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.

Thiopurines Analogues with Additional Ring: Synthesis, Spectroscopic Properties, and Anticancer Potency.

Purine scaffolds constitute a starting point for the synthesis of numerous chemotherapeutics used in treating cancer, viruses, parasites, as well as bacterial and fungal infections. In this work, we synthesized a group of guanosine analogues containing an additional five-membered ring and a sulfur atom at the C-9 position. The spectral, photophysical, and biological properties of the synthesized compounds were investigated. The spectroscopic studies revealed that a combination of the thiocarbonyl chromophore and the tricyclic structure of guanine analogues shifts the absorption region above 350 nm, allowing for selective excitation when present in biological systems. Unfortunately, due to the low fluorescence quantum yield, this process cannot be used to monitor the presence of these compounds in cells. The synthesized compounds were evaluated for their effect on the viability of human cervical carcinoma (HeLa) and mouse fibroblast (NIH/3T3) cells. It was found that all of them display anticancer activity. In vitro studies were preceded by in silico ADME and PASS analyses, which confirmed that the designed compounds are promising candidates for anticancer agents.

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.

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.

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.

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.

IgA nephropathy associated with coeliac disease.

Coeliac disease (CD) is an autoimmune disorder of the small intestine triggered by ingested gluten from barley, rye and wheat. It can be associated with other autoimmune conditions, such as type 1 diabetes, autoimmune thyroiditis and hepatitis, Sjögren's syndrome and IgA nephropathy (IgAN). We describe here a case of a 24-year-old man with the above-mentioned atypical form of coeliac disease for whom the diagnosis started with renal disorder. The diagnosis of nephrotic syndrome was established and the coexistence with CD was also suspected. In fact, immunoglobulin (Ig) A and IgG antibodies against endomysium and against gliadin were detected in serum of the patient and the endoscopic biopsy of the duodenum revealed stage 3B CD. Percutaneous kidney biopsy was also performed. Class I IgAN was diagnosed. Gluten-free diet, ACE inhibitor and oral iron were introduced to the patient. The improvement of clinical and laboratory disorders of CD as well as gradual remission of the nephrotic syndrome were observed. In conclusion, there may be a small group of patients with IgAN coexisting with CD in whom a gluten-free diet seems to be the treatment of choice for the resolution of kidney disease.

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.

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 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.

Very small embryonic-like stem cells as a novel developmental concept and the hierarchy of the stem cell compartment.

Our current understanding of stem cells suffers from a lack of precision, as the stem cell compartment is a broad continuum between early stages of development and adult postnatal tissues, and it is not fully understood how this transition occurs. The definition of stem cell pluripotency is adapted from embryology and excludes the possibility that some early-development stem cells with pluri- and/or multipotential differentiation potential may reside in postnatal tissues in a dormant state in which they are protected from uncontrolled proliferation and thus do not form teratomas or have the ability to complement blastocyst development. We will discuss the concept that a population of very small embryonic-like stem cells (VSELs) could be a link between early-development stages and adult stem cell compartments and reside in a quiescent state in adult tissues. The epigenetic mechanism identified that changes expression of certain genes involved in insulin/insulin-like growth factor signaling (IIS) in VSELs, on the one hand, keeps these cells quiescent in adult tissues and, on the other hand, provides a novel view of the stem cell compartment, IIS, tissue/organ rejuvenation, aging, and cancerogenesis.

Evidence that the population of quiescent bone marrow-residing very small embryonic/epiblast-like stem cells (VSELs) expands in response to neurotoxic treatment.

The concept that bone marrow (BM)-derived cells may participate in neural regeneration remains controversial, and the identity of the specific cell type(s) involved remains unknown. We recently reported that the adult murine BM contains a highly mobile population of Sca-1(+) Lin(-) CD45(-) cells known as very small embryonic/epiblast-like stem cells (VSELs) that express several markers of pluripotency such as Oct-4. In the BM microenvironment, these cells are kept quiescent because of epigenetic modification of certain paternally imprinted genes. However, as reported, these cells can be mobilized in mice in an experimental model of stroke and express several genes involved in neurogenesis while circulating in peripheral blood (PB). In the current work, we employed a model of toxic brain damage, which is induced by administration of kainic acid, to see not only whether VSELs can be mobilized into PB in response to this neurotoxin, but, more importantly, whether they proliferate and expand in BM tissue. We report here for the first time that brain damage leads to activation and expansion of the BM pool of quiescent VSELs, which precedes their subsequent egress into PB. Harnessing these cells in neural tissue regeneration is currently one of the challenges in regenerative medicine.

Regulation of endothelial connexin40 expression by shear stress.

Endothelial connexin (Cx)40 plays an important role in signal propagation along blood vessel walls, modulating vessel diameter and thereby blood flow. Blood flow, in turn, has been shown to alter endothelial Cx40 expression. However, the timing and shear stress dependence of this relationship have remained unclear, as have the signal transduction pathways involved and the functional implications. Therefore, the aim of this study was to quantify the effects of shear stress on endothelial Cx40 expression, to analyze the role of phosphoinositide 3-kinase (PI3K)/Akt signaling involved, and to assess the possible functional consequences for the adaptation of microvascular networks. First-passage human umbilical vein endothelial cells were exposed to defined shear stress conditions and analyzed for Cx40 using real-time RT-PCR and immunoblot analysis. Shear stress caused long-term induction of Cx40 protein expression, with two short-term mRNA peaks at 4 and 16 h, indicating the dynamic nature of the adaptation process. Maximum shear stress-dependent induction was observed at shear levels between 6 and 10 dyn/cm(2). Simulation of this pattern of shear-dependent Cx expression in a vascular adaptation model of a microvascular network led to an improved fit for the simulated results to experimental measurements. Cx40 expression was greatly reduced by inhibiting PI3K or Akt, with PI3K activity being required for basal Cx40 expression and Akt activity taking part in its shear stress-dependent induction.

Shear stress increases endothelial hyaluronan synthase 2 and hyaluronan synthesis especially in regard to an atheroprotective flow profile.

Recent studies revealed that in vivo the inner blood vessel surface is lined with an endothelial surface layer at least 0.5 µm thick, which serves as an aegis, protecting the vessel wall from arteriosclerosis. Hyaluronan seems to be a constitutive component in regard to the atheroprotective properties of this surface structure. It has been shown that arterial pulsatile laminar blood flow increases the thickness of this surface layer in vivo, while it is significantly reduced at atheroprone regions with disturbed flow. This study was undertaken to reveal whether endothelial hyaluronan synthesis via hyaluronan synthase 2 (HAS2) can be changed by different shear stress conditions in vitro, especially in regard to an undisturbed, arterial-like pulsatile flow profile. Human umbilical vein endothelial cells, exposed to constant or pulsatile shear stress in a cone-and-plate system, were analysed for HAS2 expression by real-time RT-PCR and immunoblotting, and for hyaluronan by ELISA. Hyaluronan synthase 2 mRNA and protein were found to be transiently increased in a shear stress-dependent manner via the phosphatidylinositol 3-kinase-Akt pathway. Especially pulsatile, arterial-like shear stress conditions induced enzyme and hyaluronan effectively, while lower shear stress that continuously changed its direction did not induce any differences in comparison with control cultures not exposed to shear stress. These experiments provide a link between the production of a constitutive component of the endothelial surface layer by endothelial cells and blood flow.

Expression of ADAMTS1 in endothelial cells is induced by shear stress and suppressed in sprouting capillaries.

ADAMTS1 inhibits capillary sprouting, and since capillary sprouts do not experience the shear stress caused by blood flow, this study undertook to clarify the relationship between shear stress and ADAMTS1. It was found that endothelial cells exposed to shear stress displayed a strong upregulation of ADAMTS1, dependent upon both the magnitude and duration of their exposure. Investigation of the underlying pathways demonstrated involvement of phospholipase C, phosphoinositide 3-kinase, and nitric oxide. Forkhead box protein O1 was identified as a likely inhibitor of the system, as its knockdown was followed by a slight increase in ADAMTS1 expression. In silico prediction displayed a transcriptional binding site for Forkhead box protein O1 in the promotor region of the ADAMTS1 gene, as well as sites for nuclear factor 1, SP1, and AP-1. The anti-angiogenic effects of ADAMTS1 were attributed to its cleavage of thrombospondin 1 into a 70-kDa fragment, and a significant enhancement of this fragment was indeed demonstrated by immunoblotting shear stress-treated cells. Accordingly, scratch wound closure displayed a slowdown in conditioned medium from shear stress-treated endothelial cells, an effect that could be completely blocked by a knockdown of thrombospondin 1 and partially blocked by a knockdown of ADAMTS1. Non-perfused capillary sprouts in rat mesenteries stained negative for ADAMTS1, while vessels in the microcirculation that had already experienced blood flow yielded the opposite results. The shear stress-dependent expression of ADAMTS1 in vitro was therefore also demonstrated in vivo and thereby confirmed as a mechanism connecting blood flow with the regulation of angiogenesis.