Hybrids of manganese oxide and lipid liquid crystalline nanoparticles (LLCNPs@MnO) as potential magnetic resonance imaging (MRI) contrast agents.

Due to the health risks associated with the use of Gd-chelates and the promising effects of using nanoparticles as T1 contrast agents (CAs) for MRI, Mn-based nanoparticles are considered a highly competitive alternative. The use of hybrid constructs with paramagnetic functionality of Mn-based nanoparticles is an effective approach, in particular, the use of biocompatible lipid liquid crystalline nanoparticles (LLCNPs) as a carrier of MnO nanoparticles. LLCNPs possess a unique internal structure ensuring a payload of different polarity MnO nanoparticles. In view of MRI application, the surface properties including the polarity of MnO are crucial factors determining their relaxation rate and thus the MRI efficiency. Two novel hybrid constructs consisting of LLCNPs loaded with hydrophobic MnO-oleate and hydrophilic MnO-DMSA NPs were prepared. These nanosystems were studied in terms of their physico-chemical properties, positive T1 contrast enhancement properties (in vitro and in vivo) and biological safety. LLCNPs@MnO-oleate and LLCNPs@MnO-DMSA hybrids exhibited a heterogeneous phase composition, however with differences in the inner periodic arrangement and structural parameters, as well as in the preferable localization of MnO NPs within the LLCNPs. Also, these hybrids differed in terms of particle size-related parameters and colloidal stability, which was found to be strongly dependent on the addition of differently functionalized MnO NPs. Embedding both types of MnO NPs into LLCNPs resulted in high relaxivity parameters, in comparison to bare MnO-DMSA NPs and also commercially developed CAs (e.g. Dotarem and Teslascan). Further biosafety studies revealed that cell internalization pathways were dependent on the prepared hybrid type, while viability, effects on the mitochondria membrane potential and cytoskeletal networks were rather related to the susceptibility of the particular cell line. The high relaxation rates achieved with the developed hybrid LLCNPs@MnO enable them to be possibly used as novel and biologically safe MRI T1-enhancing CAs in in vivo imaging.

The Role of Cytokines in the Metastasis of Solid Tumors to the Spine: Systematic Review.

Although many studies have investigated the role of cytokines in bone metastases, our knowledge of their function in spine metastasis is limited. Therefore, we performed a systematic review to map the available evidence on the involvement of cytokines in spine metastasis in solid tumors. A PubMed search identified 211 articles demonstrating a functional link between cytokines/cytokine receptors and bone metastases, including six articles confirming the role of cytokines/cytokine receptors in spine metastases. A total of 68 cytokines/cytokine receptors were identified to mediate bone metastases; 9 (mostly chemokines) played a role in spine metastases: CXC motif chemokine ligand (CXCL) 5, CXCL12, CXC motif chemokine receptor (CXCR) 4, CXCR6, interleukin (IL) 10 in prostate cancer, CX3C motif chemokine ligand (CX3CL) 1 and CX3C motif chemokine receptor (CX3CR) 1 in liver cancer, CC motif chemokine ligand (CCL) 2 in breast cancer, and transforming growth factor (TGF) ß in skin cancer. Except for CXCR6, all cytokines/cytokine receptors were shown to operate in the spine, with CX3CL1, CX3CR1, IL10, CCL2, CXCL12, and CXCR4 mediating bone marrow colonization, CXCL5 and TGFß promoting tumor cell proliferation, and TGFß additionally driving bone remodeling. The number of cytokines/cytokine receptors confirmed to mediate spinal metastasis is low compared with the vast spectrum of cytokines/cytokine receptors participating in other parts of the skeleton. Therefore, further research is needed, including validation of the role of cytokines mediating metastases to other bones, to precisely address the unmet clinical need associated with spine metastases.

Modeling the human heart ex vivo-current possibilities and strive for future applications.

The high organ specification of the human heart is inversely proportional to its functional recovery after damage. The discovery of induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) has accelerated research in human heart regeneration and physiology. Nevertheless, due to the immaturity of iPSC-CMs, they are far from being an representative model of the adult heart physiology. Therefore, number of laboratories strive to obtain a heart tissues by engineering methods by structuring iPSC-CMs into complex and advanced platforms. By using the iPSC-CMs and arranging them in 3D cultures it is possible to obtain a human heart muscle with physiological capabilities potentially similar to the adult heart, while remaining in vitro. Here, we attempt to describe existing examples of heart muscle either in vitro or ex vivo models and discuss potential options for the further development of such structures. This will be a crucial step for ultimate derivation of complete heart tissue-mimicking organs and their future use in drug development, therapeutic approaches testing, pre-clinical studies, and clinical applications. This review particularly aims to compile available models of advanced human heart tissue for scientists considering which model would best fit their research needs.

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

BACKGROUND: The application of a circulating miR-195 inhibitor could be a helping factor in the in vitro model of human skeletal muscle-derived stem/progenitor cells (SkMDS/PCs). Previously, microRNA-195 (miR-195) expression has been reported to be a negative factor for myogenesis. AIMS: The study aimed 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 aging), antioxidant, and myogenic gene expression analyses were performed at two time points, at the seventh and eleventh 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 aging 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 (the eleventh cell passage). CONCLUSIONS: The performed studies may lead to the preconditioning of myogenic stem cells to extend their potential for pro-regenerative activity. The miR-195 inhibitor may serve as a conditioning factor augmenting selective antioxidant gene expression and proliferative potential of SkMDS/PCs, but it does not have an impact on cell aging and/ or apoptosis.

Multiparametric Evaluation of Post-MI Small Animal Models Using Metabolic ([18F]FDG) and Perfusion-Based (SYN1) Heart Viability Tracers.

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 [18F] fluorodeoxyglucose ([18F]-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 [18F]-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 [18F]-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.

Molecular Imaging of Human Skeletal Myoblasts (huSKM) in Mouse Post-Infarction Myocardium.

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.

pNiPAM-Nanoparticle-Based Antiapoptotic Approach for Pro-Regenerative Capacity of Skeletal Myogenic Cells.

The biocompatibility of pNiPAM (Poly N-isopropylacrylamide) copolymers has been examined and they did not exert any cytotoxic effects. Their properties and vulnerable temperature characteristics make them candidates for use in medical applications. We synthesized a well-characterized nanoparticles-based cargo system that would effectively deliver a biological agent to human skeletal myogenic cells (SkMCs); among other aspects, a downregulating apoptotic pathway potentially responsible for poor regeneration of myocardium. We confirmed the size of the pNiPAM based spheres at around 100 nm and the nanomeric shape of nanoparticles (NP) obtained. We confirmed that 33 °C is the adequate temperature for phase transition. We performed the dynamics of cargo release. A small amount of examined protein was detected at 10 min after reaching LCTS (lower critical solution temperature). The presented results of the test with BSA (bovine serum albumin) and doxorubicin loaded into nanoparticles showed a similar release profile for both substances. SkMCs incubated with NP loaded with antiapoptotic agent, BCB (Bax channel blocker), significantly diminished cell apoptosis (p < 0.01). Moreover, the lowest apoptotic level was detected in SkMCs treated with camptothecin and simultaneously incubated with pNiPAMs loaded with BCB. Application of nanoparticles loaded with BCB or subjected to BCB alone did not, however, diminish the amount of apparently necrotic cells.

Molecular imaging of myogenic stem/progenitor cells with [18F]-FHBG PET/CT system in SCID mice model of post-infarction heart.

Preclinical and clinical studies have shown that stem cells can promote the regeneration of damaged tissues, but therapeutic protocols need better quality control to confirm the location and number of transplanted cells. This study describes in vivo imaging while assessing reporter gene expression by its binding to a radiolabelled molecule to the respective receptor expressed in target cells. Five mice underwent human skeletal muscle-derived stem/progenitor cell (huSkMDS/PC EF1-HSV-TK) intracardial transplantation after induction of myocardial infarction (MI). The metabolic parameters of control and post-infarction stem progenitor cell-implanted mice were monitored using 2-deoxy-18F-fluorodeoxyglucose ([18F]-FDG) before and after double promotor/reporter probe imaging with 9-(4-18F-fluoro-3-[hydroxymethyl]butyl)guanine ([18F]-FHBG) using positron emission tomography (PET) combined with computed tomography (CT). Standardized uptake values (SUVs) were then calculated based on set regions of interest (ROIs). Experimental animals were euthanized after magnetic resonance imaging (MRI). Molecular [18F]-FHBG imaging of myogenic stem/progenitor cells in control and post-infarction mice confirmed the survival and proliferation of transplanted cells, as shown by an increased or stable signal from the PET apparatus throughout the 5 weeks of monitoring. huSkMDS/PC EF1-HSV-TK transplantation improved cardiac metabolic ([18F]-FDG with PET) and haemodynamic (MRI) parameters. In vivo PET/CT and MRI revealed that the precise use of a promotor/reporter probe incorporated into stem/progenitor cells may improve non-invasive monitoring of targeted cellular therapy in the cardiovascular system.

Addition of Popular Exogenous Antioxidant Agent, PBN, to Culture Media May Be an Important Step to Optimization of Myogenic Stem/Progenitor Cell Preparation Protocol.

The aim of the study was to modify human skeletal muscle-derived stem/progenitor cells (SkMDS/PCs) and demonstrate the optimal cell preparation protocol for application in post-infarction hearts. We used conditioned SkMDS/PC culture medium with α-phenyl-N-tert-butyl nitrone (PBN). SkMDS/PCs were cultured under hypoxic conditions and the results were compared to the standard ones. We observed a significant increase of CD-56 positive phenotypic marker the ability to form functional myotubes, increase in the proportion of young cells in cell primary suspensions, and a decrease in the percentage of apoptotic cells among PBN-conditioned cells in normoxia an hypoxia. We also observed significantly higher levels of SOD3 expression; maintained expression of SOD1, SOD2, and CAT; a higher level of BCL2 gene expression; and a rather significant decrease in Hsp70 gene expression in PBN-conditioned SkMDS/PCs compared to the WT population under hypoxic conditions. In addition, significant increase of myogenic genes expression was observed after PBN addition to culture medium, compared to WT population under hypoxia. Interestingly, PBN addition significantly increased the lengths of telomeres under hypoxia. Based on the data obtained, we can postulate that PBN conditioning of human SkMDS/PCs could be a promising step in improving myogenic cell preparation protocol for pro-regenerative treatment of post-infarction hearts.

Transient and Stable Overexpression of Extracellular Superoxide Dismutase Is Positively Associated with the Myogenic Function of Human Skeletal Muscle-Derived Stem/Progenitor Cells.

In the present study, the genetic modification of human skeletal muscle-derived stem/progenitor cells (SkMDS/PCs) was investigated to identify the optimal protocol for myogenic cell preparation for use in post-infarction heart therapy. We used two types of modifications: GFP-transfection (using electroporation) and SOD3 transduction (using a lentiviral vector). SkMDS/PCs were cultured under different in vitro conditions, including standard (21% oxygen) and hypoxic (3% oxygen), the latter of which corresponded to the prevailing conditions in the post-infarction heart. Transfection/transduction efficacy, skeletal myogenic cell marker expression (CD56), cellular senescence, and apoptosis, as well as the expression of antioxidant (SOD1, SOD2, and SOD3), anti-aging (SIRT1 and FOXO), anti-apoptotic (BCL2), and myogenic (MyoD and MyoG) genes, were evaluated. The percentage of GFP-positive SkMDS/PCs was determined as an indicator of the efficacy of transfection, which reached 55%, while transduction showed better efficiency, reaching approximately 85% as estimated by fluorescence microscopy. The CD56-positive SkMDS/PCs were present in approximately 77% of the tested cells after transient transfection and approximately 96% after transduction. Under standard in vitro culture conditions, the ability of the differentiated, transfected SkMDS/PCs to form myotubes was greater than that of the wild type (WT) cell population (p < 0.001), while the cells transduced with the SOD3 gene exhibited an increase in cell fusion under both standard (p < 0.05) and hypoxic conditions (p < 0.001). In transduced SkMDS/PCs, we observed a positive influence of SOD3 overexpression on cell ageing and apoptosis. We observed an increase in the percentage of young cells under standard (p < 0.05) and hypoxic (p < 0.001) in vitro culture conditions, with a notable decrease in the percentage of senescent and advanced senescent cells in the SOD3-overexpressing cell population detected compared to that observed for the untransduced muscle-derived cells. A lower percentage of apoptotic cells was observed for transduced SkMDS/PCs than that for WT cells under hypoxic in vitro culture conditions. In transiently transfected SkMDS/PCs, we observed significantly higher gene expression levels of SOD2 (almost 40-fold) (p < 0.001) and FOXO (p < 0.05) (approximately 3-fold) under both normoxic and hypoxic culture conditions and of BCL2 under hypoxia compared to those observed in untreated cells (WT). In addition, myogenic genes showed a significant increase in MyoD (almost 18-fold) expression under standard culture conditions (p < 0.0001) and decreased MyoG expression (approximately 2-fold) after transfection (p < 0.05) compared with that detected in the WT skeletal muscle-derived cell control. Taken together, these results demonstrate that SOD3-tranduced skeletal muscle-derived cells may have potential for use in the regenerative treatment of the post-infarction heart.

Chromatin and transcriptome changes in human myoblasts show spatio-temporal correlations and demonstrate DPP4 inhibition in differentiated myotubes.

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.

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 co­transplantation of BM-MSCs and SM-SPCs was performed directly into the biceps brachii (two patients) and gastrocnemius (one patient). During a six­month follow­up, 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 BM­MSC and SM­SPC 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.

Tissue-specific promoter-based reporter system for monitoring cell differentiation from iPSCs to cardiomyocytes.

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.

Novel Mutations Segregating with Complete Androgen Insensitivity Syndrome and their Molecular Characteristics.

We analyzed three cases of Complete Androgen Insensitivity Syndrome (CAIS) and report three hitherto undisclosed causes of the disease. RNA-Seq, Real-timePCR, Western immunoblotting, and immunohistochemistry were performed with the aim of characterizing the disease-causing variants. In case No.1, we have identified a novel androgen receptor (AR) mutation (c.840delT) within the first exon in the N-terminal transactivation domain. This thymine deletion resulted in a frameshift and thus introduced a premature stop codon at amino acid 282. In case No.2, we observed a nonsynonymous mutation in the ligand-binding domain (c.2491C>T). Case No.3 did not reveal AR mutation; however, we have found a heterozygous mutation in CYP11A1 gene, which has a role in steroid hormone biosynthesis. Comparative RNA-Seq analysis of CAIS and control revealed 4293 significantly deregulated genes. In patients with CAIS, we observed a significant increase in the expression levels of PLCXD3, TM4SF18, CFI, GPX8, and SFRP4, and a significant decrease in the expression of SPATA16, TSACC, TCP10L, and DPY19L2 genes (more than 10-fold, p < 0.05). Our findings will be helpful in molecular diagnostics of patients with CAIS, as well as the identified genes could be also potential biomarkers for the germ cells differentiation process.

The impact of in vitro cell culture duration on the maturation of human cardiomyocytes derived from induced pluripotent stem cells of myogenic origin.

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 ß-adrenergic stimulation.

Potential use of superparamagnetic iron oxide nanoparticles for in vitro and in vivo bioimaging of human myoblasts.

Myocardial infarction (MI) is one of the most frequent causes of death in industrialized countries. Stem cells therapy seems to be very promising for regenerative medicine. Skeletal myoblasts transplantation into postinfarction scar has been shown to be effective in the failing heart but shows limitations such, e.g. cell retention and survival. We synthesized and investigated superparamagnetic iron oxide nanoparticles (SPIONs) as an agent for direct cell labeling, which can be used for stem cells imaging. High quality, monodisperse and biocompatible DMSA-coated SPIONs were obtained with thermal decomposition and subsequent ligand exchange reaction. SPIONs' presence within myoblasts was confirmed by Prussian Blue staining and inductively coupled plasma mass spectrometry (ICP-MS). SPIONs' influence on tested cells was studied by their proliferation, ageing, differentiation potential and ROS production. Cytotoxicity of obtained nanoparticles and myoblast associated apoptosis were also tested, as well as iron-related and coating-related genes expression. We examined SPIONs' impact on overexpression of two pro-angiogenic factors introduced via myoblast electroporation method. Proposed SPION-labeling was sufficient to visualize firefly luciferase-modified and SPION-labeled cells with magnetic resonance imaging (MRI) combined with bioluminescence imaging (BLI) in vivo. The obtained results demonstrated a limited SPIONs' influence on treated skeletal myoblasts, not interfering with basic cell functions.

Biological and Pro-Angiogenic Properties of Genetically Modified Human Primary Myoblasts Overexpressing Placental Growth Factor in In Vitro and In Vivo Studies.

Cardiovascular diseases are a growing problem in developing countries; therefore, there is an ongoing intensive search for new approaches to treat these disorders. Currently, cellular therapies are focused on healing the damaged heart by implanting stem cells modified with pro-angiogenic factors. This approach ensures that the introduced cells are capable of fulfilling the complex requirements of the environment, including the replacement of the post-infarction scar with cells that are able to contract and promote the formation of new blood vessels that can supply the ischaemic region with nutrients and oxygen. This study focused on the genetic modification of human skeletal muscle cells (SkMCs). We chose myoblast cells due to their close biological resemblance to cardiomyocytes and the placental growth factor (PlGF) gene due to its pro-angiogenic potential. In our in vitro studies, we transfected SkMCs with the PlGF gene using electroporation, which has previously been proven to be efficient and generate robust overexpression of the PlGF gene and elevate PlGF protein secretion. Moreover, the functionality of the secreted pro-angiogenic proteins was confirmed using an in vitro capillary development assay. We have also examined the influence of PlGF overexpression on VEGF-A and VEGF-B, which are well-known factors described in the literature as the most potent activators of blood vessel formation. We were able to confirm the overexpression of VEGF-A in myoblasts transfected with the PlGF gene. The results obtained in this study were further verified in an animal model. These data were able to confirm the potential therapeutic effects of the applied treatments.

Hypoxia-Inducible Factor-1 in Physiological and Pathophysiological Angiogenesis: Applications and Therapies.

The cardiovascular system ensures the delivery of oxygen and nutrients to all cells, tissues, and organs. Under extended exposure to reduced oxygen levels, cells are able to survive through the transcriptional activation of a series of genes that participate in angiogenesis, glucose metabolism, and cell proliferation. The oxygen-sensitive transcriptional activator HIF-1 (hypoxia-inducible factor-1) is a key transcriptional mediator of the response to hypoxic conditions. The HIF-1 pathway was found to be a master regulator of angiogenesis. Whether the process is physiological or pathological, HIF-1 seems to participate in vasculature formation by synergistic correlations with other proangiogenic factors such as VEGF (vascular endothelial growth factor), PlGF (placental growth factor), or angiopoietins. Considering the important contributions of HIF-1 in angiogenesis and vasculogenesis, it should be considered a promising target for treating ischaemic diseases or cancer. In this review, we discuss the roles of HIF-1 in both physiological/pathophysiological angiogenesis and potential strategies for clinical therapy.

Genetically modified human myoblasts with eNOS may improve regenerative ability of myogenic stem cells to infarcted heart.

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.