Myoblast Sheets Research Articles

A first-in-human clinical study of laparoscopic autologous myoblast sheet transplantation to prevent delayed perforation after duodenal endoscopic mucosal dissection

BackgroundThe detection rate of superficial non-ampullary duodenal epithelial tumors (SNADETs) has recently been increasing. Large tumors may contain malignant lesions and early therapeutic intervention is recommended. Endoscopic mucosal dissection (ESD) is considered a feasible treatment modality, however, the anatomical and physiological characteristics of the duodenum create a risk of postoperative perforation after ESD.MethodsTo explore whether myoblast sheet transplantation could prevent delayed perforation after ESD, a first-in-human (FIH) clinical trial of laparoscopic autologous myoblast sheet transplantation after duodenal ESD was launched. Autologous myoblast sheets fabricated from muscle tissue obtained seven weeks before ESD were transplanted laparoscopically onto the serous side of the ESD. The primary endpoints were the onset of peritonitis due to delayed perforation within three days after surgery and all adverse events during the follow-up period.ResultsThree patients with SNADETs ≥ 20 mm in size underwent transplantation of a myoblast sheet onto the serous side of the duodenum after ESD. In case 1, The patient’s postoperative course was uneventful. Endoscopy and abdominal computed tomography revealed no signs of delayed perforation. Despite incomplete mucosal closure in case 2, and multiple micro perforations during ESD in case 3, cell sheet transplantation could prevent the postoperative massive perforation after ESD, and endoscopy on day 49 after transplantation revealed no stenosis.ConclusionsThis clinical trial showed the safety, efficacy, and procedural operability of this novel regenerative medicine approach involving transplanting an autologous myoblast sheet laparoscopically onto the serosa after ESD in cases with a high risk of delayed perforation. This result indicates the potential application of cell sheet medicine in treating various abdominal organs and conditions with minimal invasiveness in the future.Trial registrationjRCT, jRCT2073210094. Registered November 8 2021,https://jrct.niph.go.jp/latest-detail/jRCT2073210094.

Effects of Myostatin on Nuclear Morphology at the Myotendinous Junction.

Myostatin (Myo) is known to suppress skeletal muscle growth, and was recently reported to control tendon homeostasis. The purpose of the present study was to investigate the regulatory involvement of Myo in the myotendinous junction (MTJ) in vivo and in vitro. After Achilles tendon injury in mice, we identified unexpected cell accumulation on the tendon side of the MTJ. At postoperative day 7 (POD7), the nuclei had an egg-like profile, whereas at POD28 they were spindle-shaped. The aspect ratio of nuclei on the tendon side of the MTJ differed significantly between POD7 and POD28 (p = 4.67 × 10-34). We then investigated Myo expression in the injured Achilles tendon. At the MTJ, Myo expression was significantly increased at POD28 relative to POD7 (p = 0.0309). To investigate the action of Myo in vitro, we then prepared laminated sheets of myoblasts (C2C12) and fibroblasts (NIH3T3) (a pseudo MTJ model). Myo did not affect the expression of Pax7 and desmin (markers of muscle development), scleraxis and temonodulin (markers of tendon development), or Sox9 (a common marker of muscle and tendon development) in the cell sheets. However, Myo changed the nuclear morphology of scleraxis-positive cells arrayed at the boundary between the myoblast sheet and the fibroblast sheet (aspect ratio of the cell nuclei, myostatin(+) vs. myostatin(-): p = 0.000134). Myo may strengthen the connection at the MTJ in the initial stages of growth and wound healing.

Crisscross multilayering of cell sheets.

Hydrostatic skeletons such as the Hydra's consist of two stacked layers of muscle cells perpendicularly oriented. In vivo, these bilayers first assemble, and then the muscle fibers of both layers develop and organize with this crisscross orientation. In the present work, we identify an alternative mechanism of crisscross bilayering of myoblasts in vitro, which results from the prior local organization of these active cells in the initial monolayer. The myoblast sheet can be described as a contractile active nematic in which, as expected, most of the +1/2 topological defects associated with this nematic order self-propel. However, as a result of the production of extracellular matrix (ECM) by the cells, a subpopulation of these comet-like defects does not show any self-propulsion. Perpendicular bilayering occurs at these stationary defects. Cells located at the head of these defects converge toward their core where they accumulate until they start migrating on top of the tail of the first layer, while the tail cells migrate in the opposite direction under the head. Since the cells keep their initial orientations, the two stacked layers end up perpendicularly oriented. This concerted process leading to a crisscross bilayering is mediated by the secretion of ECM.

Rapid and chronological expression of angiogenetic genes is a major mechanism involved in cell sheet transplantation in a rat gastric ulcer model.

IntroductionCell sheet technology has been applied in the treatment of patients with severe cardiac failure. Although the paracrine effect of cell sheets accelerating angiogenesis is thought to be the intrinsic mechanism for improvement of cardiac function, little is known about how a cell sheet would function in the abdomen.MethodsWe used acetic acid-induced gastric ulcer rat model to elucidate the mechanisms of myoblast sheet transplantation in the abdomen. Myoblast sheet was implanted onto the serosal side of the gastric ulcer and the effect of sheet transplantation was analyzed. The maximal diameter of the ulcer and the changes in the gene expression of various growth factors in transplanted site was analyzed. The progenitor marker CD34 was also examined by immunohistochemistry.ResultsCell sheet transplantation accelerated the ulcer healing. qPCR showed that angiogenic growth factors were significantly upregulated around the ulcer in the transplantation group. In addition, at first, HIF-1a and SDF-1 continued to increase from 3 h after transplantation to 72 h, then VEGF increased significantly after 24 h with a slight delay. An immunohistochemical analysis showed a statistically significant increase in CD34 positivity in the tissue around the ulcer in the transplantation group.ConclusionMyoblast sheet secreted various growth factors and cytokines immediately after transplantation onto the serosal side of artificial ulcer in the abdomen. Autonomous secretion, resulting in the time-dependent and well-orchestrated gene expression of various growth factors, plays a crucial role in the cell sheet function. Cell sheet transplantation is expected to be useful to support angiogenesis of the ischemic area in the abdominal cavity.

Gelatin MAGIC powder as nutrient-delivering 3D spacer for growing cell sheets into cost-effective cultured meat

Cell cultured meat is artificial meat obtained by culturing animal-derived cells in vitro, and received significant attention as an emerging future protein source. The mass proliferation of cells in the cultured meat production is a strenuous process that delays the commercialization of cultured meat because it requires an expensive culture medium for a long period. Herein, we report on a strategy to develop advanced cultured meat using fish gelatin mass growth-inducing culture (MAGIC) powder and myoblast sheets. The MAGIC powder had an edible gelatin microsphere (GMS) structure and exhibited different morphologies and bonding activities depending on the degree of crosslinking. We analyzed the loading and release of nutrients for each GMS with diverse surface properties, and selected the most effective GMSs to improve the proliferation of myoblasts under serum-reduced medium. The GMSs exerted four significant functions in the culture of myoblast sheets, and consequently produced cost- and time-effective meat-like cell sheets than the conventional method. We prepared cultured meats composed of cell sheet containing GMSs and evaluated the quality of the cultured meat by comparing the tissue properties with soy meat and chicken breast.

Highly feasible procedure for laparoscopic transplantation of cell sheets under pneumoperitoneum in porcine model.

Cell sheet technology is one of the most successful methodologies in regenerative medicine. Various applications of cell sheets have been introduced in first-in-human studies in several clinical fields. When transplanting a cell sheet into internal organs, a relatively large incision is required for delivery due to difficulty handling the sheet. We developed a laparoscopic delivery procedure for safe and easy transplantation of cell sheets in a porcine model. Pneumoperitoneum was established by inflation with CO2. First, to increase the strength during handling, fibrin was sprayed onto the surface of the cell sheet, and then a myoblast sheet was placed onto the newly developed carrier. The sheets were pinched with laparoscopic forceps to insert into the abdominal cavity through the laparoscopic port. Myoblast sheets were then applied to the surface of the liver, colon, small intestine, and stomach, and procedure times were measured. At three days post transplantation, a histopathological examination was performed to confirm engraftment of the sheet. The function and engraftment were also analyzed in a duodenal endoscopic submucosal dissection (ESD) model. The fibrin-processed myoblast sheet was able to be managed with conventional laparoscopic forceps without breaking. Despite the drastic change in air pressure by passing through the laparoscopic port, the sheets suffered no apparent damage. The transplantation procedure times did not markedly differ among transplant sites. A histopathological examination revealed thin-layered, desmin-positive cells at each transplant site. With transplantation following ESD, the engrafted myoblast sheets effectively prevented delayed perforation. Our procedure is simple, and the system involves a carrier made of medically fit silicon, commercially available fibrin glue and conventional laparoscopic forceps. Our procedure is a powerful tool for laparoscopical cell sheet transplantation.

Multilayered Human Skeletal Muscle Myoblast Sheets Promote the Healing Process After Colonic Anastomosis in Rats

Colorectal anastomotic leakage is one of the most feared and fatal complications of colorectal surgery. To date, no external coating material that can prevent anastomotic leakage has been developed. As myoblasts possess anti-inflammatory capacity and improve wound healing, we developed a multilayered human skeletal muscle myoblast (HSMM) sheet by periodic exposure to supraphysiological hydrostatic pressure during repeated cell seeding. We assessed whether the application of an HSMM sheet can promote the healing process after colonic anastomosis. Partial colectomy and insufficient suturing were employed to create a high-risk colo-colonic anastomosis model in 60 nude rats. Rats were divided into a control group (n = 30) and an HSMM sheet group (n = 30). Macroscopic findings, anastomotic bursting pressure, and histology at the colonic anastomotic site were evaluated on postoperative day (POD) 3, 5, 7, 14, and 28. The application of an HSMM sheet significantly suppressed abscess formation at the anastomotic site compared to the control group on POD3 and 5. The anastomotic bursting pressure in the HSMM sheet group was higher than that in the control group on POD3 and 5. Inflammatory cell infiltration in the HSMM sheet group was significantly suppressed compared to that in the control group throughout the time course. Collagen deposition in the HSMM sheet group on POD3 was significantly abundant compared to that in the control group. Regeneration of the mucosa at the colonic anastomotic site was promoted in the HSMM sheet group compared to that in the control group on POD14 and 28. Immunohistochemical analysis demonstrated that surviving cells in the HSMM sheet gradually decreased with postoperative time and none were detected on POD14. These results suggest that the application of a multilayered HSMM sheet may prevent postoperative colonic anastomotic leakage.

Autologous skeletal myoblast sheet prevents cardiomyocyte ischemia and right heart dysfunction in pressure-overloaded right heart porcine model

Abstract Introduction Severe heart failure (HF) with congenital heart disease (CHD) have demonstrated life threatening disorder despite of remarkable progress in medical therapies. Autologous skeletal myoblast sheet transplantation therapy showed clinical efficacy for left ventricular dysfunction by cytokine paracrine effects, which are expected to be sufficiently effective against right ventricular (RV) dysfunction which is often seen in end-stage of CHD patients with severe HF. Hypothesis An autologous skeletal myoblast sheet transplantation alleviates RV dysfunction in a pressure-overloaded right heart in a porcine model. Methods Five-to-six-month-old Göttingen mini-pigs underwent pulmonary artery banding with vascular occluding system. To create the porcine model of chronic pressure-overloaded right heart, vascular occluding system was gradually inflated, over a month, to make pulmonary stenosis to banding velocity >3.0 m/s measured by echocardiography (UCG), and then fixed for another month. Two months after banding, autologous skeletal myoblast sheet was placed on the epicardium of the RV free wall and followed for 2 months. Groups were as follows: control (C, n=5), sheet implantation (S, n=5). Cardiac function was measured using UCG, cardiac computed tomography (CT), and cardiac catheterization (Cath). Two months after sheet implantation, hearts were dissected for histologic analysis. Results Before sheet implantation, RV dysfunction was equal in groups; however, 2 months after sheet implantation, RV dysfunction and myocardial ischemia was significantly ameliorated in group S than group C. On CT, RV ejection fraction exacerbation were well controlled in Group S compared to Group C (S 44.9±2.2 vs C 31.9±2.1% [p=0.0042]). UCG and Cath revealed well maintained systolic and diastolic function in Group S compared to Group C (Tei index: S 0.42±0.06 vs C 0.70±0.07 [p=0.0240], Fraction Area Change: S 45.8±7.8 vs C 19.5±1.3% [p=0.0240], Isovolumic Relaxation Time; S 44.3±9.2 vs C 97.3±9.5 ms [p=0.0304]). On C11-Acetate Positron Emission Tomography, myocardial ischemia was more prominent in Group C compared to Group S (K mono-Rest/Stress: S 3.17±0.69 vs C 2.03±0.65 min-1 [p=0.0421], Myocardial Blood Flow-Rest/Stress: S 3.22±0.39 vs C 2.13±0.92 min-1 [p=0.0421]). In histologic analysis, Group S presented less progressed hypertrophic change in periodic acid-Schiff stain (S 13.5±0.9 vs C 18.0±3.0 μg [p=0.0240]), anti-fibrotic changes in picrosirius red stain (S 3.0±0.3 vs C 4.2±0.2% [p=0.0421]), more angiogenesis in CD31 expression (S 18.3±1.5 vs C 10.7±2.8 / 104 μm2 [p=0.0240]), and less production of reactive oxygen species in fluorescent immunostaining (S 5.9±1.7 vs C 18.4±1.7% [p=0.0304]). Conclusion Autologous skeletal myoblast sheet transplantation alleviates cardiomyocyte Ischemia and RV dysfunction in a porcine model of pressure-overloaded right heart. Funding Acknowledgement Type of funding source: None

Role and therapeutic effects of skeletal muscle-derived non-myogenic cells in a rat myocardial infarction model

BackgroundTransplantation of skeletal myoblast sheets is a promising strategy for the treatment of heart failure, and its therapeutic effects have already been proven in both animal disease models and clinical trials. Myoblast sheets reportedly demonstrate their therapeutic effects by producing many paracrine factors. Although the quality of processed cells for transplantation can be evaluated by the positive ratio of CD56, a myoblast marker, it is unclear which cell populations from isolated cells produce paracrine factors that have an impact on therapeutic effects, and whether these therapeutic effects are closely correlated with CD56-positive cells isolated from the skeletal muscle is also unclear. Therefore, we hypothesized that CD56-negative cells as well as CD56-positive cells isolated from the skeletal muscle produce paracrine factors and have therapeutic effects in skeletal muscle-derived cell sheet therapy for heart failure.MethodsCell surface and intracellular markers of CD56-negative non-myogenic cells (NMCs) and CD56-positive myoblasts were evaluated. We also analyzed cytokine expression, tube formation ability, and stem cell mobilization in both cell populations. Finally, we assessed the therapeutic effects of the cell populations in a rat myocardial infarction model.ResultsAnalysis of cell surface and intracellular markers revealed that CD56-negative NMCs expressed fibroblast markers and a higher level of mesenchymal cell markers, such as CD49b and CD140a, than myoblasts. Both NMCs and myoblasts expressed various cytokines in vitro with different expression patterns. In addition, NMCs induced tube formation (control vs. myoblasts vs. NMCs: 100 ± 11.2 vs. 142 ± 8.3 vs. 198 ± 7.4%) and stem cell mobilization (control vs. myoblasts vs. NMCs: 100 ± 6.8 vs. 210 ± 22.9 vs. 351 ± 36.0%) to a higher degree in vitro than did myoblasts. The effect of NMCs and myoblasts on the improvement of cardiac function and suppression of myocardial fibrosis in rat myocardial infarction model was comparable.ConclusionThese results indicate that NMCs exhibit therapeutic effects in skeletal muscle-derived cell sheet therapy for heart failure. Thus, accurate parameters correlating with therapeutic effects need to be further explored.

The Efficacy of Autologous Myoblast Sheet Transplantation to Prevent Perforation After Duodenal Endoscopic Submucosal Dissection in Porcine Model

The recent advent of endoscopy has enabled the endoscopic submucosal dissection (ESD) of superficial nonampullary duodenal epithelial tumors. However, the substantially thin wall and presence of bile and pancreatic juice make it technically difficult to perform duodenal ESD without perforation, which leads to lethal complications. The present study evaluated the efficacy of autologous myoblast sheet transplantation for the prevention of late perforation after duodenal ESD in a porcine model. Two weeks before ESD, skeletal muscle was surgically excised from the femur of pigs, and myoblasts were isolated and seeded in temperature-responsive culture dishes to prepare sheets. Immediately after ESD, the autologous myoblast sheets were attached to the serosal surface at the ESD site with omentopexy. The pigs were divided into two groups: the autologous myoblast sheet group (n = 5), where the myoblast cell sheet was attached to the ESD ulcer part from the duodenal serous side, and the Omentum group (n = 5), where only the omentum was used. The pigs were sacrificed and analyzed macroscopically and histologically on postoperative day 3. The macroscopic examination of the abdominal cavity revealed perforation in the ESD ulcer area and leakage of bile in the Omentum group but no perforation in the Sheet group. A histopathological examination revealed that continuity of the duodenal wall at the ESD site was maintained with dense connective tissue in the Sheet group. In conclusion, autologous myoblast sheets were useful for preventing perforation after duodenal ESD.

Analysis of force vector field during centrifugation for optimizing cell sheet adhesion.

A three-dimensional tissue was fabricated by layering cell sheets with centrifugation. In this system, an optimal centrifugal force promoted the adhesion between (a) a cell sheet and a culture dish, and (b) layered cell sheets, resulting in a significant decrease in the fabrication time of the tissue. However, negative effects like sliding/significant deformation of cell sheets were observed upon high rotational speed use. These negative effects inhibit the further shortening of the fabrication time. The sliding/deformation suggests that the centrifugal forces were applied on the cell sheets in unwanted directions. Studies on the force vector field applied to the object placed on the plate during centrifugation are not available, and thus, the reason for the occurrence of such negative effects is unclear. Here, we theoretically derived the spatial distribution of acceleration applied on a plate during centrifugation. Using this theory, we found that the negative effects were triggered by the centrifugal force in the direction parallel to the plate surface, which appeared due to an inclination of the plate surface against a horizontal plane. Therefore, by adding weights on the plate edge to maintain the plate surface in a horizontal position, we succeeded in eliminating the negative effects and in increasing the rotational speed, with the minimum risk of sliding/deformation of cell sheets. We succeeded in reducing the time to establish tight adhesion between a mouse myoblast sheet and a culture dish, and layered cell sheets by increasing the centrifugal force from 5 min to 1 min without significant cytotoxicity.

Long-term outcome of a dilated cardiomyopathy patient after mitral valve surgery combined with tissue-engineered myoblast sheets\u2014report of a case

BackgroundDilated cardiomyopathy (DCM) is a life-threatening heart muscle disease characterized by progressive heart failure, which often requires left ventricular assist device (LVAD) implantation or heart transplantation (HTx). A tissue engineering strategy using cell sheets for end-stage heart failure has emerged in recent years.Case presentationHere, we describe a 50-year-old DCM patient with severe symptoms of heart failure with severe mitral regurgitation (MR) who was not a candidate for LVAD or HTx. The patient underwent mitral valve replacement followed by the transplantation of autologous myoblast sheets.ConclusionThe patient’s clinical symptoms improved with a preservation of cardiac performance, and he has survived for over 6 years since the combined surgery. This combined method was feasible and appeared to be effective, and thus represents a promising strategy for treating severe heart failure in end-stage DCM and as an alternative treatment for selected patients who are not candidates for LVAD or HTx.

A novel, flexible and automated manufacturing facility for cell-based health care products: Tissue Factory.

IntroductionCurrent production facilities for Cell-Based Health care Products (CBHPs), also referred as Advanced-Therapy Medicinal Products or Regenerative Medicine Products, are still dependent on manual work performed by skilled workers. A more robust, safer and efficient manufacturing system will be necessary to meet the expected expansion of this industrial field in the future. Thus, the ‘flexible Modular Platform (fMP)’ was newly designed to be a true “factory” utilizing the state-of-the-art technology to replace conventional “laboratory-like” manufacturing methods. Then, we built the Tissue Factory as the first actual entity of the fMP.MethodsThe Tissue Factory was designed based on the fMP in which several automated modules are combined to perform various culture processes. Each module has a biologically sealed chamber that can be decontaminated by hydrogen peroxide. The asepticity of the processing environment was tested according to a pharmaceutical sterility method. Then, three procedures, production of multi-layered skeletal myoblast sheets, expansion of human articular chondrocytes and passage culture of human induced pluripotent stem cells, were conducted by the system to confirm its ability to manufacture CHBPs.ResultsFalling or adhered microorganisms were not detected either just after decontamination or during the cell culture processes. In cell culture tests, multi-layered skeletal myoblast sheets were successfully manufactured using the method optimized for automatic processing. In addition, human articular chondrocytes and human induced-pluripotent stem cells could be propagated through three passages by the system at a yield comparable to manual operations.ConclusionsThe Tissue Factory, based on the fMP, successfully reproduced three tentative manufacturing processes of CBHPs without any microbial contamination. The platform will improve the manufacturability in terms of lower production cost, improved quality variance and reduced contamination risks. Moreover, its flexibility has the potential to adapt to the modern challenges in the business environment including employment issues, low operational rates, and relocation of facilities. The fMP is expected to become the standard design basis of future manufacturing facilities for CBHPs.

250 - Effect of fibroblast population on vegf secretion in human skeletal muscle myoblast sheet

Human skeletal muscle myoblast (HSMM) sheet transplantation is a novel and promising approach to restores the function of a damaged tissue for myocardial infarction treatment. Since the HSMM sheet has ability to secrete multiple cytokines that improve heart function through the facilitation of angiogenesis, here we are focusing on understanding of cytokines regulation in the HSMM sheet, which is necessary for development of more functional and complex engineered tissue.

Effect of Mesenchymal Cells on Myoblast Sheets Embedded in Collagen Gel.

The objective of this study was to investigate the effects of mesenchymal cells on myoblasts in long-term cultivation of myoblast cell sheets. Sheets of myoblasts and mesenchymal cells from Japanese rabbit oral mucosa were generated and analyzed by histochemistry, Western blot, and reverse transcription-polymerase chain reaction. The presence of desmin and type IV collagen, which is seen in normal muscle tissue, was also confirmed in all the sheets produced. Expression of desmin and type IV collagen showed a decrease under co-culture conditions. In addition, expression of genes important in maintaining the undifferentiated state (Pax7, CD34, myogenin, MyoD) in myoblasts was observed throughout the long cultivation period. Insulin-like growth factor was expressed only when the mesenchymal cells were co-cultured with myoblasts. These data suggest that the presence of mesenchymal cells in a long-term co-culture system influences myoblast differentiation.

Peer review report 1 on “Tooth extraction in a patient after autologous skeletal myoblast sheet transplantation for severe dilated cardiomyopathy”

Peer review report 1 on “Tooth extraction in a patient after autologous skeletal myoblast sheet transplantation for severe dilated cardiomyopathy”

Peer review report 2 on “Tooth extraction in a patient after autologous skeletal myoblast sheet transplantation for severe dilated cardiomyopathy”

Peer review report 2 on “Tooth extraction in a patient after autologous skeletal myoblast sheet transplantation for severe dilated cardiomyopathy”

Proliferative activity of skeletal myoblast sheet by paracrine effects of mesenchymal stem cells

ObjectivesThe purpose of this study was to examine the proliferative activity enhancement of skeletal myoblasts in the presence and absence of mesenchymal stem cells (MSC). MethodsWe artificially fabricated two types of cell sheets by co-culturing rabbit skeletal myoblast sheets with rabbit MSCs using type IA collagen gel (MC+), and cell sheets without rabbit MSCs (MC−). ResultsThe results of hematoxylin and eosin staining revealed that MC+ was thicker than MC− on day 7. Immunohistochemical staining revealed a low level of desmin expression in both sheets on day 2. Desmin expression increased at days 7 and 12, and desmin localization was consistent with the stratified area. Reverse transcription PCR revealed the presence of MyoD and PAX7 in both sheets on days 2, 7, and 12. The presence of myogenin was confirmed in both sheets on days 7 and 12. Hepatocyte growth factor expression was evident in MC+ on day 2, and in both sheets on day 7. Measurement of cell proliferative activity based on DNA cell cycle analysis indicated that MC+ had significantly higher cell proliferative activity than MC− on day 7. There were no significant differences in cell proliferative activity between MC+ and MC− on day 12. ConclusionsThis study demonstrated that the presence of MSCs could transiently enhance the proliferative activity of myoblasts, but that this enhancement is ultimately diminished due to contact inhibition.

The Paracrine Effect of Skeletal Myoblasts Is Cardioprotective Against Oxidative Stress and Involves EGFR-ErbB4 Signaling, Cystathionase, and the Unfolded Protein Response.

Therapeutic effects of skeletal myoblast transplantation into the myocardium are mediated via paracrine factors. We investigated the ability of myoblast-derived soluble mediators to protect cardiomyocytes from oxidative stress. Fetal rat cardiac cells were treated with conditioned medium from cultures of myoblasts or cardiac fibroblasts, and oxidative stress was induced with H2O2. Myoblast-derived factors effectively prevented oxidative stress-induced cardiac cell death and loss of mitochondrial membrane potential. This protective effect was mediated via epidermal growth factor (EGF) receptor and c-Met signaling, and mimicked by neuregulin 1 but not EGF. Microarray analysis of cardiac cells treated with myoblast versus cardiac fibroblast-derived mediators revealed differential regulation of genes associated with antioxidative effects: cystathionine-γ-lyase (cst), xanthine oxidase, and thioredoxin-interacting protein as well as tribbles homolog 3 (trib3). Cardiac cell pretreatment with tunicamycin, an inducer of trib3, also protected them against H2O2-induced cell death. Epicardial transplantation of myoblast sheets in a rat model of acute myocardial infarction was used to evaluate the expression of CST and trib3 as markers of myoblasts' paracrine effect in vivo. Myoblast sheets induced expression of the CST as well as trib3 in infarcted myocardium. CST localized around blood vessels, suggesting smooth muscle cell localization. Our results provide a deeper molecular insight into the therapeutic mechanisms of myoblast-derived paracrine signaling in cardiac cells and suggest that myoblast transplantation therapy may prevent oxidative stress-induced cardiac deterioration and progression of heart failure.

Elucidation and regulation of heterogeneous cell behavior inside a multilayered skeletal muscle myoblast sheet

Elucidation and regulation of heterogeneous cell behavior inside a multilayered skeletal muscle myoblast sheet