Detection of ER Stress in iPSC-Derived Neurons Carrying the p.N370S Mutation in the GBA1 Gene.

Endoplasmic reticulum (ER) stress is involved in the pathogenesis of many human diseases, such as cancer, type 2 diabetes, kidney disease, atherosclerosis and neurodegenerative diseases, in particular Parkinson's disease (PD). Since there is currently no treatment for PD, a better understanding of the molecular mechanisms underlying its pathogenesis, including the mechanisms of the switch from adaptation in the form of unfolded protein response (UPR) to apoptosis under ER stress conditions, may help in the search for treatment methods. Genetically encoded biosensors based on fluorescent proteins are suitable tools that facilitate the study of living cells and visualization of molecular events in real time. The combination of technologies to generate patient-specific iPSC lines and genetically encoded biosensors allows the creation of cell models with new properties. Using CRISPR-Cas9-mediated homologous recombination at the AAVS1 locus of iPSC with the genetic variant p.N370S (rs76763715) in the GBA1 gene, we created a cell model designed to study the activation conditions of the IRE1-XBP1 cascade of the UPR system. The cell lines obtained have a doxycycline-dependent expression of the genetically encoded biosensor XBP1-TagRFP, possess all the properties of human pluripotent cells, and can be used to test physical conditions and chemical compounds that affect the development of ER stress, the functioning of the UPR system, and in particular, the IRE1-XBP1 cascade.

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.

A Model of iPSC-Derived Macrophages with TNFAIP3 Overexpression Reveals the Peculiarities of TNFAIP3 Protein Expression and Function in Human Macrophages.

Macrophages play a crucial role in the development and control of inflammation. Understanding the mechanisms balancing macrophage inflammatory activity is important to develop new strategies for treating inflammation-related diseases. TNF-α-induced protein 3 (TNFAIP3, A20) is a negative regulator of intracellular inflammatory cascades; its deficiency induces hyper-inflammatory reactions. Whether A20 overexpression can dampen macrophage inflammatory response remains unclear. Here, we generated human-induced pluripotent stem cells with tetracycline-inducible A20 expression and differentiated them into macrophages (A20-iMacs). A20-iMacs displayed morphology, phenotype, and phagocytic activity typical of macrophages, and they displayed upregulated A20 expression in response to doxycycline. A20 overexpression dampened the A20-iMac response to TNF-α, as shown by a decreased expression of IL1B and IL6 mRNA. A dynamic analysis of A20 expression following the generation of A20-iMacs and control iMacs showed that the expression declined in iMacs and that iMacs expressed a lower molecular weight form of the A20 protein (~70 kDa) compared with less differentiated cells (~90 kDa). A low-level expression of A20 and the predominance of a low-molecular-weight A20 form were also characteristic of monocyte-derived macrophages. The study for the first time developed a model for generating macrophages with an inducible expression of a target gene and identified the peculiarities of A20 expression in macrophages that likely underlie macrophage preparedness for inflammatory reactivity. It also suggested the possibility of mitigating inflammatory macrophage responses via A20 overexpression.

Generation of three induced pluripotent stem cell lines (RAUi001-A, RAUi001-B and RAUi001-C) from peripheral blood mononuclear cells of a healthy Armenian individual.

The study of pathological processes in cells carrying mutations should be carried out in comparison with a healthy control group. Familial Mediterranean fever (FMF), which is caused by a mutation in the MEFV gene, is predominantly found in people of Armenian nationality with the prevalence of 14-100 per 10000. We have obtained induced pluripotent stem cells (iPSCs) from Armenian healthy patient, which will be included as a control group in the study of this disease. iPSCs rapidly proliferate in colonies of cells with a typical pluripotent-like morphology, have a normal karyotype (46,XX). iPSCs express pluripotency markers (OCT4, SOX2, TRA-1-60, NANOG) and are able to give derivatives of three germ layers.

Potential Binding Sites of Pharmacological Chaperone NCGC00241607 on Mutant ß-Glucocerebrosidase and Its Efficacy on Patient-Derived Cell Cultures in Gaucher and Parkinson's Disease.

Mutations in the GBA1 gene, encoding the lysosomal enzyme glucocerebrosidase (GCase), cause Gaucher disease (GD) and are the most common genetic risk factor for Parkinson's disease (PD). Pharmacological chaperones (PCs) are being developed as an alternative treatment approach for GD and PD. To date, NCGC00241607 (NCGC607) is one of the most promising PCs. Using molecular docking and molecular dynamics simulation we identified and characterized six allosteric binding sites on the GCase surface suitable for PCs. Two sites were energetically more preferable for NCGC607 and located nearby to the active site of the enzyme. We evaluated the effects of NCGC607 treatment on GCase activity and protein levels, glycolipids concentration in cultured macrophages from GD (n = 9) and GBA-PD (n = 5) patients as well as in induced human pluripotent stem cells (iPSC)-derived dopaminergic (DA) neurons from GBA-PD patient. The results showed that NCGC607 treatment increased GCase activity (by 1.3-fold) and protein levels (by 1.5-fold), decreased glycolipids concentration (by 4.0-fold) in cultured macrophages derived from GD patients and also enhanced GCase activity (by 1.5-fold) in cultured macrophages derived from GBA-PD patients with N370S mutation (p < 0.05). In iPSC-derived DA neurons from GBA-PD patients with N370S mutation NCGC607 treatment increased GCase activity and protein levels by 1.1-fold and 1.7-fold (p < 0.05). Thus, our results showed that NCGC607 could bind to allosteric sites on the GCase surface and confirmed its efficacy on cultured macrophages from GD and GBA-PD patients as well as on iPSC-derived DA neurons from GBA-PD patients.

Biochemical Characteristics of iPSC-Derived Dopaminergic Neurons from N370S GBA Variant Carriers with and without Parkinson's Disease.

GBA variants increase the risk of Parkinson's disease (PD) by 10 times. The GBA gene encodes the lysosomal enzyme glucocerebrosidase (GCase). The p.N370S substitution causes a violation of the enzyme conformation, which affects its stability in the cell. We studied the biochemical characteristics of dopaminergic (DA) neurons generated from induced pluripotent stem cells (iPSCs) from a PD patient with the GBA p.N370S mutation (GBA-PD), an asymptomatic GBA p.N370S carrier (GBA-carrier), and two healthy donors (control). Using liquid chromatography with tandem mass spectrometry (LC-MS/MS), we measured the activity of six lysosomal enzymes (GCase, galactocerebrosidase (GALC), alpha-glucosidase (GAA), alpha-galactosidase (GLA), sphingomyelinase (ASM), and alpha-iduronidase (IDUA)) in iPSC-derived DA neurons from the GBA-PD and GBA-carrier. DA neurons from the GBA mutation carrier demonstrated decreased GCase activity compared to the control. The decrease was not associated with any changes in GBA expression levels in DA neurons. GCase activity was more markedly decreased in the DA neurons of GBA-PD patient compared to the GBA-carrier. The amount of GCase protein was decreased only in GBA-PD neurons. Additionally, alterations in the activity of the other lysosomal enzymes (GLA and IDUA) were found in GBA-PD neurons compared to GBA-carrier and control neurons. Further study of the molecular differences between the GBA-PD and the GBA-carrier is essential to investigate whether genetic factors or external conditions are the causes of the penetrance of the p.N370S GBA variant.

IPSC-Derived Astrocytes Contribute to In Vitro Modeling of Parkinson's Disease Caused by the GBA1 N370S Mutation.

Parkinson's disease (PD) is a neurodegenerative disorder that ranks second in prevalence after Alzheimer's disease. The number of PD diagnoses increases annually. Nevertheless, modern PD treatments merely mitigate symptoms rather than preventing neurodegeneration progression. The creation of an appropriate model to thoroughly study the mechanisms of PD pathogenesis remains a current challenge in biomedicine. Recently, there has been an increase in data regarding the involvement of not only dopaminergic neurons of the substantia nigra but also astrocytes in the pathogenesis of PD. Cell models based on induced pluripotent stem cells (iPSCs) and their differentiated derivatives are a useful tool for studying the contribution and interaction of these two cell types in PD. Here, we generated two iPSC lines, ICGi034-B and ICGi034-C, by reprogramming peripheral blood mononuclear cells of a patient with a heterozygous mutation c.1226A>G (p.N370S) in the GBA1 gene by non-integrating episomal vectors encoding OCT4, KLF4, L-MYC, SOX2, LIN28, and mp53DD. The iPSC lines demonstrate the expression of pluripotency markers and are capable of differentiating into three germ layers. We differentiated the ICGi034-B and ICGi034-C iPSC lines into astrocytes. This resulting cell model can be used to study the involvement of astrocytes in the pathogenesis of GBA-associated PD.

Generation of induced pluripotent stem cell line, ICGi033-A, by reprogramming peripheral blood mononuclear cells from a patient with Huntington's disease.

Huntington's disease (HD) is a hereditary autosomal dominant neurodegenerative disease caused by the polyglutamine stretch expansion in the huntingtin (HTT) protein. In HD, dysregulation of multiple cellular processes occurs, resulting in the death of medium spiny neurons of striatum. A line of induced pluripotent stem cells (iPSCs) ICGi033-A was obtained from peripheral blood mononuclear cells of a patient carrying 77 CAG repeats in the HTT gene. The iPSCs express pluripotency markers, have a normal karyotype, and differentiate into three germ layers: endoderm, ectoderm, mesoderm.

Generation of induced pluripotent stem cell line, ICGi034-A, by reprogramming peripheral blood mononuclear cells from a patient with Parkinson's disease associated with GBA mutation.

Mutation in the glucocerebrosidase encoding gene (GBA) is one of the most frequent genetic cause of Parkinson's disease. ICGi034-A induced pluripotent stem cell (iPSC) line obtained by reprogramming peripheral blood mononuclear cells (PBMCs) of a patient with heterozygous c.1226A > G (p.N370S) mutation in the GBA gene can be used for studying the fundamental mechanisms of the pathogenesis of GBA-associated Parkinson's disease, and for potential drug screening. The iPSCs express pluripotency markers (NANOG, SSEA4, TRA-1-60, OCT4, SOX2), have a normal karyotype, and are capable of producing derivatives of three germ layers.

A Human Induced Pluripotent Stem Cell-Derived Isogenic Model of Huntington's Disease Based on Neuronal Cells Has Several Relevant Phenotypic Abnormalities.

Huntington's disease (HD) is a severe neurodegenerative disorder caused by a CAG triplet expansion in the first exon of the HTT gene. Here we report the introduction of an HD mutation into the genome of healthy human embryonic fibroblasts through CRISPR/Cas9-mediated homologous recombination. We verified the specificity of the created HTT-editing system and confirmed the absence of undesirable genomic modifications at off-target sites. We showed that both mutant and control isogenic induced pluripotent stem cells (iPSCs) derived by reprogramming of the fibroblast clones can be differentiated into striatal medium spiny neurons. We next demonstrated phenotypic abnormalities in the mutant iPSC-derived neural cells, including impaired neural rosette formation and increased sensitivity to growth factor withdrawal. Moreover, using electron microscopic analysis, we detected a series of ultrastructural defects in the mutant neurons, which did not contain huntingtin aggregates, suggesting that these defects appear early in HD development. Thus, our study describes creation of a new isogenic iPSC-based cell system that models HD and recapitulates HD-specific disturbances in the mutant cells, including some ultrastructural features implemented for the first time.

Macrophages Derived From Human Induced Pluripotent Stem Cells Are Low-Activated "Naïve-Like" Cells Capable of Restricting Mycobacteria Growth.

In peripheral tissues, immune protection critically depends on the activity of tissue resident macrophages, which makes our understanding of the biology of these cells of great significance. Until recently, human macrophage studies were largely based on the analysis of monocyte-derived macrophages that differ from tissue resident macrophages by many characteristics. To model tissue resident macrophages, methods of generating macrophages from pluripotent stem cells have been developed. However, the immunological properties of macrophages derived from pluripotent stem cells remain under-investigated. In this study, we aimed to perform the multifarious immunological characteristics of macrophages generated from human induced pluripotent stem cells (iMϕs), including an analysis of their phenotype, secretory and antibacterial activities, as well as their comparison with macrophages derived from blood monocytes and infected lung tissue. We report that iMϕs displayed the morphology and the CD11b+CD45+CD14+ phenotype typical for mononuclear phagocytes. The cells co-expressed markers known to be associated with classically (CD80, CD86, CCR5) and alternatively (CD163 and CD206) activated macrophages, with a bias toward a higher expression of the latter. iMϕs secreted pro-inflammatory (IL-6, CXCL8, CCL2, CCL4, CXCL1, CXCL10) and anti-inflammatory (IL-10, IL-1RA, CCL22) cytokines with a high IL-10/IL-12p70 index (>20). iMϕs were phagocytic and restricted Mycobacterium tuberculosis growth in vitro by >75%. iMϕs differed from blood monocytes/macrophages by a lower expression level of HLA-DR and the CD14+CD16int phenotype and shared several phenotypic characteristics with lung macrophages. In response to LPS, iMϕs up-regulated HLA-DR and produced TNF-α. IFN-γ increased iMϕ reactivity to LPS, but did not increase iMϕ mycobactericidal capacity. The results characterize iMϕs as differentiated but low-activated/low-polarized "naïve-like" macrophages that are capable of mounting inflammatory and antibacterial responses when exposed to inflammatory stimuli or pathogens. iMϕs represent a valuable model for studying antibacterial responses of tissue resident macrophages and for developing approaches to modulating macrophage activity.

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.

Impact of Xist RNA on chromatin modifications and transcriptional silencing maintenance at different stages of imprinted X chromosome inactivation in vole Microtus levis.

In vole Microtus levis, cells of preimplantation embryo and extraembryonic tissues undergo imprinted X chromosome inactivation (iXCI) which is triggered by a long non-coding nuclear RNA, Xist. At early stages of iXCI, chromatin of vole inactive X chromosome is enriched with the HP1 heterochromatin-specific protein, trimethylated H3K9 and H4K20 attributable to constitutive heterochromatin. In the study, using vole trophoblast stem (TS) cells as a model of iXCI, we further investigated chromatin of the inactive X chromosome of M. levis and tried to find out the role of Xist RNA. We demonstrated that chromatin of the inactive X chromosome in vole TS cells also contained the SETDB1 histone methyltransferase and KAP1 protein. In addition, we observed that Xist RNA did not contribute significantly to maintenance of X chromosome inactive state during iXCI in vole TS cells. Xist repression affected neither transcriptional silencing caused by iXCI nor maintenance of trimethylated H3K9 and H4K20 as well as HP1, KAP1, and SETDB1 on the inactive X chromosome. Moreover, the unique repertoire of chromatin modifications on the inactive X chromosome in vole TS cells could be disrupted by a chemical compound, DZNep, and then restored even in the absence of Xist RNA. However, Xist transcript was necessary for recruitment of an additional repressive histone modification, trimethylated H3K27, to the inactive X chromosome during vole TS cell differentiation.

Dynamics of the two heterochromatin types during imprinted X chromosome inactivation in vole Microtus levis.

In rodent female mammals, there are two forms of X-inactivation - imprinted and random which take place in extraembryonic and embryonic tissues, respectively. The inactive X-chromosome during random X-inactivation was shown to contain two types of facultative heterochromatin that alternate and do not overlap. However, chromatin structure of the inactive X-chromosome during imprinted X-inactivation, especially at early stages, is still not well understood. In this work, we studied chromatin modifications associated with the inactive X-chromosome at different stages of imprinted X-inactivation in a rodent, Microtus levis. It has been found that imprinted X-inactivation in vole occurs in a species-specific manner in two steps. The inactive X-chromosome at early stages of imprinted X-inactivation is characterized by accumulation of H3K9me3, HP1, H4K20me3, and uH2A, resembling to some extent the pattern of repressive chromatin modifications of meiotic sex chromatin. Later, the inactive X-chromosome recruits trimethylated H3K27 and acquires the two types of heterochromatin associated with random X-inactivation.

Human induced pluripotent stem cells derived from fetal neural stem cells successfully undergo directed differentiation into cartilage.

Induced pluripotent stem (iPS) cells can be derived from a wide range of somatic cells via overexpression of a set of specific genes. With respect to their properties, iPS cells closely resemble embryonic stem cells. Because of their main property, pluripotency, iPS cells have excellent prospects for use in substitutive cell therapy; however, the methods of directed differentiation of iPS cells have not been yet sufficiently elaborated. In this work, we derived human iPS cells from fetal neural stem (FNS) cells by transfection with a polycistronic plasmid vector carrying the mouse Oct4, Sox2, Klf4, and c-Myc genes or a plasmid expressing the human OCT4 gene. We have shown that human FNS cells can be effectively reprogrammed despite a low transfection level (10%-15%) and that the use of 2-propylvaleric (valproic) acid and BIX-01294 increases the yield of iPS cell clones to ∼7-fold. Further, transient expression of OCT4 alone is sufficient for reprogramming. The iPS cells obtained express all the major markers of embryonic stem cells and are able to differentiate in vitro into ectodermal, mesodermal, and endodermal derivatives. In addition, we have found that the human iPS cells derived from FNS cells can be successfully subjected to in vitro directed chondrogenic differentiation to form functional cartilaginous tissue.

FGF4 independent derivation of trophoblast stem cells from the common vole.

The derivation of stable multipotent trophoblast stem (TS) cell lines from preimplantation, and early postimplantation mouse embryos has been reported previously. FGF4, and its receptor FGFR2, have been identified as embryonic signaling factors responsible for the maintenance of the undifferentiated state of multipotent TS cells. Here we report the derivation of stable TS-like cell lines from the vole M. rossiaemeridionalis, in the absence of FGF4 and heparin. Vole TS-like cells are similar to murine TS cells with respect to their morphology, transcription factor gene expression and differentiation in vitro into derivatives of the trophectoderm lineage, and with respect to their ability to invade and erode host tissues, forming haemorrhagic tumours after subcutaneous injection into nude mice. Moreover, vole TS-like cells carry an inactive paternal X chromosome, indicating that they have undergone imprinted X inactivation, which is characteristic of the trophoblast lineage. Our results indicate that an alternative signaling pathway may be responsible for the establishment and stable proliferation of vole TS-like cells.