In vitro culture of primary human myoblasts by using the dextran microcarriers Cytodex3®

Introduction. Primary cells in vitro culture scale-up is a crucial issue in cell-based tissue and organ regeneration therapy. Reducing costs and space occupied by the cells cultured in vitro has been an important target. Cells cultured in vitro with the use of bioreactor with dextran microcarriers (Cytodex®) have potentially a chance to meet many of the cell therapy requirements. Material and methods. We used collagen-coated carriers (Cytodex3®) and a spinner flask bioreactor to develop environment suitable for human myoblast proliferation. In parallel, standard adherent in vitro culture conditions for myoblasts propagation (T-flask) were conducted. Cell cycle characterization, senescence, myogenic gene ex­pression and cell apoptosis were evaluated in order to find differences between two culture systems under study. Results. The number of cells obtained in bioreactor per 106 of starting cells population was approximately ten times lower in comparison with T-flask culture system. The microcarriers cultured adult myoblasts in compari­son with the regular T-flask culture showed faster and more advanced replicative aging and lower proliferative potential. Moreover, the percentage of the cells that entailed an irreversible cell arrest (G0 phase) was also significantly (p < 0.0001) increased. Conclusions. Our results suggest that population of primary human myoblasts obtained from adult individuals and propagated on dextran microcarriers did not meet the requirements of the regenerative medicine regarding quantity and quality of the cells obtained. Nonetheless, further optimization of the cell scaling up process including both microcarriers and/or bioreactor program is still an important option

Effect of miR-195 inhibition on human skeletal muscle-derived stem/progenitor cells

BACKGROUND: Application of a circulating miR-195 inhibitor could be a helping factor in in vitro model of human skeletal muscle- derived stem/progenitor cells (SkMDS/PCs). Previously, miR-195 expression has been reported to be a negative factor for myogenesis. AIMS: The aim of the study was to obtain anti-apoptotic and anti-aging effects in in vitro cultured myoblasts and to improve their ability to form myotubes by suppressing miR-195 expression. METHODS: Human wild-type (WT) SkMDS/PC cells incubated with control (nonspecific) miRNA inhibitor and miR-195-inhibited SkMDS/PCs were studied. Functional assays (myotube formation and cell ageing), antioxidant, and myogenic gene expression analyses were performed at two time points, at the 7th and 11th cell passages. RESULTS: Myotube formation was found to be almost 2-fold higher in the miR-195-inhibited SkMDS/PCs population (p<0.05) compared to WT cells. miR-195 inhibition did not appear to affect cell ageing or rejuvenate human SkMDS/PCs. Antioxidant (SOD3 and FOXO) gene expression was augmented in the miR-195-inhibited SkMDS/PCs population, but no positive effect on the remaining antioxidant genes (SOD1, SOD2, and catalase) was observed. A significant increase in MyoD gene expression with a concomitant decrease in MyoG (p<0.05) was further documented in miR-195-inhibited SkMDS/PCs compared to WT cells (11th cell passage). CONCLUSIONS: The performed studies may lead to the preconditioning of myogenic stem cells to extend their potential for pro-regenerative activity. miR-195 inhibitor may serve as conditioning factor augmenting selective antioxidant genes expression and proliferative potential of SkMDS/PCs, but not having an impact on cell aging and/ or apoptosis

Poprawa właściwości regeneracyjnych komórek macierzystych pochodzenia miogennego poprzez zastosowanie modyfikowanych genetycznie mioblastów w terapii serca po zawale

Background: Modern therapies of post infarcted heart failure are focused on perfusion improvement of the injured myocardium. This effect can be achieved by, among other means, implanting stem cells which could be genetically modified with factors inducing the formation of new blood vessels in the post infarction scar area. Combined stem cell and gene therapy seems to be a promising strategy to heal an impaired myocardium. The creation of new blood vessels can be indirectly stimulated via factors inducing vascular endothelial growth factor synthesis, for example endothelial nitric oxide synthase (eNOS). The product of this enzyme, nitric oxide, is a molecule that can influence numerous physiological activities; it can contribute to vasodilation, stimulation of endothelial cell growth, prevention of platelet aggregation and leukocyte adhesion to the endothelium.Aim: To verify the pro-angiogenic and regenerative potential of human primary myoblasts and murine myoblast cell line C2C12 transiently transfected with eNOS gene.Methods: Stem cells (either human or murine) were maintained in standard in vitro conditions. Next, both types of myoblasts were modified using electroporation and lipofection (human and murine cells), respectively. The efficacy of the transfection method was evaluated using flow cytometry. The concentration of eNOS protein was measured by ELISA immunoassay. The biological properties of modified cells were assessed using an MTT proliferation test and DAPI cell cycle analysis. To verify the influence of oxidative stress on myoblasts, cytometric tests using Annexin V and propidium iodide were applied. To check possible alterations in myogenic gene expression of stem cells transduced by genetic modification, the myogenic regulatory factors were evaluated by real-time PCR. The function of genetic modification was confirmed by a HUVEC capillary sprouting test using myoblasts supernatants.Results: Electroporation turned out to be an efficient transfection method. High amounts of secreted protein were obtained (in the range 2,000 pg/mL) in both cell types studied. Moreover, the functionality of gene overexpression product was confirmed in capillary development assay. Human myoblasts did not exhibit any changes in cell cycle; however, eNOS transfected murine myoblasts revealed a statistically significant reduction in cell cycle ratio compared to controls (p < 0.001). In the case of myogenic gene expression, a decrease in Myogenin level was only detected in the human transfected myoblast population (p < 0.05).Conclusions: The results of our study may suggest that transplantation of myoblasts overexpressing eNOS could be promising for cell therapy in regenerating the post infarction heart.Wstęp: Nowoczesne terapie regeneracyjne stosowane w leczeniu serca po zawale skupiają się głównie na poprawie perfuzji w obszarze uszkodzonego miokardium. Efekt ten można osiągnąć m.in. poprzez wszczepienie w obszar blizny pozawałowej komórek macierzystych zmodyfikowanych czynnikami indukującymi powstawanie naczyń krwionośnych. Połączenie dwóch rodzajów terapii: komórkowej i genowej stanowi obiecującą metodę leczenia skutków zawału serca. Zapoczątkowanie tworzenia naczyń krwionośnych może się odbywać w sposób pośredni, poprzez uczestnictwo enzymu eNOS w stymulacji powstawania dużej ilości czynnika VEGF, będącego głównym induktorem angiogenezy. Dodatkowo produkt enzymu eNOS — tlenek azotu jest cząsteczką, która ma duże znaczenie dla fizjologii organizmu; przyczynia się m.in. do rozszerzania naczyń krwionośnych, stymuluje komórki śródbłonka, zapobiega agregacji płytek krwi i adhezji leukocytów w naczyniach.Cel: Głównym celem pracy było sprawdzenie potencjału regeneracyjnego i właściwości proangiogennych zmodyfikowanych genetycznie komórek macierzystych mięśni szkieletowych, zarówno pierwotnej zawiesiny ludzkiej, jak i mysich komórek linii C2C12 za pomocą konstrukcji genowej zawierającej sekwencję kodującą dla genu eNOS.Metody: Komórki macierzyste (ludzkie i mysie) były hodowane w standardowych warunkach in vitro. Następnie obydwa typy komórek zostały zmodyfikowane za pomocą elektroporacji i lipofekcji (ta ostatnia dla komórek mysich). Wydajność transfekcji sprawdzono za pomocą cytometrii przepływowej. Stężenie białka eNOS oznaczono testem immunoenzymatycznym ELISA. Podstawowe funkcje biologiczne zmodyfikowanych komórek zostały ocenione przy użyciu testu proliferacyjnego MTT oraz DAPI do analizy cyklu komórkowego. Wpływ stresu oksydacyjnego na przeżywalność komórek zbadano cytometrycznie, wykorzystując aneksynę V i jodek propidyny. Aby ocenić, czy ekspresja genów szlaku miogennego nie została zaburzona przez wniesioną modyfikację genetyczną, przeprowadzono reakcję PCR w czasie rzeczywistym. Funkcjonalność zastosowanej modyfikacji genetycznej sprawdzono za pomocą testu proliferacji komórek HUVEC z użyciem mediów zebranych znad poszczególnych populacji komórek po hodowli.Wyniki: Elektroporacja okazała się wydajną metodą transfekcji pierwotnych komórek ludzkich. Otrzymano wysokie stężenie białka eNOS w przypadku obu badanych rodzajów komórek: mysich i ludzkich (> 2000 pg/ml). Funkcjonalność modyfikacji potwierdzono w teście tworzenia kapilar przez komórki HUVEC. Ludzkie mioblasty nie wykazały żadnych istotnych zmian w cyklu komórkowym, natomiast komórki mysie wykazały istotny statystycznie spadek we współczynniku proliferacji (na poziomie istotności p < 0,001). W profilu ekspresji genów miogennych istotny statystycznie okazał się jednie spadek ekspresji miogeniny w przypadku mioblastów ludzkich (p < 0,05). Przeżywalność komórek modyfikowanych nie odbiegała znacząco od wyników uzyskanych w przypadku komórek ‘natywnych’.Wnioski: Wyniki badań wskazują na słuszność zastosowania modyfikacji genetycznej komórek macierzystych z perspektywicznym planem użycia genu eNOS w badaniach przedklinicznych na modelu mysim. Taka terapia może się okazać w przyszłości obiecującym sposobem regeneracji serca po zawale

Co-Transplantation of Bone Marrow-MSCs and Myogenic Stem/Progenitor Cells from Adult Donors Improves Muscle Function of Patients with Duchenne Muscular Dystrophy

Duchenne muscular dystrophy (DMD) is a genetic disorder associated with a progressive deficiency of dystrophin that leads to skeletal muscle degeneration. In this study, we tested the hypothesis that a co-transplantation of two stem/progenitor cell populations, namely bone marrow-derived mesenchymal stem cells (BM-MSCs) and skeletal muscle-derived stem/progenitor cells (SM-SPCs), directly into the dystrophic muscle can improve the skeletal muscle function of DMD patients. Three patients diagnosed with DMD, confirmed by the dystrophin gene mutation, were enrolled into a study approved by the local Bioethics Committee (no. 79/2015). Stem/progenitor cells collected from bone marrow and skeletal muscles of related healthy donors, based on HLA matched antigens, were expanded in a closed MC3 cell culture system. A simultaneous co-transplantation of BM-MSCs and SM-SPCs was performed directly into the biceps brachii (two patients) and gastrocnemius (one patient). During a six-month follow-up, the patients were examined with electromyography (EMG) and monitored for blood kinase creatine level. Muscle biopsies were examined with histology and assessed for dystrophin at the mRNA and protein level. A panel of 27 cytokines was analysed with multiplex ELISA. We did not observe any adverse effects after the intramuscular administration of cells. The efficacy of BM-MSC and SM-SPC application was confirmed through an EMG assessment by an increase in motor unit parameters, especially in terms of duration, amplitude range, area, and size index. The beneficial effect of cellular therapy was confirmed by a decrease in creatine kinase levels and a normalised profile of pro-inflammatory cytokines. BM-MSCs may support the pro-regenerative potential of SM-SPCs thanks to their trophic, paracrine, and immunomodulatory activity. Both applied cell populations may fuse with degenerating skeletal muscle fibres in situ, facilitating skeletal muscle recovery. However, further studies are required to optimise the dose and timing of stem/progenitor cell delivery

ESX1 gene as a potential candidate responsible for male infertility in nonobstructive azoospermia

Abstract Infertility is a problem that affects approximately 15% of couples, and male infertility is responsible for 40–50% of these cases. The cause of male infertility is still poorly diagnosed and treated. One of the prominent causes of male infertility is disturbed spermatogenesis, which can lead to nonobstructive azoospermia (NOA). Whole-genome sequencing (WGS) allows us to identify novel rare variants in potentially NOA-associated genes, among others, in the ESX1 gene. The aim of this study was to activate the ESX1 gene using CRISPRa technology in human germ cells (testicular seminoma cells—TCam-2). Successful activation of the ESX1 gene in TCam-2 cells using the CRISPRa system was achieved, and the expression level of the ESX1 gene was significantly higher in modified TCam-2 cells than in WT cells or the negative control with nontargeted gRNA (p < 0.01). Using RNA-seq, a network of over 50 genes potentially regulated by the ESX1 gene was determined. Finally, 6 genes, NANOG, CXCR4, RPS6KA5, CCND1, PDE1C, and LINC00662, participating in cell proliferation and differentiation were verified in azoospermic patients with and without a mutation in the ESX1 gene as well as in men with normal spermatogenesis, where inverse correlations in the expression levels of the observed genes were noted

Whole-genome sequencing identifies new candidate genes for nonobstructive azoospermia

Background: Genetic causes that lead to spermatogenetic failure in patients with nonobstructive azoospermia (NOA) have not been yet completely established. Objective: To identify low-frequency NOA-associated single nucleotide variants (SNVs) using whole-genome sequencing (WGS). Materials and methods: Men with various types of NOA (n = 39), including samples that had been previously tested with whole-exome sequencing (WES; n = 6) and did not result in diagnostic conclusions. Variants were annotated using the Ensembl Variant Effect Predictor, utilizing frequencies from GnomAD and other databases to provide clinically relevant information (ClinVar), conservation scores (phyloP), and effect predictions (i.e., MutationTaster). Structural protein modeling was also performed. Results: Using WGS, we revealed potential NOA-associated SNVs, such as: TKTL1, IGSF1, ZFPM2, VCX3A (novel disease causing variants), ESX1, TEX13A, TEX14, DNAH1, FANCM, QRICH2, FSIP2, USP9Y, PMFBP1, MEI1, PIWIL1, WDR66, ZFX, KCND1, KIAA1210, DHRSX, ZMYM3, FAM47C, FANCB, FAM50B (genes previously known to be associated with infertility) and ALG13, BEND2, BRWD3, DDX53, TAF4, FAM47B, FAM9B, FAM9C, MAGEB6, MAP3K15, RBMXL3, SSX3 and FMR1NB genes, which may be involved in spermatogenesis. Discussion and conclusion: In this study, we identified novel potential candidate NOA-associated genes in 29 individuals out of 39 azoospermic males. Note that in 5 out of 6 patients subjected previously to WES analysis, which did not disclose potentially causative variants, the WGS analysis was successful with NOA-associated gene findings.publishedVersio