Muscle-derived Stem Cells Transplantation Research Articles

Mechanism of Hematopoietic Reconstruction in Mice Treated with Danggui Buxue Decoction combined with Muscle-Derived Stem Cell Transplantation

To investigate the mechanism of hematopoietic reconstruction in mice treated with Danggui Buxue Decoction (DBD) combined with the muscle-derived stem cell transplantation (MDSCT). Female Kunming mice were randomly divided into the 6 groups: irradiation model, the bone marrow transplantation, the MDSC transplantation, the DBD 1 (4.5 g/kg), 2 (13.5 g/kg), and 3 (22.5 g/kg) + MDSC transplantation. After a week of oral administration of normal saline or different doses of DBD, The mice were exposied to 8 Gy 137Cs γ ray and were followed by bone marrow or MDSC transplantation. The expression levels of Notch1, Jagged1 and Hes1 in bone marrow, thymus and spleen were measured at 3 and 8 weeks after irradiation and transplantation. In the bone marrow, 3 weeks after above-mentioned treatment, the expression of Notch1 mRNA increased obviously and the expression of Jagged1, Hes1 mRNA decreased obviously in each intervention group, compared with the irradiation model group. 8th week after treatment, the expression of Notch1 mRNA decreased obviously in each intervention group, the Jagged1 mRNA expression decreased obviously except the bone marrow group, and Hes1 mRNA expression increased (P＜0.05) in each intervention group. 3 weeks after treatment, compared with the irradiation model group, the expression of Notch1 mRNA in the thymocytes increased only in DBD1+MDSC group, Jagged1, Hes1 mRNA was increased in the MDSC transplantation group and the DBD1、2+MDSC group. 8th week after treatment, the expression of Notch1, Jagged1 mRNA expression decreased in each intervention group, the expression of Hes1 mRNA increased obviously in the MDSC transplantation group and the DBD1、2+MDSC group (P＜0.05). In the spleen, 3 weeks after treatment, the expression of Notch1, Jagged1 mRNA in the spleen of each intervention group decreased obviously, compared with the irradiation model group. The expression of Jagged1, Hes1 mRNA in each intervention group were increased obviously 8th week after treatment (P＜0.05). MDSC transplantation after pretreatment of DBD can improve the hematopoietic reconstitution in mice with lethal dose radiation damage. Notch1、Jagged1 and Hes1 play different roles in this process, but the concrete mechanism needs to be further studied.

A Functional Comparison of Treatment of Intrinsic Sphincter Deficiency with Muscle-Derived and Adipose Tissue-Derived Stem Cells.

This study investigated the effect of muscle-derived stem cells (MDSCs) and adipose tissue-derived stem cells (ADSCs) in the treatment of stress urinary incontinence (SUI) and their differences in a rat model. MDSCs and ADSC were isolated from rats (n = 10), examined for their properties, and labeled with enhanced green fluorescent protein (EGFP) and β-galactosidase (β-gal) gene. Rats received bladder-neck and transurethral sphincter injection of EGFP-labeled MDSCs and β-gal gene-labeled ADSC and injection of D-Hanks as a control (n = 24 each group). At 0, 15, 30, and 60 days after cells injection, urinary voiding function was assessed by urine dynamics detector. The rats were killed to harvest their urethras for tracking of MDSCs and ADSC. Western blotting and quantitative real-time reverse transcription PCR (qRT-PCR) was performed to detect smooth muscle contents. Urodynamic test showed that MDSCs and ADSC improved the function of urination in rats with intrinsic sphincter deficiency (ISD), and effect of MDSCs-treatment was more pronounced. In addition, histologic analysis showed that the MDSCs and ADSC-treated groups had significantly higher myosin and α-smooth muscle actin (α-SMA) content than the control group. Compared with ADSC-treated groups, the MDSCs-treated groups in myosin and α-SMA content showed the tendency of increase. In summary, MDSCs and ADSCs have obvious effects in the treatment and/or prevention of ISD and transplantation of MDSCs is more effective than ADSC. © 2018 IUBMB Life, 70(10):976-984, 2018.

The Combined Use of Losartan and Muscle-Derived Stem Cells Significantly Improves the Functional Recovery of Muscle in a Young Mouse Model of Contusion Injuries

Background: Although muscle injuries tend to heal uneventfully in most cases, incomplete functional recovery commonly occurs as a result of scar tissue formation at the site of injury, even after treatment with muscle-derived stem cells (MDSCs). Hypothesis: The transplantation of MDSCs in the presence of a transforming growth factor β1 (TGF-β1) antagonist (losartan) would result in decreased scar tissue formation and enhance muscle regeneration after contusion injuries in a mouse model. Study Design: Controlled laboratory study. Methods: An animal model of muscle contusion was developed using the tibialis anterior muscle in 48 healthy mice at 8 to 10 weeks of age. After sustaining muscle contusion injuries, the mice were divided into 4 groups: (1) saline injection group (control group; n = 15), (2) MDSC transplantation group (MDSC group; n = 15), (3) MDSC transplantation plus oral losartan group (MDSC/losartan group; n = 15), and (4) healthy uninjured group (healthy group; n = 3). Losartan was administrated systemically beginning 3 days after injury and continued until the designated endpoint (1, 2, or 4 weeks after injury). MDSCs were transplanted 4 days after injury. Muscle regeneration and fibrotic scar formation were evaluated by histology, and the expression of follistatin, MyoD, Smad7, and Smad2/3 were analyzed by immunohistochemistry and reverse transcription polymerase chain reaction analysis. Functional recovery was measured via electrical stimulation of the peroneal nerve. Results: When compared with MDSC transplantation alone, MDSC/losartan treatment resulted in significantly decreased scar formation, an increase in the number of regenerating myofibers, and improved functional recovery after muscle contusions. In support of these findings, the expression levels of Smad7 and MyoD were significantly increased in the group treated with both MDSCs and losartan. Conclusion: When compared with MDSCs alone, the simultaneous treatment of muscle contusions with MDSCs and losartan significantly reduced scar formation, increased the number of regenerating myofibers, and improved the functional recovery of muscle; these effects were caused, at least in part, by the losartan-mediated upregulation of Smad7 and MyoD. Increased levels of Smad7 and MyoD together reduced the deposition of scar tissue (via the inhibition of TGF-β1 by Smad7) and committed the transplanted MDSCs toward a myogenic lineage (via Smad7-regulated MyoD expression). Clinical Relevance: The study findings contribute to the development of biological treatments to accelerate and improve the quality of muscle healing after injury.

Quality of life assessment in female patients 2 and 4 years after muscle-derived cell transplants for stress urinary incontinence treatment.

Regenerative medicine for the treatment of urinary incontinence has become a popular area of focus in the search for therapies for this disease. The paper focused on women's quality of life assessment who were subjected to transplantation of MDSC (autologous muscle derived stem cells) to the urethral sphincter. The procedure was conducted in 16 female patients who completed the observation stage. Assessment of quality of life before and after the treatment (two and four years post-operation) was conducted based on the validated I-QOL questionnaire (the Polish language version). The questionnaire study showed that autologous cell therapy significantly improves quality of life in female patients suffering from stress urinary incontinence (SUI). The total I-QOL score increased from 49 (SD ± 7.7) before therapy to 77 (SD ± 5.4) two years post-operation. Four years after the procedure, quality of life remained at a higher level than before therapy, although quality of life decreased by several points when compared with the results from the two-year follow-up - 63 (SD ± 7.2). Patients reported significantly less concern related to their ability to reach the toilet to avoid incontinence, improved sleep at night, a higher level of satisfaction with life, and more satisfaction with their sexual lives (p<0.05). The MDSC injection procedure for SUI treatment has significant improved quality of life in the majority of our patients in 2 and 4 year follow-up.

Neuromuscular Electrical Stimulation as a Method to Maximize the Beneficial Effects of Muscle Stem Cells Transplanted into Dystrophic Skeletal Muscle

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.

Mechanical Loading of Stem Cells for Improvement of Transplantation Outcome in a Model of Acute Myocardial Infarction: The Role of Loading History

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.

Role of angiogenesis after muscle derived stem cell transplantation in injured medial collateral ligament

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.

Intramuscular Transplantation of Muscle-Derived Stem Cells Accelerates Skeletal Muscle Healing After Contusion Injury via Enhancement of Angiogenesis

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.

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

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

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

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

Transplantation of Muscle-Derived Stem Cells into the Corpus Cavernosum Restores Erectile Function in a Rat Model of Cavernous Nerve Injury

PurposeMuscle-derived stem cells (MDSCs) harvested from skeletal muscles have the advantage of providing easier access and do not pose the immunogenic risks of embryonic stem cells. We investigated the effect of intracavernosal transplantation of MDSCs on erectile function in rats with bilateral cavernous nerve injury.Materials and MethodsAdult male white rats underwent experimentation in 3 groups: group I, sham operation; group II, bilateral cavernous nerve injury; group III, bilateral cavernous nerve injury with MDSC injection. MDSCs were harvested from the femoral muscle of rats and were then injected into the cavernosum. Survival of MDSCs and measurement of erectile function was studied after 4 weeks. We checked the intracavernosal pressure (ICP) and obtained penile tissue. The expression of cyclic guanosine monophosphate (cGMP) was analyzed.ResultsFour weeks after transplantation, PKH-26-labeled MDSCs were identified in the cavernosal tissues of group III. Peak ICP and the drop rate of group II were 52±8.7 mmHg and 34±6.5 mmHg/min, respectively, whereas peak ICP and the drop rate of group III were 97±15.6 mmHg and 17±4.9 mmHg/min, respectively, showing that erectile function improved after MDSC transplantation (p<0.05). The expression of cGMP was significantly lower in group II (21.9±5.8 fmol/well) than in group I and group III (70.2±10.3 and 58.9±10.5 fmol/well, respectively).ConclusionsIn a cavernous nerve injury rat model, intracavernosal transplantation of MDSCs showed acceptable survival of MDSCs as well as improvement of erectile function.

Transplantation of muscle-derived stem cells plus biodegradable fibrin glue restores the urethral sphincter in a pudendal nerve-transected rat model

We investigated whether fibrin glue (FG) could promote urethral sphincter restoration in muscle-derived stem cell (MDSC)-based injection therapies in a pudendal nerve-transected (PNT) rat, which was used as a stress urinary incontinence (SUI) model. MDSCs were purified from the gastrocnemius muscles of 4-week-old inbred female SPF Wistar rats and labeled with green fluorescent protein. Animals were divided into five groups (N = 15): sham (S), PNT (D), PNT+FG injection (F), PNT+MDSC injection (M), and PNT+MDSC+FG injection (FM). Each group was subdivided into 1- and 4-week groups. One and 4 weeks after injection into the proximal urethra, leak point pressure (LPP) was measured to assess urethral resistance function. Histology and immunohistochemistry were performed 4 weeks after injection. LPP was increased significantly in FM and M animals after implantation compared to group D (P < 0.01), but was not different from group S. LPP was slightly higher in the FM group than in the M group but there was no significant difference between them at different times. Histological and immunohistochemical examination demonstrated increased numbers of surviving MDSCs (109 ± 19 vs 82 ± 11/hpf, P = 0.026), increased muscle/collagen ratio (0.40 ± 0.02 vs 0.34 ± 0.02, P = 0.044), as well as increased microvessel density (16.9 ± 0.6 vs 14.1 ± 0.4/hpf, P = 0.001) at the injection sites in FM compared to M animals. Fibrin glue may potentially improve the action of transplanted MDSCs to restore the histology and function of the urethral sphincter in a SUI rat model. Injection of MDSCs with fibrin glue may provide a novel cellular therapy method for SUI.

Cellular Antioxidant Levels Influence Muscle Stem Cell Therapy

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.

The Synergistic Effect of Treadmill Running on Stem-Cell Transplantation to Heal Injured Skeletal Muscle

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.

Effect of VEGF on the Regenerative Capacity of Muscle Stem Cells in Dystrophic Skeletal Muscle

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.

Functional Overloading of Dystrophic Mice Enhances Muscle-Derived Stem Cell Contribution to Muscle Contractile Capacity

Ambrosio F, Ferrari RJ, Fitzgerald GK, Carvell G, Boninger ML, Huard J. Functional overloading of dystrophic mice enhances muscle-derived stem cell contribution to muscle contractile capacity. Arch Phys Med Rehabil Objectives To evaluate the effect of functional overloading on the transplantation of muscle derived stem cells (MDSCs) into dystrophic muscle and the ability of transplanted cells to increase dystrophic muscle's ability to resist overloading-induced weakness. Design Cross-sectional. Setting Laboratory. Animals Male mice (N=10) with a dystrophin gene mutation. Interventions MDSCs were intramuscularly transplanted into the extensor digitorum longus muscle (EDL). Functional overloading of the EDL was performed by surgical ablation of the EDL's synergist. Main Outcome Measures The total number of dystrophin-positive fibers/cross-section (as a measure of stem cell engraftment), the average number of CD31+ cells (as a measure of capillarity), and in vitro EDL contractile strength. Independent t tests were used to investigate the effect of overloading on engraftment, capillarity, and strength. Paired t tests were used to investigate the effect of MDSC engraftment on strength and capillarity. Results MDSC transplantation protects dystrophic muscles against overloading-induced weakness (specific twitch force: control 4.5N/cm 2±2.3; MDSC treated 7.9N/cm 2±1.4) ( P=.02). This improved force production following overloading is concomitant with an increased regeneration by transplanted MDSCs (MDSC: 26.6±20.2 dystrophin-positive fibers/cross-section; overloading + MDSC: 170.6±130.9 dystrophin-positive fibers/cross-section [ P=.03]). Overloading-induced increases in skeletal muscle capillarity is significantly correlated with increased MDSC engraftment ( R 2=.80, P=.01). Conclusions These findings suggest that the functional contribution of transplanted MDSCs may rely on activity-dependent mechanisms, possibly mediated by skeletal muscle vascularity. Rehabilitation modalities may play an important role in the development of stem cell transplantation strategies for the treatment of muscular dystrophy.

266. Transplantation of Muscle-Derived Stem Cells Genetically Engineered To Express Vascular Endothelial Growth Factor (VEGF) Decreases Fibrosis in Dystrophic Muscle

266. Transplantation of Muscle-Derived Stem Cells Genetically Engineered To Express Vascular Endothelial Growth Factor (VEGF) Decreases Fibrosis in Dystrophic Muscle

Regenerative medicine based on muscle stem cells

Members of our laboratory have isolated various populations of myogenic cells from the postnatal skeletal muscle of normal mice by examining the cells’ adhesion characteristics, proliferation behavior, and myogenic and stem cell marker expression profiles. Although most of these cell populations have displayed characteristics similar to those of satellite cells, we also have identified a unique population of muscle‐derived stem cells (MDSCs). MDSCs exhibit long‐term proliferation and high self‐renewal rates, high resistance to stress, and can differentiate toward various lineages, including muscle (skeletal and cardiac), neural, endothelial, osteogenic, and chondrogenic lineages, both in vitro and in vivo. The transplantation of MDSCs, in contrast to that of other myogenic cells, has improved the efficiency of dystrophic muscle regeneration and the delivery of dystrophin to dystrophic muscle. I will also show that transplantation of female MDSCs (F‐MDSC) rather than male MDSCs (M‐MDSCs) significantly improves skeletal muscle regeneration despite a similar myogenic and stem cells markers expression by both cell types. My presentation will also address the influence of environmental cues released within dystrophic or injured skeletal muscle upon the differentiation of MDSCs toward fibrotic cells. I will discuss potential strategies by which scar tissue formation can be prevented within injured muscle through TGF‐β1 blockade. I then will discuss the use of MDSCs in gene therapy and tissue engineering applications designed to improve bone and cartilage healing through the genetic modification of MDSCs to express osteogenic proteins (BMP‐2 and ‐4) as well as angiogenic factor (VEGF). The results outlined in my presentation open new avenues by which researchers could use muscle stem cell–based gene therapy and tissue engineering to improve tissue regeneration.

A Relationship Between Vascular Endothelial Growth Factor, Angiogenesis, and Cardiac Repair After Muscle Stem Cell Transplantation Into Ischemic Hearts

We investigated whether vascular endothelial growth factor (VEGF) was associated with the angiogenic and therapeutic effects induced after transplantation of skeletal muscle-derived stem cells (MDSCs) into a myocardial infarction (MI). Because very few MDSCs were found to differentiate into new blood vessels when injected into the heart, the mechanism underlying the occurrence of angiogenesis after MDSC transplantation is currently unknown. In the present study, we used a gain- or loss-of-VEGF function approach with skeletal MDSCs engineered to express VEGF or soluble Flt1, a VEGF-specific antagonist, to identify the involvement of VEGF in MDSC transplantation-induced neoangiogenesis. Vascular endothelial growth factor- and soluble Flt1-engineered MDSCs were injected into an acute MI. Angiogenesis and cardiac function were evaluated by immunohistochemistry and echocardiography. Both control and VEGF-overexpressing MDSCs induced angiogenesis, prevented adverse cardiac remodeling, and improved function compared with saline-injected hearts. However, these therapeutic effects were diminished in hearts transplanted with MDSCs expressing soluble Flt1 despite successful cell engraftment. In vitro experiments demonstrated that MDSCs increased secretion of VEGF in response to hypoxia and cyclic stretch (likely conditions in ischemic hearts), suggesting that transplanted MDSCs release VEGF in vivo. Our findings suggest that VEGF is essential for the induction of angiogenesis and functional improvements observed after MDSC transplantation for infarct repair.

Regeneration of dystrophin-expressing myocytes in the mdx heart by skeletal muscle stem cells

Cell transplantation holds promise as a potential treatment for cardiac dysfunction. Our group has isolated populations of murine skeletal muscle-derived stem cells (MDSCs) that exhibit stem cell-like properties. Here, we investigated the fate of MDSCs after transplantation into the hearts of dystrophin-deficient mdx mice, which model Duchenne muscular dystrophy (DMD). Transplanted MDSCs generated large grafts consisting primarily of numerous dystrophin-positive myocytes and, to a lesser degree, dystrophin-negative non-myocytes that expressed an endothelial phenotype. Most of the dystrophin-positive myocytes expressed a skeletal muscle phenotype and did not express a cardiac phenotype. However, some donor myocytes, located at the graft-host myocardium border, were observed to express cardiac-specific markers. More than half of these donor cells that exhibited a cardiac phenotype still maintained a skeletal muscle phenotype, demonstrating a hybrid state. Sex-mismatched donors and hosts revealed that many donor-derived cells that acquired a cardiac phenotype did so through fusion with host cardiomyocytes. Connexin43 gap junctions were not expressed by donor-derived myocytes in the graft. Scar tissue formation in the border region may inhibit the fusion and gap junction connections between donor and host cells. This study demonstrates that MDSC transplantation warrants further investigation as a potential therapy for cardiac dysfunction in DMD.