Casein kinase 2 activity is a host restriction factor for AAV transduction

So far, the mechanisms that impede AAV transduction, especially in the human heart, are poorly understood, hampering the introduction of new, effective gene therapy strategies. Therefore, the aim of this study was to identify and overcome the main cellular barriers to successful transduction in the heart, using iPSC-derived cardiomyocytes (iPSC-CMs), cardiac fibroblasts (iPSC-CFs), and primary endothelial cells (HAECs) to model vector-host interactions. Through phosphoproteome analysis we established that casein kinase 2 (CK2) signalling is one of the most significantly affected pathways upon AAV exposure. Transient inhibition of CK2 activity substantially enhanced the transduction rate of AAV2, AAV6 and AAV9 in all tested cell types. In particular, CK2 inhibition improved the trafficking of AAVs through the cytoplasm, impaired DNA-damage response through destabilisation of Mre11 and altered the RNA processing pathways, which were also highly responsive to AAV transduction. Also, it augmented transgene expression in already transduced iPSC-CFs, which retain AAV genomes in a functional, but probably silent form. In summary, presented study provides new insights into the current understanding of the host-AAV vector interaction, identifying CK2 activity as a key barrier to efficient transduction and transgene expression, what may translate to improvement the outcome of AAV-based therapies in the future

Effect of heme oxygenase-1 on the differentiation of human myoblasts and the regeneration of murine skeletal muscles after acute and chronic injury

Background Impaired muscle regeneration is a hallmark of Duchenne muscular dystrophy (DMD), a neuromuscular disorder caused by mutations in the DMD gene encoding dystrophin. The lack of heme oxygenase-1 (HO-1, Hmox1), a known anti-inflammatory and cytoprotective enzyme, was shown to aggravate DMD pathology. Methods We evaluated the role of HO-1 overexpression in human induced pluripotent stem cell (hiPSC)-derived skeletal muscle cells (hiPSC-SkM) in vitro and in the regeneration process in vivo in wild-type mice. Furthermore, the effect of cobalt protoporphyrin IX (CoPP), a pharmacological inducer of HO-1 expression, on regeneration markers during myogenic hiPSC differentiation and progression of the dystrophic phenotype was analysed in the mdx mouse DMD model. Results HO-1 has an impact on hiPSC-SkM generation by decreasing cell fusion capacity and the expression of myogenic regulatory factors and muscle-specific microRNAs (myomiRs). Also, strong induction of HO-1 by CoPP totally abolished hiPSC-SkM differentiation. Injection of HO-1-overexpressing hiPSC-SkM into the cardiotoxin (CTX)-injured muscle of immunodeficient wild-type mice was associated with decreased expression of miR-206 and Myh3 and lower number of regenerating fibers, suggesting some advanced regeneration. However, the very potent induction of HO-1 by CoPP did not exert any protective effect on necrosis, leukocyte infiltration, fibrosis, myofiber regeneration biomarkers, and exercise capacity of mdx mice. Conclusions In summary, HO-1 inhibits the expression of differentiation markers in human iPSC-derived myoblasts. Although moderate overexpression of HO-1 in the injected myoblast was associated with partially advanced muscle regeneration, the high systemic induction of HO-1 did not improve muscle regeneration. The appropriate threshold of HO-1 expression must be established for the therapeutic effect of HO-1 on muscle regeneration

Human induced pluripotent stem cell-derived cardiomyocytes, in contrast to adipose tissue-derived stromal cells, efficiently improve heart function in murine model of myocardial infarction

Cell therapies are extensively tested to restore heart function after myocardial infarction (MI). Survival of any cell type after intracardiac administration, however, may be limited due to unfavorable conditions of damaged tissue. Therefore, the aim of this study was to evaluate the therapeutic effect of adipose-derived stromal cells (ADSCs) and human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) overexpressing either the proangiogenic SDF-1α or anti-inflammatory heme oxygenase-1 (HO-1) in a murine model of MI. ADSCs and hiPSCs were transduced with lentiviral vectors encoding luciferase (Luc), GFP and either HO-1 or SDF-1α. hiPSCs were then differentiated to hiPSC-CMs using small molecules modulating the WNT pathway. Genetically modified ADSCs were firstly administered via intracardiac injection after MI induction in Nude mice. Next, ADSCs-Luc-GFP and genetically modified hiPSC-CMs were injected into the hearts of the more receptive NOD/SCID strain to compare the therapeutic effect of both cell types. Ultrasonography, performed on days 7, 14, 28 and 42, revealed a significant decrease of left ventricular ejection fraction (LVEF) in all MI-induced groups. No improvement of LVEF was observed in ADSC-treated Nude and NOD/SCID mice. In contrast, administration of hiPSC-CMs resulted in a substantial increase of LVEF, occurring between 28 and 42 days after MI, and decreased fibrosis, regardless of genetic modification. Importantly, bioluminescence analysis, as well as immunofluorescent staining, confirmed the presence of hiPSC-CMs in murine tissue. Interestingly, the luminescence signal was strongest in hearts treated with hiPSC-CMs overexpressing HO-1. Performed experiments demonstrate that hiPSC-CMs, unlike ADSCs, are effective in improving heart function after MI. Additionally, long-term evaluation of heart function seems to be crucial for proper assessment of the effect of cell administration

The influence of chemical composition and surface conditions of bioactive materials on early osteogenesis of human mesenchymal stem cells

W inżynierii tkanki kostnej wykorzystywane są liczne biomateriały, które stanowią biozgodne, a często też stymulujące kościotworzenie, podłoże lub rusztowanie. Celem pracy było sprawdzenie jak skład chemiczny kompozytów na bazie kopolimeru kwasu mlekowego i kwasu glikolowego i bioaktywnych szkieł, zawierających w swoim składzie tlenki: krzemu, wapnia oraz fosforu w różnych proporcjach procentowych, wpływa na wczesną osteogenezę ludzkich mezenchymalnych komórek macierzystych. W tym celu zbadano poziom mRNA dla BMP-2, RUNX2 i SP7 oraz aktywność alkalicznej fosfatazy (ALP) w komórkach hodowanych na ww. podłożach. Dodatkowo sprawdzono potencjał materiałów do stymulowania mineralizacji macierzy zewnątrzkomórkowej. Wyniki wskazują, że praktycznie każdy z badanych materiałów indukuje odpowiedź kościotwórczą komórek. Jednakże zaobserwowano znacząco wyższą ekspresję zwłaszcza BMP-2 i SP7 na poziomie mRNA w komórkach hodowanych na materiałach z dodatkiem P2O5. Preinkubacja materiałów w symulowanym osoczu (SBF), która prowadzi do wytworzenia powierzchniowej warstwy hydroksyapaptytu, niweluje większość obserwowanych różnic w odpowiedzi kościotwórczej komórek, dlatego wnioskujemy, że niewielkie zmiany w chemizmie podłoży i rusztowań mogą znacząco wpływać na odpowiedź komórek, jeśli nie zostały one uprzednio poddane oddziaływaniu sztucznego osocza celem wykrystalizowania powierzchniowej warstwy hydroksyapatytu. Potwierdzono także korzystny wpływ ekstraktów jonowych zebranych znad badanych materiałów na stymulowanie procesów mineralizacji macierzy. Wyniki tego eksperymentu sugerują, że materiały nieinkubowane w symulowanym osoczu oddziałują na komórki prawdopodobnie głównie poprzez jony uwalniane w reakcjach powierzchniowych.In bone engineering there are used different biomaterials, which are biocompatible and often stimulating osteogenesis 2D or 3D scaffolds. The aim of the study was to check how a composition of composite of a copolymer of lactic acid and glycolic acid and bioactive glasses containing oxides of: silicon, calcium and phosphorus in different proportions can affect early osteogenesis in human mesenchymal stem cells. For this purpose it was checked the mRNA level for BMP-2, RUNX2 and SP7 and the activity of alkaline phosphatase (ALP) in the cells cultured on the above substrates. In addition, it was tested the potential of the materials to stimulate mineralization of extracellular matrix. Analysis of the results of gene expression level showed that practically each of the materials induces an osteogenic response of cells. However there was a significantly higher gene expression of especially BMP-2 and SP7 in cells grown on substrates containing bioglasses with P2O5. Preincubation of materials in simulated body fluid (SBF), which leads to production of surface layer of hydroxiapatite, eliminates most of the observed differences in osteogenic response of cells and therefore we conclude that small changes in the chemistry of scaffolds can significantly affect the response of cells, particularly if they have already been subjected to the influence of SBF. It was also found beneficial effects of ions extracts, which were collected from the tested materials, on stimulation of extracellular matrix mineralization. It suggests that the materials, which were not incubated in SBF may interact with cells mainly by ions released in the surface reactions

The lipid content in the pig embryos cultured in the presence of hyaluronic acid.

Ze względu na duże wykorzystywanie zarodków w różnorodnych eksperymentach naukowych, wciąż poszukiwane są metody hodowli zarodków in vitro, umożliwiające uzyskiwanie dużego odsetka zarodków osiągających stadium blastocyty ekspandującej. Jednym z czynników wpływających pozytywnie na większy odsetek uzyskanych blastocyst jest dodanie do medium hodowlanego kwasu hialuronowego. W niniejszej pracy sprawdzono jak hialuronian sodu (HA) w różnych stężeniach wpływa na metabolizm lipidów zarodka. W tym celu analizowano w mikroskopie konfokalnym zarodki wybarwione Czerwienią Nilu, pochodzące z grup hodowanych w obecności 0,25mg/ml i 1mg/ml HA jak i grupy kontrolnej (bez HA). Na podstawie uzyskanych wyników można stwierdzić, że nie ma statystycznie istotnych różnic pomiędzy grupą zarodków hodowanych w medium z dodatkiem 1mg/ml hialuronianu sodu a grupą kontrolną. Znaleziono jednak różnice w przypadku grupy zarodków hodowanych w medium z dodatkiem 0,25 mg/ml kwasu hialuronowego a kontrolą. Okazuje się, że niższe stężenie kwasu hialuronowego spowalnia metabolizm lipidów zarodka.Due to the large use of blastocysts in different scientific experiments, researchers still seek a method of in vitro embryo culture, enabling to obtain a high percentage of embryos reaching this stage. One of the factors influencing positively on the higher percentage of obtained blastocyst is hyaluronic acid (HA) added to culture medium. In this experiment it was checked how various concentrations of sodium hyaluronate affect on embryo's lipid metabolism. For this purpose it has been analyzed in a confocal microscope embryos stained with Nile Red and derived from the three groups (0.25 mg/ml HA, 1mg/ml HA and control). Analysis of the results shows that there is no statistically significant difference between the experimental group of embryos cultured in medium containing 1mg/ml sodium hyaluronate and the control group. However, the differences were found in the case of the experimental group of embryos cultured in medium containing 0.25 mg / ml of hyaluronic acid. It appears that a lower concentration of hyaluronic acid slows down the metabolism of the embryo

Wpływ genetycznego i farmakologicznego zahamowania oksygenazy hemowej w ludzkiej linii nowotworu prostaty DU-145

Rak prostaty jest jednym z najczęściej występujących nowotworów wśród mężczyzn. Obecnie znane terapie nie są wystarczająco efektywne, szczególnie w przypadku zaawansowanych stadiów nowotworu, dlatego potrzebne są nowe strategie leczenia. Oksygenaza hemowa-1 (HO-1), cytoprotekcyjny, antyapoptotycznyi przeciwzapalny enzym, wykazuje nadekspresję w wielu typach nowotworów, dlatego sugeruje się, że może być potencjalnym celem dla terapii przeciwnowotworowych. Celem tej pracy była ocena wpływu genetycznego i farmakologicznego zahamowania HO na żywotność, potencjał klonogenny i wrażliwość na chemioterapię komórek linii ludzkiego nowotworu prostaty DU-145. Ponieważ obecnie znane inhibitory aktywności HO mają pewne wady, które zwykle wykluczają ich zastosowanie w celach klinicznych, naszym celem była ocena efektywności nowej klasy inhibitorów Na początku potwierdziliśmy ich hamujący efekt na aktywność oksygenazy, który był nawet wyższy niż w przypadku obecnie znanych inhibitorów – protoporfiryny cynowej (SnPP) i pochodnej imidazolowej QC-308. Ponadto, zastosowanie zarówno nowych, jak i komercjalnie dostępnych inhibitorów HO skutkowało obniżonym potencjałem klonogennym i silniejszym efektem cytotoksycznym znanego chemioterapeutyka: doksorubicyny. Dodatkowo, wstępne badania in vivo potwierdziły skuteczność testowanych związków w hamowaniu aktywności HO w wątrobach myszy.Jednakże, genetyczne zahamowanie zarówno HO-1, jak i konstytutywnej izoformy HO-2 lub obu enzymówjednocześnie,z użyciem swoistych shRNA nie powtórzyło w pełni wyników uzyskanych po zastosowaniu chemicznych inhibitorów w linii komórkowej DU-145. Podsumowując, wykazaliśmy efektywność nowych inhibitorów HO zarówno w badaniach in vitro, jak i in vivo, jednak ich efekt może być zależny nie tylko od zahamowania HO-1. Wyniki przeprowadzonych badań sugerują, że nowa klasa inhibitorów HO, może być w przyszłości rozpatrywana jako potencjalna strategia leczenia nowotworów prostaty, jednakże poznanie dokładnego mechanizmu wymaga dalszych badań.Prostate cancer is one of the most frequent types of cancers occurring in men. As currently known therapies are not effective enough, especially in advanced cancer stages, new treatment strategies are needed. Heme oxygenase-1 (HO-1), a cytoprotective, anti-apoptotic, and anti-inflammatory enzyme is overexpressed in many types of tumors, therefore it may be proposed as a potential target for anticancer treatment. The purpose of this work was to validate a role of genetic and pharmacological inhibition of HO on viability, clonogenic potential and sensitivity to chemotherapy in DU-145 human prostate cancer cell line.As already known HO activity inhibitors have some disadvantages, which usually exclude their usage from clinical applications, we aimed to validate an effectiveness of novel class of inhibitors. In the beginning we confirmed their inhibitory effect on HO activity, which was even higher than in case of already known inhibitors – tin protoporphyrin (SnPP) and imidazole-based derivative QC-308. Moreover, both new and commercially available HO inhibitors diminished clonogenic potential and enhanced cytotoxic effect of known chemotherapeutic agent - doxorubicin. Additionally, preliminary in vivo study showed also effectiveness of tested compounds to inhibit HO activity in mouse livers. However, genetic inhibition of HO-1 as well as constitutive isoform HO-2 or both enzymes simultaneously with a use of specific shRNA did not fullyreplicate the results obtained with chemical inhibitors in DU-145 cell line.In conclusion, we have demonstrated the effectiveness of novel HO inhibitors both in vitro and in vivo, however, their effect might be dependent not only on HO-1 inhibition. In summary, the novel class of HO inhibitors may be in future considered as a potential anticancer treatment in prostate cancer, however the exact mechanism of their action needs further investigation

Generation of DMBi002-A human induced pluripotent stem cell line from patient with Spinal muscular atrophy type 3

Spinal Muscular Atrophy (SMA) is a genetic neuromuscular disease caused by mutations in SMN1 gene encoding survival motor neuron (SMN) protein. Lack of this protein leads to progressive loss of motor neurons and therefore to gradual loss of signal transmission between motor neurons and skeletal muscle cells. As a consequence, patients develop muscle atrophy and lose the ability to move independently, what is also related to problems with breathing and swallowing. Here, we describe the generation of human induced pluripotent stem cells (hiPSC) from peripheral blood mononuclear cells (PBMC) of adult SMA type 3 patient with a use of Sendai virus vectors