Experimental interventions attenuate a conjunctival epidermal metaplasia model.

The conjunctiva is a non-keratinized, stratified columnar epithelium with characteristics different from the cornea and eyelid epidermis. From development to adulthood, a distinguishing feature of ocular versus epidermal epithelia is the expression of the master regulator PAX6. A conditionally immortalized conjunctival epithelial cell line (iHCjEC) devoid of stromal or immune cells established in our laboratory spontaneously manifested epidermal metaplasia and upregulated expression of the keratinization-related genes SPRR1A/B and the epidermal cytokeratins KRT1 and KRT10 at the expense of the conjunctival trait. In addition, iHCjEC indicated a significant decrease in PAX6 expression. Dry eye syndrome (DES) and severe ocular surface diseases, such as Sjögren's syndrome and Stevens-Johnson syndrome, cause the keratinization of the entire ocular surface epithelia. We used iHCjECs as a conjunctiva epidermal metaplasia model to test PAX6, serum, and glucocorticoid interventions. Reintroducing PAX6 to iHCjECs resulted in upregulating genes related to cell adhesion and tight junctions, including MIR200CHG and CLDN1. The administration of glucocorticoids or serum resulted in the downregulation of epidermal genes (DSG1, SPRR1A/B, and KRT1) and partially corrected epidermal metaplasia. Our results using an isolated conjunctival epidermal metaplasia model point toward the possibility of rationally "repurposing" clinical interventions, such as glucocorticoid, serum, or PAX6 administration, for treating epidermal metaplasia of the conjunctiva.

Multiple knockout mouse and embryonic stem cell models reveal the role of miR-124a in neuronal maturation.

MicroRNA-124a (miR-124a) is one of the most abundantly expressed microRNAs in the central nervous system and is encoded in mammals by the three genomic loci miR-124a-1/2/3; however, its in vivo roles in neuronal development and function remain ambiguous. In the present study, we investigated the effect of miR-124a loss on neuronal differentiation in mice and in embryonic stem (ES) cells. Since miR-124a-3 exhibits only background expression levels in the brain and we were unable to obtain miR-124a-1/2/3 triple knockout (TKO) mice by mating, we generated and analyzed miR-124a-1/2 double knockout (DKO) mice. We found that these DKO mice exhibit perinatal lethality. RNA-seq analysis demonstrated that the expression levels of proneural and neuronal marker genes were almost unchanged between the control and miR-124a-1/2 DKO brains; however, genes related to neuronal synaptic formation and function were enriched among downregulated genes in the miR-124a-1/2 DKO brain. In addition, we found the transcription regulator Tardbp/TDP-43, loss of which leads to defects in neuronal maturation and function, was inactivated in the miR-124a-1/2 DKO brain. Furthermore, Tardbp knockdown suppressed neurite extension in cultured neuronal cells. We also generated miR-124a-1/2/3 TKO ES cells using CRISPR-Cas9 as an alternative to TKO mice. Phase-contrast microscopic, immunocytochemical, and gene expression analyses showed that miR-124a-1/2/3 TKO ES cell lines were able to differentiate into neurons. Collectively, these results suggest that miR-124a plays a role in neuronal maturation rather than neurogenesis in vivo and advance our understanding of the functional roles of microRNAs in central nervous system development.

BAC-DROP: Rapid Digestion of Proteome Fractionated via Dissolvable Polyacrylamide Gel Electrophoresis and Its Application to Bottom-Up Proteomics Workflow.

The GeLC-MS workflow, which combines low-cost, easy-to-use sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (SDS-PAGE) with liquid chromatography-mass spectrometry (LC-MS), is very popular in current bottom-up proteomics. However, GeLC-MS requires that PAGE-separated proteins undergo overnight enzymatic digestion in a gel, resulting in more than 20 h of sample preparation for LC-MS. In this study, we overcame the limitations of GeLC-MS by developing a rapid digestion workflow for PAGE separation of proteins using N,N'-bis(acryloyl)cystamine (BAC) cross-linked gels that can be solubilized by reductive treatment. Making use of an established workflow called BAC-DROP (BAC-gel dissolution to digest PAGE-resolved objective proteins), crude proteome samples were fractionated based on molecular weight by BAC cross-linked PAGE. After fractionation, the gel fragments were reductively dissolved in under 5 min, and in-solution trypsin digestion of the protein released from the gel was completed in less than 1 h at 70 °C, equivalent to a 90-95% reduction in time compared to conventional in-gel trypsin digestion. The introduction of the BAC-DROP workflow to the MS assays for inflammatory biomarker CRP and viral marker HBsAg allowed for serum sample preparation to be completed in as little as 5 h, demonstrating successful marker quantification from a 0.5 µL sample of human serum.

Monoallelic, antisense and total RNA transcription in an in vitro neural differentiation system based on F1 hybrid mice.

We developed an in vitro system to differentiate embryonic stem cells (ESCs) derived from reciprocally crossed F1 hybrid mice into neurons, and used it to investigate poly(A)+ and total RNA transcription at different stages of cell differentiation. By comparing expression profiles of transcripts assembled from 20 RNA sequencing datasets [2 alleles×(2 cell lines×4 time-points+2 mouse brains)], the relative influence of strain, cell and parent specificities to overall expression could be assessed. Divergent expression profiles of ESCs converged tightly at neural progenitor stage. Patterns of temporal variation of monoallelically expressed transcripts and antisense transcripts were quantified. Comparison of sense and antisense transcript pairs within the poly(A)+ sample, within the total RNA sample, and across poly(A)+ and total RNA samples revealed distinct rates of pairs showing anti-correlated expression variation. Unique patterns of sharing of poly(A)+ and poly(A)- transcription were identified in distinct RNA species. Regulation and functionality of monoallelic expression, antisense transcripts and poly(A)- transcription remain elusive. We demonstrated the effectiveness of our approach to capture these transcriptional activities, and provided new resources to elucidate the mammalian developmental transcriptome.

Development of a High-Efficacy Reprogramming Method for Generating Human Induced Pluripotent Stem (iPS) Cells from Pathologic and Senescent Somatic Cells.

Induced pluripotent stem (iPS) cells are a type of artificial pluripotent stem cell induced by the epigenetic silencing of somatic cells by the Yamanaka factors. Advances in iPS cell reprogramming technology will allow aging or damaged cells to be replaced by a patient's own rejuvenated cells. However, tissue that is senescent or pathologic has a relatively low reprogramming efficiency as compared with juvenile or robust tissue, resulting in incomplete reprogramming; iPS cells generated from such tissue types do not have sufficient differentiation ability and are therefore difficult to apply clinically. Here, we develop a new reprogramming method and examine it using myofibroblasts, which are pathologic somatic cells, from patient skin tissue and from each of the four heart chambers of a recipient heart in heart transplant surgery. By adjusting the type and amount of vectors containing transcriptional factors for iPS cell reprogramming, as well as adjusting the transfection load and culture medium, the efficiency of iPS cell induction from aged patient skin-derived fibroblasts was increased, and we successfully induced iPS cells from myocardial fibroblasts isolated from the pathologic heart of a heart transplant recipient.

A Comprehensive Genomic Analysis Reveals the Genetic Landscape of Mitochondrial Respiratory Chain Complex Deficiencies.

Mitochondrial disorders have the highest incidence among congenital metabolic disorders characterized by biochemical respiratory chain complex deficiencies. It occurs at a rate of 1 in 5,000 births, and has phenotypic and genetic heterogeneity. Mutations in about 1,500 nuclear encoded mitochondrial proteins may cause mitochondrial dysfunction of energy production and mitochondrial disorders. More than 250 genes that cause mitochondrial disorders have been reported to date. However exact genetic diagnosis for patients still remained largely unknown. To reveal this heterogeneity, we performed comprehensive genomic analyses for 142 patients with childhood-onset mitochondrial respiratory chain complex deficiencies. The approach includes whole mtDNA and exome analyses using high-throughput sequencing, and chromosomal aberration analyses using high-density oligonucleotide arrays. We identified 37 novel mutations in known mitochondrial disease genes and 3 mitochondria-related genes (MRPS23, QRSL1, and PNPLA4) as novel causative genes. We also identified 2 genes known to cause monogenic diseases (MECP2 and TNNI3) and 3 chromosomal aberrations (6q24.3-q25.1, 17p12, and 22q11.21) as causes in this cohort. Our approaches enhance the ability to identify pathogenic gene mutations in patients with biochemically defined mitochondrial respiratory chain complex deficiencies in clinical settings. They also underscore clinical and genetic heterogeneity and will improve patient care of this complex disorder.

Mutations of optineurin in amyotrophic lateral sclerosis.

Amyotrophic lateral sclerosis (ALS) has its onset in middle age and is a progressive disorder characterized by degeneration of motor neurons of the primary motor cortex, brainstem and spinal cord. Most cases of ALS are sporadic, but about 10% are familial. Genes known to cause classic familial ALS (FALS) are superoxide dismutase 1 (SOD1), ANG encoding angiogenin, TARDP encoding transactive response (TAR) DNA-binding protein TDP-43 (ref. 4) and fused in sarcoma/translated in liposarcoma (FUS, also known as TLS). However, these genetic defects occur in only about 20-30% of cases of FALS, and most genes causing FALS are unknown. Here we show that there are mutations in the gene encoding optineurin (OPTN), earlier reported to be a causative gene of primary open-angle glaucoma (POAG), in patients with ALS. We found three types of mutation of OPTN: a homozygous deletion of exon 5, a homozygous Q398X nonsense mutation and a heterozygous E478G missense mutation within its ubiquitin-binding domain. Analysis of cell transfection showed that the nonsense and missense mutations of OPTN abolished the inhibition of activation of nuclear factor kappa B (NF-kappaB), and the E478G mutation revealed a cytoplasmic distribution different from that of the wild type or a POAG mutation. A case with the E478G mutation showed OPTN-immunoreactive cytoplasmic inclusions. Furthermore, TDP-43- or SOD1-positive inclusions of sporadic and SOD1 cases of ALS were also noticeably immunolabelled by anti-OPTN antibodies. Our findings strongly suggest that OPTN is involved in the pathogenesis of ALS. They also indicate that NF-kappaB inhibitors could be used to treat ALS and that transgenic mice bearing various mutations of OPTN will be relevant in developing new drugs for this disorder.

ES cell differentiation system recapitulates the establishment of imprinted gene expression in a cell-type-specific manner.

Genomic imprinting is a phenomenon whereby monoallelic gene expression occurs in a parent-of-origin-specific manner. A subset of imprinted genes acquires a tissue-specific imprinted status during the course of tissue development, and this process can be analyzed by means of an in vitro differentiation system utilizing embryonic stem (ES) cells. In neurons, the gene Ube3a is expressed from the maternal allele only, and a paternally expressed non-coding, antisense RNA has been implicated in the imprinting process in mice and humans. Here, to study the genomic imprinting mechanism, we established F1 hybrid ES cells derived from two sub-species of Mus musculus and established an in vitro neuronal differentiation system in which neuron-specific imprinting of Ube3a was recapitulated. With this system, we revealed that the switch from biallelic expression to maternal, monoallelic expression of Ube3a occurs late in neuronal development, during the neurite outgrowth period, and that the expression of endogenous antisense transcript from the Ube3a locus is up-regulated several hundred-fold during the same period. Our results suggest that evaluation of the quality of ES cells by studying their differentiation in vitro should include evaluation of epigenetic aspects, such as a comparison with the genomic imprinting status found in tissues in vivo, in addition to the evaluation of differentiation gene markers and morphology. Our F1 hybrid ES cells and in vitro differentiation system will allow researchers to investigate complex end-points such as neuron-specific genomic imprinting, and our F1 hybrid ES cells are a useful resource for other tissue-specific genomic imprinting and epigenetic analyses.

Enhancing the neural differentiation capabilities of genetically asymmetric mouse F1 hybrid embryonic stem cell lines.

Allele-specific, monoallelic expression in diploid organisms represents an extreme case of allelic imbalance resulting from incompatibility between cis- and trans-elements. Due to haploinsufficiency, such monoallelic expression can lead to sporadic genetic diseases. In mice, allelic imbalances can be introduced into F1 offspring from inbred strains. Previously, we established F1 hybrid embryonic stem (ES) cell lines derived from four different mouse strains, each belonging to a different subspecies with substantial genetic polymorphisms. In this study, we investigated the neural differentiation capacity of the established ES cell lines. By introducing different culture conditions, which kept the ES cells undifferentiated under various pluripotencies, we succeeded in differentiating the majority of ES cell lines (eight out of eleven) with our default neural differentiation paradigm. Still, three lines exhibited insufficient differentiation despite combining culture conditions promoting undifferentiated as well as differentiated status. In addition, Ube3a imprinting was seen in two lines. Our findings contribute to the methodological understanding of mouse ES cell pluripotency and lead to the practical utility of F1 hybrid ES cells as a model for studying phenotypes resulting from gene locus interactions.

Inter-subspecies mouse F1 hybrid embryonic stem cell lines newly established for studies of allelic imbalance in gene expression.

Allele-specific monoallelic gene expression is a unique phenomenon and a great resource for analyzing gene regulation. To study this phenomenon, we established new embryonic stem (ES) cell lines derived from F1 hybrid blastocysts from crosses between four mouse subspecies (Mus musculus domesticus, C57BL/6; M. musculus molossinus, MSM/Ms; M. musculus musculus, PWK; M. musculus castaneus, HMI/Ms) and analyzed the expression levels of undifferentiated pluripotent stem cell markers and karyotypes of each line. To demonstrate the utility of our cell lines, we analyzed the allele-specific expression pattern of the Inpp5d gene as an example. The allelic expression depended on the parental alleles; this dependence could be a consequence of differences in compatibility between cis- and trans-elements of the Inpp5d gene from different subspecies. The use of parental mice from four subspecies greatly enhanced genetic polymorphism. The F1 hybrid ES cells retained this polymorphism not only in the Inpp5d gene, but also at a genome-wide level. As we demonstrated for the Inpp5d gene, the established cell lines can contribute to the analysis of allelic expression imbalance based on the incompatibility between cis- and trans-elements and of phenotypes related to this incompatibility.

Perspectives on frailty as a total life-course disease with consideration of the fetal environment.

Frailty attracts research as it represents a significant target for intervention to extend the healthy life span. An unanswered question in this field is the time point during the life-course at which an individual becomes predisposed to frailty. Here, we propose that frailty has a fetal origin and should be regarded as part of the spectrum of the developmental origins of health and disease. The developmental origins of health and disease theory originated from findings linking the fetal environment to lifestyle-related disorders such as hypertension and diabetes. Coincidentally, a recent trend in frailty research also centers on vascular dysfunction and metabolic alterations as the causality of lifestyle-related disorders such as sarcopenia and dementia. Here, we explore the relationship between fetal programming, frailty-related disorders (sarcopenia and dementia), and other age-related diseases mainly based on reports on intrauterine growth restriction. We propose a "total" life-course approach to combat frailty. With this viewpoint, not only physicians and gerontologists but also obstetricians and pediatricians should team up to overcome age-related diseases in the elderly. Geriatr Gerontol Int 2023; 23: 263-269.

Isolation of alveolar epithelial type II progenitor cells from adult human lungs.

Resident stem/progenitor cells in the lung are important for tissue homeostasis and repair. However, a progenitor population for alveolar type II (ATII) cells in adult human lungs has not been identified. The aim of this study is to isolate progenitor cells from adult human lungs with the ability to differentiate into ATII cells. We isolated colony-forming cells that had the capability for self-renewal and the potential to generate ATII cells in vitro. These undifferentiated progenitor cells expressed surface markers of mesenchymal stem cells (MSCs) and surfactant proteins associated with ATII cells, such as CD90 and pro-surfactant protein-C (pro-SP-C), respectively. Microarray analyses indicated that transcripts associated with lung development were enriched in the pro-SP-C(+)/CD90(+) cells compared with bone marrow-MSCs. Furthermore, pathological evaluation indicated that pro-SP-C and CD90 double-positive cells were present within alveolar walls in normal lungs, and significantly increased in ATII cell hyperplasias contributing to alveolar epithelial repair in damaged lungs. Our findings demonstrated that adult human lungs contain a progenitor population for ATII cells. This study is a first step toward better understanding of stem cell biology in adult human lung alveoli.

Genome-wide analysis of expression modes and DNA methylation status at sense-antisense transcript loci in mouse.

The functionality of sense-antisense transcripts (SATs), although widespread throughout the mammalian genome, is largely unknown. Here, we analyzed the SATs expression and its associated promoter DNA methylation status by surveying 12 tissues of mice to gain insights into the relationship between expression and DNA methylation of SATs. We have found that sense and antisense expression positively correlate in most tissues. However, in some SATs with tissue-specific expression, the expression level of a transcript from a CpG island-bearing promoter is low when the promoter DNA methylation is present. In these circumstances, the expression level of its opposite-strand transcript, especially when it is poly(A)-negative was coincidentally higher. These observations suggest that, albeit the general tendency of sense-antisense simultaneous expression, some antisense transcripts have coordinated expression with its counterpart sense gene promoter methylation. This cross-strand relationship is not a privilege of imprinted genes but seems to occur widely in SATs.

Differential requirement for nucleostemin in embryonic stem cell and neural stem cell viability.

Stem cells have the remarkable ability to self-renew and to generate multiple cell types. Nucleostemin is one of proteins that are enriched in many types of stem cells. Targeted deletion of nucleostemin in the mouse results in developmental arrest at the implantation stage, indicating that nucleostemin is crucial for early embryogenesis. However, the molecular basis of nucleostemin function in early mouse embryos remains largely unknown, and the role of nucleostemin in tissue stem cells has not been examined by gene targeting analyses due to the early embryonic lethality of nucleostemin null animals. To address these questions, we generated inducible nucleostemin null embryonic stem (ES) cells in which both alleles of nucleostemin are disrupted, but nucleostemin cDNA under the control of a tetracycline-responsive transcriptional activator is introduced into the Rosa26 locus. We show that loss of nucleostemin results in reduced cell proliferation and increased apoptosis in both ES cells and ES cell-derived neural stem/progenitor cells. The reduction in cell viability is much more profound in ES cells than in neural stem/progenitor cells, an effect that is mediated at least in part by increased induction and accumulation of p53 and/or activated caspase-3 in ES cells than in neural stem/progenitor cells.

Rapid induction of the immediate early gene c-fos in a chick forebrain system involved in memory.

Previous work has shown that expression of Fos protein in neurons of the intermediate and medial mesopallium (IMM), a memory region in the forebrain of the domestic chick, increases in a learning-related manner after behavioural imprinting. We show here, using in situ hybridisation, that when chicks are trained for 15 min with an imprinting stimulus, expression of c-fos mRNA in the IMM rises to a maximum at or before the end of this training period. The results suggest that the learning-related increase in Fos protein production, which occurs in identifiable neuronal sub-populations in the IMM, reflects events that make an early contribution to learning and/or memory processing.

Role of SoxB1 transcription factors in development.

SoxB1 factors, which include Sox1, 2, and 3, share more than 90% amino acid identity in their DNA binding HMG box and participate in diverse developmental events. They are known to exert cell-type-specific functions in concert with other transcription factors on Sox factor-dependent regulatory enhancers. Due to the high degree of sequence similarity both within and outside the HMG box, SoxB1 members show almost identical biological activities. As a result, they exhibit strong functional redundancy in regions where SoxB1 members are coexpressed, such as neural stem/progenitor cells in the developing central nervous system.

The N-end rule pathway enzyme Naa10 supports epiblast specification in mouse embryonic stem cells by modulating FGF/MAPK.

N-terminal acetylation (Nt-acetylation) refers to the acetylation of the free α-amino group at the N-terminus of a polypeptide. While the effects of Nt-acetylation are multifaceted, its most known function is in the acetylation-dependent N-end rule protein degradation pathway (Ac/N-end rule pathway), where Nt-acetylation is recognized as a degron by designated E3 ligases, eventually leading to target degradation by the ubiquitin-proteasome system. Naa10 is the catalytic subunit of the major Nt-acetylation enzyme NatA, which Nt-acetylates proteins whose second amino acid has a small side chain. In humans, NAA10 is the responsible mutated gene in Ogden syndrome and is thought to play important roles in development. However, it is unclear how the Ac/N-end rule pathway affects the differentiation ability of mouse embryonic stem cells (mESCs). We hypothesized that the balance of pluripotency factors may be maintained by the Ac/N-end rule pathway. Thus, we established Naa10 knockout mESCs to test this hypothesis. We found that Naa10 deficiency attenuated differentiation towards the epiblast lineage, deviating towards primitive endoderm. However, this was not caused by disturbing the balance of pluripotency factors, rather by augmenting FGF/MAPK signaling.

Markers associated with neuron-specific Ube3a imprinting during neuronal differentiation of mouse embryonic stem cells.

Understanding gene expression in the brain requires allele-specific transcriptome analysis because of the presence of neuron-specific imprinted genes, which are expressed in a neuron-specific and parent-of-origin-specific manner. Ube3a is a neuron-specific imprinted gene with an expression pattern that changes from biallelic to maternal only (Ube3a imprinting) during differentiation. Because Ube3a imprinting occurs only in neurons, it has the potential to be a marker to assess the quality of neurons produced by in vitro neuronal differentiation of embryonic stem cells (ESCs). For the analysis of Ube3a imprinting, genetic polymorphisms between the two alleles are necessary to identify the parental origin of each. However, ESCs derived from commonly used inbred mouse strains have no genetic polymorphisms. To overcome this problem, we examined 10 markers of neurogenesis to determine whether they were associated with Ube3a imprinting. We measured the relative expression levels of these 10 gene markers and assessed the Ube3a imprinting status of 54 neuron samples differentiated under various in vitro conditions. Then we divided the samples into two groups depending on their Ube3a imprinting status and selected markers statistically associated with Ube3a imprinting. The identified markers included the antisense noncoding transcript of Ube3a and a mature neuron marker Mtap2, consistent with the markers we used empirically in our previous study to assess the quality of differentiated neurons. These findings provide new quality control criteria for differentiated neurons, and could also be applied to human ESCs and induced pluripotent stem cells.

Oral Administration of Red Ginseng Extract Promotes Neurorestoration after Compressive Spinal Cord Injury in Rats.

Red ginseng and its active ingredients have been shown to decrease neuron death after brain ischemia in experimental animals. However, little is known about the effects of orally administered ginseng extract on spinal cord injury. We orally gave red ginseng extract (RGE) to rats with compressed spinal cord injury (SCI). Open-field locomotor scores were measured as indices of motor function. Histopathological changes and cytokine expressions in situ after SCI were evaluated. Compared to vehicle treatment, RGE treatment (350 mg/kg/day) significantly improved locomotor score up to levels close to those pre-SCI, prevented neuron loss, and facilitated the restoration of white matter in the spinal cord at 14 days after SCI. Treatment with RGE caused less aggregation of Iba-1-positive microglia in grey and white matter at 7 days after SCI, upregulated the expression levels of VEGF and Bcl-xL, and reduced IL-1ß and TNFα expressions in the spinal cord at 7 and 14 days after SCI. We concluded that oral administration of RGE facilitates almost complete functional recovery from motor and behavioral abnormalities in rats with SCI and prevents neuron death in situ, possibly through inhibition of inflammation and upregulation of neuroprotective factors in the injured spinal cord.