Identification of Quiescent LGR5+ Stem Cells in the Human Colon.

BACKGROUND & AIMS: In the mouse intestinal epithelium, Lgr5+ stem cells are vulnerable to injury, owing to their predominantly cycling nature, and their progenies de-differentiate to replenish the stem cell pool. However, how human colonic stem cells behave in homeostasis and during regeneration remains unknown. METHODS: Transcriptional heterogeneity among colonic epithelial cells was analyzed by means of single-cell RNA sequencing analysis of human and mouse colonic epithelial cells. To trace the fate of human colonic stem or differentiated cells, we generated LGR5-tdTomato, LGR5-iCasase9-tdTomato, LGR5-split-Cre, and KRT20-ERCreER knock-in human colon organoids via genome engineering. p27+ dormant cells were further visualized with the p27-mVenus reporter. To analyze the dynamics of human colonic stem cells in vivo, we orthotopically xenotransplanted fluorescence-labeled human colon organoids into immune-deficient mice. The cell cycle dynamics in xenograft cells were evaluated using 5-ethynyl-2'-deoxyuridine pulse-chase analysis. The clonogenic capacity of slow-cycling human stem cells or differentiated cells was analyzed in the context of homeostasis, LGR5 ablation, and 5-fluorouracil-induced mucosal injury. RESULTS: Single-cell RNA sequencing analysis illuminated the presence of nondividing LGR5+ stem cells in the human colon. Visualization and lineage tracing of slow-cycling LGR5+p27+ cells and orthotopic xenotransplantation validated their homeostatic lineage-forming capability in vivo, which was augmented by 5-FU-induced mucosal damage. Transforming growth factor-ß signaling regulated the quiescent state of LGR5+ cells. Despite the plasticity of differentiated KRT20+ cells, they did not display clonal growth after 5-FU-induced injury, suggesting that occupation of the niche environment by LGR5+p27+ cells prevented neighboring differentiated cells from de-differentiating. CONCLUSIONS: Our results highlight the quiescent nature of human LGR5+ colonic stem cells and their contribution to post-injury regeneration.

Loss of TET proteins in regulatory T cells promotes abnormal proliferation, Foxp3 destabilization and IL-17 expression.

Ten-eleven translocation (TET) proteins regulate DNA methylation and gene expression by converting 5-methylcytosine (5mC) to 5-hydroxymethylcytosine (5hmC). Although Tet2/Tet3 deficiency has been reported to lead to myeloid cell, B-cell and invariant natural killer T (iNKT) cell malignancy, the effect of TET on regulatory T cells (Tregs) has not been elucidated. We found that Tet2/Tet3 deficiency in Tregs led to lethal hyperproliferation of CD4+Foxp3+ T cells in the spleen and mesenteric lymph nodes after 5 months of age. Additionally, in aged Treg-specific Tet2/Tet3-deficient mice, serum IgG1, IgG3, IgM and IgE levels were markedly elevated. High IL-17 expression was observed in both Foxp3+ and Fopx3- CD4+ T cells, and adoptive transfer of Tet2/Tet3-deficient Tregs into lymphopenic mice inhibited Foxp3 expression and caused conversion into IL-17-producing cells. However, the conserved non-coding DNA sequence-2 (CNS2) region of the Foxp3 gene locus, which has been shown to be particularly important for stable Foxp3 expression, was only partly methylated. We identified novel TET-dependent demethylation sites in the Foxp3 upstream enhancer, which may contribute to stable Foxp3 expression. Together, these data indicate that Tet2 and Tet3 are involved in Treg stability and immune homeostasis in mice.

Transient ectopic expression of the histone demethylase JMJD3 accelerates the differentiation of human pluripotent stem cells.

Harnessing epigenetic regulation is crucial for the efficient and proper differentiation of pluripotent stem cells (PSCs) into desired cell types. Histone H3 lysine 27 trimethylation (H3K27me3) functions as a barrier against cell differentiation through the suppression of developmental gene expression in PSCs. Here, we have generated human PSC (hPSC) lines in which genome-wide reduction of H3K27me3 can be induced by ectopic expression of the catalytic domain of the histone demethylase JMJD3 (called JMJD3c). We found that transient, forced demethylation of H3K27me3 alone triggers the upregulation of mesoendodermal genes, even when the culture conditions for the hPSCs are not changed. Furthermore, transient and forced expression of JMJD3c followed by the forced expression of lineage-defining transcription factors enabled the hPSCs to activate tissue-specific genes directly. We have also shown that the introduction of JMJD3c facilitates the differentiation of hPSCs into functional hepatic cells and skeletal muscle cells. These results suggest the utility of the direct manipulation of epigenomes for generating desired cell types from hPSCs for cell transplantation therapy and platforms for drug screenings.

Selective modulation of local linkages between active transcription and oxidative demethylation activity shapes cardiomyocyte-specific gene-body epigenetic status in mice.

BACKGROUND: Cell-type-specific genes exhibit heterogeneity in genomic contexts and may be subject to different epigenetic regulations through different gene transcriptional processes depending on the cell type involved. The gene-body regions (GBRs) of some cardiomyocyte (CM)-specific genes are long and highly hypomethylated in CMs. To explore the cell-type specificities of epigenetic patterns and functions, multiple epigenetic modifications of GBRs were compared among CMs, liver cells and embryonic stem cells (ESCs). RESULTS: We found that most genes show a moderately negative correlation between transcript levels and gene lengths. As CM-specific genes are generally longer than other cell-type-specific genes, we hypothesized that the gene-body epigenetic features of CMs may support the transcriptional regulation of CM-specific genes. We found gene-body DNA hypomethylation in a CM-specific gene subset co-localized with rare gene-body marks, including RNA polymerase II (Pol II) and p300. Interestingly, 5-hydroxymethylcytosine (5hmC) within the gene body marked cell-type-specific genes at neonatal stages and active gene-body histone mark H3K36 trimethylation declined and overlapped with cell-type-specific gene-body DNA hypomethylation and selective Pol II/p300 accumulation in adulthood. Different combinations of gene-body epigenetic modifications were also observed with genome-wide scale cell-type specificity, revealing the occurrence of dynamic epigenetic rearrangements in GBRs across different cell types. CONCLUSIONS: As 5hmC enrichment proceeded to hypomethylated GBRs, we considered that hypomethylation may not represent a static state but rather an equilibrium state of turnover due to the balance between local methylation linked to transcription and Tet oxidative modification causing demethylation. Accordingly, we conclude that demethylation in CMs can be a used to establish such cell-type-specific epigenetic domains in relation to liver cells. The establishment of cell-type-specific epigenetic control may also change genomic contexts of evolution and may contribute to the development of cell-type-specific transcriptional coordination.

[Survey on Information Sharing and Approaches to Cooperation between Hospitals and Community Pharmacies in the Care of Outpatients Receiving Chemotherapy].

Outpatients undergoing chemotherapy receive oral anticancer drugs, supportive care medicine, and drugs for complications from health insurance pharmacies(ie, drugstores). Therefore, drugstore personnel and patients were surveyed using a questionnaire to ascertain the current conditions of information sharing between drugstores and hospitals. Only 31% of the patients surveyed responded that they received cancer chemotherapy via the drugstores, while a few of them understood the need for information sharing with the drugstore. We also found that the drugstores required a considerable amount of patient information including prescribed therapeutic drugs, treatment regimens, disease conditions, and test value. Therefore, we held a study session and clinical conference to facilitate the creation of an information-sharing system. In conclusion, it is imperative for drugstores and hospitals to cooperate and establish a strategy for information sharing in the future.

Biomineralization-inspired synthesis of functional organic/inorganic hybrid materials: organic molecular control of self-organization of hybrids.

Organisms produce various organic/inorganic hybrid materials, which are called biominerals. They form through the self-organization of organic molecules and inorganic elements under ambient conditions. Biominerals often have highly organized and hierarchical structures from nanometer to macroscopic length scales, resulting in their remarkable physical and chemical properties that cannot be obtained by simple accumulation of their organic and inorganic constituents. These observations motivate us to create novel functional materials exhibiting properties superior to conventional materials--both synthetic and natural. Herein, we introduce recent progress in understanding biomineralization processes at the molecular level and the development of organic/inorganic hybrid materials by these processes. We specifically outline fundamental molecular studies on silica, iron oxide, and calcium carbonate biomineralization and describe material synthesis based on these mechanisms. These approaches allow us to design a variety of advanced hybrid materials with desired morphologies, sizes, compositions, and structures through environmentally friendly synthetic routes using functions of organic molecules.

Late-replicating heterochromatin is characterized by decreased cytosine methylation in the human genome.

Heterochromatin is believed to be associated with increased levels of cytosine methylation. With the recent availability of genome-wide, high-resolution molecular data reflecting chromatin organization and methylation, such relationships can be explored systematically. As well-defined surrogates for heterochromatin, we tested the relationship between DNA replication timing and DNase hypersensitivity with cytosine methylation in two human cell types, unexpectedly finding the later-replicating, more heterochromatic regions to be less methylated than early replicating regions. When we integrated gene-expression data into the study, we found that regions of increased gene expression were earlier replicating, as previously identified, and that transcription-targeted cytosine methylation in gene bodies contributes to the positive correlation with early replication. A self-organizing map (SOM) approach was able to identify genomic regions with early replication and increased methylation, but lacking annotated transcripts, loci missed in simple two variable analyses, possibly encoding unrecognized intergenic transcripts. We conclude that the relationship of cytosine methylation with heterochromatin is not simple and depends on whether the genomic context is tandemly repetitive sequences often found near centromeres, which are known to be heterochromatic and methylated, or the remaining majority of the genome, where cytosine methylation is targeted preferentially to the transcriptionally active, euchromatic compartment of the genome.

Genome of the marsupial Monodelphis domestica reveals innovation in non-coding sequences.

We report a high-quality draft of the genome sequence of the grey, short-tailed opossum (Monodelphis domestica). As the first metatherian ('marsupial') species to be sequenced, the opossum provides a unique perspective on the organization and evolution of mammalian genomes. Distinctive features of the opossum chromosomes provide support for recent theories about genome evolution and function, including a strong influence of biased gene conversion on nucleotide sequence composition, and a relationship between chromosomal characteristics and X chromosome inactivation. Comparison of opossum and eutherian genomes also reveals a sharp difference in evolutionary innovation between protein-coding and non-coding functional elements. True innovation in protein-coding genes seems to be relatively rare, with lineage-specific differences being largely due to diversification and rapid turnover in gene families involved in environmental interactions. In contrast, about 20% of eutherian conserved non-coding elements (CNEs) are recent inventions that postdate the divergence of Eutheria and Metatheria. A substantial proportion of these eutherian-specific CNEs arose from sequence inserted by transposable elements, pointing to transposons as a major creative force in the evolution of mammalian gene regulation.

Genotype-phenotype mapping of a patient-derived lung cancer organoid biobank identifies NKX2-1-defined Wnt dependency in lung adenocarcinoma.

Human lung cancer is a constellation of tumors with various histological and molecular properties. To build a preclinical platform that covers this broad disease spectrum, we obtained lung cancer specimens from multiple sources, including sputum and circulating tumor cells, and generated a living biobank consisting of 43 lines of patient-derived lung cancer organoids. The organoids recapitulated the histological and molecular hallmarks of the original tumors. Phenotypic screening of niche factor dependency revealed that EGFR mutations in lung adenocarcinoma are associated with the independence from Wnt ligands. Gene engineering of alveolar organoids reveals that constitutive activation of EGFR-RAS signaling provides Wnt independence. Loss of the alveolar identity gene NKX2-1 confers Wnt dependency, regardless of EGFR signal mutation. Sensitivity to Wnt-targeting therapy can be stratified by the expression status of NKX2-1. Our results highlight the potential of phenotype-driven organoid screening and engineering for the fabrication of therapeutic strategies to combat cancer.

Genetic analysis of essential cardiac transcription factors in 256 patients with non-syndromic congenital heart defects.

BACKGROUND: The genetic basis of most congenital heart defects (CHDs), especially non-syndromic and non-familial conditions, remains largely unknown. METHODS AND RESULTS: DNA samples were collected from immortalized cell lines and original genomes of 256 non-syndromic, non-familial patients with cardiac outflow tract (OFT) defects. Genes encoding NKX2.5, GATA4, GATA6, MEF2C, and ISL1, essential for heart development, were analyzed using PCR-based bidirectional sequencing. The transcriptional activity of proteins with identified sequence variations was analyzed using a luciferase assay. A novel sequence variant (A103V in MEF2C) was identified, in addition to 4 unreported non-synonymous sequence variants in 3 known causative genes (A6V in NKX2.5, T330R and S339R in GATA4, and E142K in GATA6) in 5 individuals. None of these was found in 500 controls without CHDs. In vitro functional assay showed that all proteins with identified sequence variations exhibited significant changes in transcriptional activity and/or synergistic activity with other transcription factors. Furthermore, overexpression of the A103V MEF2C variant in a fish system disturbed early cardiac development. CONCLUSIONS: New mutations in the transcription factors NKX2.5, GATA4, GATA6, and MEF2C that affect their protein function were identified in 2.3% (6/256) of patients with OFT defects. Our results provide the first demonstration of MEF2C mutation and suggest that disturbances in the regulatory circuits involving these cardiac transcription factors may cause a subset of non-syndromic and non-familial CHDs.

Establishment of trophoblast stem cell lines from somatic cell nuclear-transferred embryos.

Placental abnormalities occur frequently in cloned animals. Here, we attempted to isolate trophoblast stem (TS) cells from mouse blastocysts produced by somatic cell nuclear transfer (NT) at the blastocyst stage (NT blastocysts). Despite the predicted deficiency of the trophoblast cell lineage, we succeeded in isolating cell colonies with typical morphology of TS cells and cell lines from the NT blastocysts (ntTS cell lines) with efficiency as high as that from native blastocysts. The established 10 ntTS cell lines could be maintained in the undifferentiated state and induced to differentiate into several trophoblast subtypes in vitro. A comprehensive analysis of the transcriptional and epigenetic traits demonstrated that ntTS cells were indistinguishable from control TS cells. In addition, ntTS cells contributed exclusively to the placenta and survived until term in chimeras, indicating that ntTS cells have developmental potential as stem cells. Taken together, our data show that NT blastocysts contain cells that can produce TS cells in culture, suggesting that proper commitment to the trophoblast cell lineage in NT embryos occurs by the blastocyst stage.

Intestinal epithelial organoids: regeneration and maintenance of the intestinal epithelium.

Vital functions of the intestines: digestion, absorption, and surface barrier are performed by the intestinal epithelium, which consists of various differentiated cells and intestinal stem cells. Recent technological advances in sequencing technology, including single-cell transcriptomics and epigenetic analysis, have facilitated the genetic characterization of diverse intestinal epithelial cell types and surrounding mesenchymal niche environments. Organoids have allowed biological analysis of the human intestinal epithelium in coordination with genome engineering, genetic lineage tracing, and transplantation into orthotopic tissue. Together, these technologies have prompted the development of organoid-based regenerative therapies for intestinal diseases, including short-bowel syndrome. This article provides an overview of the current understanding of intestinal epithelial self-renewal during homeostasis and regeneration and provides a perspective for future organoid medicine.

Effective treatment with mitotane for a canine case of presumed ectopic Cushing's syndrome-related pheochromocytoma.

Background: In humans, ectopic Cushing's syndrome (ECS) is characterized by hypercortisolemia, which is caused by small lung carcinoma, bronchial carcinoids, and pheochromocytoma. In dogs, only a few cases of ECS associated with pheochromocytoma have been reported to date. Case Description: Herein, we describe a canine case of malignant pheochromocytoma that is presumed to be the cause of ECS. An 11-year-old, castrated, male Toy Poodle with hypercortisolemia was diagnosed with an adrenal tumor (AT) and treated with mitotane. Although repeated adrenocorticotropic hormone stimulation tests revealed improvement in the dog's condition by mitotane treatment, its condition started declining 197 days post-diagnosis, and he died on day 280. The necropsy revealed the AT was a pheochromocytoma, not an adrenocortical tumor. However, because of no pathological change in the pituitary gland and the other adrenal gland, pheochromocytoma was presumed to be the cause of ECS. Conclusion: This is the first report that describes the effectiveness of mitotane against presumed ECS-related pheochromocytoma.

High-resolution genome-wide cytosine methylation profiling with simultaneous copy number analysis and optimization for limited cell numbers.

Many genome-wide assays involve the generation of a subset (or representation) of the genome following restriction enzyme digestion. The use of enzymes sensitive to cytosine methylation allows high-throughput analysis of this epigenetic regulatory process. We show that the use of a dual-adapter approach allows us to generate genomic representations that includes fragments of <200 bp in size, previously not possible when using the standard approach of using a single adapter. By expanding the representation to smaller fragments using HpaII or MspI, we increase the representation by these isoschizomers to more than 1.32 million loci in the human genome, representing 98.5% of CpG islands and 91.1% of refSeq promoters. This advance allows the development of a new, high-resolution version of our HpaII-tiny fragment Enrichment by Ligation-mediated PCR (HELP) assay to study cytosine methylation. We also show that the MspI representation generates information about copy-number variation, that the assay can be used on as little as 10 ng of DNA and that massively parallel sequencing can be used as an alternative to microarrays to read the output of the assay, making this a powerful discovery platform for studies of genomic and epigenomic abnormalities.

OGT Regulates Hematopoietic Stem Cell Maintenance via PINK1-Dependent Mitophagy.

O-linked N-acetylglucosamine (O-GlcNAc) transferase (OGT) is a unique enzyme introducing O-GlcNAc moiety on target proteins, and it critically regulates various cellular processes in diverse cell types. However, its roles in hematopoietic stem and progenitor cells (HSPCs) remain elusive. Here, using Ogt conditional knockout mice, we show that OGT is essential for HSPCs. Ogt is highly expressed in HSPCs, and its disruption induces rapid loss of HSPCs with increased reactive oxygen species and apoptosis. In particular, Ogt-deficient hematopoietic stem cells (HSCs) lose quiescence, cannot be maintained in vivo, and become vulnerable to regenerative and competitive stress. Interestingly, Ogt-deficient HSCs accumulate defective mitochondria due to impaired mitophagy with decreased key mitophagy regulator, Pink1, through dysregulation of H3K4me3. Furthermore, overexpression of PINK1 restores mitophagy and the number of Ogt-deficient HSCs. Collectively, our results reveal that OGT critically regulates maintenance and stress response of HSCs by ensuring mitochondrial quality through PINK1-dependent mitophagy.

Trophoblast cell lineage in cloned mouse embryos.

Most conceptuses derived by somatic cell nuclear transfer (SCNT) in mice undergo developmental arrest as a result of embryonic or extraembryonic defects. Even when fetuses survive to term, prominent placental overgrowth or placentomegaly is often present, indicating that SCNT affects the development of trophoblast cell lineage. The trophoblast cell lineage is established at the blastocyst stage when the stem cell population of the trophoblast cell lineage resides in the polar trophectoderm. Therefore, it is possible that the developmental arrest and placentomegaly that accompany SCNT are induced by insufficient reprogramming of the donor somatic nucleus to enable the cells to acquire full potency as stem cells of the trophoblast cell lineage. Despite the abnormalities of the extraembryonic tissues of SCNT embryos, trophoblast stem (TS) cell lines have been successfully isolated from SCNT blastocysts and their properties appear to be indistinguishable from those of TS cells derived from native blastocysts. This suggests that SCNT does not affect the emergence and autonomous properties of TS cells. In this review, we discuss specification of cell lineage and the extent of reprogramming of TS cells in SCNT blastocysts.

Consultation on the Functional Assessment of Students with Severe Challenging Behavior in a Japanese Special School for Intellectual Disabilities.

BACKGROUND: It is important to intervene early and treat children and individuals with behavioral disorders. We conducted a functional assessment-based consultation for teachers of several students with severe behavioral disorders and examined the effects of the consultation. METHODS: Eight students with severe behavioral disorders were selected from two special schools for intellectual disabilities in western Japan. An external consultant team conducted a functional assessment-based consultation in cooperation with a team of teachers. Consultations were held once a month, and comprised three to six sessions per student. RESULTS: As a result of the functional assessment, only 8 out of 10 behaviors with some communication function, and 2 with only sensory enhancements were estimated. The Effects of consultations based on functional assessment were presented. It was found that 6 out of 10 target behaviors had obtained high effects. The total score for each behavioral scale showed a statistically significant improvement. CONCLUSION: Although consultations lasted for only six months and occurred from three to six times for each student, scale scores for problem behavior before and after intervention were improved, overall. Each case report suggested that many factors influence the difference in the effects of consultation among individual students. This study is significant in that it provides a model for the consultation system that operates on a short-term basis, and presents a means for small-scale group consultations for students with intellectual disabilities and autism in cooperation with external specialized institutions in special schools in Japan.

Transcriptomic features of trophoblast lineage cells derived from human induced pluripotent stem cells treated with BMP 4.

INTRODUCTION: Early development of the human placenta remains poorly understood due to the lack of proper model systems. Previous reports have demonstrated that human induced pluripotent stem cells (hiPSCs) treated with bone morphogenetic protein 4 (BMP4) can differentiate into extraembryonic tissues as useful models of the early stage of trophoblast (TB) differentiation. In our previous study, we optimized the culture conditions of hiPSC-derived TB lineages, but the differentiated cells were heterogeneous. METHODS: In order to characterize the hiPSC-derived TB lineage cells, four types of hiPSCs were treated with 50 ng/mL of BMP4 for 10 days. Subsequently, cells that were positive for the pan-TB marker keratin 7(KRT7) were purified from the differentiated cells using flow cytometry and identified with a DNA microarray. RESULTS: Comparisons of our microarray data with the human transcriptome in a previous large-scale analysis showed that the gene expression patterns of KRT7+ cells were similar to the placenta. In total, 259 upregulated genes were commonly expressed in all four KRT7+ groups, including well-known TB markers. Among these upregulated genes, several with poorly investigated expression patterns and functions were confirmed as expressed in the primary placenta. While only XAGE2 and KCNQ2 were expressed in TB layers, XAGE2 was expressed throughout pregnancy and KCNQ2 was expressed only in cytotrophoblasts of the first trimester placenta. CONCLUSION: BMP4-treated KRT7+ cells were in the course of the human placental development. In addition, this approach allowed the identification of new genes that might be involved in placentation. However, further studies are needed to confirm their functions.

Generation and Profiling of 2,135 Human ESC Lines for the Systematic Analyses of Cell States Perturbed by Inducing Single Transcription Factors.

Transcription factors (TFs) play a pivotal role in determining cell states, yet our understanding of the causative relationship between TFs and cell states is limited. Here, we systematically examine the state changes of human pluripotent embryonic stem cells (hESCs) by the large-scale manipulation of single TFs. We establish 2,135 hESC lines, representing three clones each of 714 doxycycline (Dox)-inducible genes including 481 TFs, and obtain 26,998 microscopic cell images and 2,174 transcriptome datasets-RNA sequencing (RNA-seq) or microarrays-48 h after the presence or absence of Dox. Interestingly, the expression of essentially all the genes, including genes located in heterochromatin regions, are perturbed by these TFs. TFs are also characterized by their ability to induce differentiation of hESCs into specific cell lineages. These analyses help to provide a way of classifying TFs and identifying specific sets of TFs for directing hESC differentiation into desired cell types.

The HELP assay.

Genomic representations using ligation-mediated PCR have been used successfully as the foundation for a number of high-throughput assays. HpaII tiny fragment enrichment by ligation-mediated PCR (HELP) is an example of the use of such representations to study cytosine methylation in the genome. The HELP assay differs from most other assays testing cytosine methylation because of its positive representation of hypomethylated DNA in the genome, whereas other assays infer the presence of hypomethylated sequences by the absence of signal, for which there can be confounding technical reasons. Hypomethylated sequences represent the minority of the genome and tend to be located at unique sequences with functionally interesting properties such as transcription start sites. By performing a comparative genomic hybridization using an MspI representation from the same DNA sample, we represent all potential loci that could be generated by HpaII in the situation of global hypomethylation; in practice, HpaII generates a subset of loci from this population, allowing us to discriminate hypomethylated loci (represented by both HpaII and MspI) from methylated loci (represented by MspI only).