robust angiogenesis using an injectable polyvalent coacervate of a polycation, heparin, and fibroblast growth factor-2 (FGF2). FGF2 was loaded into the coacervate at nearly 100% efficiency. In vitro assays demonstrated that the matrix protected FGF2 from proteolytic degradations. FGF2 released from the coacervate was more effective in the differentiation of endothelial cells and chemotaxis of pericytes than free FGF2. One injection of 500 ng of FGF2 in the coacervate elicited comprehensive angiogenesis in vivo. The number of endothelial and mural cells increased significantly, and the local tissue contained more and larger blood vessels with increased circulation. Mural cells actively participated during the whole angiogenic process: Within 7 d of the injection, pericytes were recruited to close proximity of the endothelial cells. Mature vasculature stabilized by vascular smooth muscle cells persisted till at least 4 wk. On the other hand, bolus injection of an identical amount of free FGF2 induced weak angiogenic responses. These results demonstrate the potential of polyvalent coacervate as a new controlled delivery platform.

Recovery from skeletal muscle injury is often incomplete because of the formation of fibrosis and inadequate myofiber regeneration; therefore, injured muscle could benefit significantly from therapies that both stimulate muscle regeneration and inhibit fibrosis. To this end, we focused on blocking myostatin, a member of the transforming growth factor-β superfamily and a negative regulator of muscle regeneration, with the myostatin antagonist follistatin. In vivo, follistatin-overexpressing transgenic mice underwent significantly greater myofiber regeneration and had less fibrosis formation compared with wild-type mice after skeletal muscle injury. Follistatin's mode of action is likely due to its ability to block myostatin and enhance neovacularization. Furthermore, muscle progenitor cells isolated from follistatin-overexpressing mice were significantly superior to muscle progenitors isolated from wild-type mice at regenerating dystrophin-positive myofibers when transplanted into the skeletal muscle of dystrophic mdx/severe combined immunodeficiency mice. In vitro, follistatin stimulated myoblasts to express MyoD, Myf5, and myogenin, which are myogenic transcription factors that promote myogenic differentiation. Moreover, follistatin's ability to enhance muscle differentiation is at least partially due to its ability to block myostatin, activin A, and transforming growth factor-β1, all of which are negative regulators of muscle cell differentiation. The findings of this study suggest that follistatin is a promising agent for improving skeletal muscle healing after injury and muscle diseases, such as the muscular dystrophies., (Copyright © 2011 American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.)

We have found that when muscle-derived stem cells (MDSCs) are implanted into a variety of tissues only a small fraction of the donor cells can be found within the regenerated tissues and the vast majority of cells are host derived. This observation has also been documented by other investigators using a variety of different stem cell types. It is speculated that the transplanted stem cells release factors that modulate repair indirectly by mobilizing the host's cells and attracting them to the injury site in a paracrine manner. This process is loosely called a 'paracrine mechanism', but its effects are not necessarily restricted to the injury site. In support of this speculation, it has been reported that increasing angiogenesis leads to an improvement of cardiac function, while inhibiting angiogenesis reduces the regeneration capacity of the stem cells in the injured vascularized tissues. This observation supports the finding that most of the cells that contribute to the repair process are indeed chemo-attracted to the injury site, potentially through host neo-angiogenesis. Since it has recently been observed that cells residing within the walls of blood vessels (endothelial cells and pericytes) appear to represent an origin for post-natal stem cells, it is tempting to hypothesize that the promotion of tissue repair, via neo-angiogenesis, involves these blood vessel-derived stem cells. For non-vascularized tissues, such as articular cartilage, the regenerative property of the injected stem cells still promotes a paracrine, or bystander, effect, which involves the resident cells found within the injured microenvironment, albeit not through the promotion of angiogenesis. In this paper, we review the current knowledge of post-natal stem cell therapy and demonstrate the influence that implanted stem cells have on the tissue regeneration and repair process. We argue that the terminal differentiation capacity of implanted stem cells is not the major determinant of the cells regenerative potential and that the paracrine effect imparted by the transplanted cells plays a greater role in the regeneration process.

Background: The ultrasonic Harmonic Blade cuts and coagulates soft tissue at temperatures lower than conventional electrosurgery. This study investigated whether improved hemostatic control and reduced collateral damage in skeletal muscle incisions translates into improved myofiber regeneration, reduced fibrosis and faster muscle recovery., Materials and Methods: Transections in the left gastrocnemius muscles of mice were made with the Harmonic Blade, and contralaterally, with either cold steel scissors or electrosurgery. Histology up to 8 wk after surgery was performed to evaluate myofiber regeneration and fibrosis. Tissue inflammation (Gr1+ neutrophils) and vascularization (CD31+ capillaries) were assessed immunohistochemically at 1 wk ., Results: Overall the Harmonic Blade showed significantly higher level of muscle regeneration than cold steel. Fibrosis for both the Harmonic Blade and cold steel decreased three-fold over the 8 wk period, while electrosurgery yielded significantly increasing fibrosis through wk 4 before declining. At 1 wk post-surgery the Harmonic Blade induced less inflammation than electrosurgery, and higher vascularization than electrosurgery and cold steel., Conclusions: Harmonic Blade-incised tissue showed accelerated vascularization, slight reduction of inflammation, enhanced muscle regeneration and decreased scarring, demonstrating a more effective healing process than electrosurgery., (Copyright © 2011 Elsevier Inc. All rights reserved.)

The capability to engineer microenvironmental cues to direct a stem cell population toward multiple fates, simultaneously, in spatially defined regions is important for understanding the maintenance and repair of multi-tissue units. We have previously developed an inkjet-based bioprinter to create patterns of solid-phase growth factors (GFs) immobilized to an extracellular matrix (ECM) substrate, and applied this approach to drive muscle-derived stem cells toward osteoblasts 'on-pattern' and myocytes 'off-pattern' simultaneously. Here this technology is extended to spatially control osteoblast, tenocyte and myocyte differentiation simultaneously. Utilizing immunofluorescence staining to identify tendon-promoting GFs, fibroblast growth factor-2 (FGF-2) was shown to upregulate the tendon marker Scleraxis (Scx) in C3H10T1/2 mesenchymal fibroblasts, C2C12 myoblasts and primary muscle-derived stem cells, while downregulating the myofibroblast marker α-smooth muscle actin (α-SMA). Quantitative PCR studies indicated that FGF-2 may direct stem cells toward a tendon fate via the Ets family members of transcription factors such as pea3 and erm. Neighboring patterns of FGF-2 and bone morphogenetic protein-2 (BMP-2) printed onto a single fibrin-coated coverslip upregulated Scx and the osteoblast marker ALP, respectively, while non-printed regions showed spontaneous myotube differentiation. This work illustrates spatial control of multi-phenotype differentiation and may have potential in the regeneration of multi-tissue units., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

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.

Perivascular multipotent mesenchymal progenitors exist in a variety of tissues, including the placenta. Here, we suggest that the abundant vasculature present in the human placenta can serve as a source of myogenic cells to regenerate skeletal muscle. Chorionic villi dissected from the mid-gestation human placenta were first transplanted intact into the gastrocnemius muscles of SCID/mdx mice, where they participated in muscle regeneration by producing myofibers expressing human dystrophin and spectrin. In vitro-cultured placental villi released rapidly adhering and migratory CD146+CD34⁻CD45⁻CD56⁻ cells of putative perivascular origin that expressed mesenchymal stem cell markers. CD146+CD34⁻CD45⁻CD56⁻ perivascular cells isolated and purified from the placental villi by flow cytometry were indeed highly myogenic in culture, and generated dystrophin-positive myofibers, and they promoted angiogenesis after transplantation into SCID/mdx mouse muscles. These observations confirm the existence of mesenchymal progenitor cells within the walls of human blood vessels, and suggest that the richly vascularized human placenta is an abundant source of perivascular myogenic cells able to migrate within dystrophic muscle and regenerate myofibers.

Background: Dedifferentiation of muscle cells in the tissue of mammals has yet to be observed. One of the challenges facing the study of skeletal muscle cell dedifferentiation is the availability of a reliable model that can confidentially distinguish differentiated cell populations of myotubes and non-fused mononuclear cells, including stem cells that can coexist within the population of cells being studied., Methodology/principal Findings: In the current study, we created a Cre/Lox-β-galactosidase system, which can specifically tag differentiated multinuclear myotubes and myotube-generated mononuclear cells based on the activation of the marker gene, β-galactosidase. By using this system in an adult mouse model, we found that β-galactosidase positive mononuclear cells were generated from β-galactosidase positive multinuclear myofibers upon muscle injury. We also demonstrated that these mononuclear cells can develop into a variety of different muscle cell lineages, i.e., myoblasts, satellite cells, and muscle derived stem cells., Conclusions/significance: These novel findings demonstrated, for the first time, that cellular dedifferentiation of skeletal muscle cells actually occurs in mammalian skeletal muscle following traumatic injury in vivo.

Background: Despite the initial promise of myoblast transfer therapy to restore dystrophin in Duchenne muscular dystrophy patients, clinical efficacy has been limited, primarily by poor cell survival post-transplantation. Murine muscle derived stem cells (MDSCs) isolated from slowly adhering cells (SACs) via the preplate technique, induce greater muscle regeneration than murine myoblasts, primarily due to improved post-transplantation survival, which is conferred by their increased stress resistance capacity. Aldehyde dehydrogenase (ALDH) represents a family of enzymes with important morphogenic as well as oxidative damage mitigating roles and has been found to be a marker of stem cells in both normal and malignant tissue. In this study, we hypothesized that elevated ALDH levels could identify murine and human muscle derived cell (hMDC) progenitors, endowed with enhanced stress resistance and muscle regeneration capacity., Methodology/principal Findings: Skeletal muscle progenitors were isolated from murine and human skeletal muscle by a modified preplate technique and unfractionated enzymatic digestion, respectively. ALDH(hi) subpopulations isolated by fluorescence activate cell sorting demonstrated increased proliferation and myogenic differentiation capacities compared to their ALDH(lo) counterparts when cultivated in oxidative and inflammatory stress media conditions. This behavior correlated with increased intracellular levels of reduced glutathione and superoxide dismutase. ALDH(hi) murine myoblasts were observed to exhibit an increased muscle regenerative potential compared to ALDH(lo) myoblasts, undergo multipotent differentiation (osteogenic and chondrogenic), and were found predominately in the SAC fraction, characteristics that are also observed in murine MDSCs. Likewise, human ALDH(hi) hMDCs demonstrated superior muscle regenerative capacity compared to ALDH(lo) hMDCs., Conclusions: The methodology of isolating myogenic cells on the basis of elevated ALDH activity yielded cells with increased stress resistance, a behavior that conferred increased regenerative capacity of dystrophic murine skeletal muscle. This result demonstrates the critical role of stress resistance in myogenic cell therapy as well as confirms the role of ALDH as a marker for rapid isolation of murine and human myogenic progenitors for cell therapy.

Background: Based on growing evidence that some adult multipotent cells necessary for tissue regeneration reside in the walls of blood vessels and the clinical success of vein wrapping for functional repair of nerve damage, we hypothesized that the repair of nerves via vein wrapping is mediated by cells migrating from the implanted venous grafts into the nerve bundle., Methodology/principal Findings: To test the hypothesis, severed femoral nerves of rats were grafted with venous grafts from animals of the opposite sex. Nerve regeneration was impaired when decellularized or irradiated venous grafts were used in comparison to untreated grafts, supporting the involvement of venous graft-derived cells in peripheral nerve repair. Donor cells bearing Y chromosomes integrated into the area of the host injured nerve and participated in remyelination and nerve regeneration. The regenerated nerve exhibited proper axonal myelination, and expressed neuronal and glial cell markers., Conclusions/significance: These novel findings identify the mechanism by which vein wrapping promotes nerve regeneration.

Current advances in stem cell research and innovative biological approaches in the field of tissue engineering and regenerative medicine could eventually translate into prospective clinical applications. Various adult organs and tissues harbor stem and progenitor cells that could potentially be used to repair, regenerate, and restore a variety of different tissues following acute injury or tissue destructive diseases. Skeletal muscle is a very convenient and plentiful source of somatic stem cells. It contains several distinct populations of myogenic stem cells including satellite cells that are mainly responsible for muscle growth and regeneration, and multipotent muscle-derived stem cells (MDSCs). Although both cell populations share some phenotypic similarities, MDSCs display a much greater differentiation potential in vitro and are capable of regenerating various tissues in vivo. Furthermore, these cells not only participate in the regeneration process by differentiating into tissue-specific cell types, but also promote endogenous tissue repair by secreting a multitude of trophic factors. In this article, we describe the biological aspects of MDSC isolation and characterization and provide an overview of potential therapeutic application of these cells for the treatment of cardiac and skeletal muscle injuries and diseases, urological dysfunction, and bone and cartilage defects. We also discuss major challenges and limitations currently faced by MDSC-based therapies that await resolution before these techniques can be applied clinically.

Intervertebral disc degeneration (IDD) is a common and debilitating disorder that results in reduced flexibility of the spine, pain, and reduced mobility. Risk factors for IDD include age, genetic predisposition, injury, and other environmental factors such as smoking. Loss of proteoglycans (PGs) contributes to IDD with advancing age. Currently there is a lack of a model for rapid investigation of disc aging and evaluation of therapeutic interventions. Here we examined progression of disc aging in a murine model of a human progeroid syndrome caused by deficiency of the DNA repair endonuclease, ERCC1-XPF (Ercc1(-/Δ) mice). The ERCC1-deficient mice showed loss of disc height and degenerative structural changes in their vertebral bodies similar to those reported for old rodents. Compared to their wild-type littermates, Ercc1(-/Δ) mice also exhibit other age-related IDD characteristics, including premature loss of disc PG, reduced matrix PG synthesis, and enhanced apoptosis and cell senescence. Finally, the onset of age-associated disc pathologies was further accelerated in Ercc1(-/Δ) mice following chronic treatment with the chemotherapeutic agent mechlorethamine. These results demonstrate that Ercc1(-/Δ) mice represent an accurate and rapid model of disc aging and provide novel evidence that DNA damage negatively impacts PG synthesis., (© 2010 Orthopaedic Research Society. Published by Wiley Periodicals, Inc.)

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.

The IκB kinase (IKKα, β and the regulatory subunit IKKγ) complex regulates nuclear factor of κB (NF-κB) transcriptional activity, which is upregulated in many chronic inflammatory diseases. NF-κB signaling promotes inflammation and limits muscle regeneration in Duchenne muscular dystrophy (DMD), resulting in fibrotic and fatty tissue replacement of muscle that exacerbates the wasting process in dystrophic muscles. Here, we examined whether dominant-negative forms of IKKα (IKKα-dn) and IKKβ (IKKβ-dn) delivered by adeno-associated viral (AAV) vectors to the gastrocnemius (GAS) and tibialis anterior (TA) muscles of 1, 2 and 11-month-old mdx mice, a murine DMD model, block NF-κB activation and increase muscle regeneration. At 1 month post-treatment, the levels of nuclear NF-κB in locally treated muscle were decreased by gene transfer with either AAV-CMV-IKKα-dn or AAV-CMV-IKKβ-dn, but not by IKK wild-type controls (IKKα and β) or phosphate-buffered saline (PBS). Although treatment with AAV-IKKα-dn or AAV-IKKβ-dn vectors had no significant effect on muscle regeneration in young mdx mice treated at 1 and 2 months of age and collected 1 month later, treatment of old (11 months) mdx with AAV-CMV-IKKα-dn or AAV-CMV-IKKβ-dn significantly increased levels of muscle regeneration. In addition, there was a significant decrease in myofiber necrosis in the AAV-IKKα-dn- and AAV-IKKβ-dn-treated mdx muscle in both young and old mice. These results demonstrate that inhibition of IKKα or IKKβ in dystrophic muscle reduces the adverse effects of NF-κB signaling, resulting in a therapeutic effect. Moreover, these results clearly demonstrate the therapeutic benefits of inhibiting NF-κB activation by AAV gene transfer in dystrophic muscle to promote regeneration, particularly in older mdx mice, and block necrosis.

The success of small-diameter tissue engineered vascular grafts (TEVGs) greatly relies on an appropriate cell source and an efficient cellular delivery and carrier system. Pericytes have recently been shown to express mesenchymal stem cell features. Their relative availability and multipotentiality make them a promising candidate for TEVG applications. The objective of this study was to incorporate pericytes into a biodegradable scaffold rapidly, densely and efficiently, and to assess the efficacy of the pericyte-seeded scaffold in vivo. Bi-layered elastomeric poly(ester-urethane)urea scaffolds (length = 10 mm; inner diameter = 1.3 mm) were bulk seeded with 3 x 10(6) pericytes using a customized rotational vacuum seeding device in less than 2 min (seeding efficiency > 90%). The seeded scaffolds were cultured in spinner flasks for 2 days and then implanted into Lewis rats as aortic interposition grafts for 8 weeks. Results showed pericytes populated the porous layer of the scaffolds evenly and maintained their original phenotype after the dynamic culture. After implantation, pericyte-seeded TEVGs showed a significant higher patency rate than the unseeded control: 100% versus 38% (p < 0.05). Patent pericyte-seeded TEVGs revealed extensive tissue remodeling with collagen and elastin present. The remodeled tissue consisted of multiple layers of alpha-smooth muscle actin- and calponin-positive cells, and a von Willebrand factor-positive monolayer in the lumen. These results demonstrate the feasibility of a pericyte-based TEVG and suggest that the pericytes play a role in maintaining patency of the TEVG as an arterial conduit., (Copyright © 2010 Elsevier Ltd. All rights reserved.)

Although cellular transplantation has been shown to promote improvements in cardiac function following injury, poor cell survival following transplantation continues to limit the efficacy of this therapy. We have previously observed that transplantation of muscle-derived stem cells (MDSCs) improves cardiac function in an acute murine model of myocardial infarction to a greater extent than myoblasts. This improved regenerative capacity of MDSCs is linked to their increased level of antioxidants such as glutathione (GSH) and superoxide dismutase. In the current study, we demonstrated the pivotal role of antioxidant levels on MDSCs survival and cardiac functional recovery by either reducing the antioxidant levels with diethyl maleate or increasing antioxidant levels with N-acetylcysteine (NAC). Both the anti- and pro-oxidant treatments dramatically influenced the survival of the MDSCs in vitro. When NAC-treated MDSCs were transplanted into infarcted myocardium, we observed significantly improved cardiac function, decreased scar tissue formation, and increased numbers of CD31(+) endothelial cell structures, compared to the injection of untreated and diethyl maleate-treated cells. These results indicate that elevating the levels of antioxidants in MDSCs with NAC can significantly influence their tissue regeneration capacity.

Injectable biomaterials have been recently investigated as a therapeutic approach for cardiac repair. Porcine-derived small intestinal submucosa (SIS) material is currently used in the clinic to promote accelerated wound healing for a variety of disorders. In this study, we hypothesized that gels derived from SIS extracellular matrix would be advantageous as an injectable material for cardiac repair. We evaluated 2 forms of SIS gel, types B (SIS-B) and C (SIS-C), for their ability to provide a therapeutic effect when injected directly into ischemic myocardium using a murine model of an acute myocardial infarction. Echocardiography analysis at both 2 and 6 weeks after infarction demonstrated preservation of end-systolic left ventricular geometry and improvement of cardiac contractility in the hearts injected with SIS-B when compared with control hearts injected with saline. However, the SIS-C gel provided no functional efficacy in comparison with control. Histological analysis revealed that SIS-B reduced infarct size and induced angiogenesis relative to control, whereas injection of SIS-C had minimal effect on these histological parameters. Characterization of both gels revealed differential growth factor content with SIS-B exhibiting higher levels of basic fibroblast growth factor than SIS-C, which may explain, at least in part, the differential histological and functional results. This study suggests that SIS gel offers therapeutic potential as an injectable material for the repair of ischemic myocardium. Further understanding of SIS gel characteristics, such as biological and physical properties, that are critical determinants of efficacy would be important for optimization of this biomaterial for cardiac repair., (Copyright 2010 Elsevier Ltd. All rights reserved.)

Adult stem cells have long been discussed in regards to their application in regenerative medicine. Adult stem cells have generated a great deal of excitement for treating injured and diseased tissues due to their impressive capabilities to undergo multi-lineage cell differentiation and their self-renewal ability. Most importantly, these qualities have made them advantageous for use in autologous cell transplantation therapies. The current protocol will introduce the readers to the modified preplate technique where soft tissues of the musculoskeletal system, e.g. tendon and muscle, are 1(st) enzymatically dissociated and then placed in collagen coated flasks with medium. The supernatant, which is composed of medium and the remaining floating cells, is serially transferred daily to new flasks. The stem cells are the slowest to adhere to the flasks which is usually takes 5-7 days (serial transfers or preplates). By using this technique, adult stem cells present in these tissues can be easily harvested through fairly non-invasive procedures.

Background: There is a clinical need for bone replacement strategies because of the shortfalls endemic to autologous bone grafting, especially in the pediatric patient population. For the past 25 years, the animal model that has been used to test bone replacement strategies has been the calvarial critical-size defect, based on the initial size of the bone defect. This study was undertaken to test the concept of the critical size in several different models. A review of the theoretical and scientific bases for the critical-size defect was also undertaken., Methods: Two different rodent species (including 28 adult mice and six adult rats) were used to assess bone healing by means of two-dimensional radiographic analysis after creating small bone defects using different surgical techniques., Results: Defects in mice that were smaller than critical-size defects (1.8-mm diameter) were shown to heal a maximum of 50 percent 1 year postoperatively. Small defects (2.3-mm diameter) in the rat skull showed approximately 35 percent healing after 6 weeks. Neither the choice of rodent species nor the maintenance of the dura mater significantly affected calvarial bone healing., Conclusions: These results suggest that calvarial bone healing is not well described and much more data need to be collected. Also, after a review of the existing literature and a critique of the clinical applicability of the model, it is suggested that the use of the term "critical-size defect" be discontinued.

Skeletal muscle-derived stem cells (MDSCs) are able to differentiate into cardiomyocytes (CMs). However, it remains to be investigated whether differentiated CMs contract similar to native CMs. Here, we developed a three-dimensional collagen gel bioreactor (3DGB) that induces a working CM phenotype from MDSCs, and the contractile properties are directly measured as an engineered cardiac tissue. Neonate rat MDSCs were isolated from hind-leg muscles via the preplate technique. Isolated MDSCs were approximately 60% positive to Sca-1 and negative to CD34, CD45, or c-kit antigens. We sorted Sca-1(-) MDSCs and constructed MDSC-3DGBs by mixing MDSCs with acid soluble rat tail collagen type-I and matrix factors. MDSC-3DGB exhibited spontaneous cyclic contraction by culture day 7. MDSC-3DGB expressed cardiac-specific genes and proteins. Histological assessment revealed that cardiac-specific troponin-T and -I expressed in a typical striation pattern and connexin-43 was expressed similar to the native fetal ventricular papillary muscle. beta-Adrenergic stimulation increased MDSC-3DGB spontaneous beat frequency. MDSC-3DGB generated contractile force and intracellular calcium ion transients similar to engineered cardiac tissue from native cardiac cells. Results suggest that MDSC-3DGB induces a working CM phenotype in MDSCs and is a useful 3D culture system to directly assess the contractile properties of differentiated CMs in vitro.

The purpose of this study was to demonstrate spatial control of osteoblast differentiation in vitro and bone formation in vivo using inkjet bioprinting technology and to create three-dimensional persistent bio-ink patterns of bone morphogenetic protein-2 (BMP-2) and its modifiers immobilized within microporous scaffolds. Semicircular patterns of BMP-2 were printed within circular DermaMatrix human allograft scaffold constructs. The contralateral halves of the constructs were unprinted or printed with BMP-2 modifiers, including the BMP-2 inhibitor, noggin. Printed bio-ink pattern retention was validated using fluorescent or (125)I-labeled bio-inks. Mouse C2C12 progenitor cells cultured on patterned constructs differentiated in a dose-dependent fashion toward an osteoblastic fate in register to BMP-2 patterns. The fidelity of spatial restriction of osteoblastic differentiation at the boundary between neighboring BMP-2 and noggin patterns improved in comparison with patterns without noggin. Acellular DermaMatrix constructs similarly patterned with BMP-2 and noggin were then implanted into a mouse calvarial defect model. Patterns of bone formation in vivo were comparable with patterned responses of osteoblastic differentiation in vitro. These results demonstrate that three-dimensional biopatterning of a growth factor and growth factor modifier within a construct can direct cell differentiation in vitro and tissue formation in vivo in register to printed patterns.

Limited autologous vascular graft availability and poor patency rates of synthetic grafts for bypass or replacement of small-diameter arteries remain a concern in the surgical community. These limitations could potentially be improved by a tissue engineering approach. We report here our progress in the development and in vivo testing of a stem-cell-based tissue-engineered vascular graft for arterial applications. Poly(ester urethane)urea scaffolds (length = 10 mm; inner diameter = 1.2 mm) were created by thermally induced phase separation (TIPS). Compound scaffolds were generated by reinforcing TIPS scaffolds with an outer electrospun layer of the same biomaterial (ES-TIPS). Both TIPS and ES-TIPS scaffolds were bulk-seeded with 10 x 10(6) allogeneic, LacZ-transfected, muscle-derived stem cells (MDSCs), and then placed in spinner flask culture for 48 h. Constructs were implanted as interposition grafts in the abdominal aorta of rats for 8 weeks. Angiograms and histological assessment were performed at the time of explant. Cell-seeded constructs showed a higher patency rate than the unseeded controls: 65% (ES-TIPS) and 53% (TIPS) versus 10% (acellular TIPS). TIPS scaffolds had a 50% mechanical failure rate with aneurysmal formation, whereas no dilation was observed in the hybrid scaffolds. A smooth-muscle-like layer of cells was observed near the luminal surface of the constructs that stained positive for smooth muscle alpha-actin and calponin. LacZ+ cells were shown to be engrafted in the remodeled construct. A confluent layer of von Willebrand Factor-positive cells was observed in the lumen of MDSC-seeded constructs, whereas acellular controls showed platelet and fibrin deposition. This is the first evidence that MDSCs improve patency and contribute to the remodeling of a tissue-engineered vascular graft for arterial applications.

The liver is unique for its ability to regenerate after injury, however, critical injuries or disease cause it to lose this quality. Stem cells have been explored as a possibility to restore the function of seriously damaged livers, based on their self-renewability and multiple differentiation capacity. These experiments examine the ability of muscle derived stem cells (MDSCs) to differentiate into hepatocyte-like cells in vitro and acquire functional liver attributes for repairing damaged livers. In vitro experiments were performed using MDSCs from postnatal mice and mouse hepatocyte cell lines. Our data revealed that MDSCs differentiated into hepatocyte-like cells and expressed liver cell markers, albumin, hepatocyte nuclear factor 4 α, and alpha feto-protein, both at the RNA and protein level. Additionally, in vivo studies showed successful engraftment of MDSCs into hepatectomized mouse livers of mice. These results provide evidence suggesting that MDSCs have the capacity to differentiate into liver cell-like cells and may serve as potential candidates to aid in liver regeneration.

Importance of the Field: Muscle-directed gene therapy for genetic muscle diseases can be performed by the recombinant adeno-associated viral (rAAV) vector delivery system to achieve long-term therapeutic gene transfer in all affected muscles., Areas Covered in This Review: Recent progress in rAAV-vector-mediated muscle-directed gene transfer and associated techniques for the treatment of muscular dystrophies (MD). The review covers literature from the past 2 - 3 years., What the Reader Will Gain: rAAV-directed muscular dystrophy gene therapy can be achieved by mini-dystrophin replacement and exon-skipping strategies. The additional strategies of enhancing muscle regeneration and reducing inflammation in the muscle micro-environment should be useful to optimize therapeutic efficacy. This review compares the merits and shortcomings of different administration methods, promoters and experimental animals that will guide the choice of the appropriate strategy for clinical trials., Take Home Message: Restoration of muscle histopathology and function has been performed using rAAV systemic gene delivery. In addition, the combination of gene replacement and adjuvant therapies in the future may be beneficial with regard to improving muscle regeneration and decreasing myofiber necrosis. The challenges faced by large animal model studies and in human trials arise from gene transfer efficiency and immune response, which may be overcome by optimizing the rAAV vectors utilized and the administration methods.

Muscle-derived stem-cell (MDSC) transplantation presents a promising method for the treatment of muscle injuries. This study investigated the ability of exercise to enhance MDSC transplantation into the injured muscle. Mice were divided into four groups: contusion + phosphate-buffered saline (C + PBS; n = 14 muscles), C + MDSC transplantation (n = 12 muscles), C + PBS + treadmill running (C + PBS + TM; n = 17 muscles), and C + MDSC + TM (n = 13 muscles). One day after injury, the TM groups began running for 1 or 5 weeks. Two days after injury, muscles of C + MDSC and C + MDSC + TM groups were injected with MDSCs. One or 5 weeks later, the number and differentiation of transplanted MDSCs, myofiber regeneration, collagen I formation, and vascularity were assessed histologically. In vitro, MDSCs were subjected to mechanical stimulation, and growth kinetics were quantified. In vitro, mechanical stimulation decreased the MDSC population doubling time (18.6 +/- 1.6 h) and cell division time (10.9 +/- 0.7 h), compared with the controls (population doubling time: 23.0 +/- 3.4 h; cell division time: 13.3 +/- 1.1 h) (p = 0.01 and 0.03, respectively). In vivo, 5 weeks of TM increased the myogenic contribution of transplanted MDSCs, compared with the controls (p = 0.02). C + MDSC, C + PBS + TM, and C + MDSC + TM demonstrated decreased fibrosis at 5 weeks, compared with the C + PBS controls (p = 0.00, p = 0.03, and p = 0.02, respectively). Results suggest that the mechanical stimulation favors MDSC proliferation, both in vitro and in vivo, and that exercise enhances MDSC transplantation after injury.

Platelets, as the first cellular response after disruption of vascular and/or tissue integrity, cover any existing injury within our body. But is the regenerative potential of platelets limited to providing a cellular patch for wounds? This review highlights the recent advance in our understanding of platelets being distinctly regulated and regulating cells that contribute immensely to the healing process from the very initial stage to the late events of tissue regeneration. For instance, the intrinsic actions of platelets as a regenerative cell, the participation of platelets in angiogenic processes, and the interplay of platelets and circulating stem and progenitor cells, as well as potential therapeutic implications, are addressed. Although we are starting to understand the underlying mechanisms connecting platelets to the components of tissue regeneration just mentioned, many aspects remain to be elucidated. The demand to invest research in this area is underscored by the fact that platelets or platelet-derived molecules are already applied in clinical contexts such as connective tissue regeneration, whereas other research fields have largely neglected platelet effects going beyond their participation in the coagulation cascade. Understanding the mechanisms connecting platelets to tissue regeneration, however, will inevitably open novel options in regenerative medicine., (Thieme Medical Publishers.)

We have isolated a population of muscle-derived stem cells (MDSCs) that, when compared with myoblasts, display an improved regeneration capacity, exhibit better cell survival, and improve myogenesis and angiogenesis. In addition, we and others have observed that the origin of the MDSCs may reside within the blood vessel walls (endothelial cells and pericytes). Here, we investigated the role of vascular endothelial growth factor (VEGF)-mediated angiogenesis in MDSC transplantation-based skeletal muscle regeneration in mdx mice (an animal model of muscular dystrophy). We studied MDSC and MDSC transduced to overexpress VEGF; no differences were observed in vitro in terms of phenotype or myogenic differentiation. However, after in vivo transplantation, we observe an increase in angiogenesis and endogenous muscle regeneration as well as a reduction in muscle fibrosis in muscles transplanted with VEGF-expressing cells when compared to control cells. In contrast, we observe a significant decrease in vascularization and an increase in fibrosis in the muscles transplanted with MDSCs expressing soluble forms-like tyrosine kinase 1 (sFlt1) (VEGF-specific antagonist) when compared to control MDSCs. Our results indicate that VEGF-expressing cells do not increase the number of dystrophin-positive fibers in the injected mdx muscle, when compared to the control MDSCs. Together the results suggest that the transplantation of VEGF-expressing MDSCs improved skeletal muscle repair through modulation of angiogenesis, regeneration and fibrosis in the injected mdx skeletal muscle.