Expression Pattern of Trace Amine-Associated Receptors during Differentiation of Human Pluripotent Stem Cells to Dopaminergic Neurons.

Trace amine-associated receptors (TAARs), which were discovered only in 2001, are known to be involved in the regulation of a spectrum of neuronal processes and may play a role in the pathogenesis of a number of neuropsychiatric diseases, such as schizophrenia and others. We have previously shown that TAARs also have interconnections with the regulation of neurogenesis and, in particular, with the neurogenesis of dopamine neurons, but the exact mechanisms of this are still unknown. In our work we analyzed the expression of TAARs (TAAR1, TAAR2, TAAR5, TAAR6, TAAR8 and TAAR9) in cells from the human substantia nigra and ventral tegmental areas and in human pluripotent stem cells at consecutive stages of their differentiation to dopaminergic neurons, using RNA sequencing data from open databases, and TaqMan PCR data from the differentiation of human induced pluripotent stem cells in vitro. Detectable levels of TAARs expression were found in cells at the pluripotent stages, and the dynamic of their expression had a trend of increasing with the differentiation and maturation of dopamine neurons. The expression of several TAAR types (particularly TAAR5) was also found in human dopaminergic neuron-enriched zones in the midbrain. This is the first evidence of TAARs expression during neuronal differentiation, which can help to approach an understanding of the role of TAARs in neurogenesis.

Antibacterial Activity and Cytocompatibility of Electrospun PLGA Scaffolds Surface-Modified by Pulsed DC Magnetron Co-Sputtering of Copper and Titanium.

Biocompatible poly(lactide-co-glycolide) scaffolds fabricated via electrospinning are having promising properties as implants for the regeneration of fast-growing tissues, which are able to degrade in the body. The hereby-presented research work investigates the surface modification of these scaffolds in order to improve antibacterial properties of this type of scaffolds, as it can increase their application possibilities in medicine. Therefore, the scaffolds were surface-modified by means of pulsed direct current magnetron co-sputtering of copper and titanium targets in an inert atmosphere of argon. In order to obtain different amounts of copper and titanium in the resulting coatings, three different surface-modified scaffold samples were produced by changing the magnetron sputtering process parameters. The success of the antibacterial properties' improvement was tested with the methicillin-resistant bacterium Staphylococcus aureus. In addition, the resulting cell toxicity of the surface modification by copper and titanium was examined using mouse embryonic and human gingival fibroblasts. As a result, the scaffold samples surface-modified with the highest copper to titanium ratio show the best antibacterial properties and no toxicity against mouse fibroblasts, but have a toxic effect to human gingival fibroblasts. The scaffold samples with the lowest copper to titanium ratio display no antibacterial effect and toxicity. The optimal poly(lactide-co-glycolide) scaffold sample is surface-modified with a medium ratio of copper and titanium that has antibacterial properties and is non-toxic to both cell cultures.

Modulation of Notch Signaling at Early Stages of Differentiation of Human Induced Pluripotent Stem Cells to Dopaminergic Neurons.

Elaboration of protocols for differentiation of human pluripotent stem cells to dopamine neurons is an important issue for development of cell replacement therapy for Parkinson's disease. A number of protocols have been already developed; however, their efficiency and specificity still can be improved. Investigating the role of signaling cascades, important for neurogenesis, can help to solve this problem and to provide a deeper understanding of their role in neuronal development. Notch signaling plays an essential role in development and maintenance of the central nervous system after birth. In our study, we analyzed the effect of Notch activation and inhibition at the early stages of differentiation of human induced pluripotent stem cells to dopaminergic neurons. We found that, during the first seven days of differentiation, the cells were not sensitive to the Notch inhibition. On the contrary, activation of Notch signaling during the same time period led to significant changes and was associated with an increase in expression of genes, specific for caudal parts of the brain, a decrease of expression of genes, specific for forebrain, as well as a decrease of expression of genes, important for the formation of axons and dendrites and microtubule stabilizing proteins.

Surface Modification of Electrospun Bioresorbable and Biostable Scaffolds by Pulsed DC Magnetron Sputtering of Titanium for Gingival Tissue Regeneration.

In this study, polymer scaffolds were fabricated from biodegradable poly(lactide-co-glycolide) (PLGA) and from non-biodegradable vinylidene fluoride-tetrafluoroethylene (VDF-TeFE) by electrospinning. These polymer scaffolds were subsequently surface-modified by sputtering titanium targets in an argon atmosphere. Direct current pulsed magnetron sputtering was applied to prevent a significant influence of discharge plasma on the morphology and mechanical properties of the nonwoven polymer scaffolds. The scaffolds with initially hydrophobic properties show higher hydrophilicity and absorbing properties after surface modification with titanium. The surface modification by titanium significantly increases the cell adhesion of both the biodegradable and the non-biodegradable scaffolds. Immunocytochemistry investigations of human gingival fibroblast cells on the surface-modified scaffolds indicate that a PLGA scaffold exhibits higher cell adhesion than a VDF-TeFE scaffold.

Crenigacestat (LY3039478) inhibits osteogenic differentiation of human valve interstitial cells from patients with aortic valve calcification in vitro.

Calcific aortic valve disease (CAVD) is one of the dangerous forms of vascular calcification. CAVD leads to calcification of the aortic valve and disturbance of blood flow. Despite high mortality, there is no targeted therapy against CAVD or vascular calcification. Osteogenic differentiation of valve interstitial cells (VICs) is one of the key factors of CAVD progression and inhibition of this process seems a fruitful target for potential therapy. By our previous study we assumed that inhibitors of Notch pathway might be effective to suppress aortic valve leaflet calcification. We tested CB-103 and crenigacestat (LY3039478), two selective inhibitors of Notch-signaling, for suppression of osteogenic differentiation of VICs isolated from patients with CAVD in vitro. Effect of inhibitors were assessed by the measurement of extracellular matrix calcification and osteogenic gene expression. For effective inhibitor (crenigacestat) we also performed MTT and proteomics study for better understanding of its effect on VICs in vitro. CB-103 did not affect osteogenic differentiation. Crenigacestat completely inhibited osteogenic differentiation (both matrix mineralization and Runx2 expression) in the dosages that had no obvious cytotoxicity. Using proteomics analysis, we found several osteogenic differentiation-related proteins associated with the effect of crenigacestat on VICs differentiation. Taking into account that crenigacestat is FDA approved for clinical trials for anti-tumor therapy, we argue that this drug could be considered as a potential inhibitor of cardiovascular calcification.

Mechanisms of Regenerative Potential Activation in Cardiac Mesenchymal Cells.

Recovery of the contractile function of the heart and the regeneration of the myocardium after ischemic injury are contemporary issues in regenerative medicine and cell biology. This study aimed to analyze early transcriptional events in cardiac tissue after infarction and to explore the cell population that can be isolated from myocardial tissue. We induced myocardial infarction in Wistar rats by permanent ligation of the left coronary artery and showed a change in the expression pattern of Notch-associated genes and Bmp2/Runx2 in post-MI tissues using RNA sequencing and RT-PCR. We obtained primary cardiac mesenchymal cell (CMC) cultures from postinfarction myocardium by enzymatic dissociation of tissues, which retained part of the activation stimulus and had a pronounced proliferative potential, assessed using a "xCELLigence" real-time system. Hypoxia in vitro also causes healthy CMCs to overexpress Notch-associated genes and Bmp2/Runx2. Exogenous activation of the Notch signaling pathway by lentiviral transduction of healthy CMCs resulted in a dose-dependent activation of the Runx2 transcription factor but did not affect the activity of the Bmp2 factor. Thus, the results of this study showed that acute hypoxic stress could cause short-term activation of the embryonic signaling pathways Notch and Bmp in CMCs, and this interaction is closely related to the processes of early myocardial remodeling after a heart attack. The ability to correctly modulate and control the corresponding signals in the heart can help increase the regenerative capacity of the myocardium before the formation of fibrotic conditions.

Comparative Analysis of Dental Pulp and Periodontal Stem Cells: Differences in Morphology, Functionality, Osteogenic Differentiation and Proteome.

Dental stem cells are heterogeneous in their properties. Despite their common origin from neural crest stem cells, they have different functional capacities and biological functions due to niche influence. In this study, we assessed the differences between dental pulp stem cells (DPSC) and periodontal ligament stem cells (PDLSC) in their pluripotency and neuroepithelial markers transcription, morphological and functional features, osteoblast/odontoblast differentiation and proteomic profile during osteogenic differentiation. The data were collected in paired observations: two cell cultures, DPSC and PDLSC, were obtained from each donor. Both populations had the mesenchymal stem cells surface marker set exposed on their membranes but differed in Nestin (a marker of neuroectodermal origin) expression, morphology, and proliferation rate. OCT4 mRNA was revealed in DPSC and PDLSC, while OCT4 protein was present in the nuclei of DPSC only. However, transcription of OCT4 mRNA was 1000-10,000-fold lower in dental stem cells than in blastocysts. DPSC proliferated at a slower rate and have a shape closer to polygonal but they responded better to osteogenic stimuli as compared to PDLSC. RUNX2 mRNA was detected by qPCR in both types of dental stem cells but RUNX2 protein was detected by LC-MS/MS shotgun proteomics only in PDLSC suggesting the posttranscriptional regulation. DSPP and DMP1, marker genes of odontoblastic type of osteogenic differentiation, were transcribed in DPSC but not in PDLSC samples. Our results prove that DPSC and PDLSC are different in their biology and therapeutic potential: DPSC are a good candidate for osteogenic or odontogenic bone-replacement cell-seeded medicines, while fast proliferating PDLSC are a prospective candidate for other cell products.

Proteomic Profiling of the Human Fetal Multipotent Mesenchymal Stromal Cells Secretome.

Secretome of multipotent mesenchymal stromal cells (MSCs) is actively used in biomedical applications such as alveolar bone regeneration, treatment of cardiovascular disease, and neurodegenerative disorders. Nevertheless, hMSCs have low proliferative potential and production of the industrial quantity of their secretome might be challenging. Human fetal multipotent mesenchymal stromal cells (FetMSCs) isolated from early human embryo bone marrow are easy to expand and might be a potential source for pharmaceutical substances production based on their secretome. However, the secretome of FetMSCs was not previously analyzed. Here, we describe the secretome of FetMSCs using LC-MALDI shotgun proteomics. We identified 236 proteins. Functional annotation of the identified proteins revealed their involvement in angiogenesis, ossification, regulation of apoptosis, and immune response processes, which made it promising for biomedical applications. The proteins identified in the FetMSCs secretome are involved in the same biological processes as proteins from previously described adult hMSCs secretomes. Nevertheless, many of the common hMSCs secretome components (such as VEGF, FGF, Wnt and TGF-ß) have not been identified in the FetMSCs secretome.

Modification of PCL Scaffolds by Reactive Magnetron Sputtering: A Possibility for Modulating Macrophage Responses.

Direct current (DC) reactive magnetron sputtering is as an efficient method for enhancing the biocompatibility of poly(ε-caprolactone) (PCL) scaffolds. However, the PCL chemical bonding state, the composition of the deposited coating, and their interaction with immune cells remain unknown. Herein, we demonstrated that the DC reactive magnetron sputtering of the titanium target in a nitrogen atmosphere leads to the formation of nitrogen-containing moieties and the titanium dioxide coating on the scaffold surface. We have provided the possible mechanism of PCL fragmentation and coating formation supported by XPS results and DFT calculations. Our preliminary biological studies suggest that DC reactive magnetron sputtering of the titanium target could be an effective tool to control macrophage functional responses toward PCL scaffolds as it allows to inhibit respiratory burst while retaining cell viability and scavenging activity.

Transfer of Synthetic Human Chromosome into Human Induced Pluripotent Stem Cells for Biomedical Applications.

AlphoidtetO-type human artificial chromosome (HAC) has been recently synthetized as a novel class of gene delivery vectors for induced pluripotent stem cell (iPSC)-based tissue replacement therapeutic approach. This HAC vector was designed to deliver copies of genes into patients with genetic diseases caused by the loss of a particular gene function. The alphoidtetO-HAC vector has been successfully transferred into murine embryonic stem cells (ESCs) and maintained stably as an independent chromosome during the proliferation and differentiation of these cells. Human ESCs and iPSCs have significant differences in culturing conditions and pluripotency state in comparison with the murine naïve-type ESCs and iPSCs. To date, transferring alphoidtetO-HAC vector into human iPSCs (hiPSCs) remains a challenging task. In this study, we performed the microcell-mediated chromosome transfer (MMCT) of alphoidtetO-HAC expressing the green fluorescent protein into newly generated hiPSCs. We used a recently modified MMCT method that employs an envelope protein of amphotropic murine leukemia virus as a targeting cell fusion agent. Our data provide evidence that a totally artificial vector, alphoidtetO-HAC, can be transferred and maintained in human iPSCs as an independent autonomous chromosome without affecting pluripotent properties of the cells. These data also open new perspectives for implementing alphoidtetO-HAC as a gene therapy tool in future biomedical applications.

In vitro toxicity of FemOn, FemOn-SiO2 composite, and SiO2-FemOn core-shell magnetic nanoparticles.

Over the last decade, magnetic iron oxide nanoparticles (IONPs) have drawn much attention for their potential biomedical applications. However, serious in vitro and in vivo safety concerns continue to exist. In this study, the effects of uncoated, FemOn-SiO2 composite flake-like, and SiO2-FemOn core-shell IONPs on cell viability, function, and morphology were tested 48 h postincubation in human umbilical vein endothelial cell culture. Cell viability and apoptosis/necrosis rate were determined using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay and annexin V-phycoerythrin kit, respectively. Cell morphology was evaluated using bright-field microscopy and forward and lateral light scattering profiles obtained with flow cytometry analysis. All tested IONP types were used at three different doses, that is, 0.7, 7.0, and 70.0 µg. Dose-dependent changes in cell morphology, viability, and apoptosis rate were shown. At higher doses, all types of IONPs caused formation of binucleated cells suggesting impaired cytokinesis. FemOn-SiO2 composite flake-like and SiO2-FemOn core-shell IONPs were characterized by similar profile of cytotoxicity, whereas bare IONPs were shown to be less toxic. The presence of either silica core or silica nanoflakes in composite IONPs can promote cytotoxic effects.

Notch-dependent EMT is attenuated in patients with aortic aneurysm and bicuspid aortic valve.

Bicuspid aortic valve is the most common congenital heart malformation and the reasons for the aortopathies associated with bicuspid aortic valve remain unclear. NOTCH1 mutations are associated with bicuspid aortic valve and have been found in individuals with various left ventricular outflow tract abnormalities. Notch is a key signaling during cardiac valve formation that promotes the endothelial-to-mesenchymal transition. We address the role of Notch signaling in human aortic endothelial cells from patients with bicuspid aortic valve and aortic aneurysm. Aortic endothelial cells were isolated from tissue fragments of bicuspid aortic valve-associated thoracic aortic aneurysm patients and from healthy donors. Endothelial-to-mesenchymal transition was induced by activation of Notch signaling. Effectiveness of the transition was estimated by loss of endothelial and gain of mesenchymal markers by immunocytochemistry and qPCR. We show that aortic endothelial cells from the patients with aortic aneurysm and bicuspid aortic valve have down regulated Notch signaling and fail to activate Notch-dependent endothelial-to-mesenchymal transition in response to its stimulation by different Notch ligands. Our findings support the idea that bicuspid aortic valve and associated aortic aneurysm is associated with dysregulation of the entire Notch signaling pathway independently on the specific gene mutation.

Self-renewal of murine embryonic stem cells is supported by the serine/threonine kinases Pim-1 and Pim-3.

pim-1 and pim-3 encode serine/threonine kinases involved in the regulation of cell proliferation and apoptosis in response to cytokine stimulation. We analyzed the regulation of pim-1 and pim-3 by the leukemia inhibitory factor (LIF)/gp130/signal transducer and activator of transcription-3 (STAT3) pathway and the role of Pim-1 and Pim-3 kinases in mouse embryonic stem (ES) cell self-renewal. Making use of ES cells expressing a granulocyte colony-stimulating factor:gp130 chimeric receptor and a hormone-dependent signal transducer and activator of transcription-3 estrogen receptor (STAT3-ER(T2)), we showed that expression of pim-1 and pim-3 was upregulated by LIF/gp130-dependent signaling and the STAT3 transcription factor. ES cells overexpressing pim-1 and pim-3 had a greater capacity to self-renew and displayed a greater resistance to LIF starvation based on a clonal assay. In contrast, knockdown of pim-1 and pim-3 increased the rate of spontaneous differentiation in a self-renewal assay. Knockdown of pim-1 and pim-3 was also detrimental to the growth of undifferentiated ES cell colonies and increased the rate of apoptosis. These findings provide a novel role of Pim-1 and Pim-3 kinases in the control of self-renewal of ES cells. Disclosure of potential conflicts of interest is found at the end of this article.