Losartan enhances the success of myoblast transplantation

Duchenne muscular dystrophy is a recessive X-linked genetic disease caused by dystrophin gene mutations. Cell therapy can be a potential approach aiming to introduce a functional dystrophin in the dystrophic patient myofibers. However, this strategy produced so far limited results. Transforming growth factor-β (TGF-β) is a negative regulator of skeletal muscle development and is responsible for limiting myogenic regeneration. The combination of TGF-β signaling inhibition with myoblast transplantation can be an effective therapeutic approach in dystrophin-deficient patients. Our aim was to verify whether the success of human myoblast transplantation in immunodeficient dystrophic mice is enhanced with losartan, a molecule that downregulates TGF-β expression. In vitro, blocking TGF-β activity with losartan increased proliferation and fusion and decreased apoptosis in human myoblasts. In vivo, human myoblasts were transplanted in mice treated with oral losartan. Immunodetection of human dystrophin in tibialis anterior cross sections 1 month posttransplantation revealed more human dystrophin-positive myofibers in these mice than in nontreated dystrophic mice. Thus, blocking the TGF-β signal with losartan treatment improved the success of myoblast transplantation probably by increasing myoblast proliferation and fusion, decreasing macrophage activation, and changing the expression of myogenic regulator factors.     

Differential effect of BMP4 on NIH/3T3 and C2C12 cells: implications for endochondral bone formation.

After intramuscular implantation, BMP4-expressing NIH/3T3 fibroblasts and BMP4-expressing C2C12 myoblasts can promote ectopic cartilage and bone formation. Fibroblasts tend to undergo chondrogenesis, whereas myoblasts primarily undergo osteogenesis. These results suggest that endochondral bone formation may involve different cell types, a finding that could have major implications for the tissue engineering of bone and cartilage. INTRODUCTION: The delivery of BMP4 through cell-based gene therapy can trigger ectopic endochondral bone formation in skeletal muscle. We hypothesized that, when stimulated with or transduced to express BMP4, different types of cells residing within skeletal muscle might participate in different stages of endochondral bone formation. MATERIALS AND METHODS: We compared the responses of a fibroblast cell line (NIH/3T3), a myoblast cell line (C2C12), primary fibroblasts, and primary myoblasts to BMP4 stimulation in vitro. We then transduced the four cell populations to express BMP4 and compared their ability to promote ectopic endochondral bone formation in skeletal muscle. RESULTS: Under the influence of BMP4 in vitro and in vivo, NIH/3T3 cells differentiated toward both chondrogenic and osteogenic lineages, whereas most C2C12 cells underwent primarily osteogenic differentiation. NIH/3T3 cells genetically modified to express BMP4 induced delayed but more robust cartilage formation than did genetically modified C2C12 cells, which promoted rapid ossification. These differences in terms of the timing and amount of cartilage and bone formation persisted even after we introduced a retrovirus encoding dominant negative Runx2 (DNRunx2) into the C2C12 cells, which interferes with the function of Runx2. Superior osteogenic potential was also displayed by the primary myoblasts in vitro and in vivo compared with the primary fibroblasts. The different proliferation abilities and differentiation potentials exhibited by these cells when influenced by BMP4 may at least partially explain the differing roles that BMP4-expressing myogenic cells and BMP4-expressing fibroblastic cells play in endochondral bone formation. CONCLUSIONS: Our findings suggest that the process of endochondral bone formation in skeletal muscle after delivery of BMP4 involves different cell types, including fibroblastic cells, which are more involved in the chondrogenic phases, and myoblastic cells, which are primarily involved in osteogenesis. These findings could have important implications for the development of tissue engineering applications focused on bone and cartilage repair.     

Enhanced antitumor effects of bone marrow transplantation in combination with fibroblast-mediated IL-2 and IL-3 gene therapy.

BACKGROUND: Bone marrow transplantation (BMT) and gene therapy are potent approaches to the recovery of bone marrow depression and induction of antitumor immunity after chemotherapy for the treatment of malignancies. In the present study, enhanced antitumor effect of BMT in combination with fibroblast-mediated interleukin (IL)-2 and IL-3 gene therapy was observed in tumor-bearing mice after chemotherapy. METHODS: BALB/c mice were inoculated s.c. with J558L plasmacytoma cells and injected i.p. with cyclophosphamide 300 mg/kg 3 days later. 24 hours after chemotherapy syngeneic bone marrow cells in combination with NIH3T3 fibroblast cells engineered to produce IL-2 (NIH3T3-IL-2) and/or NIH3T3 cells engineered to produce IL-3 (NIH3T3-IL-3) were implanted into the tumor-bearing mice. RESULTS: BMT in combination with implantation of either NIH3T3-IL-2 or NIH3T3-IL-3 cells exerted significant inhibition on the growth of J558L tumors and prolonged the survival period of the tumor-bearing mice as compared with the treatments with Hanks solution, BMT alone, or BMT plus implantation of NIH3T3 cells transduced with Neo gene. Synergistic antitumor effect was observed in mice after combined BMT and cytokine gene therapy. The cytotoxicities of natural killer cells, cytotoxic T lymphocytes, and macrophages in mice increased markedly after the combined treatment. Recovery of CFU-GM, CFU-MK and CFU-E formation in mice after combined therapy was accelerated obviously in mice after combined therapy. CONCLUSIONS: BMT in combination with fibroblast-mediated IL-2 and IL-3 gene therapy elicited augmented antitumor effects synergistically in tumor-bearing mice after chemotherapy mainly through induction of antitumor immune response and accelerated recovery of hematopoiesis.     

Long‐Term Evaluation of Myoblast Seeded Patches Implanted on Infarcted Rat Hearts

Cell transplantation presents great potential for treatment of patients with severe heart failure. However, its clinical application was revealed to be more challenging than initially expected in experimental studies. Further investigations need to be undertaken to define the optimal treatment conditions. We previously reported on the epicardial implantation of a bio‐engineered construct of skeletal myoblast‐seeded polyurethane and its preventive effect on progression toward heart failure. In the present study, we present a long‐term evaluation of this functional outcome. Left anterior descending coronary ligation was performed in female Lewis rats. Two weeks later, animals were treated with either epicardial implantation of biograft, acellular scaffold, sham operation, or direct intramyocardial skeletal myoblast injection. Functional assessments were performed with serial echocardiographies every 3 months and end point left ventricle pressure was assessed. Hearts were then harvested for histological examinations. Myocardial infarction induced a slow and progressive reduction in fractional shortening after 3 months. Progression toward heart failure was significantly prevented for up to 6 months after injection of myoblasts and for up to 9 months following biograft implantation. Nevertheless, this effect vanished after 12 months, with immunohistological examinations revealing an absence of the transplanted myoblasts within the scaffold. We demonstrated that tissue therapy is superior to cell therapy for stabilization of heart function. However, beneficial effects are transient.     

Adjuvant immunotherapy using fibroblasts genetically engineered to secrete interleukin 12 prevents recurrence after surgical resection of established tumors in a murine adenocarcinoma model

BACKGROUND: To explore effective therapeutic strategy against cancer of the gastrointestinal tract, tumor vaccination using fibroblasts secreting interleukin-12 (IL-12) was developed as an adjuvant therapy against murine tumor after surgical resection. METHODS: Initially, IL-12 was genetically engineered into fibroblasts (IL-12/3T3 cells), and then we evaluated in vivo and in vitro antitumor effects. In the vaccination model, irradiated C-26 tumor mass was reinoculated intradermally with IL-12/3T3 cells in mice as a tumor vaccine to examine how much it suppresses tumor recurrence. RESULTS: IL-12/3T3 cells producing 7.2 ng/10(6) cells/24 h murine IL-12 in vitro exerted dose-dependent potent tumor suppression when coinoculated with C-26 cells in vivo. Specific immunity was also acquired in 63% of mice in vivo. In the vaccination model, protective immunity was developed in 70% of mice that were inoculated with irradiated tumor mass and IL-12/3T3 cells. In addition, local recurrence was not observed in vaccinated mice, although 44% of control mice had recurrence. CONCLUSIONS: Coinoculation of genetically engineered fibroblasts secreting IL-12 with irradiated tumor mass was proved to be an effective tumor vaccine. This system of vaccination is easily applicable to clinical situations, particularly to human gastrointestinal tract cancers.     

Selective cell dissemination into the heart by retrograde intracoronary infusion in the rat

Cell transplantation through the intracoronary route has theoretical advantages over direct intramuscular injection in global cell dissemination into the myocardium with less injury. The authors have established a novel experimental model of retrograde intracoronary infusion in the rat by which the feasibility of this infusion for cell transplantation and the dynamics of myoblasts grafted by this route were investigated. After retrograde intracoronary implantation of beta-galactosidase-expressing myoblasts, myoblasts were observed to disseminate to all layers of only the left ventricular free wall, with little myocardial damage, and to differentiate into multinuclear myotubes by day 28. Beta-galactosidase enzyme activity estimated that 31.4% of initially grafted myoblasts existed within the heart at 10 min after implantation. The number slightly increased by day 3, increased to 117.3% at day 7, and reached a plateau of 132.1% at day 14. These data suggest the utility of retrograde intracoronary infusion for selective cell dissemination into the myocardium.     

Transplantation of myocyte precursors derived from embryonic stem cells transfected with IGFII gene in a mouse model of muscle injury

BACKGROUND: Reconstruction of skeletal muscle tissue is hampered by the lack of availability of functional substitution of the tissue. METHODS: Embryonic stem (ES) cells were transfected with the insulin-like growth factor (IGF) II gene and were selected with G418. The resultant cell clones were analyzed regarding their myogenic differentiation in vitro and in vivo. RESULTS: The cells expressed early and late myogenic differentiation markers, including myoD, myogenin, and dystrophin in vitro. They had phosphorylated Akt within the cells, suggesting their activation by the secreted IGFII. Transplantation of the cells to injured anterior tibial muscle of mice significantly improved their motor functions compared to injured mice transplanted with undifferentiated ES cells and injured mice given vehicle alone. The transfected cells adapted to the injured muscle, formed myofibers positive for dystrophin and negative for MyoD and myogenin. Trichrome staining and toluidine blue staining support myofiber formation in vivo. The enzymatic activity of acetylcholine esterase suggested the functional activity of the regenerated motor units. The evoked electromyogram of anterior tibial muscle transplanted with the transfected cells showed significantly higher potentials compared to that transplanted with undifferentiated ES cells and that injected with phosphate-buffered saline (control injury). Electron microscopic examination confirmed the myofiber formation in the cells in vivo. CONCLUSIONS: Transfection of IGFII gene into ES cells may be applicable for transplantation therapy of muscle damage due to injury and myopathies.     

Transplantation of virally transduced cells into the dermis of immunocompetent and immunodeficient (SCID) mice to determine gene expression profile and differential donor cell survival.

Cell therapy and bioengineering hold great promise as therapeutic approaches using cells and cell-derived factors to treat various pathologic or trauma-induced states. One possible application is the transplantation of cells into wounded tissue to help regulate tissue repair. Cells engineered for optimal wound healing may help to minimize scarring following surgery or to enhance the rate of healing of chronic wounds. The purpose of the current study was to determine the effect of a viral insert, the LacZ-bearing, first generation adenovirus AdRGD, on the survival of dermally transplanted murine skin allogenic fibroblasts. The LacZ insert facilitated quantitation of both cell survival and gene expression and was used here to measure viable cell number. In addition to bearing the LacZ marker, the AdRGD vector is capable of carrying therapeutic gene inserts, so this study tested the feasibility of gene therapy for wound healing. Murine skeletal muscle PP6 (i.e., Pre-Plate 6) myogenic stem cells served as an alternate donor cell type. Cells were labeled with the LacZ-bearing AdRGD adenovirus vector and injected (50,000 cells/site) into the dorsal skin of adult normal, immunocompetent mice as well as in immunodeficient SCID mice. Skin biopsies were taken on days 0, 1, 2, 3, and 7 post-transplant, and assayed for LacZ expression. Soon after transplant (day 1), cell numbers underwent a transient decrease, but by day 2 post-transplant they were present in appreciable numbers. Between days 2-7 post-transplant, both allogenic fibroblasts and PP6 myogenic stem cells maintained survivability in similar numbers. Further, survival of transplanted cell types was similar in both normal, immunocompetent as well as SCID mice during this time period. There were no signs of acute inflammation or rejection in any of the samples. This study shows that AdRGD-transduced cells are not immunogenic in the mouse skin model and the cells show similar survival for the first 7 days post-transplantation independent of the cell type or immunocompetence of the host.     

Dose-related mechanisms of immunosuppression mediated by murine anti-CD3 monoclonal antibody in pancreatic islet cell transplantation and delayed-type hypersensitivity

Although anti-CD3 mAb therapy is used extensively in clinical transplantation, the dose-related effects and mechanisms of action are not clearly defined. We have examined the dose-related effects of an antimurine CD3 mAb, 145-2C11, in pancreatic islet cell allograft and the delayed type hypersensitivity reaction models of T-cell-dependent immunity. Low-dose anti-CD3 therapy (0.5 micrograms/day) administered over several days mediated superficially equal, effective clinical immunosuppression as a single high-dose intravenous injection (400 micrograms). T cells harvested from animals treated with high-dose anti-CD3 were unresponsive to in vitro restimulation. In contrast, T cells isolated from low-dose treated animals retained in vitro proliferative capacity when restimulated with polyvalent anti-CD3 mAb. The terminal complement components were not required to support in vivo immunosuppression mediated by anti-CD3 mAb as C5 deficient mice were immunosuppressed by the administration of this mAb. In some pancreatic islet cell allograft recipients, permanent engraftment, but not tolerance, was achieved. Replacement of donor leukocytes produced acute rejection in hosts bearing long-term, well-accepted grafts. Prolonged anti-CD3 mAb treatment may provide sufficient time for replacement or inactivation of donor leukocytes.     

Increased myogenic potential and fusion of matrilysin-expressing myoblasts transplanted in mice

The success of myoblast transplantation in clinical trials has been limited in part by the low dispersion of grafted cells outside the injection site. Our research group previously reported that the culture of myoblasts with concanavalin A, a stimulator of metalloproteinase production, increased their migration. Several lines of evidence also suggested that muscle cell fusion involves metalloproteinase-sensitive mechanisms. To determine whether the increased expression of metalloproteinases had an influence on myoblast fusion and dispersion through the muscle following transplantation, we generated a myoblast cell line expressing human matrilysin (MMP-7). The MMP-7-expressing myoblasts were obtained by the stable transfection of a matrilysin expression vector in a TnILacZ immortomouse myoblast clone. Matrilysin-expressing myoblasts showed a highly increased in vitro fusion index, forming seven times (p < 0.001) more myotubes than the control cell line and three times (p < 0.001) more myotubes than the Immortomyoblast parental clone. Single-site transplantation of matrilysin-expressing myoblasts generated more fibers (p < 0.001), over a greater surface (p < 0.001) than the control cell line. The cotransplantation of matrilysin-expressing myoblasts and of normal human myoblasts in SCID mice increased the number of human dystrophin-positive fibers and myotubes by sixfold. Although no significant increased migration of myoblasts outside the injection sites was observed, our results show that the metalloproteinase activity can improve the myogenic potential of myoblasts in vitro and the fusion of myoblasts with host fibers in vivo. MMP-7 expression may be useful in increasing myoblast transplantation success.     

成纤维细胞介导的人α—干扰素基因疗法治疗人黑色素瘤？…

目的 以人α－干扰素（ＩＦＮ－α）基因为治疗性目的基因,建立了ＩＦＮ－α基因疗法治疗人黑色素瘤的实验模型并探讨其免疫学机理。方法 将人ＩＦＮ－α基因体外转染成纤维细胞ＮＩＨ３Ｔ３中,获得一株高分泌ＩＦＮ－α的细胞克隆株ＮＩＨ３Ｔ３－ＩＦＮ－α＾＋。将该高分泌ＩＦＮ－α细胞克隆株在体外大量扩增后回植裸鼠腹腔内,测定裸鼠血清中ＩＦＮ－α的含量并观察对荷瘤裸鼠的治疗效果。结果 １２ｈ后裸鼠血清中即能测到     

Complement deposition and cell death after myoblast transplantation

One of the problems limiting myoblast transplantation (MT) is the early death of the transplanted cells. Because complement can be fixed by myoblasts in vitro, and because it has the capacity to induce cell lysis, its possible role in the early death of transplanted myoblasts was investigated. CD1 mice and Macaca mulata monkeys were used as recipients for MT. In some mice, C3 was depleted before MT using Cobra Venom Factor. Mice were sacrificed during the first hour and up to 3 days after MT. Monkeys were biopsied 1 to 4 h after MT. Myoblast necrosis was assessed by the presence of intracellular calcium. Complement deposition was demonstrated by immunohistochemistry with anti-C3 and anti-C5b-9 neoantigen antibodies. In mice, C3 deposition was observed in damaged muscle fibers and in regions containing necrosed myoblasts. Complement depletion did not diminish the proportion of necrosed cells. In monkeys, only a small percentage of transplanted myoblasts showed C3 or C5b-9 deposition, mostly intracellular. Complement activation seems not to be implicated in directly damaging the transplanted cells, but seems secondary to cellular death. Taking into account its chemotactic functions, complement could be implicated in the migration of neutrophils and macrophages into the clusters of transplanted cells.     

Noncultured cell transplantation in an ovine model of right ventricular preparation

OBJECTIVE: Beyond the first 2 months of life, pulmonary artery banding is warranted before two-stage arterial switch operation. The aim of this study was to explore whether myogenic cell transplantation could contribute to right ventricular function during pulmonary artery constriction in an ovine model. METHODS: Sixteen rams were assigned to one of the following groups: group 1, simple pulmonary artery banding (n = 5); group 2, pulmonary artery banding and cell implantation in the right ventricle (n = 7); and group 3, pulmonary artery banding and placebo injection in the right ventricle (n = 4). Hemodynamic assessment with pressure-volume loops was performed on days 0 and 60. The pulmonary artery banding and the injections were achieved through a left fourth intercostal thoracotomy. Autologous myogenic cell implantation was carried out with a noncultured cell preparation, as previously described by our group. Implanted sites were processed with monoclonal antibodies to a fast skeletal-specific isoform of myosin heavy chain (MY32). RESULTS: Skeletal myosin heavy chain expression was detected at 2 months after noncultured cell implantation in all grafted animals. Right ventricular training resulted in statistically significant increased signs of contractility in all three groups. There was no observed difference, however, between the cell therapy group and the other two groups with respect to signs of cardiac function. CONCLUSION: Successful engraftment of noncultured cells into right ventricular myocardium did not translate into a functional benefit that we could demonstrate in our ovine model. Cellular therapy thus is probably not useful to strengthen a left ventricle being retrained through pulmonary artery banding before arterial switch operation. However, cell transplantation may affect the outcome of right ventricular failure long term after atrial switch operation. Although preliminary, this investigation paves the way for further research into cellular cardiomyoplasty, right ventricular failure, and congenital heart disease.     

T-cell depletion and graft survival induced by anti-human CD3 immunotoxins in human CD3epsilon transgenic mice

BACKGROUND: Anti-CD3 immunotoxins are broad-spectrum immunosuppressive agents in a wide range of organ transplantation animal models with potential use in eliciting antigen-specific tolerance. However, the anti-CD3 immunotoxins used in animal studies do not cross-react with human T cells, limiting extrapolation to humans and hindering clinical development. METHODS: Three anti-human CD3-directed immunotoxins, DT389-scFv(UCHT1), scFv(UCHT1)-PE38, and UCHT1-CRM9, were compared in vitro and in transgenic mice, tg(epsilon)600+/-, that have T cells expressing both human and murine CD3epsilon antigens. RESULTS: These immunotoxins were extraordinarily potent in vitro against human or transgenic mouse T cells, with IC50 values in cellular assays ranging from pM to fM. Systemic administration of these immunotoxins dose-dependently depleted >99% of tg(epsilon)600+/- lymph node and spleen T cells in vivo. Depletion was specific for T cells. The loss of the concanavalin A-induced, but not the lipopolysaccharide-induced, splenic proliferative response from immunotoxin-treated animals further demonstrated specific loss of T-cell function. Immunotoxin treatment prolonged fully allogeneic skin graft survival in tg(epsilon)600+/- recipients to 25 days from 10 days in untreated animals. T-cells recovered to approximately 50% of normal levels after approximately 22 days in animals with or without skin grafts; T-cell recovery correlated with skin graft rejection. All three immunotoxins elicited >100 day median survival of fully allogeneic heterotopic heart grafts. By 100 days, T cells recovered to normal numbers in these animals, but the grafts showed chronic rejection. CONCLUSION: These immunotoxins profoundly deplete T cells in vivo and effectively prolong allogeneic graft survival.     

Comparison of benefits on myocardial performance of cellular cardiomyoplasty with skeletal myoblasts and fibroblasts

Cellular cardiomyoplasty (CCM), or introduction of immature cells into terminally injured heart, can mediate repair of chronically injured myocardium. Several different cell types, ranging from embryonic stem cells to autologous skeletal myoblasts, have been successfully propagated within damaged heart and shown to improve myocardial performance. However, it is unclear if the functional advantages associated with CCM depend upon the use of myogenic cells or if similar results can be seen with other cell types. Thus, we compared indices of regional contractile (systolic) and diastolic myocardial performance following transplantation of either autologous skeletal myoblasts (Mb) or dermal fibroblasts (Fb) into chronically injured rabbit heart. In vivo left ventricular (LV) pressure (P) and regional segment length (SL) were determined in 15 rabbits by micromanometry and sonomicrometry 1 week following LV cryoinjury (CRYO) and again 3 weeks after autologous skeletal Mb or dermal Fb transplantation. Quantification of systolic performance was based on the linear regression of regional stroke work and end-diastolic (ED) SL. Regional diastolic properties were assessed using the curvilinear relationships between LVEDP and strain (epsilon) as well as LVEDP and EDSL. At study termination, cellular engraftment was characterized histologically in a blinded fashion. Indices of diastolic performance were improved following CCM with either Mb or Fb. However, only Mb transplantation improved systolic performance; Fb transfer actually resulted in a significant decline in systolic performance. These data suggest that both contractile and noncontractile cells can improve regional material properties or structural integrity of terminally injured heart, as reflected by improvements in diastolic performance. However, only Mb improved systolic performance in the damaged region, supporting the role of myogenic cells in augmenting contraction. Further studies are needed to define the mechanism by which these effects occur and to evaluate the long-term safety and efficacy of CCM with any cell type.     

Embryonic stem cells differentiated in vitro as a novel source of cells for transplantation

The controlled differentiation of mouse embryonic stem (ES) cells into near homogeneous populations of both neurons and skeletal muscle cells that can survive and function in vivo after transplantation is reported. We show that treatment of pluripotent ES cells with retinoic acid (RA) and dimethylsulfoxide (DMSO) induce differentiation of these cells into highly enriched populations of γ-aminobutyric acid (GABA) expressing neurons and skeletal myoblasts, respectively. For neuronal differentiation, RA alone is sufficient to induce ES cells to differentiate into neuronal cells that show properties of postmitotic neurons both in vitro and in vivo. In vivo function of RA-induced neuronal cells was demonstrated by transplantation into the quinolinic acid lesioned striatum of rats (a rat model for Huntington's disease), where cells integrated and survived for up to 6 wk. The response of embryonic stem cells to DMSO to form muscle was less dramatic than that observed for RA. DMSO-induced ES cells formed mixed populations of muscle cells composed of cardiac, smooth, and skeletal muscle instead of homogeneous populations of a single muscle cell type. To determine whether the response of ES cells to DMSO induction could be further controlled, ES cells were stably transfected with a gene coding for the muscle-specific regulatory factor, MyoD. When induced with DMSO, ES cells constitutively expressing high levels of MyoD differentiated exclusively into skeletal myoblasts (no cardiac or smooth muscle cells) that fused to form myotubes capable of spontaneous contraction. Thus, the specific muscle cell type formed was controlled by the expression of MyoD. These results provided evidence that the specific cell type formed (whether it be muscle, neuronal, or other cell types) can be controlled in vitro. Further, these results demonstrated that ES cells can provide a source of multiple differentiated cell types that can be used for transplantation.     

A factor implicated in the myogenic conversion of nonmuscle cells derived from the mouse dermis

Using the mdx mouse model for human Duchenne muscular dystrophy we have shown that a cell population residing in the dermis of C57B1/10ScSn mouse skin is capable of converting to a myogenic lineage when implanted into the mdx muscle environment. It was important to determine the characteristics of the converting cell. A previous in vitro study indicated that 10% of cells underwent conversion but only when the cells were grown in medium previously harvested from a myogenic culture. In the present study we cloned cells derived from the dermis to identify the converting cells. Clones grown in normal growth medium showed no conversion, but when grown in medium conditioned by muscle cells around 40% conversion was achieved in several individual clones. We investigated whether the protein beta-galactoside binding protein (betaGBP), which is secreted by myoblasts and acts as a cell growth regulator of fibroblasts. could be a candidate factor responsible for conversion. Medium harvested from COS-1 cells infected with a construct containing betaGBP has been used for this investigation. Growth of dermal fibroblasts in medium enriched with this factor showed a high rate of conversion to cells expressing muscle-specific factors.     

Use of repeating dispensers to increase the efficiency of the intramuscular myogenic cell injection procedure

Intramuscular myoblast transplantation in humans and nonhuman primates requires precise repetitive cell injections very close to each other. Performed with syringes operated manually throughout large regions, this procedure takes a lot of time, becoming tiring and thus imprecise. We tested two repetitive dispensers with Hamilton syringes as cell injection devices to facilitate this procedure. Monkeys received intramuscular allotransplantations of beta-galactosidase-labeled myoblasts, using either a monosyringe or a multisyringe repeating dispenser. The monosyringe repeating dispenser allowed performing cell injections faster and easier than with a manually operated syringe. The multisyringe dispenser accelerated the procedure still more, but it was not ergonomic. Biopsies of the myoblast-injected sites 1 month later showed abundant beta-galactosidase-positive myofibers, with the same density and morphological pattern observed following myoblast transplantation with a syringe operated manually. We recommend the monosyringe repeating dispenser for myoblast transplantation in skeletal muscles and maybe in the heart.