Congenital anaemia associated with loss-of-function variants in DNA polymerase epsilon 1.

DNA polymerase epsilon (Pol ε), a component of the core replisome, is involved in DNA replication. Although genetic defects of Pol ε have been reported to cause immunodeficiency syndromes, its role in haematopoiesis remains unknown. Here, we identified compound heterozygous variants (p.[Asp1131fs];[Thr1891del]) in POLE, encoding Pol ε catalytic subunit A (POLE1), in siblings with a syndromic form of severe congenital transfusion-dependent anaemia. In contrast to Diamond-Blackfan anaemia, marked reticulocytopenia or marked erythroid hypoplasia was not found. Their bone marrow aspirates during infancy revealed erythroid dysplasia with strongly positive TP53 in immunostaining. Repetitive examinations demonstrated trilineage myelodysplasia within 2 years from birth. They had short stature and facial dysmorphism. HEK293 cell-based expression experiments and analyses of patient-derived induced pluripotent stem cells (iPSCs) disclosed a reduced mRNA level of Asp1131fs-POLE1 and defective nuclear translocation of Thr1891del-POLE1. Analysis of iPSCs showed compensatory mRNA upregulation of the other replisome components and increase of the TP53 protein, both suggesting dysfunction of the replisome. We created Pole-knockout medaka fish and found that heterozygous fishes were viable, but with decreased RBCs. Our observations expand the phenotypic spectrum of the Pol ε defect in humans, additionally providing unique evidence linking Pol ε to haematopoiesis.

Low-density cell culture enhances hepatic function through tight junction formation in HepG2 cells.

BACKGROUND INFORMATION: An in vitro evaluation system using cultured hepatocytes is the most useful method in preclinical research, such as drug metabolism and toxicity test. Human hepatocytes should be used in an in vitro evaluation system because the expression of drug-metabolizing enzymes varies among animal species. HepG2 cells, a liver cancer-derived cell line, are widely used as a human hepatocyte model; however, their hepatic functions are generally weak. RESULTS: In this study, we showed that low-density HepG2 cell culture induces hepatic function. The morphology of HepG2 cells was altered depending on the cell density at the time of seeding. Low-density cultured HepG2 cells proliferated as tightly packed colonies. The HepG2 cell colonies in low-density culture demonstrated enhanced tight junction formation. Tight junction protein gene expression levels, such as those of zonula occludens-1 (ZO-1), junctional adhesion molecule 1 (JAM), claudin, occludin, and tricellulin, increased in low-density cultured HepG2 cells. Phases I and II metabolic enzymes, phase III transporter gene expression, and CYP3A4 activity also increased in low-density cultured HepG2 cells. Occludin and tricellulin knockdown inhibited the increased hepatic function in low-density cultures. Tricellulin knockdown reduced the expression of hepatocyte nuclear factor 6 (HNF6), CCAAT/enhancer-binding protein alpha (CEBPA), and aryl hydrocarbon receptor (AHR). In addition, the expression of nuclear receptor subfamily 1 group h member 2 (NR1H2) increased in low-density cultures, canceled by occludin and tricellulin knockdown. CONCLUSIONS: Our results suggest that low-density HepG2 cell cultures enhance hepatic function by promoting tight junction formation and demonstrate the importance of cell density in drug evaluation using hepatocyte cell lines.

Exploring trophoblast-specific Tead4 enhancers through chromatin conformation capture assays followed by functional screening.

Tead4 is critical for blastocyst development and trophoblast differentiation. We assayed long-range chromosomal interactions on the Tead4 promoter in mouse embryonic stem (ES) cells and trophoblast stem (TS) cells. Using luciferase reporter assays with ES and TS cells for 34 candidate enhancer regions, we identified five genomic fragments that increased Tead4 promoter activity in a TS-specific manner. The five loci consisted of three intra- and two inter-chromosomal loci relative to Tead4 on chromosome 6. We established five mouse lines with one of the five enhancer elements deleted and evaluated the effect of each deletion on Tead4 expression in blastocysts. By quantitative RT-PCR, we measured a 42% decrease in Tead4 expression in the blastocysts with a homozygous deletion with a 1.5 kb genomic interval on chromosome 19 (n = 14) than in wild-type blastocysts. By conducting RNA-seq analysis, we confirmed the trans effect of this enhancer deletion on Tead4 without significant cis effects on its neighbor genes at least within a 1.7 Mb distance. Our results demonstrated that the genomic interval on chromosome 19 is required for the appropriate level of Tead4 expression in blastocysts and suggested that an inter-chromosomal enhancer-promoter interaction may be the underlying mechanism.

Mitochondrial replacement by genome transfer in human oocytes: Efficacy, concerns, and legality.

BACKGROUND: Pathogenic mitochondrial (mt)DNA mutations, which often cause life-threatening disorders, are maternally inherited via the cytoplasm of oocytes. Mitochondrial replacement therapy (MRT) is expected to prevent second-generation transmission of mtDNA mutations. However, MRT may affect the function of respiratory chain complexes comprised of both nuclear and mitochondrial proteins. METHODS: Based on the literature and current regulatory guidelines (especially in Japan), we analyzed and reviewed the recent developments in human models of MRT. MAIN FINDINGS: MRT does not compromise pre-implantation development or stem cell isolation. Mitochondrial function in stem cells after MRT is also normal. Although mtDNA carryover is usually less than 0.5%, even low levels of heteroplasmy can affect the stability of the mtDNA genotype, and directional or stochastic mtDNA drift occurs in a subset of stem cell lines (mtDNA genetic drift). MRT could prevent serious genetic disorders from being passed on to the offspring. However, it should be noted that this technique currently poses significant risks for use in embryos designed for implantation. CONCLUSION: The maternal genome is fundamentally compatible with different mitochondrial genotypes, and vertical inheritance is not required for normal mitochondrial function. Unresolved questions regarding mtDNA genetic drift can be addressed by basic research using MRT.

Mitochondrial Genetic Drift after Nuclear Transfer in Oocytes.

Mitochondria are energy-producing intracellular organelles containing their own genetic material in the form of mitochondrial DNA (mtDNA), which codes for proteins and RNAs essential for mitochondrial function. Some mtDNA mutations can cause mitochondria-related diseases. Mitochondrial diseases are a heterogeneous group of inherited disorders with no cure, in which mutated mtDNA is passed from mothers to offspring via maternal egg cytoplasm. Mitochondrial replacement (MR) is a genome transfer technology in which mtDNA carrying disease-related mutations is replaced by presumably disease-free mtDNA. This therapy aims at preventing the transmission of known disease-causing mitochondria to the next generation. Here, a proof of concept for the specific removal or editing of mtDNA disease-related mutations by genome editing is introduced. Although the amount of mtDNA carryover introduced into human oocytes during nuclear transfer is low, the safety of mtDNA heteroplasmy remains a concern. This is particularly true regarding donor-recipient mtDNA mismatch (mtDNA-mtDNA), mtDNA-nuclear DNA (nDNA) mismatch caused by mixing recipient nDNA with donor mtDNA, and mtDNA replicative segregation. These conditions can lead to mtDNA genetic drift and reversion to the original genotype. In this review, we address the current state of knowledge regarding nuclear transplantation for preventing the inheritance of mitochondrial diseases.

Autonomous trisomic rescue of Down syndrome cells.

Down syndrome is the most frequent chromosomal abnormality among live-born infants. All Down syndrome patients have mental retardation and are prone to develop early onset Alzheimer's disease. However, it has not yet been elucidated whether there is a correlation between the phenotype of Down syndrome and the extra chromosome 21. In this study, we continuously cultivated induced pluripotent stem cells (iPSCs) with chromosome 21 trisomy for more than 70 weeks, and serendipitously obtained revertant cells with normal chromosome 21 diploids from the trisomic cells during long-term cultivation. Repeated experiments revealed that this trisomy rescue was not due to mosaicism of chromosome 21 diploid cells and occurred at an extremely high frequency. We herewith report the spontaneous correction from chromosome 21 trisomy to disomy without genetic manipulation, chemical treatment or exposure to irradiation. The revertant diploid cells will possibly serve a reference for drug screening and a raw material of regenerative medicinal products for cell-based therapy.

Maintenance of Xist Imprinting Depends on Chromatin Condensation State and Rnf12 Dosage in Mice.

In female mammals, activation of Xist (X-inactive specific transcript) is essential for establishment of X chromosome inactivation. During early embryonic development in mice, paternal Xist is preferentially expressed whereas maternal Xist (Xm-Xist) is silenced. Unlike autosomal imprinted genes, Xist imprinting for Xm-Xist silencing was erased in cloned or parthenogenetic but not fertilized embryos. However, the molecular mechanism underlying the variable nature of Xm-Xist imprinting is poorly understood. Here, we revealed that Xm-Xist silencing depends on chromatin condensation states at the Xist/Tsix genomic region and on Rnf12 expression levels. In early preimplantation, chromatin decondensation via H3K9me3 loss and histone acetylation gain caused Xm-Xist derepression irrespective of embryo type. Although the presence of the paternal genome during pronuclear formation impeded Xm-Xist derepression, Xm-Xist was robustly derepressed when the maternal genome was decondensed before fertilization. Once Xm-Xist was derepressed by chromatin alterations, the derepression was stably maintained and rescued XmXpΔ lethality, indicating that loss of Xm-Xist imprinting was irreversible. In late preimplantation, Oct4 served as a chromatin opener to create transcriptional permissive states at Xm-Xist/Tsix genomic loci. In parthenogenetic embryos, Rnf12 overdose caused Xm-Xist derepression via Xm-Tsix repression; physiological Rnf12 levels were essential for Xm-Xist silencing maintenance in fertilized embryos. Thus, chromatin condensation and fine-tuning of Rnf12 dosage were crucial for Xist imprint maintenance by silencing Xm-Xist.

[Genome editing in human embryos: ethical concerns and practical applications].

As genome editing techniques are developed, they have been applied to somatic cells in clinical use. Regarding the adaptation of these techniques in human zygotes, advances in single-cell analysis technology are expected to enhance knowledge at the molecular level, even in human preimplantation embryo development, and the application of genome editing technology for molecular functional analysis is expected. However, the use of genetic modification techniques on germlines, including zygotes, spreads to all cells of an individual and is subsequently passed down through generations. Here we explore the possibilities and challenges of applying genome editing techniques to better understand the scientific aspects of the human germline.

Chromatin condensation of Xist genomic loci during oogenesis in mice.

Repression of maternal Xist (Xm-Xist) during preimplantation in mouse embryos is essential for establishing imprinted X chromosome inactivation. Nuclear transplantation (NT) studies using nuclei derived from non-growing (ng) and full-grown (fg) oocytes have indicated that maternal-specific repressive modifications are imposed on Xm-Xist during oogenesis, as well as on autosomal imprinted genes. Recent studies have revealed that histone H3 lysine 9 trimethylation (H3K9me3) enrichments on Xm-Xist promoter regions are involved in silencing at the preimplantation stages. However, whether H3K9me3 is imposed on Xm-Xist during oogenesis is not known. Here, we dissected the chromatin states in ng and fg oocytes and early preimplantation stage embryos. Chromatin immunoprecipitation experiments against H3K9me3 revealed that there was no significant enrichment within the Xm-Xist region during oogenesis. However, NT embryos with ng nuclei (ngNT) showed extensive Xm-Xist derepression and H3K9me3 hypomethylation of the promoter region at the 4-cell stage, which corresponds to the onset of paternal Xist expression. We also found that the chromatin state at the Xist genomic locus became markedly condensed as oocyte growth proceeded. Although the condensed Xm-Xist genomic locus relaxed during early preimplantation phases, the extent of the relaxation across Xm-Xist loci derived from normally developed oocytes was significantly smaller than those of paternal-Xist and ngNT-Xist genomic loci. Furthermore, Xm-Xist from 2-cell metaphase nuclei became derepressed following NT. We propose that chromatin condensation is associated with imprinted Xist repression and that skipping of the condensation step by NT leads to Xist activation during the early preimplantation phase.

Efficient production of trophoblast lineage cells from human induced pluripotent stem cells.

Human induced pluripotent stem cells (hiPSCs) are potentially useful in both clinical applications and basic biological research. hiPSCs can differentiate into extra-embryonic cells in the presence of BMP4. However, the differentiation potential of hiPSCs can be affected by culture conditions or genetic variation. In this study, we investigated the effect of various BMP4 concentrations on the expression states of trophoblast markers and the optimal conditions for trophoblast induction. A high-fidelity gene expression assay using hiPSC lines showed that the expression levels of various trophoblast marker genes, such as KRT7, GCM1, CGB, and HLA-G, were upregulated by BMP4 in a dose-dependent manner in all types of hiPSCs used in this study. Treatment with high doses of BMP4 for prolonged periods increased the ratio of cells with trophoblast markers irrespective of the presence of bFGF. We found that the expression states of major pluripotency- and differentiation-related protein-coding genes in BMP4-treated cells depended on culture conditions rather than donor cell types. However, miRNA expression states were affected by donor cell types rather than BMP4 dose. Furthermore, the effect of the presence of bFGF on differentiation potential of KRT7-positive cells differed among iPSC types. Mechanistically, chromatin states around KRT7 promoter regions were comparable among the iPSC types used in this study, indicating that hiPSC chromatin state at these regions is not a parameter for cytotrophoblast differentiation potential. In conclusion, the optimal conditions for trophoblast differentiation from hiPSCs differ according to parental cell line.

Human cytomegalovirus downregulates SLITRK6 expression through IE2.

Congenital human cytomegalovirus (HCMV) infection causes sensorineural hearing loss (SNHL) and other neurological disorders, although the neuropathogenesis of HCMV infection is not well understood. Here, we show that the expression of SLITRK6, one of causative genes for hereditary SNHL, was robustly downregulated by HCMV infection in cultured neural cells. We also show that HCMV-encoded immediate-early 2 (IE2) proteins mediate this downregulation and their carboxy-terminal region, especially amino acid residue Gln548, has a critical role. These findings suggest that the downregulation of SLITRK6 expression by IE2 may have a role in HCMV-induced SNHL and other neurological disorders.

The serine 106 residue within the N-terminal transactivation domain is crucial for Oct4 function in mice.

Pou5f1/Oct4 is a key transcription factor for the induction of pluripotency and totipotency in preimplantation mouse embryos. In mice, loss or gain of function experiments have demonstrated an important role for Oct4 in preimplantation and developmental ability. In this study, using mouse preimplantation embryos as a model for the evaluation of Oct4 function, we constructed Oct4 overexpression embryos with various mutations at the N-terminal transactivation domain. Developmental competency and molecular biological phenotypes depended on the type of mutation. The replacement of serine 106 with alanine resulted in more severe phenotypes similar to that of wild type Oct4, indicating that this alteration using alanine is negligible for Oct4 function. In contrast, we found that Oct4-specific antibodies could not recognize Oct4 protein when this residue was replaced by aspartic acid (Oct4-S106D). Oct4-S106D overexpressing embryos did not show developmental arrest and aberrant chromatin structure. Thus, these results demonstrated that the Ser-106 residue within the N-terminal transactivation domain is crucial for Oct4 function and suggested that this mutation might affect Oct4 protein conformation.

α2-6 sialylation is a marker of the differentiation potential of human mesenchymal stem cells.

Human somatic stem cells such as human mesenchymal stem cells (hMSCs) are considered attractive cell sources for stem cell-based therapy. However, quality control issues have been raised concerning their safety and efficacy. Here we used lectin microarray technology to identify cell surface glycans as markers of the differentiation potential of stem cells. We found that α2-6Sia-specific lectins show stronger binding to early passage adipose-derived hMSCs (with differentiation ability) than late passage cells (without the ability to differentiate). Flow cytometry analysis using α2-6Sia-specific lectins supported the results obtained by lectin microarray. Similar results were obtained for bone marrow-derived hMSCs and cartilage tissue-derived chondrocytes. Little or no binding of α2-6Sia-specific lectins was observed for human dermal fibroblasts, which are unable to differentiate, suggesting that the binding of α2-6Sia-specific lectins is associated with the differentiation ability of cells, but not to their capacity to proliferate. Quantitative analysis of the linkage mode of Sia using anion-exchange chromatography showed that the percentage of α2-6Sia linkage type was higher in early passage adipose-derived hMSCs than late passage cells. Integrinα5 was found to be a carrier protein of α2-6Sia. Sialidase treatment significantly reduced the differentiation efficiency of bone marrow-derived hMSCs. Based on these findings, we propose that α2-6sialylation is a marker of differentiation potential in stem cells such as adipose-derived hMSCs, bone marrow-derived hMSCs, and cartilage tissue-derived chondrocytes.

Spatiotemporal dynamics of OCT4 protein localization during preimplantation development in mice.

Spatiotemporal expression of transcription factors is crucial for genomic reprogramming. Pou5f1 (Oct4) is an essential transcription factor for reprogramming. A recent study reported that OCT4A, which is crucial for establishment and maintenance of pluripotent cells, is expressed in oocytes, but maternal OCT4A is dispensable for totipotency induction. Whereas another study reported that OCT4B, which is not related to pluripotency, is predominantly expressed instead of OCT4A during early preimplantation phases in mice. To determine the expression states of OCT4 in murine preimplantation embryos, we conducted in-depth expression and functional analyses. We found that pluripotency-related OCT4 mainly localizes to the cytoplasm in early preimplantation phases, with no major nuclear localization until the 8-16-cell stage despite high expression in both oocytes and early embryos. RNA-sequencing analysis using oocytes and early preimplantation embryos could not identify the splice variants creating alternative forms of OCT4 protein. Forced expression of OCT4 in zygotes by the injection of polyadenylated mRNA clearly showed nuclear localization of OCT4 protein around 3-5-fold greater than physiological levels and impaired developmental competency in a dose-dependent manner. Embryos with modest overexpression of OCT4 could develop to the 16-cell stage; however, more than 50% of the embryos were arrested at this stage, similar to the results for OCT4 depletion. In contrast, extensive overexpression of OCT4 resulted in complete arrest at the 2-cell stage accompanied by downregulation of zygotically activated genes and repetitive elements related to the totipotent state. These results demonstrated that OCT4 protein localization was spatiotemporally altered during preimplantation development, and strict control of Oct4 protein levels was essential for proper totipotential reprogramming.

Notch inhibition allows oncogene-independent generation of iPS cells.

The reprogramming of somatic cells to pluripotency using defined transcription factors holds great promise for biomedicine. However, human reprogramming remains inefficient and relies either on the use of the potentially dangerous oncogenes KLF4 and CMYC or the genetic inhibition of the tumor suppressor gene p53. We hypothesized that inhibition of signal transduction pathways that promote differentiation of the target somatic cells during development might relieve the requirement for non-core pluripotency factors during induced pluripotent stem cell (iPSC) reprogramming. Here, we show that inhibition of Notch greatly improves the efficiency of iPSC generation from mouse and human keratinocytes by suppressing p21 in a p53-independent manner and thereby enriching for undifferentiated cells capable of long-term self-renewal. Pharmacological inhibition of Notch enabled routine production of human iPSCs without KLF4 and CMYC while leaving p53 activity intact. Thus, restricting the development of somatic cells by altering intercellular communication enables the production of safer human iPSCs.

Aberrant silencing of imprinted genes on chromosome 12qF1 in mouse induced pluripotent stem cells.

Induced pluripotent stem cells (iPSCs) have been generated by enforced expression of defined sets of transcription factors in somatic cells. It remains controversial whether iPSCs are molecularly and functionally equivalent to blastocyst-derived embryonic stem (ES) cells. By comparing genetically identical mouse ES cells and iPSCs, we show here that their overall messenger RNA and microRNA expression patterns are indistinguishable with the exception of a few transcripts encoded within the imprinted Dlk1-Dio3 gene cluster on chromosome 12qF1, which were aberrantly silenced in most of the iPSC clones. Consistent with a developmental role of the Dlk1-Dio3 gene cluster, these iPSC clones contributed poorly to chimaeras and failed to support the development of entirely iPSC-derived animals ('all-iPSC mice'). In contrast, iPSC clones with normal expression of the Dlk1-Dio3 cluster contributed to high-grade chimaeras and generated viable all-iPSC mice. Notably, treatment of an iPSC clone that had silenced Dlk1-Dio3 with a histone deacetylase inhibitor reactivated the locus and rescued its ability to support full-term development of all-iPSC mice. Thus, the expression state of a single imprinted gene cluster seems to distinguish most murine iPSCs from ES cells and allows for the prospective identification of iPSC clones that have the full development potential of ES cells.

A synthetic nanofibrillar matrix promotes in vitro hepatic differentiation of embryonic stem cells and induced pluripotent stem cells.

Embryonic stem (ES) cells recapitulate normal developmental processes and serve as an attractive source for routine access to a large number of cells for research and therapies. We previously reported that ES cells cultured on M15 cells, or a synthesized basement membrane (sBM) substratum, efficiently differentiated into an endodermal fate and subsequently adopted fates of various digestive organs, such as the pancreas and liver. Here, we established a novel hepatic differentiation procedure using the synthetic nanofiber (sNF) as a cell culture scaffold. We first compared endoderm induction and hepatic differentiation between murine ES cells grown on sNF and several other substrata. The functional assays for hepatocytes reveal that the ES cells grown on sNF were directed into hepatic differentiation. To clarify the mechanisms for the promotion of ES cell differentiation in the sNF system, we focused on the function of Rac1, which is a Rho family member protein known to regulate the actin cytoskeleton. We observed the activation of Rac1 in undifferentiated and differentiated ES cells cultured on sNF plates, but not in those cultured on normal plastic plates. We also show that inhibition of Rac1 blocked the potentiating effects of sNF on endoderm and hepatic differentiation throughout the whole differentiation stages. Taken together, our results suggest that morphological changes result in cellular differentiation controlled by Rac1 activation, and that motility is not only the consequence, but is also able to trigger differentiation. In conclusion, we believe that sNF is a promising material that might contribute to tissue engineering and drug delivery.

Establishment and directed differentiation of induced pluripotent stem cells from glycogen storage disease type Ib patient.

Glycogen storage disease type Ib (GSDIb) is caused by a deficiency in the glucose-6-phosphate transporter (G6PT), which leads to neutrophil dysfunction. However, the underlying causes of these dysfunctions and their relationship with glucose homeostasis are unclear. Induced pluripotent stem cells (iPSCs) hold a great promise for advances in developmental biology, cell-based therapy and modeling of human disease. Here, we examined the use of iPSCs as a model for GSDIb. In this study, one 2-year-old patient was genetically screened and diagnosed with GSDIb. We established iPSCs and differentiated these cells into hepatocytes and neutrophils, which comprise the main pathological components of GSDIb. Cells that differentiated into hepatocytes exhibited characteristic albumin secretion and indocyanine green uptake. Moreover, iPSC-derived cells generated from patients with GSDIb metabolic abnormalities recapitulated key pathological features of the diseases affecting the patients from whom they were derived, such as glycogen, lactate, pyruvate and lipid accumulation. Cells that were differentiated into neutrophils also showed the GSDIb pathology. In addition to the expression of neutrophil markers, we showed increased superoxide anion production, increased annexin V binding and activation of caspase-3 and caspase-9, consistent with the GSDIb patient's neutrophils. These results indicate valuable tools for the analysis of this pathology and the development of future treatments.

DNA methylation dynamics in human induced pluripotent stem cells over time.

Epigenetic reprogramming is a critical event in the generation of induced pluripotent stem cells (iPSCs). Here, we determined the DNA methylation profiles of 22 human iPSC lines derived from five different cell types (human endometrium, placental artery endothelium, amnion, fetal lung fibroblast, and menstrual blood cell) and five human embryonic stem cell (ESC) lines, and we followed the aberrant methylation sites in iPSCs for up to 42 weeks. The iPSCs exhibited distinct epigenetic differences from ESCs, which were caused by aberrant methylation at early passages. Multiple appearances and then disappearances of random aberrant methylation were detected throughout iPSC reprogramming. Continuous passaging of the iPSCs diminished the differences between iPSCs and ESCs, implying that iPSCs lose the characteristics inherited from the parent cells and adapt to very closely resemble ESCs over time. Human iPSCs were gradually reprogrammed through the "convergence" of aberrant hyper-methylation events that continuously appeared in a de novo manner. This iPS reprogramming consisted of stochastic de novo methylation and selection/fixation of methylation in an environment suitable for ESCs. Taken together, random methylation and convergence are driving forces for long-term reprogramming of iPSCs to ESCs.