EBV load is associated with cfDNA fragmentation and renal damage in SLE patients.

BACKGROUND: For long Epstein-Barr virus (EBV) has been suspected to be involved in the pathogenesis of systemic lupus erythematosus (SLE). The aim of this study was to verify the association between EBV, cell-free DNA (cfDNA) and kidney disease in SLE. METHODS: Blood samples were obtained from 43 SLE patients and 50 healthy individuals. EBV load was measured via real-time PCR assay. Sizing and quantification of plasma cfDNA was performed on Bioanalyzer. We proposed that the uniformity of cfDNA fragmentation can be described using cfDNA fragmentation index. RESULTS: SLE patients with chronic kidney disease (CKD +) had higher EBV load compared to CKD(-) patients (P = 0.042). Patients with high cfDNA level had higher EBV load (P = 0.041) and higher cfDNA fragmentation index (P < 0.001) compared to patients with low cfDNA level. Among patients with high cfDNA level, EBV load was higher in CKD(+) group compared to CKD(-) group (P = 0.035). EBV load was positively correlated with the fragmentation index in all SLE patients (P = 0.028, R2 = 0.13), and the correlation was even more pronounced in CKD (+) patients (P < 0.001, R2 = 0.20). CONCLUSIONS: We showed that EBV load was associated with non-uniform cfDNA fragmentation, higher cfDNA levels, and kidney disease in SLE patients. Although the causality of this relationship could not be determined with the current study, it brings rationale for further investigations on the role of EBV and cfDNA interplay in SLE pathogenesis.

A novel JAK/ROCK inhibitor, CPL409116, demonstrates potent efficacy in the mouse model of systemic lupus erythematosus.

Systemic lupus erythematosus is a chronic inflammatory disease, in which treatment is still limited due to suboptimal efficacy and toxicities associated with the available therapies. JAK kinases are well known to play an important role in systemic lupus erythematous. There is growing evidence that ROCK kinases are also important in disease development. In this paper, we present the results of the development of CPL409116, a dual JAK and ROCK inhibitor. The studies we performed demonstrate that this molecule is an effective JAK and ROCK inhibitor which efficiently blocks disease progression in NZBWF1/J mouse models of systemic lupus erythematous.

Serum galectin-3 and galectin-3 binding protein levels in systemic lupus erythematosus and cutaneous lupus erythematosus.

INTRODUCTION: The roles of galectin-3 (Gal-3) and galectin-3 binding protein (G3BP) in systemic lupus erythematosus (SLE) are of ongoing interest, but the data are insufficient due to highly limited available studies. There are no data on cutaneous lupus erythematosus (CLE). AIM: To assess serum Gal-3 and G3BP concentrations in SLE patients with and without LE-specific skin lesions, CLE patients and to correlate levels of proteins with clinical and laboratory parameters. MATERIAL AND METHODS: The study included 71 SLE patients with and without LE-specific skin lesions, 23 CLE patients and 40 controls. Gal-3 and G3BP were measured by specific enzyme-linked immunosorbent assays (ELISA). RESULTS: Serum Gal-3 and G3BP concentrations were significantly higher in SLE with and without LE-specific lesions compared to controls, but without differences between SLE groups. Gal-3 and G3BP levels were also elevated in CLE compared to controls (p = 0.001, p = 0.005; respectively). There was a positive correlation between G3BP level and CLASI activity score both in CLE (r = 0.55, p = 0.006) and in SLE patients with LE-specific lesions (r = 0.36, p = 0.02). G3BP and Gal-3 levels did not differ in SLE with LE-specific lesions and CLE. There was a positive correlation between serum G3BP level and the SLEDAI score in SLE patients (r = 0.26, p = 0.03). CONCLUSIONS: Our findings indicate that serum G3BP and Gal-3 are elevated in CLE. Additionally, G3BP might be associated with the extent of skin lesions. There are no differences between G3BP and Gal-3 concentrations in SLE with and without LE-specific skin lesions.

Cell-free DNA profiling in patients with lupus nephritis.

BACKGROUND: Increased level of cell-free DNA (cf-DNA) is associated with systemic lupus erythematosus (SLE) and might be related to disease activity. The aim of this study was to evaluate whether cfDNA integrity, size distribution and concentration of different cfDNA fractions is associated with lupus activity and kidney involvement. METHODS: Blood samples were collected from 43 SLE patients and 50 healthy controls. Nuclear and mitochondrial fractions of cfDNA and intracellular DNA were quantified by real-time qPCR. Sizing and quantification of total cfDNA level was performed on Bioanalyzer. RESULTS: We determined four parameters that characterized cfDNA profile: fragmentation index, ratio of intra- to extracellular mtDNA copy number, cfDNA concentration, and presence of 54-149 bp and 209-297 bp fragments. Patients with healthy-like cfDNA profile had higher eGFR (P = 0.009) and more often no indications for kidney biopsy or less advanced lupus nephritis (LN) (P = 0.037). In contrary, SLE patients with distinct cfDNA profile (characterized by increased cfDNA concentration and fragmentation, higher discrepancy between intra- to extracellular mtDNA copy number, and the presence of 54-149 bp and 209-297 bp fragments) had lower eGFR (P = 0.005) and more often advanced LN or history of renal transplantation (P = 0.001). CONCLUSIONS: We showed that cfDNA profiling may help to distinguish SLE patients with renal involvement and severe disease course from patients with more favorable outcomes. We suggest cfDNA profile a promising SLE biomarker.

Scalp involvement in pemphigus: a prognostic marker.

INTRODUCTION: Scalp involvement in the course of pemphigus is observed in 16-60% of patients. AIM: To determine the prognostic significance of scalp involvement in pemphigus vulgaris and pemphigus foliaceus. MATERIAL AND METHODS: A total of 75 patients (46 with pemphigus vulgaris, 29 with pemphigus foliaceus) were included into this prospective study. The following clinical data were analyzed: Pemphigus Disease Area Index, time to complete clinical remission and duration of complete clinical remission. Indirect immunofluorescence and enzyme-linked immunosorbent assay were performed to monitor serum pemphigus antibodies. RESULTS: Scalp involvement was observed in 30/46 (65.2%) patients with pemphigus vulgaris and 28/29 (96.6%) patients with pemphigus foliaceus. A positive correlation was found between scalp involvement and general disease severity as measured by the Pemphigus Disease Area Index (r = 0.7, p < 0.05). The time required to achieve a complete clinical remission in patients with and without scalp involvement was 39.1 ±47.1 and 9.1 ±7.8 months, respectively. The duration of complete clinical remission was 14.1 ±17.4 and 105.7 ±108.8 months, respectively. The respective time required to achieve serological remission was 37.7 ±58.5 and 15.5 ±18.8 months, whereas the duration of serological remission was 9.2 ±18.8 and 39.1 ±60.1 months, respectively. The average concentration of anti-desmoglein 1 autoantibodies was significantly higher in patients with scalp involvement compared to patients without scalp involvement (109.9 ±68.0 U/ml and 21.3 ±39.4 U/ml). CONCLUSIONS: Scalp involvement in pemphigus is associated with a higher disease severity, longer time required to achieve complete clinical and serological remission and may indicate the need for a more aggressive therapeutical approach.

Bmp-12 activates tenogenic pathway in human adipose stem cells and affects their immunomodulatory and secretory properties.

BACKGROUND: Cell-based therapy is a treatment method in tendon injuries. Bone morphogenic protein 12 (BMP-12) possesses tenogenic activity and was proposed as a differentiating factor for stem cells directed to transplantation. However, BMPs belong to pleiotropic TGF-ß superfamily and have diverse effect on cells. Therefore, the aim of this study was to determine if BMP-12 induces tenogenic differentiation of human adipose stem cells (hASCs) and how it affects other features of this population. RESULTS: Human ASCs from 6 healthy donors were treated or not with BMP-12 (50 or 100 ng/ml, 7 days) and tested for gene expression (COLL1, SCX, MKH, DCN, TNC, RUNX2), protein expression (COLL1, COLL3, MKH), proliferation, migration, secretory activity, immunomodulatory properties and susceptibility to oxidative stress. RT-PCR revealed up-regulation of SCX, MKH and RUNX2 genes in BMP-12 treated cells (2.05, 2.65 and 1.87 fold in comparison to control, respectively, p < 0.05) and Western Blot revealed significant increase of COLL1 and MHK expression after BMP-12 treatment. Addition of BMP-12 significantly enhanced secretion of VEGF, IL-6, MMP-1 and MPP-8 by hASCs while had no effect on TGF-ß, IL-10, EGF and MMP-13. Moreover, BMP-12 presence in medium attenuated inhibitory effect of hASCs on allo-activated lymphocytes proliferation. At the same time BMP-12 displayed no influence on hASCs proliferation, migration and susceptibility to oxidative stress. CONCLUSION: BMP-12 activates tenogenic pathway in hASCs but also affects secretory activity and impairs immunomodulatory potential of this population that can influence the clinical outcome after cell transplantation.

Comparison of the paracrine activity of mesenchymal stem cells derived from human umbilical cord, amniotic membrane and adipose tissue.

AIM: The study was conducted to investigate secretory activity and define the paracrine potential of mesenchymal stem cells from human umbilical cord and amniotic membrane (UC-MSCs and AM-MSCs, respectively). METHODS: UC-MSCs (n = 6) were obtained from tissue explants using an adherent method after two weeks of incubation. AM-MSCs (n = 6) were obtained by digestion with tripsin and collagenase. MSC phenotype was confirmed in vitro by performing flow cytometry, differentiation assays and vimentin staining. Supernatants were collected after 48 h culturing in serum-free conditions and the following concentrations were determined: epidermal growth factor (EGF), interleukin (IL)-6, IL-10, tumor necrosis factor-α, transforming growth factor-ß (TGF-ß), vascular endothelial growth factor-α (VEGF-α) and metalloproteinase (MMP) 1, 8 and 13, using multiplex supernatant cytokine assay. Data were compared with adipose tissue derived MSCs (AD-MSCs, n = 6). RESULTS: Both UC-MSC and AM-MSC populations were positively identified as MSCs by flow cytometry and differentiation potential into bone, cartilage and adipose tissue. Using a multiple cytokine detection assay, we proved that both UC-MSCs and AM-MSCs show high secretive capacity. However, the secretion profile differed between cells from various sources. UC-MSCs showed significantly higher production of TGF-ß and lower production of VEGF-α, compared to AD-MSCs (P = 0.004) and AM-MSCs (P = 0.039) and lower levels of EGF (P = 0005). AM-MSCs showed significantly lower levels of MMP-8 than UC-MSCs (P = 0.024); however, there was no difference in levels of released cytokines compared to AD-MSCs. CONCLUSION: AM-MSCs show similar IL production as AD-MSCs, while UC-MSCs have a significantly different profile, which suggests diverse biological potential of both cell types for immunomodulative and regenerative therapy.

Dynamics of acute local inflammatory response after autologous transplantation of muscle-derived cells into the skeletal muscle.

The vast majority of myoblasts transplanted into the skeletal muscle die within the first week after injection. Inflammatory response to the intramuscular cell transfer was studied in allogeneic but not in autologous model. The aim of this study was to evaluate immune reaction to autotransplantation of myogenic cells and to assess its dynamics within the first week after injection. Muscle-derived cells or medium alone was injected into the intact skeletal muscles in autologous model. Tissue samples were collected 1, 3, and 7 days after the procedure. Our analysis revealed the peak increase of the gene expression of all evaluated cytokines (Il-1α, Il-1 ß, Il-6, Tgf-ß, and Tnf-α) at day 1. The mRNA level of analyzed cytokines normalized in subsequent time points. The increase of Il- ß gene expression was further confirmed at the protein level. Analysis of the tissue sections revealed rapid infiltration of injected cell clusters with neutrophils and macrophages. The inflammatory infiltration was almost completely resolved at day 7. The survived cells were able to participate in the muscle regeneration process. Presented results demonstrate that autotransplanted muscle-derived cells induce classical early immune reaction in the site of injection which may contribute to cellular graft elimination.

Transplantation of mesenchymal stem cells into the skeletal muscle induces cytokine generation.

Mesenchymal stem cells due to the high proliferative potential, capacity of multilineage differentiation became a hope of regenerative medicine. However, the organism's response to the transplantation of MSCs is not fully elucidated. The aim of the present study was to evaluate the acute local tissue response to syngeneic MSCs administration into the muscle. Rat syngeneic MSCs were transplanted into the skeletal muscle and the tissue surrounding the injection site was collected after 24h. Analogous samples from untreated and PBS treated muscles served as controls. The analysis of genes expression using real-time PCR revealed significant up-regulation of proinflammatory cytokines: IL-1α, IL-1ß, IL-6 in MSCs treated muscles in comparison to the PBS group. The evaluation of protein concentration (ELISA) in collected samples showed that injection of MSCs caused significant elevation of IL-1ß. Immunofluorescent assessment of the tissue revealed infiltration of leukocytes and macrophages. Quantitative analysis of the samples immunostained for CD68 and CD43 antigens revealed that the number of phagocytes was significantly higher in MSC treated muscle when compared to the samples from PBS group. To conclude, the administration of mesenchymal stem cells into the muscle in syngeneic model induces the features of acute inflammation that affects cell engraftment.

Exercise differentially regulates renalase expression in skeletal muscle and kidney.

Renalase is a newly discovered amine oxidase and may lower blood pressure by metabolizing catecholamines. We have hypothesized that exercise and training may regulate renalase expression to control blood pressure. In this study, we investigated changes in renalase expression after exercise and training in white and red portion of the gastrocnemius muscle, kidney, and serum in rats. Rats were either untrained or subjected to six weeks of endurance training, which predominantly recruits red fibers. Rats from each group were sacrificed before (n = 10), immediately after (n = 10), or three hours (n = 10) following exercise. Renalase mRNA and protein levels were measured by RT-PCR and ELISA, respectively. There were no significant changes in renalase expression after prolonged training or acute exercise in the serum or red muscle of rats. However, in white muscle, renalase mRNA and protein levels decreased after acute exercise in untrained rats, whereas, in trained rats, its protein level remained unchanged, despite a decrease in mRNA. Thus, exercise influenced renalase expression only in white muscle fibers that are not predominantly recruited during exercise. The reduction of renalase protein in white muscle suggests that renalase may contribute to blood redistribution between contracting and non-contracting fibers during exercise. In the kidney, renalase protein levels decreased after training, while mRNA levels increased. The reduction in renalase protein levels may contribute to functional kidney hypoperfusion, which has been observed after training. In conclusion, exercise differentially regulates renalase expression in skeletal muscle and kidney.

Characterization of bone-marrow-derived rat mesenchymal stem cells depending on donor age.

It is generally accepted that autologous transfers, as non-immunogenic, constitute the safest approach in cellular transplantations. However, this attitude is often associated with the need for isolation and extracorporeal propagation of cells derived from aged patients. Thus the knowledge about relationship between aging and the properties of MSCs (mesenchymal stem cells) is crucial in developing new clinical strategies. The aim of this study was to perform complex comparison of MSC derived from young and aged individuals, which included phenotype, proliferating rate, osteogenic and adipogenic potential and secretory activity. Evaluated populations were isolated from bone marrow of 3-month-old and 24-month-old rats. There was no significant difference in membrane antigen expression and PDT (population doubling time). Additionally, the adipogenic and osteogenic potential did not vary between studied populations. The reaction of MSCs to either mitogen [bFGF (basic fibroblas t growth factor)] or oxidative stress (H2O2) in vitro displayed a very similar pattern in both analysed populations. There was no difference in TGFß1 (transforming growth factor ß1) and VEGF (vascular endothelial growth factor) secretion measured by ELISA test and gene expression evaluated by real-time PCR. However, the expression of the gene for IL-1α (interleukin-1α) was 8-fold lower in oMSC (MSC isolated from old rats). These results indicate that aging individuals can be considered as candidates for autologous transplantation of bone-marrow-derived MSCs.

Preclinical characterization of CPL302-253, a selective inhibitor of PI3Kδ, as the candidate for the inhalatory treatment and prevention of Asthma.

Asthma is a common chronic inflammatory disease. Although effective asthma therapies are available, part of asthmatic population do not respond to these treatment options. In this work we present the result of development of CPL302-253 molecule, a selective PI3Kδ inhibitor. This molecule is intended to be a preclinical candidate for dry powder inhalation in asthma treatment. Studies we performed showed that this molecule is safe and effective PI3Kδ inhibitor that can impact many immune functions. We developed a short, 15-day HDM induced asthma mouse model, in which we showed that CPL302-253 is able to block inflammatory processes leading to asthma development in vivo.

The Influence of Cell Source and Donor Age on the Tenogenic Potential and Chemokine Secretion of Human Mesenchymal Stromal Cells.

BACKGROUND: Cellular therapy is proposed for tendinopathy treatment. Bone marrow- (BM-MSC) and adipose tissue- (ASC) derived mesenchymal stromal cells are candidate populations for such a therapy. The first aim of the study was to compare human BM-MSCs and ASCs for their basal expression of factors associated with tenogenesis as well as chemotaxis. The additional aim was to evaluate if the donor age influences these features. METHODS: Cells were isolated from 24 human donors, 8 for each group: hASC, hBM-MSC Y (age ≤ 45), and hBM-MSC A (age > 45). The microarray analysis was performed on RNA isolated from hASC and hBM-MSC A cells. Based on microarray results, 8 factors were chosen for further evaluation. Two genes were additionally included in the analysis: SCLERAXIS and PPARγ. All these 10 factors were tested for gene expression by the qRT-PCR method, and all except of RUNX2 were additionally evaluated for protein expression or secretion. RESULTS: Microarray analysis showed over 1,400 genes with a significantly different expression between hASC and hBM-MSC groups. Eight of these genes were selected for further analysis: CXCL6, CXCL12, CXCL16, TGF-ß2, SMAD3, COLLAGEN 14A1, MOHAWK, and RUNX2. In the subsequent qRT-PCR analysis, hBM-MSCs showed a significantly higher expression than did hASCs in following genes: CXCL12, CXCL16, TGF-ß2, SMAD3, COLLAGEN 14A1, and SCLERAXIS (p < 0.05, regardless of BM donor age). In the case of CXCL12, the difference between hASC and hBM-MSC was significant only for younger BM donors, whereas for COLLAGEN 14A1-only for elder BM donors. PPARγ displayed a higher expression in hASCs compared to hBM-MSCs. In regard to CXCL6, MOHAWK, and RUNX2 gene expression, no statistically significant differences between groups were observed. CONCLUSIONS: In the context of cell-based therapy for tendinopathies, bone marrow appears to be a more attractive source of MSCs than does adipose tissue. The age of cell donors seems to be less important than cell source, although cells from elder donors show slightly higher basal tenogenic potential than do cells from younger donors.

Increased Activity of the Intracardiac Oxytocinergic System in the Development of Postinfarction Heart Failure.

Aim. The present study was designed to test the hypothesis that the development of postinfarction heart failure is associated with a change of activity of the intracardiac oxytocinergic system. Methods. Experiments were performed on male Sprague-Dawley rats subjected to myocardial infarction or sham surgery. Four weeks after the surgery, blood samples were collected and the samples of the left ventricle (LV) and right ventricle (RV) were harvested for evaluation of the mRNA expression (RT-PCR) of oxytocin (OT), oxytocin receptor (OTR), natriuretic peptides, and the level of OT and OTR protein (ELISA). The concentration of N-terminal B-type natriuretic peptide was measured to determine the presence of heart failure. Results. Plasma NT-proBNP concentration was higher in the infarcted rats. In the infarcted rats, the expression of OT mRNA and the OT protein level were higher in the RV. There were no significant differences between infarcted and noninfarcted rats in the expression of OT mRNA and in the OT protein level in the fragments of the LV. In both the left and the right ventricles, OTR mRNA expression was lower but the level of OTR protein was higher in the infarcted rats. Conclusions. In the present study, we indicate that postinfarction heart failure is associated with an increased activity of the intracardiac oxytocinergic system.

Uremic toxins impair human bone marrow-derived mesenchymal stem cells functionality in vitro.

Mesenchymal stem cells (MSCs) are becoming therapeutic agents of interest in many areas of medicine, including renal diseases and kidney transplantations. However, the effect of uremia on cell properties is still unclear. Therefore, we examined the in vitro influence of uremic toxins, p-cresol (PC) and indoxyl sulfate (IS), on human bone marrow-derived MSC functionality. Cultured MSCs were treated with PC and IS at concentrations corresponding to subsequent stages of chronic kidney disease. Cell viability was characterized by metabolic activity (MTT assay) and proliferation rate (BrdU assay). Apoptosis (Annexin V test) and cell membrane damage (LDH assay) were also tested. MSC secretory properties were determined by measuring cytokine/growth factor levels in media from toxin-treated cells (ELISA). Uremic concentrations of PC and IS resulted in significant inhibition of MSC metabolic activity and proliferation. Toxins did not induce apoptosis, but damaged cell membranes. MSC paracrine activity was also altered - a decrease of VEGF and TGF-ß1 levels and an increase in IGF-1 and IL-8 secretion was detected. Presented data indicate a negative influence of uremic toxins on functional characteristics of human bone marrow-derived MSCs. Therefore, their use as autologous therapeutic agents for kidney disease may be questionable and requires further investigations. The observed phenomenon may be attributable to many other MSC therapies, because of the high prevalence of chronic kidney disease in adult population.

Complement components, proteolysis­related, and cell communication­related proteins detected in urine proteomics are associated with IgA nephropathy.

INTRODUCTION: IgA nephropathy (IgAN) is the most common primary glomerulonephritis. The first symptoms of IgAN are erytrocyturia or hematuria, proteinuria, and decline in renal function, or any combination of the above. One of the promising diagnostic methods is urine proteomics. OBJECTIVES: We studied urine proteomics in patients with IgAN and age- and sex­matched healthy controls. To minimize the risk of protein degradation, we proposed a new protocol for urine collection and preparation. PATIENTS AND METHODS: A total of 30 patients with IgAN and 30 controls were enrolled into the study. Thirty urine samples of the IgAN group were divided into 3 disease pooled samples (DPS I, II, and III) and 30 urine samples of the control group were divided into 3 control pooled samples (CPS I, II, and III). We used isoelectric focusing/liquid chromatography-mass spectrometry/mass spectrometry (IEF/LC­MS/MS) to detect all proteins larger than 10 kDa. RESULTS: Using qualitative analysis, we identified 761, 951, and 956 proteins in each of the 3 IEF/LC­MS/MS experiments. The results were combined, yielding a dataset with 1238 proteins identified by at least 2 peptides. The statistical analysis of the quantitative results revealed 18 proteins that were differently populated in the urine of IgAN patients compared with healthy controls. We found increased urinary concentrations of complement components, coagulation factors, extracellular matrix, intracellular, transmembrane, and other proteins in patients with IgAN. Some of them have never been linked to IgAN before. CONCLUSIONS: We demonstrated that urine proteomics is a promising tool for diagnosing and monitoring patients with IgAN.

The effect of endoscopic administration of autologous porcine muscle-derived cells into the urethral sphincter.

OBJECTIVE: To verify the fate of autologous porcine myogenic cells after endoscopic administration into the urethral sphincter. METHODS: This study was performed on pig animal models. The muscle-derived cells (MDCs) were isolated and identified. After the third passage, the 6 × 10(7) of PKH26 labeled cells were injected into the urethral sphincter using a urethrocystoscope. The urethras were collected after 28 days. To analyze the fate of injected cells, the PKH26 presence, the desmin expression, and the distribution of acetylcholine receptors were evaluated in the tissue sections. Moreover, the maximal urethral closure pressure (MUCP) was assessed in experimental and control groups at day 1 and day 28. RESULTS: The isolated porcine MDCs expressed desmin and were able to differentiate into myotubes in vitro. At day 28 after the transplantation, the depots of PKH26-positive cells were observed in the muscular layer, but also in the submucosa. The staining for desmin revealed that cells located in the muscle layer were integrated with muscle fibers that possessed acetylcholine receptors. However, cells administered into nonmuscle tissue did not express desmin. Urethral pressure profilometry demonstrated no significant differences between MUCP in the transplanted group in comparison to the control group at day 28. CONCLUSION: The present study demonstrates the successful endoscopic transplantation of myogenic cells into the urethral sphincter. The experiments indicated the key importance of precise cell administration in terms of their fate after the injection.

Myogenic stem cells.

Both skeletal muscle and bone marrow tissue contain myogenic stem cells. The population residing in muscles is heterogenic. Predominant in number are "typical" satellite cells - muscle progenitors migrating from somites during embryonic life. Another population is group of multipotent muscle stem cells which, at least in part, are derived from bone marrow. These cells are tracked by gradient of growth factors releasing from muscle during injury or exercise. Recruited bone marrow-derived cells gradually change their phenotype becoming muscle stem cells and eventually can attain satellite cell position and express Pax7 protein. Mesenchymal stem cells (MSC) isolated directly from bone marrow also display myogenic potential, although methods of induction of myogenic differentiation in vitro have not been optimized yet. Concerning efforts of exploiting myogenic stem cells in cell-mediated therapies it is important to understand the cause of impaired regenerative potential of aged muscle. Up to now, most of research data suggest that majority of age related changes in skeletal muscles are reversible, thus depending on extrinsic factors. However, irreversible intrinsic features of muscle stem cells are also taken into consideration.