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Mucopolysaccharidosis II (MPS II) is a lysosomal storage disorder (LSD), caused by iduronate 2-sulphatase (IDS) enzyme dysfunction. The neuropathology of the disease is not well understood, although the neural symptoms are currently incurable. MPS II-patient derived iPSC lines were established and differentiated to neuronal lineage. The disease phenotype was confirmed by IDS enzyme and glycosaminoglycan assay. MPS II neuronal precursor cells (NPCs) showed significantly decreased self-renewal capacity, while their cortical neuronal differentiation potential was not affected. Major structural alterations in the ER and Golgi complex, accumulation of storage vacuoles, and increased apoptosis were observed both at protein expression and ultrastructural level in the MPS II neuronal cells, which was more pronounced in GFAP + astrocytes, with increased LAMP2 expression but unchanged in their RAB7 compartment. Based on these finding we hypothesize that lysosomal membrane protein (LMP) carrier vesicles have an initiating role in the formation of storage vacuoles leading to impaired lysosomal function. In conclusion, a novel human MPS II disease model was established for the first time which recapitulates the in vitro neuropathology of the disorder, providing novel information on the disease mechanism which allows better understanding of further lysosomal storage disorders and facilitates drug testing and gene therapy approaches.

Introduction Stem cell-derived cardiac myocytes are potential sources for testing cardiocytoprotective molecules against ischemia/reperfusion injury in vitro. Materials and methods Here we performed a systematic analysis of two different induced pluripotent stem cell lines (iPSC 3.4 and 4.1) and an embryonic stem cell (ESC) line-derived cardiac myocytes at two different developmental stages. Cell viability in simulated ischemia/reperfusion (SI/R)-induced injury and a known cardiocytoprotective NO-donor, S-nitroso-n-acetylpenicillamine (SNAP) was tested. Results After analysis of full embryoid bodies (EBs) and cardiac marker (VCAM and cardiac troponin I) positive cells of three lines at 6 conditions (32 different conditions altogether), we found significant SI/R injury-induced cell death in both full EBs and VCAM+ cardiac cells at later stage of their differentiation. Moreover, full EBs of the iPS 4.1 cell line after oxidative stress induction by SNAP was protected at day-8 samples. Conclusion We have shown that one iPS-derived cardiomyocyte line could serve as a testing platform for cardiocytoprotection.

Background Genomic imprinting, resulting in parent-of-origin specific gene expression, plays a critical role in mammalian development. Here, we apply allele-specific RNA-seq on isogenic B6D2F1 mice to assay imprinted genes in tissues from early embryonic tissues between E3.5 and E7.25 and in pluripotent cell lines to evaluate maintenance of imprinted gene expression. For the cell lines, we include embryonic stem cells (ESCs) and epiblast stem cells (EpiSCs) derived from fertilized embryos and from embryos obtained after nuclear transfer (NT) or parthenogenetic activation (PGA). Results As homozygous genomic regions of PGA-derived cells are not compatible with allele-specific RNA-seq, we developed an RNA-seq-based genotyping strategy allowing identification of informative heterozygous regions. Global analysis shows that proper imprinted gene expression as observed in embryonic tissues is largely lost in the ESC lines included in this study, which mainly consisted of female ESCs. Differentiation of ESC lines to embryoid bodies or NPCs does not restore monoallelic expression of imprinted genes, neither did reprogramming of the serum-cultured ESCs to the pluripotent ground state by the use of 2 kinase inhibitors. Fertilized EpiSC and EpiSC-NT lines largely maintain imprinted gene expression, as did EpiSC-PGA lines that show known paternally expressed genes being silent and known maternally expressed genes consistently showing doubled expression. Notably, two EpiSC-NT lines show aberrant silencing of Rian and Meg3, two critically imprinted genes in mouse iPSCs. With respect to female EpiSC, most of the lines displayed completely skewed X inactivation suggesting a (near) clonal origin. Conclusions Altogether, our analysis provides a comprehensive overview of imprinted gene expression in pluripotency and provides a benchmark to allow identification of cell lines that faithfully maintain imprinted gene expression and therefore retain full developmental potential. Electronic supplementary material The online version of this article (10.1186/s13072-019-0259-8) contains supplementary material, which is available to authorized users.

Peripheral blood was collected from a 3-year-old male patient with an X-linked recessive mutation of Iduronate 2-sulfatase (IDS) gene (NM_000202.7(IDS):c.85C>T) causing MPS II (OMIM 309900). Peripheral blood mononuclear cells (PBMCs) were reprogrammed by lentiviral delivery of a self-silencing hOKSM polycistronic vector. The pluripotency of the iPSC line was confirmed by the expression of pluripotency-associated markers and in vitro spontaneous differentiation towards the 3 germ layers. The iPSC line showed normal karyotype. The cell line offers a good platform to study MPS II pathophysiology, for drug testing, early biomarker discovery and gene therapy studies.

During early mammalian development, transient pools of pluripotent cells emerge that can be immortalised upon stem cell derivation. The pluripotent state, ‘naïve’ or ‘primed’, depends on the embryonic stage and derivation conditions used. Here we analyse the temporal gene expression patterns of mouse, cattle and porcine embryos at stages that harbour different types of pluripotent cells. We document conserved and divergent traits in gene expression, and identify predictor genes shared across the species that are associated with pluripotent states in vivo and in vitro. Amongst these are the pluripotency-linked genes Klf4 and Lin28b. The novel genes discovered include naïve- (Spic, Scpep1 and Gjb5) and primed-associated (Sema6a and Jakmip2) genes as well as naïve to primed transition genes (Dusp6 and Trip6). Both Gjb5 and Dusp6 play a role in pluripotency since their knockdown results in differentiation and downregulation of key pluripotency genes. Our interspecies comparison revealed new insights of pluripotency, pluripotent stem cell identity and a new molecular criterion for distinguishing between pluripotent states in various species, including human., Summary: Interspecies comparison of mouse, bovine and pig embryos revealed conserved genes which distinguish between naïve and primed pluripotency states, including in human. Some of these genes interfere with the pluripotency network and lead to differentiation.

The aim of the present study was to establish an in vitro Kleefstra syndrome (KS) disease model using the human induced pluripotent stem cell (hiPSC) technology. Previously, an autism spectrum disorder (ASD) patient with Kleefstra syndrome (KS-ASD) carrying a deleterious premature termination codon mutation in the EHMT1 gene was identified. Patient specific hiPSCs generated from peripheral blood mononuclear cells of the KS-ASD patient were differentiated into post-mitotic cortical neurons. Lower levels of EHMT1 mRNA as well as protein expression were confirmed in these cells. Morphological analysis on neuronal cells differentiated from the KS-ASD patient-derived hiPSC clones showed significantly shorter neurites and reduced arborization compared to cells generated from healthy controls. Moreover, density of dendritic protrusions of neuronal cells derived from KS-ASD hiPSCs was lower than that of control cells. Synaptic connections and spontaneous neuronal activity measured by live cell calcium imaging could be detected after 5 weeks of differentiation, when KS-ASD cells exhibited higher sensitivity of calcium responses to acetylcholine stimulation indicating a lower nicotinic cholinergic tone at baseline condition in KS-ASD cells. In addition, gene expression profiling of differentiated neuronal cells from the KS-ASD patient revealed higher expression of proliferation-related genes and lower mRNA levels of genes involved in neuronal maturation and migration. Our data demonstrate anomalous neuronal morphology, functional activity and gene expression in KS-ASD patient-specific hiPSC-derived neuronal cultures, which offers an in vitro system that contributes to a better understanding of KS and potentially other neurodevelopmental disorders including ASD.

Peripheral blood was collected from a 1-year-old male patient with an X-linked recessive mutation of Iduronate 2-sulfatase (IDS) gene (NM_000202.7(IDS):c.85C>T) causing MPS II (OMIM 309900). Peripheral blood mononuclear cells (PBMCs) were reprogrammed by lentiviral delivery of a self-silencing hOKSM polycistronic vector. The pluripotency of the iPSC line was confirmed by the expression of pluripotency-associated markers and in vitro spontaneous differentiation towards the 3 germ layers. The iPSC line showed normal karyotype. The cell line offers a good platform to study MPS II pathophysiology, for drug testing, early biomarker discovery and gene therapy studies.

Autism spectrum disorder (ASD) is a complex neurodevelopmental disorder with unknown genetic and environmental causation in most of the affected individuals. On the other hand, there are a growing number of ASD-associated syndromes, where the exact genetic origin can be revealed. Here we report a method, which included the targeted next generation sequencing (NGS) and filtering of 101 ASD associated genes, followed by database search. Next, RNA sequencing was used to study the region of interest at the transcriptional level. Using this workflow, we identified a de novo mutation in the euchromatic histone-lysine N-methyltransferase 1 gene (EHMT1) of an autistic patient with dysmorphisms. Sequencing of EHMT1 transcripts showed that the premature termination codon (Trp1138Ter) created by a single nucleotide change elicited nonsense-mediated mRNA decay, which led to haploinsufficiency already at the transcriptional level. Database and literature search provided evidence that this mutation caused Kleefstra syndrome (KS), which was confirmed by the presence of the disorder-specific phenotype in the patient. We provide a proof of principle that the implemented method is capable to elucidate the genetic etiology of individuals with syndromic autism. The novel mutation detected in the EHMT1 gene is responsible for KS's symptoms. In addition, further genetic factors might be involved in the ASD pathogenesis of the patient including a missense DPP6 mutation (Arg322Cys), which segregated with the autistic phenotype within the family.

While the rabbit (Oryctolagus cuniculus) is an important research model for aspects of human development and disease that cannot be studied in rodents, the lack of data on the genetic regulation of rabbit preimplantation development is a limitation. To assist in the understanding of this process, our aim was to isolate and characterize genes necessary for the induction and maintenance of cellular pluripotency. We are the first to report the isolation of complete coding regions of rabbit SOX2, KLF4, C-MYC and NANOG, which encode transcription factors that play crucial regulatory roles during early mammalian embryonic development. We determined the exon–intron boundaries and chromosomal localization of these genes using computational analysis. The sequences of mRNA and translated protein of the newly identified genes and those of POU5F1 were aligned to their mammalian orthologs to determine the degree of evolutionary conservation. Furthermore, the expression of these genes in embryonic and adult cells was studied at the mRNA and protein levels. We found the sequences and the expression pattern of these pluripotency-associated genes to be highly conserved between human and rabbit, indicating that the rabbit would be a valuable model for human preimplantation development. Implementing the newly identified genes either as biomarkers or as reprogramming factors might also pave the way towards the creation of stable pluripotent rabbit cell lines.

The efficiency of various assisted reproductive techniques can be improved by preconditioning the gametes and embryos with sublethal hydrostatic pressure treatment. However, the underlying molecular mechanism responsible for this protective effect remains unknown and requires further investigation. Here, we studied the effect of optimised hydrostatic pressure treatment on the global gene expression of mouse oocytes after embryonic genome activation. Based on a gene expression microarray analysis, a significant effect of treatment was observed in 4-cell embryos derived from treated oocytes, revealing a transcriptional footprint of hydrostatic pressure-affected genes. Functional analysis identified numerous genes involved in protein synthesis that were downregulated in 4-cell embryos in response to hydrostatic pressure treatment, suggesting that regulation of translation has a major role in optimised hydrostatic pressure-induced stress tolerance. We present a comprehensive microarray analysis and further delineate a potential mechanism responsible for the protective effect of hydrostatic pressure treatment.

Embryoid body (EB) formation is a common intermediate during in vitro differentiation of pluripotent stem cells into specialized cell types. We have optimized the slow-turning, lateral vessel (STLV) for large scale and homogenous EB production from mouse embryonic stem cells. The effects of inoculating different cell numbers, time of EB adherence to gelatin-coated dishes, and rotation speed for optimal EB formation and cardiac differentiation were investigated. Using 3 × 10(5) cells/ml, 10 rpm rotary speed and plating of EBs onto gelatin-coated surfaces three days after culture, were the best parameters for optimal size and EB quality on consequent cardiac differentiation. These optimized parameters enrich cardiac differentiation in ES cells when using the STLV method.

AIM: To compare gene targeting efficiencies, expression profiles, and Ca2+ handling potentials in two widely used mouse embryonic stem cell lines. METHODS: The two widely used mouse embryonic stem cell lines, R1 and HM-1, were cultured and maintained on Mitomycin C treated mouse embryonic fibroblast feeder cell layers, following standard culture procedures. Cells were incubated with primary and secondary antibodies before fluorescence activated cell sorting analysis to compare known pluripotency markers. Moreover, cells were harvested by trypsinization and transfected with a kinase-inactive murine Tyk2 targeting construct, following the BioRad and Amaxa transfection procedures. Subsequently, the cells were cultured and neomycin-resistant cells were picked after 13 d of selection. Surviving clones were screened twice by polymerase chain reaction (PCR) and finally confirmed by Southern blot analysis before comparison. Global gene expression profiles of more than 20 400 probes were also compared and significantly regulated genes were confirmed by real time PCR analysis. Calcium handling potentials of these cell lines were also compared using various agonists. RESULTS: We found significant differences in transfection efficiencies of the two cell lines (91% ± 6.1% vs 75% ± 4.2%, P = 0.01). Differences in the targeting efficiencies were also significant whether the Amaxa or BioRad platforms were used for comparison. We did not observe significant differences in the levels of many known pluripotency markers. However, our genome-wide expression analysis using more than 20 400 spotted cDNA arrays identified 55 differentially regulated transcripts (P < 0.05) implicated in various important biological processes, including binding molecular functions (particularly Ca2+ binding roles). Subsequently, we measured Ca2+ signals in these cell lines in response to various calcium agonists, both in high and low Ca2+ solutions, and found significant differences (P < 0.05) in the regulation of Ca2+ homeostasis between the investigated cell lines. Then we further compared the detection and expression of various membrane and intracellular Ca2+ receptors and similarly found significant (P < 0.05) variations in a number of calcium receptors between these cell lines. CONCLUSION: Results of this study emphasize the importance of considering intrinsic cellular variations, during selection of cell lines for experiments and interpretations of experimental results.

Background The POU5F1 gene encodes the octamer-binding transcription factor-4 (Oct4). It is crucial in the regulation of pluripotency during embryonic development and widely used as molecular marker of embryonic stem cells (ESCs). The objective of this study was to identify and to analyse the promoter region of rabbit POU5F1 gene; furthermore to examine its expression pattern in preimplantation stage rabbit embryos. Results The upstream region of rabbit POU5F1 was subcloned sequenced and four highly conserved promoter regions (CR1-4) were identified. The highest degree of similarity on sequence level was found among the conserved domains between rabbit and human. Among the enhancers the proximal enhancer region (PE-1A) exhibited the highest degree of homology (96.4%). Furthermore, the CR4 regulator domain containing the distal enhancer (DE-2A) was responsible for stem cell-specific expression. Also, BAC library screen revealed the existence of a processed pseudogene of rabbit POU5F1. The results of quantitative real-time PCR experiments showed that POU5F1 mRNA was abundantly present in oocytes and zygotes, but it was gradually reduced until the activation of the embryonic genome, thereafter a continuous increase in POU5F1 mRNA level was observed until blastocyst stage. By using the XYClone laser system the inner cell mass (ICM) and trophoblast portions of embryos were microdissected and examined separately and POU5F1 mRNA was detected in both cell types. Conclusion In this study we provide a comparative sequence analysis of the regulatory region of rabbit POU5F1 gene. Our data suggest that the POU5F1 gene is strictly regulated during early mammalian development. We proposed that the well conserved CR4 region containing the DE-2A enhancer is responsible for the highly conserved ESC specific gene expression. Notably, we are the first to report that the rabbit POU5F1 is not restricted to ICM cells only, but it is expressed in trophoblast cells as well. This information may be well applicable to investigate further the possible phylogenetic role and the regulation of POU5F1 gene.

Sublethal stress treatment [namely hydrostatic pressure (HP)] has been reported to improve the cryotolerance and developmental competence of mammalian gametes and embryos at cryopreservation, in vitro maturation, parthenogenetic activation and somatic cell NT (Pribenszky and Vajta 2011 Reprod. Fertil. Dev. 23, 48–55). In our previous study mouse oocytes were HP stress treated and then vitrified, warmed, fertilized by intracytoplasmic sperm injection (ICSI), cultured and transferred. Blastocyst rate, inner cell mass cell number and birth rate were significantly increased in the treated groups (Pribenszky et al. 2010 Fertil. Steril. 94, S32). In our present study we implemented gene expression microarray experiments to analyse the transcriptome of HP-treated eggs and 4-cell stage embryos developed from these oocytes. At first, 4 pools of 50 mouse oocytes were treated with 20 MPa for 60 min at 37°C in a programmable HP machine (Cryo-Innovation Inc., Budapest, Hungary). Second, mouse oocytes were treated with HP likewise, fertilized by ICSI and cultured to 4-cell stage. The controls were handled identically with no HP treatment. After ICSI, 78% of the treated and 82% of the control oocytes survived the procedure, of which 76 and 65% developed to 4-cell stage; furthermore 3–3 pools of 30 to 44 and 24 to 36 embryos were used for RNA isolation, respectively. The labelled cRNA target generated from 240 pg of total RNA was hybridized to 4 × 44 K whole mouse genome chips (Agilent Technologies, Palo Alto, CA, USA). All the microarray data have been deposited in the NCBI Gene Expression Omnibus database (accession GSE28443). The results were validated using RT-qPCR by analysing 9 genes per experiment. The GeneSpring GX 11 analysis of the normalized data showed no change in the global gene expression profile of the oocytes after the HP treatment; however, in the 4-cell embryo study, the analysis identified 676 significantly changed genes, confirmed by the similar expression of 8 genes by RT-qPCR. To characterise the affected pathways among these genes, we used the functional annotation tools of DAVID Bioinformatics Resources (Huang et al. 2009 Nat. Protoc. 4, 44–57). The cluster with the most outstanding gene ontology terms contained the ribosome, structural constituent of ribosome, ribonucleoprotein complex, translation and structural molecule activity (P