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.

Establishment of the Primary Avian Gonadal Somatic Cell Lines for Cytogenetic Studies.

The last decade was marked by a steep rise in avian studies at genomic and cellular levels. Cell lines are important tools for in vitro studies in cell biology and cytogenetics. We developed a simple method of primary somatic cell culture establishment from the ovaries of the great tits (Parus major) and testes of ten Passerine species, characterized the cellular composition of the ovary-derived lines using RT-PCR and immunolocalization of the tissue-specific markers and tested the efficiency of two methods of genetic transformation of the ovary-derived cell line. We found that the ovary-derived cell cultures of the great tit were composed of fibroblasts mainly, but also contained interstitial and granulosa cells. They were cultivated until the 10th passage without any noticeable decrease in their proliferative activity. The testis-derived cell cultures had lower proliferative potential. However, both ovary- and testis-derived cell cultures provided enough material for high quality mitotic metaphase chromosome preparations. The efficiency of its transduction with lentivirus containing a GFP reporter was very low, while electroporation with episomal vectors expressing GFP resulted in a high yield of GFP-positive cells. The proposed method could be used for the generation of high quality material for various cytogenetic and genomic studies.

Germline-Restricted Chromosomes and Autosomal Variants Revealed by Pachytene Karyotyping of 17 Avian Species.

Karyotypes of less than 10% of bird species are known. Using immunolocalization of the synaptonemal complex, the core structure of meiotic chromosomes at the pachytene stage, and centromere proteins, we describe male pachytene karyotypes of 17 species of birds. This method enables higher resolution than the conventional analyses of metaphase chromosomes. We provide the first descriptions of the karyotypes of 3 species (rook, Blyth's reed warbler, and European pied flycatcher), correct the published data on the karyotypes of 10 species, and confirm them for 4 species. All passerine species examined have highly conservative karyotypes, 2n = 80-82 with 7 pairs of macrochromosomes (including the ZZ sex chromosome pair which was not unambiguously distinguished from other macrochromosomes in most species) and 33-34 pairs of microchromosomes. In all of them, but not in the common cuckoo, we revealed single copies of the germline-restricted chromosomes varying in size and morphology even between closely related species. This indicates a fast evolution of this additional chromosome. The interspecies differences concern the sizes of the macrochromosomes, morphology of the microchromosomes, and sizes of the centromeres. The pachytene cells of the gouldian finch, brambling, and common linnet contain heteromorphic synaptonemal complexes indicating heterozygosity for inversions or centromere shifts. The European pied flycatcher, gouldian finch, and domestic canary have extended centromeres in several macro- and microchromosomes.

Time origin and structural analysis of the induced CRISPR/cas9 megabase-sized deletions and duplications involving the Cntn6 gene in mice.

In a previous study using one-step CRISPR/Cas9 genome editing in mouse zygotes, we created five founders carrying a 1,137 kb deletion and two founders carrying the same deletion, plus a 2,274 kb duplication involving the Cntn6 gene (encoding contactin-6). Using these mice, the present study had the following aims: (i) to establish stage of origin of these rearrangements; (ii) to determine the fate of the deleted DNA fragments; and (iii) to estimate the scale of unpredicted DNA changes accompanying the rearrangements. The present study demonstrated that all targeted deletions and duplications occurred at the one-cell stage and more often in one pronucleus only. FISH analysis revealed that there were no traces of the deleted DNA fragments either within chromosome 6 or on other chromosomes. These data were consistent with the Southern blot analysis showing that chromosomes with deletion often had close to expected sizes of removed DNA fragments. High-throughput DNA sequencing of two homozygotes for duplication demonstrated that there were no unexpected significant or scale DNA changes either at the gRNA and joint sites or other genome sites. Thus, our data suggested that CRISPR/Cas9 technology could generate megabase-sized deletions and duplications in mouse gametes at a reasonably specific level.

Germline-restricted chromosome (GRC) is widespread among songbirds.

An unusual supernumerary chromosome has been reported for two related avian species, the zebra and Bengalese finches. This large, germline-restricted chromosome (GRC) is eliminated from somatic cells and spermatids and transmitted via oocytes only. Its origin, distribution among avian lineages, and function were mostly unknown so far. Using immunolocalization of key meiotic proteins, we found that GRCs of varying size and genetic content are present in all 16 songbird species investigated and absent from germline genomes of all eight examined bird species from other avian orders. Results of fluorescent in situ hybridization of microdissected GRC probes and their sequencing indicate that GRCs show little homology between songbird species and contain a variety of repetitive elements and unique sequences with paralogs in the somatic genome. Our data suggest that the GRC evolved in the common ancestor of all songbirds and underwent significant changes in the extant descendant lineages.

Targeted genomic integration of EGFP under tubulin beta 3 class III promoter and mEos2 under tryptophan hydroxylase 2 promoter does not produce sufficient levels of reporter gene expression.

Neuronal tracing is a modern technology that is based on the expression of fluorescent proteins under the control of cell type-specific promoters. However, random genomic integration of the reporter construct often leads to incorrect spatial and temporal expression of the marker protein. Targeted integration (or knock-in) of the reporter coding sequence is supposed to provide better expression control by exploiting endogenous regulatory elements. Here we describe the generation of two fluorescent reporter systems: enhanced green fluorescent protein (EGFP) under pan-neural marker class III ß-tubulin (Tubb3) promoter and mEos2 under serotonergic neuron-specific tryptophan hydroxylase 2 (Tph2) promoter. Differentiation of Tubb3-EGFP embryonic stem (ES) cells into neurons revealed that though Tubb3-positive cells express EGFP, its expression level is not sufficient for the neuronal tracing by routine fluorescent microscopy. Similarly, the expression levels of mEos2-TPH2 in differentiated ES cells was very low and could be detected only on messenger RNA level using polymerase chain reaction-based methods. Our data shows that the use of endogenous regulatory elements to control transgene expression is not always beneficial compared with the random genomic integration.

Allele-Specific Biased Expression of the CNTN6 Gene in iPS Cell-Derived Neurons from a Patient with Intellectual Disability and 3p26.3 Microduplication Involving the CNTN6 Gene.

Copy number variations (CNVs) of the human CNTN6 gene caused by megabase-scale microdeletions or microduplications in the 3p26.3 region are often the cause of neurodevelopmental disorders, including intellectual disability and developmental delay. Surprisingly, patients with different copy numbers of this gene display notable overlapping of neuropsychiatric symptoms. The complexity of the study of human neuropathologies is associated with the inaccessibility of brain material. This problem can be overcome through the use of reprogramming technologies that permit the generation of induced pluripotent stem (iPS) cells from fibroblasts and their subsequent in vitro differentiation into neurons. We obtained a set of iPS cell lines derived from a patient carrier of the CNTN6 gene duplication and from two healthy donors. All iPS cell lines displayed the characteristics of pluripotent cells. Some iPS cell lines derived from the patient and from healthy donors were differentiated in vitro by exogenous expression of the Ngn2 transcription factor or by spontaneous neural differentiation of iPS cells through the neural rosette stage. The obtained neurons showed the characteristics of mature neurons as judged by the presence of neuronal markers and by their electrophysiological characteristics. Analysis of allele-specific expression of the CNTN6 gene in these neuronal cells by droplet digital PCR demonstrated that the level of expression of the duplicated allele was significantly reduced compared to that of the wild-type allele. Importantly, according to the sequencing data, both copies of the CNTN6 gene, which were approximately 1 Mb in size, showed no any additional structural rearrangements.

Ring chromosomes: from formation to clinical potential.

Ring chromosomes (RCs) are circular DNA molecules, which occur rarely in eukaryotic nuclear genomes. Lilian Vaughan Morgan first described them in the fruit fly. Human embryos very seldom have RCs, about 1:50,000. Carriers of RCs may have varying degrees of symptoms, from healthy phenotype to serious pathologies in physical and intellectual development. Many authors describe common symptoms of RC presence: short stature and some developmental delay that could be described as a "ring chromosome syndrome." As a rule, RCs arise de novo through the end-joining of two DNA double-strand breaks, telomere-subtelomere junction, or inv dup del rearrangement in both meiosis and mitosis. There are family cases of RC inheritance. The presence of RCs causes numerous secondary chromosome rearrangements in vivo and in vitro. RCs can change their size, become lost, or increase their copy number and cause additional deletions, duplication, and translocations, affecting both RCs and other chromosomes. In this review, we examine RC inheritance, instability, mechanisms of formation, and potential clinical applications of artificially created RCs for large-scale chromosome rearrangement treatment.

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.

Cytogenetic analysis and Dlk1-Dio3 locus epigenetic status of mouse embryonic stem cells during early passages.

Mouse embryonic stem (ES) cells are widely used in early development studies and for transgenic animal production; however, a stable karyotype is a prerequisite for their use. We derived 32 ES cell lines of outbred mice (129 × BALB (1B), C57BL × 1B, and DD × 1B F1 hybrids). Pluripotency was assessed by utilizing stem-cell-marker gene expression, teratoma formation assays and the formation of chimeras. It was shown that only 21 of the 32 ES cell lines had a diploid modal number of chromosomes of 40. In these lines, the percentage of diploid cells varied from 30.3 to 78.9 %, and trisomy of chromosomes 1, 8 and 11 was observed in some cells in 16.7, 36.7 and 20.0 % of the diploid ES cell lines, respectively. Some cells had trisomy of chromosomes 6, 9, 12, 14, 18 and 19. In situ hybridization with an X chromosome paint probe revealed that 7 of the 11 XX-cell lines had X chromosome rearrangements in some cells. Analysis of the methylation status of the Dlk1-Dio3 locus showed that imprinting was altered in 4 of the 18 ES cell lines. Thus, mouse ES cell lines are prone to chromosome abnormalities even at early passages. Therefore, routine cytogenetic and imprinting analyses are necessary for ES cell characterization.

Comparison of American mink embryonic stem and induced pluripotent stem cell transcriptomes.

BACKGROUND: Recently fibroblasts of many mammalian species have been reprogrammed to pluripotent state using overexpression of several transcription factors. This technology allows production of induced pluripotent stem (iPS) cells with properties similar to embryonic stem (ES) cells. The completeness of reprogramming process is well studied in such species as mouse and human but there is not enough data on other species. We produced American mink (Neovison vison) ES and iPS cells and compared these cells using transcriptome analysis. RESULTS: We report the generation of 10 mink ES and 22 iPS cell lines. The majority of the analyzed cell lines had normal diploid chromosome number. The only ES cell line with XX chromosome set had both X-chromosomes in active state that is characteristic of pluripotent cells. The pluripotency of ES and iPS cell lines was confirmed by formation of teratomas with cell types representing all three germ layers. Transcriptome analysis of mink embryonic fibroblasts (EF), two ES and two iPS cell lines allowed us to identify 11831 assembled contigs which were annotated. These led to a number of 6891 unique genes. Of these 3201 were differentially expressed between mink EF and ES cells. We analyzed expression levels of these genes in iPS cell lines. This allowed us to show that 80% of genes were correctly reprogrammed in iPS cells, whereas approximately 6% had an intermediate expression pattern, about 7% were not reprogrammed and about 5% had a "novel" expression pattern. We observed expression of pluripotency marker genes such as Oct4, Sox2 and Rex1 in ES and iPS cell lines with notable exception of Nanog. CONCLUSIONS: We had produced and characterized American mink ES and iPS cells. These cells were pluripotent by a number of criteria and iPS cells exhibited effective reprogramming. Interestingly, we had showed lack of Nanog expression and consider it as a species-specific feature.

Allelic expression and DNA methylation profiles of promoters at the parental Oct4 and Nanog genes in Mus musculus ES cell/Mus caroli splenocyte hybrid cells.

Expression of the parental Oct4 and Nanog alleles and DNA methylation of their promoters were studied in a set of Mus musculus embryonic stem (ES) cell/M. caroli splenocyte hybrid cells containing a variable ratio of parental chromosomes 6 and 17. The transcripts of the reactivated splenocyte Oct4 and Nanog genes were revealed in all hybrid cell clones positive for M. caroli chromosomes 6 and 17. We found that 11 CpG sites in the Oct4 promoter were heavily methylated in M. caroli splenocytes (>80%), whereas M. musculus ES cells were essentially unmethylated (<1%). Analysis of the methylation status of the Oct4 promoter in seven hybrid cell clones showed that the splenocyte-derived promoter sequence lost DNA methylation so that its methylation level was comparable with that of the ES cells. Additionally, no preferential de novo methylation was seen in the Oct4 promoters of M. musculus and M. caroli in teratomas developed from two independent hybrid clones. The upstream region of Nanog was heavily methylated in mouse embryonic fibroblasts (66%) and less methylated in M. caroli splenocytes (24%). The Nanog promoter region was completely unmethylated in M. musculus ES cells. We found that both parental alleles of the Nanog gene promoter were essentially unmethylated in five examined hybrid clones. Thus, we have demonstrated that (1) the Oct4 and Nanog genes of splenocytes are activated, and their promoters undergo demethylation in ES cell hybrids; (2) these events are independent of the number and ratio of parental chromosomes carrying these genes.

Unequal segregation of parental chromosomes in embryonic stem cell hybrids.

Chromosome segregation was studied in 14 intra- and 20 inter-specific hybrid clones generated by fusion of Mus musculus embryonic stem (ES) cells with fibroblasts or splenocytes of DD/c mice or Mus caroli. As a control for in vitro evolution of tetraploid karyotype we used a set of hybrid clones obtained by fusion of ES cells (D3) with ES cells (TgTP6.3). Identification of the parental chromosomes in the clones was performed by microsatellite analysis and in situ hybridization with labeled species-specific probes. Both analyses have revealed three types of clones: (i) stable tetraploid, observed only for ES x ES cell hybrids; (ii) bilateral loss of chromosomes of both ES and somatic partners; (iii) unilateral segregation of chromosomes of the somatic partner. Observed unilateral segregation was extensive in ES-splenocyte cell hybrids, but lower in ES-fibroblast hybrid clones. Developmental state of the somatic partner is presumably responsible for directional chromosome loss. Nonrandom segregation implies that initial differences in the parental homologous chromosomes were not immediately equalized implying at least transient persistence of the differentiated epigenotype.

Generation of fertile and diploid fish, medaka (Oryzias latipes), from nuclear transplantation of blastula and four-somite-stage embryonic cells into nonenucleated unfertilized eggs.

In two experimental series of transplantation of embryonic cell nuclei into nonenucleated unfertilized eggs in medaka (Oryzias latipes), fertile and diploid nuclear transplants were successfully generated. In the first experiment, nuclei from blastula cells of a medaka stock with the wild-type body color were transplanted into 1722 eggs from the orange-red variety. Of 26 adult nuclear transplants with the wild-type body color, 22 were, as expected, triploid and sterile, but the other four were fertile. Three of the four were diploid, and the last one was tetraploid. They transmitted the wild-type body color to the F1 and F2 progenies in a Mendelian fashion. In the second experiment, cell nuclei from four-somite-stage embryos of the orangered variety carrying the green fluorescent protein (GFP) transgene were transplanted into 1688 recipients of the same strain. Three adult nuclear transplants expressing GFP were obtained. Two of them were triploid and sterile, but the remaining one was fertile and diploid. The transgene of the donor nuclei was transmitted to the F(1) and F(2) offspring in a Mendelian fashion. These observations that diploid and fertile nuclear transplants could be obtained without enucleation of the recipient eggs may have important implications for future nuclear transplantation in medaka.

Development and gene expression of nuclear transplants generated by transplantation of cultured cell nuclei into non-enucleated eggs in the medaka Oryzias latipes.

To develop nuclear transplantation techniques for the medaka Oryzias latipes, nuclei of cultured cells from transgenic fish were transplanted into unfertilized eggs of the orange-red variety of O. latipes, without enucleation, in two experimental series. In the first experimental series, fibroblast cells cultured from the adult caudal fin were used as donors, which carried the green fluorescent protein (GFP) gene driven by the promoter of the medaka elongation factor 1alpha-A gene. Wild-type body color was another donor genetic marker used in this experimental series. In the second experimental series, cells cultured from 6-day-old embryos were used as donors, which carried the GFP genetic marker driven by the promoter of the medaka beta-actin gene. From more than 1000 eggs transplanted in each experiment, a considerable number of nuclear transplants developed to various embryonic stages showing stage- and tissue-specific expression of the donor genetic markers, although the expression was mosaic in many cases. Three and six of the transplanted eggs in the first and second experimental series (0.3 and 0.5%, respectively) hatched, and the hatchlings expressing the genetic markers survived for up to 3 weeks. The chromosome number varied among cells in a single transplant embryo. The results obtained in these experiments may help future cloning efforts in fish.