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.

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.

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.

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.

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.

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.

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.

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.

Perturbation of the titin/MURF1 signaling complex is associated with hypertrophic cardiomyopathy in a fish model and in human patients.

Hypertrophic cardiomyopathy (HCM) is a hereditary disease characterized by cardiac hypertrophy with diastolic dysfunction. Gene mutations causing HCM have been found in about half of HCM patients, while the genetic etiology and pathogenesis remain unknown for many cases of HCM. To identify novel mechanisms underlying HCM pathogenesis, we generated a cardiovascular-mutant medaka fish, non-spring heart (nsh), which showed diastolic dysfunction and hypertrophic myocardium. The nsh homozygotes had fewer myofibrils, disrupted sarcomeres and expressed pathologically stiffer titin isoforms. In addition, the nsh heterozygotes showed M-line disassembly that is similar to the pathological changes found in HCM. Positional cloning revealed a missense mutation in an immunoglobulin (Ig) domain located in the M-line-A-band transition zone of titin. Screening of mutations in 96 unrelated patients with familial HCM, who had no previously implicated mutations in known sarcomeric gene candidates, identified two mutations in Ig domains close to the M-line region of titin. In vitro studies revealed that the mutations found both in medaka fish and in familial HCM increased binding of titin to muscle-specific ring finger protein 1 (MURF1) and enhanced titin degradation by ubiquitination. These findings implicate an impaired interaction between titin and MURF1 as a novel mechanism underlying the pathogenesis of HCM.

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.

Induction of human pluripotent stem cells into kidney tissues by synthetic mRNAs encoding transcription factors.

The derivation of kidney tissues from human pluripotent stem cells (hPSCs) and its application for replacement therapy in end-stage renal disease have been widely discussed. Here we report that consecutive transfections of two sets of synthetic mRNAs encoding transcription factors can induce rapid and efficient differentiation of hPSCs into kidney tissues, termed induced nephron-like organoids (iNephLOs). The first set - FIGLA, PITX2, ASCL1 and TFAP2C, differentiated hPSCs into SIX2+SALL1+ nephron progenitor cells with 92% efficiency within 2 days. Subsequently, the second set - HNF1A, GATA3, GATA1 and EMX2, differentiated these cells into PAX8+LHX1+ pretubular aggregates in another 2 days. Further culture in both 2-dimensional and 3-dimensional conditions produced iNephLOs containing cells characterized as podocytes, proximal tubules, and distal tubules in an additional 10 days. Global gene expression profiles showed similarities between iNephLOs and the human adult kidney, suggesting possible uses of iNephLOs as in vitro models for kidneys.

Tbx6 Induces Nascent Mesoderm from Pluripotent Stem Cells and Temporally Controls Cardiac versus Somite Lineage Diversification.

The mesoderm arises from pluripotent epiblasts and differentiates into multiple lineages; however, the underlying molecular mechanisms are unclear. Tbx6 is enriched in the paraxial mesoderm and is implicated in somite formation, but its function in other mesoderms remains elusive. Here, using direct reprogramming-based screening, single-cell RNA-seq in mouse embryos, and directed cardiac differentiation in pluripotent stem cells (PSCs), we demonstrated that Tbx6 induces nascent mesoderm from PSCs and determines cardiovascular and somite lineage specification via its temporal expression. Tbx6 knockout in mouse PSCs using CRISPR/Cas9 technology inhibited mesoderm and cardiovascular differentiation, whereas transient Tbx6 expression induced mesoderm and cardiovascular specification from mouse and human PSCs via direct upregulation of Mesp1, repression of Sox2, and activation of BMP/Nodal/Wnt signaling. Notably, prolonged Tbx6 expression suppressed cardiac differentiation and induced somite lineages, including skeletal muscle and chondrocytes. Thus, Tbx6 is critical for mesoderm induction and subsequent lineage diversification.

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.

Epigenetic barrier against the propagation of fluctuating gene expression in embryonic stem cells.

The expression of pluripotency genes fluctuates in a population of embryonic stem (ES) cells and the fluctuations in the expression of some pluripotency genes correlate. However, no correlation in the fluctuation of Pou5f1, Zfp42, and Nanog expression was observed in ES cells. Correlation between Pou5f1 and Zfp42 fluctuations was demonstrated in ES cells containing a knockout in the NuRD component Mbd3. ES cells containing a triple knockout in the DNA methyltransferases Dnmt1, Dnmt3a, and Dnmt3b showed correlation between the fluctuation of Pou5f1, Zfp42, and Nanog gene expression. We suggest that an epigenetic barrier is key to preventing the propagation of fluctuating pluripotency gene expression in ES cells.

Epigenetic Manipulation Facilitates the Generation of Skeletal Muscle Cells from Pluripotent Stem Cells.

Human pluripotent stem cells (hPSCs) have the capacity to differentiate into essentially all cell types in the body. Such differentiation can be directed to specific cell types by appropriate cell culture conditions or overexpressing lineage-defining transcription factors (TFs). Especially, for the activation of myogenic program, early studies have shown the effectiveness of enforced expression of TFs associated with myogenic differentiation, such as PAX7 and MYOD1. However, the efficiency of direct differentiation was rather low, most likely due to chromatin features unique to hPSCs, which hinder the access of TFs to genes involved in muscle differentiation. Indeed, recent studies have demonstrated that ectopic expression of epigenetic-modifying factors such as a histone demethylase and an ATP-dependent remodeling factor significantly enhances myogenic differentiation from hPSCs. In this article, we review the recent progress for in vitro generation of skeletal muscles from hPSCs through forced epigenetic and transcriptional manipulation.

Salt suppresses IFNγ inducible chemokines through the IFNγ-JAK1-STAT1 signaling pathway in proximal tubular cells.

The mechanisms of immunoactivation by salt are now becoming clearer. However, those of immunosuppression remain unknown. Since clinical evidence indicates that salt protects proximal tubules from injury, we investigated mechanisms responsible for salt causing immunosuppression in proximal tubules. We focused on cytokine-related gene expression profiles in kidneys of mice fed a high salt diet using microarray analysis and found that both an interferon gamma (IFNγ) inducible chemokine, chemokine (C-X-C motif) ligand 9 (CXCL9), and receptor, CXCR3, were suppressed. We further revealed that a high salt concentration suppressed IFNγ inducible chemokines in HK2 proximal tubular cells. Finally, we demonstrated that a high salt concentration decreased IFNGR1 expression in the basolateral membrane of HK2 cells, leading to decreased phosphorylation of activation sites of Janus kinase 1 (JAK1) and Signal Transducers and Activator of Transcription 1 (STAT1), activators of chemokines. JAK inhibitor canceled the effect of a high salt concentration on STAT1 and chemokines, indicating that the JAK1-STAT1 signaling pathway is essential for this mechanism. In conclusion, a high salt concentration suppresses IFNγ-JAK1-STAT1 signaling pathways and chemokine expressions in proximal tubules. This finding may explain how salt ameliorates proximal tubular injury and offer a new insight into the linkage between salt and immunity.

Violet Light Exposure Can Be a Preventive Strategy Against Myopia Progression.

Prevalence of myopia is increasing worldwide. Outdoor activity is one of the most important environmental factors for myopia control. Here we show that violet light (VL, 360-400nm wavelength) suppresses myopia progression. First, we confirmed that VL suppressed the axial length (AL) elongation in the chick myopia model. Expression microarray analyses revealed that myopia suppressive gene EGR1 was upregulated by VL exposure. VL exposure induced significantly higher upregulation of EGR1 in chick chorioretinal tissues than blue light under the same conditions. Next, we conducted clinical research retrospectively to compare the AL elongation among myopic children who wore eyeglasses (VL blocked) and two types of contact lenses (partially VL blocked and VL transmitting). The data showed the VL transmitting contact lenses suppressed myopia progression most. These results suggest that VL is one of the important outdoor environmental factors for myopia control. Since VL is apt to be excluded from our modern society due to the excessive UV protection, VL exposure can be a preventive strategy against myopia progression.

Expression analysis of the endogenous Zscan4 locus and its coding proteins in mouse ES cells and preimplantation embryos.

Mouse Zinc finger and SCAN domain containing 4 (Zscan4) is encoded in multiple copies of Zscan4 genes, which are expressed in late two-cell stage preimplantation embryos and in 1-5% of the embryonic stem (ES) cell population at a given time. Due to the highly identical nucleotide sequences of multiple copies of Zscan4 paralogs and pseudogenes in the mouse Zscan4 genomic cluster, previous analyses have been done using exogenous transgenes under the regulation of Zscan4c promoter. In this manuscript, we generated knock-in mouse ES cell lines and mouse lines, in which the expression of endogenous Zscan4c, one of the Zscan4 genes, can be specifically monitored with a green fluorescent protein variant, Emerald. Interestingly, we found that only ∼30% of Zscan4-immunopositive ES cells were Emerald positive, suggesting that even when the Zscan4 locus is active, not all Zscan4 genes are expressed synchronously. We also carried out mass spectrometry of protein complexes associated with endogenous Zscan4 proteins. Taken together, our genetic engineering at an endogenous Zscan4c gene provides the first clue for the expression and function of each gene copy of Zscan4 locus in a physiological context.

[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.

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.