Search

Cardiovascular diseases (CVD), with myocardial infarction (MI) being one of the crucial components, wreak havoc in developed countries. Advanced imaging technologies are required to obtain quick and widely available diagnostic data. This paper describes a multimodal approach to in vivo perfusion imaging using the novel SYN1 tracer based on the fluorine-18 isotope. The NOD-SCID mice were injected intravenously with SYN1 or [ 18 F] fluorodeoxyglucose ([ 18 F]-FDG) radiotracers after induction of the MI. In all studies, the positron emission tomography-computed tomography (PET/CT) technique was used. To obtain hemodynamic data, mice were subjected to magnetic resonance imaging (MRI). Finally, the biodistribution of the SYN1 compound was performed using Wistar rat model. SYN1 showed normal accumulation in mouse and rat hearts, and MI hearts correctly indicated impaired cardiac segments when compared to [ 18 F]-FDG uptake. In vivo PET/CT and MRI studies showed statistical convergence in terms of the size of the necrotic zone and cardiac function. This was further supported with RNAseq molecular analyses to correlate the candidate function genes' expression, with Serpinb1c , Tnc and Nupr1 , with Trem2 and Aldolase B functional correlations showing statistical significance in both SYN1 and [ 18 F]-FDG. Our manuscript presents a new fluorine-18-based perfusion radiotracer for PET/CT imaging that may have importance in clinical applications. Future research should focus on confirmation of the data elucidated here to prepare SYN1 for first-in-human trials.

Current treatment protocols for myocardial infarction improve the outcome of disease to some extent but do not provide the clue for full regeneration of the heart tissues. An increasing body of evidence has shown that transplantation of cells may lead to some organ recovery. However, the optimal stem cell population has not been yet identified. We would like to propose a novel pro-regenerative treatment for post-infarction heart based on the combination of human skeletal myoblasts (huSkM) and mesenchymal stem cells (MSCs). huSkM native or overexpressing gene coding for Cx43 (huSKMCx43) alone or combined with MSCs were delivered in four cellular therapeutic variants into the healthy and post-infarction heart of mice while using molecular reporter probes. Single-Photon Emission Computed Tomography/Computed Tomography (SPECT/CT) performed right after cell delivery and 24 h later revealed a trend towards an increase in the isotopic uptake in the post-infarction group of animals treated by a combination of huSkMCx43 with MSC. Bioluminescent imaging (BLI) showed the highest increase in firefly luciferase (fluc) signal intensity in post-infarction heart treated with combination of huSkM and MSCs vs. huSkM alone ( p < 0.0001). In healthy myocardium, however, nanoluciferase signal (nanoluc) intensity varied markedly between animals treated with stem cell populations either alone or in combinations with the tendency to be simply decreased. Therefore, our observations seem to show that MSCs supported viability, engraftment, and even proliferation of huSkM in the post-infarction heart.

Mesenchymal stromal/stem cells (MSCs) are a unique population of cells that play an important role in the regeneration potential of the body. MSCs exhibit a characteristic phenotype and are capable of modulating the immune response. MSCs can be isolated from various tissues such as: bone marrow, adipose tissue, placenta, umbilical cord and others. The umbilical cord as a source of MSCs, has strong advantages, such as no-risk procedure of tissue retrieval after birth and easiness of the MSCs isolation. As the umbilical cord (UC) is a complex organ and we decided to evaluate, whether the cells derived from different regions of umbilical cord show similar or distinct properties. In this study we characterized and compared MSCs from three regions of the umbilical cord: Wharton's Jelly (WJ), the perivascular space (PRV) and the umbilical membrane (UCM). The analysis was carried out in terms of morphology, phenotype, immunomodulation potential and secretome. Based on the obtained results, we were able to conclude, that MSCs derived from distinct UC regions differ in their properties. According to our result WJ-MSCs have high and stabile proliferation potential and phenotype, when compare with other MSCs and can be treated as a preferable source of cells for medical application., (© 2021. The Author(s).)

Although less attention was paid to understanding physical localization changes in cell nuclei recently, depicting chromatin interaction maps is a topic of high interest. Here, we focused on defining extensive physical changes in chromatin organization in the process of skeletal myoblast differentiation. Based on RNA profiling data and 3D imaging of myogenic (NCAM1, DES, MYOG, ACTN3, MYF5, MYF6, ACTN2, and MYH2) and other selected genes (HPRT1, CDH15, DPP4 and VCAM1), we observed correlations between the following: (1) expression change and localization, (2) a gene and its genomic neighbourhood expression and (3) intra-chromosome and microscopical locus-centromere distances. In particular, we demonstrated the negative regulation of DPP4 mRNA (p < 0.001) and protein (p < 0.05) in differentiated myotubes, which coincided with a localization change of the DPP4 locus towards the nuclear lamina (p < 0.001) and chromosome 2 centromere (p < 0.001). Furthermore, we discuss the possible role of DPP4 in myoblasts (supported by an inhibition assay). We also provide positive regulation examples (VCAM1 and MYH2). Overall, we describe for the first time existing mechanisms of spatial gene expression regulation in myoblasts that might explain the issue of heterogenic responses observed during muscle regenerative therapies.

The possibility of using stem cell-derived cardiomyocytes opens a new platform for modeling cardiac cell differentiation and disease or the development of new drugs. Progress in this field can be accelerated by high-throughput screening (HTS) technology combined with promoter reporter system. The goal of the study was to create and evaluate a responsive promoter reporter system that allows monitoring of iPSC differentiation towards cardiomyocytes. The lentiviral promoter reporter system was based on troponin 2 (TNNT2) and alpha cardiac actin (ACTC) with firefly luciferase and mCherry, respectively. The system was evaluated in two in vitro models. First, system followed the differentiation of TNNT2-luc-T2A-Puro-mCMV-GFP and hACTC-mcherry-WPRE-EF1-Neo from transduced iPSC line towards cardiomyocytes and revealed the significant decrease in both inserts copy number during the prolonged in vitro cell culture (confirmed by I-FISH, ddPCR, qPCR). Second, differentiated and contracting control cardiomyocytes (obtained from control non-reporter transduced iPSCs) were subsequently transduced with TNNT2-luc-T2A-Puro-CMV-GFP and hACTC-mcherry-WPRE-EF1-Neo lentiviruses to observe the functionality of obtained cardiomyocytes. Our results indicated that the reporter modified cell lines can be used for HTS applications, but it is essential to monitor the stability of the reporter sequence during extended cell in vitro culture.

The lack of a fully developed human cardiac model in vitro hampers the progress of many biomedical research fields including pharmacology, developmental biology, and disease modeling. Currently, available methods may only differentiate human induced pluripotent stem cells (iPSCs) into immature cardiomyocytes. To achieve cardiomyocyte maturation, appropriate modulation of cellular microenvironment is needed. This study aims to optimize a microfluidic system that enhances maturation of human iPSC-derived cardiomyocytes (iPSC-CMs) through cyclic pulsatile hemodynamic forces. Human iPSC-CMs cultured in the microfluidic system show increased alignment and contractility and appear more rod-like shaped with increased cell size and increased sarcomere length when compared to static cultures. Increased complexity and density of the mitochondrial network in iPSC-CMs cultured in the microfluidic system are in line with expression of mitochondrial marker genes MT-CO1 and OPA1. Moreover, the optimized microfluidic system is capable of stably maintaining controlled oxygen levels and inducing hypoxia, revealed by increased expression of HIF1α and EGLN2 as well as changes in contraction parameters in iPSC-CMs. In summary, this microfluidic system boosts the structural maturation of iPSC-CM culture and could serve as an advanced in vitro cardiac model for biomedical research in the future. STATEMENT OF SIGNIFICANCE: The availability of in vitro human cardiomyocytes generated from induced pluripotent stem cells (iPSCs) opens the possibility to develop human in vitro heart models for disease modeling and drug testing. However, iPSC-derived cardiomyocytes remain structurally and functionally immature, which hinders their application. In this manuscript, we present an optimized and complete microfluidic system that enhances maturation of iPSC-derived cardiomyocytes through physiological cyclic pulsatile hemodynamic forces. Furthermore, we improved our microfluidic system by using a closed microfluidic recirculation and oxygen exchangers to achieve and maintain low oxygen in the culture chambers, which is suitable for mimicking the hypoxic condition and studying the pathophysiological mechanisms of human diseases in vitro. In the future, a variety of technologies including 3D tissue engineering could be integrated into our system, which may greatly extend the use of iPSC-derived cardiac models in drug development and disease modeling., (Copyright © 2019 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.)

Ischemic heart disease, also known as coronary artery disease (CAD), poses a challenge for regenerative medicine. iPSC technology might lead to a breakthrough due to the possibility of directed cell differentiation delivering a new powerful source of human autologous cardiomyocytes. One of the factors supporting proper cell maturation is in vitro culture duration. In this study, primary human skeletal muscle myoblasts were selected as a myogenic cell type reservoir for genetic iPSC reprogramming. Skeletal muscle myoblasts have similar ontogeny embryogenetic pathways (myoblasts vs. cardiomyocytes), and thus, a greater chance of myocardial development might be expected, with maintenance of acquired myogenic cardiac cell characteristics, from the differentiation process when iPSCs of myoblastoid origin are obtained. Analyses of cell morphological and structural changes, gene expression (cardiac markers), and functional tests (intracellular calcium transients) performed at two in vitro culture time points spanning the early stages of cardiac development (day 20 versus 40 of cell in vitro culture) confirmed the ability of the obtained myogenic cells to acquire adult features of differentiated cardiomyocytes. Prolonged 40-day iPSC-derived cardiomyocytes (iPSC-CMs) revealed progressive cellular hypertrophy; a better-developed contractile apparatus; expression of marker genes similar to human myocardial ventricular cells, including a statistically significant CX43 increase, an MHC isoform switch, and a troponin I isoform transition; more efficient intercellular calcium handling; and a stronger response to β-adrenergic stimulation.

In vitro generation of cardiomyocytes (CMs) from human cells opens the possibility to develop patient-specific therapies to various cardiomyopathies. By establishing the in vitro reprograming methods that produce human CMs, we learn about what is involved in the development of specific CM subtypes. In this review, we summarize the latest achievements in CM generation technologies, emphasizing the differentiation methods of specific CM subtypes. We also relate the biological properties and functions of the in vitro-generated CMs to those of their in vivo counterparts. Furthermore, we describe the main problem of current CM derivation methods - maturation of CMs. We subsequently discuss biochemical and physical stimuli that are used to overcome the maturation problems of in vitro-derived CMs. As a result, a more holistic approach with controllable environment and timing of specific stimuli for creation of more mature engineered heart tissues is described as well. Finally, we propose a novel approach in which enhancing energy transfer mechanisms in the immature CMs might help to overcome the current hurdle of incomplete in vitro differentiation., (Copyright © 2017 The Authors. Published by Elsevier B.V. All rights reserved.)

The derivation of pluripotent stem cells from human embryos and the generation of induced pluripotent stem cells (iPSCs) from somatic cells opened a new chapter in studies on the regeneration of the post-infarction heart and regenerative medicine as a whole. Thus, protocols for obtaining iPSCs were enthusiastically adopted and widely used for further experiments on cardiac differentiation. iPSC-mediated cardiomyocytes (iPSC-CMs) under in vitro culture conditions are generated by simulating natural cardiomyogenesis and involve the wingless-type mouse mammary tumour virus integration site family (WNT), transforming growth factor beta (TGF-β) and fibroblast growth factor (FGF) signalling pathways. New strategies have been proposed to take advantage of small chemical molecules, organic compounds and even electric or mechanical stimulation. There are three main approaches to support cardiac commitment in vitro: embryoid bodis (EBs), monolayer in vitro cultures and inductive co-cultures with visceral endoderm-like (END2) cells. In EB technique initial uniform size of pluripotent stem cell (PSC) colonies has a pivotal significance. Hence, some methods were designed to support cells aggregation. Another well-suited procedure is based on culturing cells in monolayer conditions in order to improve accessibility of growth factors and nutrients. Other distinct tactics are using visceral endoderm-like cells to culture them with PSCs due to secretion of procardiac cytokines. Finally, the appropriate purification of the obtained cardiomyocytes is required prior to their administration to a patient under the prospective cellular therapy strategy. This goal can be achieved using non-genetic methods, such as the application of surface markers and fluorescent dyes. Copyright © 2016 John Wiley & Sons, Ltd., (Copyright © 2016 John Wiley & Sons, Ltd.)

Aims: To assess the safety and efficacy of transendocardial delivery of muscle-derived stem/progenitor cells with connexin-43 overexpression (Cx-43-MDS/PC) in advanced heart failure (HF)., Methods and Results: Thirteen subjects with advanced HF, New York Heart Association (NYHA) class II-III were enrolled and treated with targeted injection of Cx-43-MDS/PCs and then monitored for at least 6 months. Overexpression of Cx43 (Cx43+) was significantly higher in all but one subject (Cx43-). Injection of MDS/PCs was associated with significant improvement of exercise capacity: NYHA (3 ± 0 vs. 1.8 ± 0.7, P = 0.003), exercise duration (388.69 ± 141.83 s vs. 462.08 ± 176.69 s, P = 0.025), peak oxygen consumption (14.38 ± 3.97 vs. 15.83 ± 3.74 ml/kg.min, P = 0.022) and oxygen pulse (10.58 ± 2.89 vs. 18.88 ± 22.63 mLO 2 /heart rate, P = 0.012). Levels of BNP, left ventricular (LV) ejection fraction and LV end-diastolic volumes tended to improve. There was a significant improvement of the mean unipolar voltage amplitudes measured for the injected segments and the entire left ventricle (9.62 ± 2.64 vs. 11.62 ± 3.50 mV, P = 0.014 and 8.83 ± 2.80 vs. 10.22 ± 3.41 mV, P = 0.041, respectively). No deaths were documented, Cx43+ (n = 12) subjects presented no significant ventricular arrhythmia; one Cx43- subject suffered from ventricular tachycardia (successfully treated with amiodarone)., Conclusions: Injection of Cx-43-MDS/PCs in patients with severe HF led to significant improvement in exercise capacity and myocardial viability of the injected segments while inducing no significant ventricular arrhythmia. This may arise from improved electrical coupling of the injected cells and injured myocardium and thus better in-situ mechanical cooperation of both cell types. Therefore, further clinical studies with Cx43+ MDS/PCs are warranted., (© 2017 The Authors. European Journal of Heart Failure © 2017 European Society of Cardiology.)

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 ex-pression 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 compari-son 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.

Purpose: To evaluate whether ejaculated human spermatozoa undergo complete apoptosis or necrosis during experimental semen bacterial infection in vitro., Methods: Apoptotic markers, including mitochondrial transmembrane potential (ΔΨm), phosphatidylserine (PS) externalization, and DNA fragmentation, have been detected simultaneously in ejaculated human sperm after their incubation with a known pathogenic (Escherichia coli), as well as with conditionally pathogenic bacterial strains (Staphylococcus haemolyticus, Bacteroides ureolyticus) and/or leukocytes. The ΔΨm and translocation of PS was evaluated using the JC-1 and Annexin V binding tests, respectively. A modified TUNEL assay with additional staining for sperm viability was used to detect the DNA fragmentation level., Results: The exposure of ejaculated spermatozoa to bacterial strains was associated with a simultaneous decrease in the percentage of sperm with normal ΔΨm and an increase in the proportion of Annexin V-positive sperm. Additionally, in the presence of S. haemolyticus, B. ureolyticus and/or leukocytes, a significant increase in the percentage of live TUNEL-positive (apoptotic) as well as dead TUNEL-positive (necrotic) sperm cells was also observed., Conclusions: The cellular death observed in spermatozoa in the presence of inflammatory mediators may be due to both apoptosis and necrosis. Here, we demonstrate for the first time that direct contact of conditionally pathogenic bacteria with ejaculated human sperm may play an even greater role in the promotion of apoptosis than in case of some pathogenic bacterial strains. These findings suggest that significant bacteriospermia and leukocytospermia may be direct causes of subfertility or additional negative factors worsening the prognosis of fertility in natural and assisted procreation.

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., (Copyright © 2014 Elsevier Ireland Ltd. All rights reserved.)