iPSC-Derived Endothelial Cells Reveal LDLR Dysfunction and Dysregulated Gene Expression Profiles in Familial Hypercholesterolemia.

Defects in the low-density lipoprotein receptor (LDLR) are associated with familial hypercholesterolemia (FH), manifested by atherosclerosis and cardiovascular disease. LDLR deficiency in hepatocytes leads to elevated blood cholesterol levels, which damage vascular cells, especially endothelial cells, through oxidative stress and inflammation. However, the distinctions between endothelial cells from individuals with normal and defective LDLR are not yet fully understood. In this study, we obtained and examined endothelial derivatives of induced pluripotent stem cells (iPSCs) generated previously from conditionally healthy donors and compound heterozygous FH patients carrying pathogenic LDLR alleles. In normal iPSC-derived endothelial cells (iPSC-ECs), we detected the LDLR protein predominantly in its mature form, whereas iPSC-ECs from FH patients have reduced levels of mature LDLR and show abolished low-density lipoprotein uptake. RNA-seq of mutant LDLR iPSC-ECs revealed a unique transcriptome profile with downregulated genes related to monocarboxylic acid transport, exocytosis, and cell adhesion, whereas upregulated signaling pathways were involved in cell secretion and leukocyte activation. Overall, these findings suggest that LDLR defects increase the susceptibility of endothelial cells to inflammation and oxidative stress. In combination with elevated extrinsic cholesterol levels, this may result in accelerated endothelial dysfunction, contributing to early progression of atherosclerosis and other cardiovascular pathologies associated with FH.

Calling and Phasing of Single-Nucleotide and Structural Variants of the LDLR Gene Using Oxford Nanopore MinION.

The LDLR locus has clinical significance for lipid metabolism, Mendelian familial hypercholesterolemia (FH), and common lipid metabolism-related diseases (coronary artery disease and Alzheimer's disease), but its intronic and structural variants are underinvestigated. The aim of this study was to design and validate a method for nearly complete sequencing of the LDLR gene using long-read Oxford Nanopore sequencing technology (ONT). Five PCR amplicons from LDLR of three patients with compound heterozygous FH were analyzed. We used standard workflows of EPI2ME Labs for variant calling. All rare missense and small deletion variants detected previously by massively parallel sequencing and Sanger sequencing were identified using ONT. One patient had a 6976 bp deletion (exons 15 and 16) that was detected by ONT with precisely located breakpoints between AluY and AluSx1. Trans-heterozygous associations between mutation c.530C>T and c.1054T>C, c.2141-966_2390-330del, and c.1327T>C, and between mutations c.1246C>T and c.940+3_940+6del of LDLR, were confirmed. We demonstrated the ability of ONT to phase variants, thereby enabling haplotype assignment for LDLR with personalized resolution. The ONT-based method was able to detect exonic variants with the additional benefit of intronic analysis in one run. This method can serve as an efficient and cost-effective tool for diagnosing FH and conducting research on extended LDLR haplotype reconstruction.

Constitutive heterochromatin propagation contributes to the X chromosome inactivation.

Imprinted X chromosome inactivation (iXCI) balances the expression of X-linked genes in preimplantation embryos and extraembryonic tissues in rodents. Long noncoding Xist RNA drives iXCI, silencing genes and recruiting Xist-dependent chromatin repressors. Some domains on the inactive X chromosome include repressive modifications specific to constitutive heterochromatin, which show no direct link to Xist RNA. We explored the relationship between Xist RNA and chromatin silencing during iXCI in vole Microtus levis. We performed locus-specific activation of Xist transcription on the only active X chromosome using the dCas9-SAM system in XO vole trophoblast stem cells (TSCs), which allow modeling iXCI events to some extent. The artificially activated endogenous vole Xist transcript is truncated and restricted ~ 6.6 kb of the exon 1. Ectopic Xist RNA accumulates on the X chromosome and recruits Xist-dependent modifications during TSC differentiation, yet is incapable by itself repressing X-linked genes. Transcriptional silencing occurs upon ectopic Xist upregulation only when repressive marks spread from the massive telomeric constitutive heterochromatin to the X chromosome region containing genes. We hypothesize that the Xist RNA-induced propagation of repressive marks from the constitutive heterochromatin could be a mechanism involved in X chromosome inactivation.

Induced pluripotent stem cell line ICGi038-A, obtained by reprogramming peripheral blood mononuclear cells from a patient with familial hypercholesterolemia due to compound heterozygous c.1246C > T/c.940 + 3_940 + 6del mutations in LDLR.

The development of cellular models for familial hypercholesterolemia (FH) is an important direction for creating new approaches to atherosclerosis treatment. Pathogenic mutations in the LDLR gene are the main FH source. We generated an iPSC line from peripheral blood mononuclear cells of the patient with compound heterozygous c.1246C > T/c.940 + 3_940 + 6del LDLR mutation. The resulting iPSC line with confirmed patient-specific mutations maintains a normal karyotype and a typical undifferentiated state, including morphology, pluripotent gene expression, and in vitro differentiation potential. This iPSC line can be further differentiated toward relevant cells to better understand FH pathogenesis.

Induced pluripotent stem cell line ICGi037-A, obtained by reprogramming peripheral blood mononuclear cells from a patient with familial hypercholesterolemia due to heterozygous p.Trp443Arg mutations in LDLR.

Familial hypercholesterolemia (FH) is an autosomal dominant disorder increasing premature cardiovascular diseases risk due to atherosclerosis. Pathogenic mutations in the LDLR gene cause most FH cases. Available treatments are effective not for all LDLR mutations. Testing drugs on FH cell models help develop new efficient treatments. We obtained an iPSC line from peripheral blood mononuclear cells of the patient with heterozygous p.Trp443Arg LDLR mutation. The iPSCs with confirmed patient-specific mutations express pluripotency markers, spontaneously differentiate into three germ layers and demonstrate normal karyotype. Patient-specific iPSCs-derived hepatocyte-like and endothelial cells are promising to develop new targeted therapies for FH.

Induced pluripotent stem cell line ICGi036-A generated by reprogramming peripheral blood mononuclear cells from a patient with familial hypercholesterolemia caused due to compound heterozygous p.Ser177Leu/p.Cys352Arg mutations in LDLR.

Familial hypercholesterolemia (FH) is a monogenic disease, leading to atherosclerosis due to a high level of low-density lipoprotein cholesterol. Most cases of the disease are based on pathological variants in the LDLR gene. Hepatocyte-like and endothelial cells derived from individual iPSCs are a good model for developing new approaches to therapy. We obtained an iPSC line from peripheral blood mononuclear cells of the patient with compound heterozygous p.Ser177Leu/p.Cys352Arg mutation in LDLR using non-integrating vectors. The iPSCs with a confirmed patient-specific mutation demonstrate pluripotency markers, normal karyotype, and the ability to differentiate into derivatives of three germ layers.

Etiopathogenesis of adolescent idiopathic scoliosis: Review of the literature and new epigenetic hypothesis on altered neural crest cells migration in early embryogenesis as the key event.

Adolescent idiopathic scoliosis (AIS) affects 2-3% of children. Numerous hypotheses on etiologic/causal factors of AIS were investigated, but all failed to identify therapeutic targets and hence failed to offer a cure. Therefore, currently there are only two options to minimize morbidity of the patients suffering AIS: bracing and spinal surgery. From the beginning of 1960th, spinal surgery, both fusion and rod placement, became the standard of management for progressive adolescent idiopathic spine deformity. However, spinal surgery is often associated with complications. These circumstances motivate AIS scientific community to continue the search for new etiologic and causal factors of AIS. While the role of the genetic factors in AIS pathogenesis was investigated intensively and universally recognized, these studies failed to nominate mutation of a particular gene or genes combination responsible for AIS development. More recently epigenetic factors were suggested to play causal role in AIS pathogenesis. Sharing this new approach, we investigated scoliotic vertebral growth plates removed during vertebral fusion (anterior surgery) for AIS correction. In recent publications we showed that cells from the convex side of human scoliotic deformities undergo normal chondrogenic/osteogenic differentiation, while cells from the concave side acquire a neuronal phenotype. Based on these facts we hypothesized that altered neural crest cell migration in early embryogenesis can be the etiological factor of AIS. In particular, we suggested that neural crest cells failed to migrate through the anterior half of somites and became deposited in sclerotome, which in turn produced chondrogenic/osteogenic-insufficient vertebral growth plates. To test this hypothesis we conducted experiments on chicken embryos with arrest neural crest cell migration by inhibiting expression of Paired-box 3 (Pax3) gene, a known enhancer and promoter of neural crest cells migration and differentiation. The results showed that chicken embryos treated with Pax3 siRNA (microinjection into the neural tube, 44 h post-fertilization) progressively developed scoliotic deformity during maturation. Therefore, this analysis suggests that although adolescent idiopathic scoliosis manifests in children around puberty, the real onset of the disease is of epigenetic nature and takes place in early embryogenesis and involves altered neural crest cells migration. If these results confirmed and further elaborated, the hypothesis may shed new light on the etiology and pathogenesis of AIS.

Diverse developmental strategies of X chromosome dosage compensation in eutherian mammals.

In eutherian mammals, dosage compensation arose to balance X-linked gene expression between sexes and relatively to autosomal gene expression in the evolution of sex chromosomes. Dosage compensation occurs in early mammalian development and comprises X chromosome upregulation and inactivation that are tightly coordinated epigenetic processes. Despite a uniform principle of dosage compensation, mechanisms of X chromosome inactivation and upregulation demonstrate a significant variability depending on sex, developmental stage, cell type, individual, and mammalian species. The review focuses on relationships between X chromosome inactivation and upregulation in mammalian early development.

A New Look at Causal Factors of Idiopathic Scoliosis: Altered Expression of Genes Controlling Chondroitin Sulfate Sulfation and Corresponding Changes in Protein Synthesis in Vertebral Body Growth Plates.

Background: In a previous report, we demonstrated the presence of cells with a neural/glial phenotype on the concave side of the vertebral body growth plate in Idiopathic Scoliosis (IS) and proposed this phenotype alteration as the main etiological factor of IS. In the present study, we utilized the same specimens of vertebral body growth plates removed during surgery for Grade III-IV IS to analyse gene expression. We suggested that phenotype changes observed on the concave side of the vertebral body growth plate can be associated with altered expression of particular genes, which in turn compromise mechanical properties of the concave side. Methods: We used a Real-Time SYBR Green PCR assay to investigate gene expression in vertebral body growth plates removed during surgery for Grade III-IV IS; cartilage tissues from human fetal spine were used as a surrogate control. Special attention was given to genes responsible for growth regulation, chondrocyte differentiation, matrix synthesis, sulfation and transmembrane transport of sulfates. We performed morphological, histochemical, biochemical, and ultrastructural analysis of vertebral body growth plates. Results: Expression of genes that control chondroitin sulfate sulfation and corresponding protein synthesis was significantly lower in scoliotic specimens compared to controls. Biochemical analysis showed 1) a decrease in diffused proteoglycans in the total pool of proteoglycans; 2) a reduced level of their sulfation; 3) a reduction in the amount of chondroitin sulfate coinciding with raising the amount of keratan sulfate; and 4) reduced levels of sulfation on the concave side of the scoliotic deformity. Conclusion: The results suggested that altered expression of genes that control chondroitin sulfate sulfation and corresponding changes in protein synthesis on the concave side of vertebral body growth plates could be causal agents of the scoliotic deformity.

A New Look at Etiological Factors of Idiopathic Scoliosis: Neural Crest Cells.

Idiopathic scoliosis is one of the most common disabling pathologies of children and adolescents. Etiology and pathogenesis of idiopathic scoliosis remain unknown. To study the etiology of this disease we identified the cells' phenotypes in the vertebral body growth plates in patients with idiopathic scoliosis. Materials and methods: The cells were isolated from vertebral body growth plates of the convex and concave sides of the deformity harvested intraoperatively in 50 patients with scoliosis. Cells were cultured and identified by methods of common morphology, neuromorphology, electron microscopy, immunohistochemistry and PCR analysis. Results: Cultured cells of convex side of deformation were identified as chondroblasts. Cells isolated from the growth plates of the concave side of the deformation showed numerous features of neuro- and glioblasts. These cells formed synapses, contain neurofilaments, and expressed neural and glial proteins. Conclusion: For the first time we demonstrated the presence of cells with neural/glial phenotype in the concave side of the vertebral body growth plate in scoliotic deformity. We hypothesized that neural and glial cells observed in the growth plates of the vertebral bodies represent derivatives of neural crest cells deposited in somites due to alterations in their migratory pathway during embryogenesis. We also propose that ectopic localization of cells derived from neural crest in the growth plate of the vertebral bodies is the main etiological factor of the scoliotic 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.

Alternative dominance of the parental genomes in hybrid cells generated through the fusion of mouse embryonic stem cells with fibroblasts.

For the first time, two types of hybrid cells with embryonic stem (ES) cell-like and fibroblast-like phenotypes were produced through the fusion of mouse ES cells with fibroblasts. Transcriptome analysis of 2,848 genes differentially expressed in the parental cells demonstrated that 34-43% of these genes are expressed in hybrid cells, consistent with their phenotypes; 25-29% of these genes display intermediate levels of expression, and 12-16% of these genes maintained expression at the parental cell level, inconsistent with the phenotype of the hybrid cell. Approximately 20% of the analyzed genes displayed unexpected expression patterns that differ from both parents. An unusual phenomenon was observed, namely, the illegitimate activation of Xist expression and the inactivation of one of two X-chromosomes in the near-tetraploid fibroblast-like hybrid cells, whereas both Xs were active before and after in vitro differentiation of the ES cell-like hybrid cells. These results and previous data obtained on heterokaryons suggest that the appearance of hybrid cells with a fibroblast-like phenotype reflects the reprogramming, rather than the induced differentiation, of the ES cell genome under the influence of a somatic partner.

Transcriptome Characteristics and X-Chromosome Inactivation Status in Cultured Rat Pluripotent Stem Cells.

Rat pluripotent stem cells, embryonic stem cells (ESCs), and induced pluripotent stem cells (iPSCs) as mouse and human ones have a great potential for studying mammalian early development, disease modeling, and evaluation of regenerative medicine approaches. However, data on pluripotency realization and self-renewal maintenance in rat cells are still very limited, and differentiation protocols of rat ESCs (rESCs) and iPSCs to study development and obtain specific cell types for biomedical applications are poorly developed. In this study, the RNA-Seq technique was first used for detailed transcriptome characterization in rat pluripotent cells. The rESC and iPSC transcriptomes demonstrated a high similarity and were significantly different from those in differentiated cells. Additionally, we have shown that reprogramming of rat somatic cells to a pluripotent state was accompanied by X-chromosome reactivation. There were two active X chromosomes in XX rESCs and iPSCs, which is one of the key attributes of the pluripotent state. Differentiation of both rESCs and iPSCs led to X-chromosome inactivation (XCI). The dynamics of XCI in differentiating rat cells was very similar to that in mice. Two types of facultative heterochromatin described in various mammalian species were revealed on the rat inactive X chromosome. To explore XCI dynamics, we established a new monolayer differentiation protocol for rESCs and iPSCs that may be applied to study different biological processes and optimized for directed derivation of specific cell types.

Mapping of Replication Origins in the X Inactivation Center of Vole Microtus levis Reveals Extended Replication Initiation Zone.

DNA replication initiates at specific positions termed replication origins. Genome-wide studies of human replication origins have shown that origins are organized into replication initiation zones. However, only few replication initiation zones have been described so far. Moreover, few origins were mapped in other mammalian species besides human and mouse. Here we analyzed pattern of short nascent strands in the X inactivation center (XIC) of vole Microtus levis in fibroblasts, trophoblast stem cells, and extraembryonic endoderm stem cells and confirmed origins locations by ChIP approach. We found that replication could be initiated in a significant part of XIC. We also analyzed state of XIC chromatin in these cell types. We compared origin localization in the mouse and vole XIC. Interestingly, origins associated with gene promoters are conserved in these species. The data obtained allow us to suggest that the X inactivation center of M. levis is one extended replication initiation zone.

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.

Epigenetic landscape for initiation of DNA replication.

The key genetic process of DNA replication is initiated at specific sites referred to as replication origins. In eukaryotes, origins of DNA replication are not specified by a defined nucleotide sequence. Recent studies have shown that the structural context and topology of DNA sequence, chromatin features, and its transcriptional activity play an important role in origin choice. During differentiation and development, significant changes in chromatin organization and transcription occur, influencing origin activity and choice. In the last few years, a number of different genome-wide studies have broadened the understanding of replication origin regulation. In this review, we discuss the epigenetic factors and mechanisms that modulate origin choice and firing.

A regulatory potential of the Xist gene promoter in vole M. rossiaemeridionalis.

X chromosome inactivation takes place in the early development of female mammals and depends on the Xist gene expression. The mechanisms of Xist expression regulation have not been well understood so far. In this work, we compared Xist promoter region of vole Microtus rossiaemeridionalis and other mammalian species. We observed three conserved regions which were characterized by computational analysis, DNaseI in vitro footprinting, and reporter construct assay. Regulatory factors potentially involved in Xist activation and repression in voles were determined. The role of CpG methylation in vole Xist expression regulation was established. A CTCF binding site was found in the 5' flanking region of the Xist promoter on the active X chromosome in both males and females. We suggest that CTCF acts as an insulator which defines an inactive Xist domain on the active X chromosome in voles.

Variability of sequence surrounding the Xist gene in rodents suggests taxon-specific regulation of X chromosome inactivation.

One of the two X chromosomes in female mammalian cells is subject to inactivation (XCI) initiated by the Xist gene. In this study, we examined in rodents (voles and rat) the conservation of the microsatellite region DXPas34, the Tsix gene (antisense counterpart of Xist), and enhancer Xite that have been shown to flank Xist and regulate XCI in mouse. We have found that mouse regions of the Tsix gene major promoter and minisatellite repeat DXPas34 are conserved among rodents. We have also shown that in voles and rat the region homologous to the mouse Tsix major promoter, initiates antisense to Xist transcription and terminates around the Xist gene start site as is observed with mouse Tsix. A conservation of Tsix expression pattern in voles, rat and mice suggests a crucial role of the antisense transcription in regulation of Xist and XIC in rodents. Most surprisingly, we have found that voles lack the regions homologous to the regulatory element Xite, which is instead replaced with the Slc7a3 gene that is unassociated with the X-inactivation centre in any other eutherians studied. Furthermore, we have not identified any transcription that could have the same functions as murine Xite in voles. Overall, our data show that not all the functional elements surrounding Xist in mice are well conserved even within rodents, thereby suggesting that the regulation of XCI may be at least partially taxon-specific.

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.