Ultrastructural Abnormalities in Induced Pluripotent Stem Cell-Derived Neural Stem Cells and Neurons of Two Cohen Syndrome Patients.

Cohen syndrome is an autosomal recessive disorder caused by VPS13B (COH1) gene mutations. This syndrome is significantly underdiagnosed and is characterized by intellectual disability, microcephaly, autistic symptoms, hypotension, myopia, retinal dystrophy, neutropenia, and obesity. VPS13B regulates intracellular membrane transport and supports the Golgi apparatus structure, which is critical for neuron formation. We generated induced pluripotent stem cells from two patients with pronounced manifestations of Cohen syndrome and differentiated them into neural stem cells and neurons. Using transmission electron microscopy, we documented multiple new ultrastructural changes associated with Cohen syndrome in the neuronal cells. We discovered considerable disturbances in the structure of some organelles: Golgi apparatus fragmentation and swelling, endoplasmic reticulum structural reorganization, mitochondrial defects, and the accumulation of large autophagosomes with undigested contents. These abnormalities underline the ultrastructural similarity of Cohen syndrome to many neurodegenerative diseases. The cell models that we developed based on patient-specific induced pluripotent stem cells can serve to uncover not only neurodegenerative processes, but the causes of intellectual disability in general.

Ultrastructural heterogeneity of the mitochondrial population in rat embryonic and induced pluripotent stem cells.

Even though rats are popular model animals, the ultrastructure of their pluripotent cells, that is, embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs), remains unexplored, although fine structure of pluripotent stem cells of mice and humans and its changes during differentiation have been investigated well. In the present study, we carried out ultrastructural and morphometric analyses of three lines of rat ESCs and two lines of rat iPSCs. The rat pluripotent stem cells were found to have the main typical morphological features of pluripotent cells: large nuclei of irregular or nearly round shape, scanty cytoplasm with few membrane organelles, and a poorly developed Golgi apparatus and endoplasmic reticulum. The cytoplasm of the rat pluripotent cells contains clusters of glycogen, previously described in human ESCs. To identify possible differences between rat ESCs and iPSCs, we performed a morphometric analysis of cell parameters. The mean area of cells and nuclei, the nuclear/cytoplasmic ratio, distributions of glycogen and diversity of mitochondria showed marked variations among the lines of rat pluripotent stem cells and were more pronounced than variations between rat ESCs and iPSCs as separate types of pluripotent stem cells. We noted morphological heterogeneity of the mitochondrial population in the rat pluripotent stem cells. The cells contained three types of mitochondria differing in the structure of cristae and in matrix density, and our morphometric analysis revealed differences in cristae structure.

'Trojan-Horse' stress-granule formation mediated by manganese oxide nanoparticles.

Exposure to nanomaterials is considered as one of the risk factors for neurodegenerative pathology. In vitro inorganic nanoparticles (NPs) absorb intrinsically disordered proteins, many of which are the constituents of stress-granules (SGs). SGs normally form in response to cellular stress and, here, we addressed whether selected inorganic NPs could trigger SGs formation in cells. To this end, we have tested a series of inorganic NPs for their ability to induce SGs formation in human glioblastoma and fibroblast cell lines. Among tested NPs, only Mn3O4 NPs triggered SGs formation in cell-type-specific and metabolic-dependent manner. In human glioblastoma U87 MG cell line, Mn3O4 NPs entered cells within minutes and resided inside intracellular vesicles for at least 48 h. Mn3O4 NPs induced a strong reduction in oxidative phosphorylation rate, but not glycolysis. We showed that Mn3O4 NPs slowly dissolve producing a local net of Mn2+ cations, which are known to inhibit oxidative phosphorylation. Indeed, direct incubation of cells with equimolar amounts of Mn2+ cations triggered SGs formation and reduced cellular respiration rate. However, while SGs formed in response to Mn3O4 NPs persisted for hours, SGs formation by Mn2+ peaked and dropped within minutes. Finally, Mn3O4 NPs mediated SGs formation via the phosphorylation of eIF2α. Thus, we conclude that exposure of U87 MG cells to Mn3O4 NPs caused a 'Trojan-horse' prolonged SGs response.

Mucin-2 knockout is a model of intercellular junction defects, mitochondrial damage and ATP depletion in the intestinal epithelium.

The disruption of the protective intestinal barrier-the 'leaky gut'-is a common complication of the inflammatory bowel disease. There is limited data on the mechanisms of the intestinal barrier disruption upon low-grade inflammation characteristic of patients with inflammatory bowel disease in clinical remission. Thus, animal models that recapitulate the complexity of chronic intestinal inflammation in vivo are of particular interest. In this study, we used Mucin-2 (Muc2) knockout mice predisposed to colitis to study intestinal barrier upon chronic inflammation. We used 4-kDa FITC-Dextran assay and transmission electron microscopy to demonstrate the increased intestinal permeability and morphological defects in intercellular junctions in Muc2 knockout mice. Confocal microscopy revealed the disruption of the apical F-actin cytoskeleton and delocalization of tight junction protein Claudin-3 from the membrane. We further demonstrate mitochondrial damage, impaired oxygen consumption and the reduction of the intestinal ATP content in Muc2 knockout mice. Finally, we show that chemically induced mitochondrial uncoupling in the wild type mice mimics the intestinal barrier disruption in vivo and causes partial loss of F-actin and membrane localization of Claudin-3. We propose that mitochondrial damage and metabolic shifts during chronic inflammation contribute to the leaky gut syndrome in Muc2 knockout animal model of colitis.

Generation of GABAergic striatal neurons by a novel iPSC differentiation protocol enabling scalability and cryopreservation of progenitor cells.

Cell models are promising tools for studying hereditary human neurodegenerative diseases. Neuronal derivatives of pluripotent stem cells provide the opportunity to investigate different stages of the neurodegeneration process. Therefore, easy and large-scale production of relevant cell types is a crucial barrier to overcome. In this work, we present an alternative protocol for iPSC differentiation into GABAergic medium spiny neurons (MSNs). The first stage involved dual-SMAD signalling inhibition through treatment with SB431542 and LDN193189, which results in the generation of neuroectodermal cells. Moreover, we used bFGF as a neuronal survival factor and dorsomorphin to inhibit BMP signalling. The combined treatment of dorsomorphin and SB431542 significantly enhanced neuronal induction, which was confirmed by the increased expression of the telencephalic-specific markers SOX1 and OTX2 as well as the forebrain marker PAX6. The next stage involved the derivation of actively proliferating MSN progenitor cells. An important feature of our protocol at this stage is the ability to perform prolonged cultivation of precursor cells at a high density without losing phenotypic properties. Moreover, the protocol enables multiple expansion steps (> 180 days cultivation) and cryopreservation of MSN progenitors. Therefore, this method allows quick production of a large number of neurons that are relevant for basic research, large-scale drug screening, and toxicological studies.

Introducing an expanded CAG tract into the huntingtin gene causes a wide spectrum of ultrastructural defects in cultured human cells.

Modeling of neurodegenerative diseases in vitro holds great promise for biomedical research. Human cell lines harboring a mutations in disease-causing genes are thought to recapitulate early stages of the development an inherited disease. Modern genome-editing tools allow researchers to create isogenic cell clones with an identical genetic background providing an adequate "healthy" control for biomedical and pharmacological experiments. Here, we generated isogenic mutant cell clones with 150 CAG repeats in the first exon of the huntingtin (HTT) gene using the CRISPR/Cas9 system and performed ultrastructural and morphometric analyses of the internal organization of the mutant cells. Electron microscopy showed that deletion of three CAG triplets or an HTT gene knockout had no significant influence on the cell structure. The insertion of 150 CAG repeats led to substantial changes in quantitative and morphological parameters of mitochondria and increased the association of mitochondria with the smooth and rough endoplasmic reticulum while causing accumulation of small autolysosomes in the cytoplasm. Our data indicate for the first time that expansion of the CAG repeat tract in HTT introduced via the CRISPR/Cas9 technology into a human cell line initiates numerous ultrastructural defects that are typical for Huntington's disease.

Mitochondria structural reorganization during mouse embryonic stem cell derivation.

Mouse embryonic stem (ES) cells are widely used in developmental biology and transgenic research. Despite numerous studies, ultrastructural reorganization of inner cell mass (ICM) cells during in vitro culture has not yet been described in detail. Here, we for the first time performed comparative morphological and morphometric analyses of three ES cell lines during their derivation in vitro. We compared morphological characteristics of blastocyst ICM cells at 3.5 and 4.5 days post coitum on feeder cells (day 6, passage 0) with those of ES cells at different passages (day 19, passage 2; day 25, passage 4; and passage 15). At passage 0, there were 23-36% of ES-like cells with various values of the medium cross-sectional area and nucleocytoplasmic parameters, 55% of fibroblast-like (probably trophoblast derivatives), and ~ 19% of dying cells. ES-like cells at passage 0 contained autolysosomes and enlarged mitochondria with reduced numerical density per cell. There were three types of mitochondria that differed in matrix density and cristae width. For the first time, we revealed cells that had two and sometimes three morphologically distinct mitochondria types in the cytoplasm. At passage 2, there were mostly ES cells with a high nucleocytoplasmic ratio and a cytoplasm depleted of organelles. At passage 4, ES cell morphology and morphometric parameters were mostly stable with little heterogeneity. According to our data, cellular structures of ICM cells undergo destabilization during derivation of an ES cell line with subsequent reorganization into the structures typical for ES cells. On the basis of ultrastructural analysis of mitochondria, we believe that the functional activity of these organelles changes during early stages of ES cell formation from the ICM.

Dominance of parental genomes in embryonic stem cell/fibroblast hybrid cells depends on the ploidy of the somatic partner.

Two dozen hybrid clones were produced by fusion of diploid embryonic stem (ES) cells positive for green fluorescent protein (GFP) with tetraploid fibroblasts derived from DD/c and C57BL-I(I)1RK mice. Cytogenetic analysis demonstrated that most cells from these hybrid clones contained near-hexaploid chromosome sets. Additionally, the presence of chromosomes derived from both parental cells was confirmed by polymerase chain reaction (PCR) analysis of polymorphic microsatellites. All hybrid cells were positive for GFP and demonstrated growth characteristics and fibroblast-like morphology. In addition, most hybrid cells were positive for collagen type I, fibronectin, and lamin A/C but were negative for Oct4 and Nanog proteins. Methylation status of the Oct4 and Nanog gene promoters was evaluated by bisulfite genomic sequencing analysis. The methylation sites (CpG-sites) of the Oct4 and Nanog gene promoters were highly methylated in hybrid cells, whereas the CpG-sites were unmethylated in the parental ES cells. Thus, the fibroblast genome dominated the ES genome in the diploid ES cell/tetraploid fibroblast hybrid cells. Immunofluorescent analysis of the pluripotent and fibroblast markers demonstrated that establishment of the fibroblast phenotype occurred shortly after fusion and that the fibroblast phenotype was further maintained in the hybrid cells. Fusion of karyoplasts and cytoplast derived from tetraploid fibroblasts with whole ES cells demonstrated that karyoplasts were able to establish the fibroblast phenotype of the reconstructed cells but not fibroblast cytoplasts. Thus, these data suggest that the dominance of parental genomes in hybrid cells of ES cell/somatic cell type depends on the ploidy of the somatic partner.

Reticulon 4a/NogoA locates to regions of high membrane curvature and may have a role in nuclear envelope growth.

Reticulon 4a (Rtn4a) is a membrane protein that shapes tubules of the endoplasmic reticulum (ER). The ER is attached to the nuclear envelope (NE) during interphase and has a role in post mitotic/meiotic NE reassembly. We speculated that Rtn4a has a role in NE dynamics. Using immuno-electron microscopy we found that Rtn4a is located at junctions between membranes in the cytoplasm, and between cytoplasmic membranes and the outer nuclear membrane in growing Xenopus oocyte nuclei. We found that during NE assembly in Xenopus egg extracts, Rtn4a localises to the edges of membranes that are flattening onto the chromatin. These results demonstrate that Rtn4a locates to regions of high membrane curvature in the ER and the assembling NE. Previously it was shown that incubation of egg extracts with antibodies against Rtn4a caused ER to form into large vesicles instead of tubules. To test whether Rtn4a contributes to NE assembly, we added the same Rtn4a antibody to nuclear assembly reactions. Chromatin was enclosed by membranes containing nuclear pore complexes, but nuclei did not grow. Instead large sacs of ER membranes attached to, but did not integrate into the NE. It is possible therefore that Rtn4a may have a role in NE assembly.