PAPP-A-Specific IGFBP-4 Proteolysis in Human Induced Pluripotent Stem Cell-Derived Cardiomyocytes.

The insulin-like growth factors IGF-I and IGF-II-as well as their binding proteins (IGFBPs), which regulate their bioavailability-are involved in many pathological and physiological processes in cardiac tissue. Pregnancy-associated plasma protein A (PAPP-A) is a metalloprotease that preferentially cleaves IGFBP-4, releasing IGF and activating its biological activity. Previous studies have shown that PAPP-A-specific IGFBP-4 proteolysis is involved in the pathogenesis of cardiovascular diseases, such as ischemia, heart failure, and acute coronary syndrome. However, it remains unclear whether PAPP-A-specific IGFBP-4 proteolysis participates in human normal cardiomyocytes. Here, we report PAPP-A-specific IGFBP-4 proteolysis occurring in human cardiomyocytes derived from two independent induced pluripotent cell lines (hiPSC-CMs), detected both on the cell surface and in the cell secretome. PAPP-A was measured by fluoroimmune analysis (FIA) in a conditioned medium of hiPSC-CMs and was detected in concentrations of up to 4.3 ± 1.33 ng/mL and 3.8 ± 1.1 ng/mL. The level of PAPP-A-specific IGFBP-4 proteolysis was determined as the concentration of NT-IGFBP-4 proteolytic fragments using FIA for a proteolytic neo-epitope-specific assay. We showed that PAPP-A-specific IGFBP-4 proteolysis is IGF-dependent and inhibited by EDTA and 1,10-phenanthroline. Therefore, it may be concluded that PAPP-A-specific IGFBP-4 proteolysis functions in human normal cardiomyocytes, and hiPSC-CMs contain membrane-bound and secreted forms of proteolytically active PAPP-A.

An Efficient 2D Protocol for Differentiation of iPSCs into Mature Postmitotic Dopaminergic Neurons: Application for Modeling Parkinson's Disease.

About 15% of patients with parkinsonism have a hereditary form of Parkinson's disease (PD). Studies on the early stages of PD pathogenesis are challenging due to the lack of relevant models. The most promising ones are models based on dopaminergic neurons (DAns) differentiated from induced pluripotent stem cells (iPSCs) of patients with hereditary forms of PD. This work describes a highly efficient 2D protocol for obtaining DAns from iPSCs. The protocol is rather simple, comparable in efficiency with previously published protocols, and does not require viral vectors. The resulting neurons have a similar transcriptome profile to previously published data for neurons, and have a high level of maturity marker expression. The proportion of sensitive (SOX6+) DAns in the population calculated from the level of gene expression is higher than resistant (CALB+) DAns. Electrophysiological studies of the DAns confirmed their voltage sensitivity and showed that a mutation in the PARK8 gene is associated with enhanced store-operated calcium entry. The study of high-purity DAns differentiated from the iPSCs of patients with hereditary PD using this differentiation protocol will allow for investigators to combine various research methods, from patch clamp to omics technologies, and maximize information about cell function in normal and pathological conditions.

Comparison of the calcium signaling alterations in GABA-ergic medium spiny neurons produced from iPSCs of different origins.

Disease models based on induced pluripotent stem cells (iPSCs) are in high demand because of their physiological adequacy and well-reproducibility of the pathological phenotype. Nowadays, the most common approach to generate iPSCs is the reprogramming of somatic cells using vectors based on lentivirus or Sendai virus. We have previously shown impairments of calcium signaling including store-operated calcium entry in Huntington's disease-specific iPSCs-based GABA-ergic medium spiny neurons. However, different approaches for iPSCs generation make it difficult to compare the models since the mechanism of reprogramming may influence the electrophysiological properties of the terminally differentiated neurons. Here, we have studied the features of calcium homeostasis in GABA-ergic medium spiny neurons differentiated from iPSCs obtained from fibroblasts of the same donor using different methods. Our data demonstrated that there were no significant differences neither in calcium influx through the store-operated channels, nor in the levels of proteins activating this type of calcium entry in neurons differentiated from iPSCs generated with lenti- and Sendai viruses-based approaches. We also found no differences in voltage-gated calcium entry for these neurons. Thus, we clearly showed that various methods of cell reprogramming result in similar deregulations in neuronal calcium signaling which substantiates the ability to combine the experimental data on functional studies of ion channels in models based on iPSCs obtained by different methods and expands the prospects for the use of biobanking.

Therapy-induced secretion of spliceosomal components mediates pro-survival crosstalk between ovarian cancer cells.

Ovarian cancer often develops resistance to conventional therapies, hampering their effectiveness. Here, using ex vivo paired ovarian cancer ascites obtained before and after chemotherapy and in vitro therapy-induced secretomes, we show that molecules secreted by ovarian cancer cells upon therapy promote cisplatin resistance and enhance DNA damage repair in recipient cancer cells. Even a short-term incubation of chemonaive ovarian cancer cells with therapy-induced secretomes induces changes resembling those that are observed in chemoresistant patient-derived tumor cells after long-term therapy. Using integrative omics techniques, we find that both ex vivo and in vitro therapy-induced secretomes are enriched with spliceosomal components, which relocalize from the nucleus to the cytoplasm and subsequently into the extracellular vesicles upon treatment. We demonstrate that these molecules substantially contribute to the phenotypic effects of therapy-induced secretomes. Thus, SNU13 and SYNCRIP spliceosomal proteins promote therapy resistance, while the exogenous U12 and U6atac snRNAs stimulate tumor growth. These findings demonstrate the significance of spliceosomal network perturbation during therapy and further highlight that extracellular signaling might be a key factor contributing to the emergence of ovarian cancer therapy resistance.

iPSC-derived cells lack immune tolerance to autologous NK-cells due to imbalance in ligands for activating and inhibitory NK-cell receptors.

BACKGROUND: Dozens of transplants generated from pluripotent stem cells are currently in clinical trials. The creation of patient-specific iPSCs makes personalized therapy possible due to their main advantage of immunotolerance. However, some reports have claimed recently that aberrant gene expression followed by proteome alterations and neoantigen formation can result in iPSCs recognition by autologous T-cells. Meanwhile, the possibility of NK-cell activation has not been previously considered. This study focused on the comparison of autologous and allogeneic immune response to iPSC-derived cells and isogeneic parental somatic cells used for reprogramming. METHODS: We established an isogeneic cell model consisting of parental dermal fibroblasts, fibroblast-like iPSC-derivatives (iPS-fibro) and iPS-fibro lacking beta-2-microglobulin (B2M). Using the cells obtained from two patients, we analyzed the activation of autologous and allogeneic T-lymphocytes and NK-cells co-cultured with target cells. RESULTS: Here we report that cells differentiated from iPSCs can be recognized by NK-cells rather than by autologous T-cells. We observed that iPS-fibro elicited a high level of NK-cell degranulation and cytotoxicity, while isogeneic parental skin fibroblasts used to obtain iPSCs barely triggered an NK-cell response. iPSC-derivatives with B2M knockout did not cause an additional increase in NK-cell activation, although they were devoid of HLA-I, the major inhibitory molecules for NK-cells. Transcriptome analysis revealed a significant imbalance of ligands for activating and inhibitory NK-cell receptors in iPS-fibro. Compared to parental fibroblasts, iPSC-derivatives had a reduced expression of HLA-I simultaneously with an increased gene expression of major activating ligands, such as MICA, NECTIN2, and PVR. The lack of inhibitory signals might be due to insufficient maturity of cells differentiated from iPSCs. In addition, we showed that pretreatment of iPS-fibro with proinflammatory cytokine IFNγ restored the ligand imbalance, thereby reducing the degranulation and cytotoxicity of NK-cells. CONCLUSION: In summary, we showed that iPSC-derived cells can be sensitive to the cytotoxic potential of autologous NK-cells regardless of HLA-I status. Thus, the balance of ligands for NK-cell receptors should be considered prior to iPSC-based cell therapies. Trial registration Not applicable.

Cerebral Organoids-Challenges to Establish a Brain Prototype.

The new cellular models based on neural cells differentiated from induced pluripotent stem cells have greatly enhanced our understanding of human nervous system development. Highly efficient protocols for the differentiation of iPSCs into different types of neural cells have allowed the creation of 2D models of many neurodegenerative diseases and nervous system development. However, the 2D culture of neurons is an imperfect model of the 3D brain tissue architecture represented by many functionally active cell types. The development of protocols for the differentiation of iPSCs into 3D cerebral organoids made it possible to establish a cellular model closest to native human brain tissue. Cerebral organoids are equally suitable for modeling various CNS pathologies, testing pharmacologically active substances, and utilization in regenerative medicine. Meanwhile, this technology is still at the initial stage of development.

Ouabain-Induced Gene Expression Changes in Human iPSC-Derived Neuron Culture Expressing Dopamine and cAMP-Regulated Phosphoprotein 32 and GABA Receptors.

Cardiotonic steroids (CTS) are specific inhibitors and endogenous ligands of a key enzyme in the CNS-the Na+, K+-ATPase, which maintains and creates an ion gradient on the plasma membrane of neurons. CTS cause the activation of various signaling cascades and changes in gene expression in neurons and other cell types. It is known that intracerebroventricular injection of cardiotonic steroid ouabain causes mania-like behavior in rodents, in part due to activation of dopamine-related signaling cascades in the dopamine and cAMP-regulated phosphoprotein 32 (DARPP-32) expressing medium spiny neurons in the striatum. Dopaminergic projections in the striatum innervate these GABAergic medium spiny neurons. The objective of this study was to assess changes in the expression of all genes in human iPSC-derived expressing DARPP-32 and GABA receptors neurons under the influence of ouabain. We noted a large number of statistically significant upregulated and downregulated genes after a 16-h incubation with non-toxic concentration (30 nM) of ouabain. These changes in the transcriptional activity were accomplished with activation of MAP-kinase ERK1/2 and transcriptional factor cAMP response element-binding protein (CREB). Thus, it can be concluded that 30 nM ouabain incubated for 16 h with human iPSC-derived expressing DARPP-32 and GABA receptors neurons activates genes associated with neuronal maturation and synapse formation, by increasing the expression of genes associated with translation, vesicular transport, and increased electron transport chain function. At the same time, the expression of genes associated with proliferation, migration, and early development of neurons decreases. These data indicate that non-toxic concentrations of ouabain may induce neuronal maturation, neurite growth, and increased synaptogenesis in dopamine-receptive GABAergic neurons, suggesting formation of plasticity and the establishment of new neuronal junctions.

STIM2 Mediates Excessive Store-Operated Calcium Entry in Patient-Specific iPSC-Derived Neurons Modeling a Juvenile Form of Huntington's Disease.

Huntington's disease (HD) is a severe autosomal-dominant neurodegenerative disorder caused by a mutation within a gene, encoding huntingtin protein. Here we have used the induced pluripotent stem cell technology to produce patient-specific terminally differentiated GABA-ergic medium spiny neurons modeling a juvenile form of HD (HD76). We have shown that calcium signaling is dramatically disturbed in HD76 neurons, specifically demonstrating higher levels of store-operated and voltage-gated calcium uptakes. However, comparing the HD76 neurons with the previously described low-repeat HD models, we have demonstrated that the severity of calcium signaling alterations does not depend on the length of the polyglutamine tract of the mutant huntingtin. Here we have also observed greater expression of huntingtin and an activator of store-operated calcium channels STIM2 in HD76 neurons. Since shRNA-mediated suppression of STIM2 decreased store-operated calcium uptake, we have speculated that high expression of STIM2 underlies the excessive entry through store-operated calcium channels in HD pathology. Moreover, a previously described potential anti-HD drug EVP4593 has been found to attenuate high levels of both huntingtin and STIM2 that may contribute to its neuroprotective effect. Our results are fully supportive in favor of the crucial role of calcium signaling deregulation in the HD pathogenesis and indicate that the cornerstone of excessive calcium uptake in HD-specific neurons is a calcium sensor and store-operated calcium channels activator STIM2, which should become a molecular target for medical treatment and novel neuroprotective drug development.

The effect of isomorphic impurities on the elastic conductivity of Dirac structures.

Results of a theoretical study of piezoresistance properties of Dirac structures on example graphene nanoribbons with different types of a conductivity, both ideal and doped with point substitution defects of various concentrations uniformly distributed in the crystal lattice, are presented. Boron and nitrogen atoms are chosen as acceptor and donor impurities respectively. Using the tight-binding method and the Anderson's model, the band structure of the nanoparticles under study is simulated. The longitudinal component of the elastic conductivity tensor is analytically calculated. Its dependence on the relative strain of longitudinal compression and tension, the concentration of impurities and the width of the nanoribbon are studied. The physical substantiation of the results obtained is given.

Manifestation of Huntington's disease pathology in human induced pluripotent stem cell-derived neurons.

BACKGROUND: Huntington's disease (HD) is an incurable hereditary neurodegenerative disorder, which manifests itself as a loss of GABAergic medium spiny (GABA MS) neurons in the striatum and caused by an expansion of the CAG repeat in exon 1 of the huntingtin gene. There is no cure for HD, existing pharmaceutical can only relieve its symptoms. RESULTS: Here, induced pluripotent stem cells were established from patients with low CAG repeat expansion in the huntingtin gene, and were then efficiently differentiated into GABA MS-like neurons (GMSLNs) under defined culture conditions. The generated HD GMSLNs recapitulated disease pathology in vitro, as evidenced by mutant huntingtin protein aggregation, increased number of lysosomes/autophagosomes, nuclear indentations, and enhanced neuronal death during cell aging. Moreover, store-operated channel (SOC) currents were detected in the differentiated neurons, and enhanced calcium entry was reproducibly demonstrated in all HD GMSLNs genotypes. Additionally, the quinazoline derivative, EVP4593, reduced the number of lysosomes/autophagosomes and SOC currents in HD GMSLNs and exerted neuroprotective effects during cell aging. CONCLUSIONS: Our data is the first to demonstrate the direct link of nuclear morphology and SOC calcium deregulation to mutant huntingtin protein expression in iPSCs-derived neurons with disease-mimetic hallmarks, providing a valuable tool for identification of candidate anti-HD drugs. Our experiments demonstrated that EVP4593 may be a promising anti-HD drug.