Human skeletal muscle-derived stem/progenitor cells modified with connexin-43 prevent arrhythmia in rat post-infarction hearts and influence gene expression in the myocardium.

Stem cell therapy in combination with genetic modification (e.g., transfection with the coding sequence for the connexion 43 gene, GJA1) may solve the problems associated with the occurrence of additional (secondary) stimulation in the post-infarcted heart (arrhythmia). Human skeletal muscle-derived stem/progenitor cells (SkMDS/PCs) were transfected with the pCiNeo-GJA1 plasmid at an efficiency of approximately 96%. Gene overexpression was assessed using qPCR, and subsequent analysis revealed that GJA1 expression increased more than 40-fold in SkMDS/PCs transfected with the appropriate coding sequence (SkMDS/PCsCX43) compared to that of the 'native' SkMDS/PCs control (SkMDS/PCsWT). Enhanced (4-fold) protein expression of connexin-43 was also confirmed by Western immunoblotting. Furthermore, using the arrhythmic score, we demonstrated the positive effects of SkMDS/PCsCX43 cell intervention in reducing additional secondary stimulations in rat post-infarcted hearts compared with that of wild-type cell delivery. Selected gene responses (Kcnq1, Cacna1c, Ncx1, Serca2a, and Tgfb1) showed significantly altered expression profiles in the rat myocardium upon intervention with SkMDS/PCsCX43. The genetic modification of human skeletal muscle-derived stem/progenitor cells with connexin-43 prevented the pro-arrhythmic effects of myogenic implanted stem cells on the host myocardium and positively influenced myocardial gene expression profiles in respect to myocardium conductivity.

Influence of hypoxia prevailing in post-infarction heart on proangiogenic gene expression and biological features of human myoblast cells applied as a pro-regenerative therapeutic tool.

Cardiovascular diseases along with MI (myocardial infarction) lead to regional ischaemia and hypoxic conditions, which prevail after infarction. Diminished O2 saturation which is related to elevated level of hypoxia inducible factor 1 (HIF-1) transcription factor, may switch the expression of many genes. To maximize effect of therapies proposed by regenerative medicine, it is essential to verify (within different time points after MI) the expression of proangiogenic genes and their receptors that are regulated, along with the expression of HIF-1α. We demonstrated a connection between the expression of Hif-1α (in murine post infarcted heart model) and the proangiogenic genes Vegf-a; and Plgf and their receptors during myocardial hypoxia. The innovative part of the study required establishment of the most accurate in vitro O2 level corresponding to the hypoxia level prevailing in myocardium after MI. We determined the influence of hypoxia on the biology of human myoblasts in in vitro oxygen conditions (3%), corresponding to those prevailing in the heart after an infarction using a murine model. We also tested myoblasts that were genetically modified with VEGF-A/FGF-4 and PlGF under hypoxic conditions and compared their characteristics with cells cultured under normoxia and hyperoxia (standard in vitro conditions) with respect to myogenic gene expression, cell proliferation, fusion potential and proangiogenic function. The examination of genetically modified myoblasts under optimized in vitro hypoxia conditions led to the conclusion that hypoxia did not negatively influence the biological functions of the myoblasts, such as cell proliferation and/or proangiogenic characteristics. These results support the expected increased proregenerative effects of such genetically modified human myoblasts.

Human myoblast transplantation in mice infarcted heart alters the expression profile of cardiac genes associated with left ventricle remodeling.

BACKGROUND: Myocardial infarction (MI) and left ventricle remodeling (LVR) are two of the most challenging disease entities in developed societies. Since conventional treatment cannot fully restore heart function new approaches were attempted to develop new strategies and technologies that could be used for myocardial regeneration. One of these strategies pursued was a cell therapy--particularly applying skeletal muscle stem cells (SkMCs). METHODS AND RESULTS: Using NOD-SCID murine model of MI and human skeletal myoblast transplantation we were able to show that SkMC administration significantly affected gene expression profile (p<0.05) (NPPB, CTGF, GATA4, SERCA2a, PLB) of the heart ventricular tissue and this change was beneficial for the heart function. We have also shown, that the level of heart biomarker, NT-proBNP, decreased in animals receiving implanted cells and that the NT-proBNP level negatively correlated with left ventricle area fraction change (LVFAC) index which makes NT-proBNP an attractive tool in assessing the efficacy of cell therapy both in the animal model and prospectively in clinical trials. CONCLUSIONS: The results obtained suggest that transplanted SkMCs exerted beneficial effect on heart regeneration and were able to inhibit LVR which was confirmed on the molecular level, giving hope for new ways of monitoring novel cellular therapies for MI.

Implantation of autologous muscle-derived stem cells in treatment of fecal incontinence: results of an experimental pilot study.

BACKGROUND: The aim of this study is to present results of the implantation of autologous myoblasts into the external anal sphincter (EAS) in ten patients with fecal incontinence. METHODS: After anatomical and functional assessment of the patients' EAS, a vastus lateralis muscle open biopsy was performed. Stem cells were extracted from the biopsy specimens and cultured in vitro. Cell suspensions were then administered to the EAS. Patients were scheduled for follow-up visits in 6-week intervals. Total follow-up was 12 months. RESULTS: All biopsy and cell implantation procedures were performed without complications. Nine of the patients completed a full 12-month follow-up. There was subjective improvement in six patients (66.7 %). In manometric examinations 18 weeks after implantation, squeeze anal pressures and high-pressure zone length increased in all patients, with particularly significant sphincter function recovery in five patients (55.6 %). Electromyographic (EMG) examination showed an increase in signal amplitude in all patients, detecting elevated numbers of propagating action potentials. Twelve months after implantation two patients experienced deterioration of continence, which was also reflected in the deterioration of manometric and EMG parameters. The remaining four patients (44.4 %) still described their continence as better than before implantation and retained satisfactory functional examination parameters. CONCLUSIONS: Implantation of autologous myoblasts gives good short-term results not only in a subjective assessment, but also in objective functional tests. It seems that this promising technology can improve the quality of life of patients with fecal incontinence, but further study is required to achieve better and more persistent results.

Biological properties of human skeletal myoblasts genetically modified to simultaneously overexpress the pro-angiogenic factors vascular endothelial growth factor-A and fibroblast growth factor-4.

Myocardial infarction results in cardiomyocyte loss and may eventually lead to cardiac failure. Skeletal myoblast transplantation into the scar area may compensate for this observed cell loss by strengthening the weakened myocardium and inducing myogenesis. Moreover, skeletal myoblasts may serve as potential transgene carriers for the myocardium (i.e., delivering pro-angiogenic factors, which may potentially improve blood perfusion in infarcted heart). We examined the influence of the simultaneous overexpression of two potent pro-angiogenic factors, fibroblast growth factor-4 (FGF-4) and vascular endothelial growth factor (VEGF), on human primary myoblast proliferation, cell cycle, resistance to hypoxic stress conditions and myogenic gene expression, as well as the induction of pro-angiogenic activities. We used a bicistronic plasmid vector encoding two factors introduced via an efficient myoblast electroporation method. The levels of overexpressed proteins were assessed, and their functionality at capillary formation was evaluated. This combined approach led to a high level of non-viral transient overexpression of both pro-angiogenic proteins, which proved to be potent regulators of blood vessel development assayed in capillary formation tests. We demonstrated in in vitro conditions that the transfection of human skeletal myoblasts with both FGF-4 and VEGF did not affect their basic biological properties such as the cell cycle, proliferation or expression of myogenic lineage-specific genes, and the modified cells adapted to oxidative stress conditions. Overall, the results obtained suggest that the applied combined approach with the use of two pro-angiogenic genes overexpressed in skeletal muscle stem cells may be an interesting alternative for the effective therapy of myocardial infarction in animal models and/or prospective clinical trials.

In vitro and in vivo characteristics of connexin 43-modified human skeletal myoblasts as candidates for prospective stem cell therapy for the failing heart.

BACKGROUND: Previously, connexin 43-modified skeletal myoblasts (MbCx) were shown to reduce the pro-arrhythmic effect during the regeneration of heart tissue in an animal model of infarction. To increase the relevance to clinical implementation, in this study, we introduced connexin 43 into human myoblasts using a highly safe non-viral vector and demonstrated that their transplantation had a positive effect on the function of the injured heart. METHODS AND RESULTS: Myoblasts were efficiently transfected with a pCiNeo-GJA1 plasmid (65.72%). qPCR analysis revealed over 32-fold higher expression of the connexin 43 gene in the MbCx cell population compared to 'native' controls. The susceptibility of the myoblasts to oxidative stress conditions (p<0.001) and the fusion index (p<0.01) were increased in the MbCx cells. Additionally, we observed changes in the MYOG and MYH2 gene expression levels in the GJA1-modified myoblasts. Finally, we observed a significant improvement in the post-infarction echocardiographic parameters after intervention using MbCx cells compared with non-transfected myoblasts (MbWt) and the control (0.9% NaCl), wherein a significant decrease in the left ventricular area change in the short axis (SAX AC%) was observed at the two-month follow-up (p<0.05 and p<0.01, respectively). CONCLUSIONS: We demonstrated the positive effect of connexin 43 overexpression on the biology and function of human skeletal myoblasts in the context of their potential clinical applications. Our preclinical studies using a mouse infarction model indicated the positive effect of MbCx implantation on the function of the injured heart.

Transient and stable transfections of mouse myoblasts with genes coding for pro-angiogenic factors.

Cardiomyocyte loss in the ischaemic heart can be the reason of many complications, eventually being even the cause of patient's death. Despite many promises, cell therapy with the use of skeletal muscle stem cells (SMSC) still remains to be modified and improved. Combined cell and gene therapy seems to be a promising strategy to heal damaged myocardium. In the present study we have investigated the influence of a simultaneous overexpression of two potent pro-angiogenic genes encoding the fibroblast growth factor-4 (FGF-4) and the vascular endothelial growth factor-A (VEGF-A) on a myogenic murine C2C12 cell line. We have demonstrated in in vitro conditions that myoblasts which overexpressed these factors exhibited significant changes in the cell cycle and pro-angiogenic potential with only slight differences in the expression of the myogenic genes. There was not observed the influence of transient or stable overexpression of FGF-4 and VEGF on cell apoptosis/necrosis in standard or oxidative stress conditions comparing to non transfected controls. Overall, our results suggest that the possible transplantation of myoblasts overexpressing pro-angiogenic factors may potentially improve the functionality of the injured myocardium although the definite proof must originate from in situ conducted pre-clinical studies.

SCF and c-kit expression profiles in male individuals with normal and impaired spermatogenesis.

The transcription levels of stem cell factor (SCF) and c-kit were examined using real-time RT PCR in interstitial and intratubular cell fractions, as well as in tissue homogenates from normal, azoospermic and neoplasmic patients. Peripheral blood mononuclear cells (PBMC) were used as a systemic control. The observed level of c-kit expression in all investigated groups was generally higher than the expression of SCF. The highest (statistically significant) level of c-kit was noted in testicular tumours (the greater part of which were represented by seminomas) in contrast to SCF mRNA, which may indicate an association between c-kit overexpression and seminoma development. In Sertoli cell only syndrome, almost equal levels of SCF and c-kit transcripts were noted. These results may indicate Leydig cells as the alternative source of c-kit gene transcription. SCF transcript values were low and comparable among the analysed subgroups except that in maturation arrest at spermatocyte stage, the SCF gene expression was statistically higher than in testicular tumours. It appears from the study that c-kit has been a dynamic gene, changing its activity in a variety of testicular pathologies while being expressed in all testicular compartments but clearly overexpressed in testicular tumours of seminomatous origin.

Interleukin-1 superfamily genes expression in normal or impaired human spermatogenesis.

Interleukin-1 (IL-1) is a pleiotropic cytokine that may play a role in contributing to the specific immune environment of mammalian testis and in regulating cell differentiation. We have determined the transcription activity of the IL-1 gene family (using real-time polymerase chain reaction (PCR)) in two main functional testicular compartments (interstitial and intratubular ones), and in tissue homogenates obtained from patients with fertility disorders (spermatogenic arrest and testicular tumors). We observed the prominent expression of gene coding for IL-1 receptor antagonist (IL-1RA) in a purified fraction of gametogenic cells (normal gonad). Caspase-1 (ICE - IL-1beta-converting enzyme) was highly expressed (on mRNA level) in interstitial compartments as well in testicular tumors (immune enhancement?). In addition we found, that the activity of IL-1RA gene decreased along spermatogenic alteration in an inversely related manner with IL-1alpha (from normal gonad through spermatogenic arrest to Sertoli cell only syndrome). Therefore, the quotient value of IL-1alpha/IL-1RA could potentially serve as the diagnostic molecular probe for spermatogenesis assessment. The precise level of mRNA for IL-1-IL-18 cytokines and their receptors, and specifically of the receptor antagonist in immune privileged gonad, could be one of the main factors responsible for maintaining testicular homeostasis, thus enabling generation of the mature spermatozoa.

Quantitative mRNA analysis of IL-1 gene system in human testis.

PROBLEM: There is a growing body of evidence that interleukins exhibit modulatory activity on development of reproductive cells. In this context, there appears to be a role for IL-1, which is also produced in human testis. We have analysed transcripts of IL-1 gene system (IL-1alpha, IL-1beta, IL-1RI, IL-1RII and IL-1RA) to evaluate the possible link between the level of gene(s) transcription and their function. METHOD OF STUDY: To determine the activity of gene transcription, a quantitative PCR with isotopic and/or nonisotopic detection was applied. RESULTS AND CONCLUSIONS: We have detected differential expression of IL-1alpha and IL-1beta genes in separate functional compartments of a male gonad. A strong expression of IL-1alpha gene in an intratubular cell fraction was shown, while the IL-1beta expression seemed to be dominant in extratubular compartment of the male gonad. Abundant amounts of IL-1RA mRNA in gametogenic cells fraction slightly higher than in interstitium have also been found. IL-1RA is the most important regulatory molecule in IL-1 system, which down-regulates activity of both interleukins. Looking more closely at gene(s) differential expression it appears that IL-1alpha can be preferentially down-regulated by IL-1RA gene in intratubular fraction while the IL-1beta, through the "false" IL-1RII receptor in the interstitium. Genes coding for both receptors (IL-1RI and IL-1RII) showed, however, relatively low levels of transcription in both studied compartments. IL-1 genes system creates a complex intragonadal environment and the function of these genes is reflected by their respective distribution in the two main functional compartments of the testis.