Spatial heterogeneity of bone marrow endothelial cells unveils a distinct subtype in the epiphysis.

Bone marrow endothelial cells (BMECs) play a key role in bone formation and haematopoiesis. Although recent studies uncovered the cellular taxonomy of stromal compartments in the bone marrow (BM), the complexity of BMECs is not fully characterized. In the present study, using single-cell RNA sequencing, we defined a spatial heterogeneity of BMECs and identified a capillary subtype, termed type S (secondary ossification) endothelial cells (ECs), exclusively existing in the epiphysis. Type S ECs possessed unique phenotypic characteristics in terms of structure, plasticity and gene expression profiles. Genetic experiments showed that type S ECs atypically contributed to the acquisition of bone strength by secreting type I collagen, the most abundant bone matrix component. Moreover, these cells formed a distinct reservoir for haematopoietic stem cells. These findings provide the landscape for the cellular architecture in the BM vasculature and underscore the importance of epiphyseal ECs during bone and haematopoietic development.

Generation of a tyrosine hydroxylase-2A-Cre knockin non-human primate model by homology-directed-repair-biased CRISPR genome editing.

Non-human primates (NHPs) are the closest animal model to humans; thus, gene engineering technology in these species holds great promise for the elucidation of higher brain functions and human disease models. Knockin (KI) gene targeting is a versatile approach to modify gene(s) of interest; however, it generally suffers from the low efficiency of homology-directed repair (HDR) in mammalian cells, especially in non-expressed gene loci. In the current study, we generated a tyrosine hydroxylase (TH)-2A-Cre KI model of the common marmoset monkey (marmoset; Callithrix jacchus) using an HDR-biased CRISPR-Cas9 genome editing approach using Cas9-DN1S and RAD51. This model should enable labeling and modification of a specific neuronal lineage using the Cre-loxP system. Collectively, the current study paves the way for versatile gene engineering in NHPs, which may be a significant step toward further biomedical and preclinical applications.

Mesenchymal properties of iPSC-derived neural progenitors that generate undesired grafts after transplantation.

Although neural stem/progenitor cells derived from human induced pluripotent stem cells (hiPSC-NS/PCs) are expected to be a cell source for cell-based therapy, tumorigenesis of hiPSC-NS/PCs is a potential problem for clinical applications. Therefore, to understand the mechanisms of tumorigenicity in NS/PCs, we clarified the cell populations of NS/PCs. We established single cell-derived NS/PC clones (scNS/PCs) from hiPSC-NS/PCs that generated undesired grafts. Additionally, we performed bioassays on scNS/PCs, which classified cell types within parental hiPSC-NS/PCs. Interestingly, we found unique subsets of scNS/PCs, which exhibited the transcriptome signature of mesenchymal lineages. Furthermore, these scNS/PCs expressed both neural (PSA-NCAM) and mesenchymal (CD73 and CD105) markers, and had an osteogenic differentiation capacity. Notably, eliminating CD73+ CD105+ cells from among parental hiPSC-NS/PCs ensured the quality of hiPSC-NS/PCs. Taken together, the existence of unexpected cell populations among NS/PCs may explain their tumorigenicity leading to potential safety issues of hiPSC-NS/PCs for future regenerative medicine.

Chromatin remodeler CHD7 targets active enhancer region to regulate cell type-specific gene expression in human neural crest cells.

A mutation in the chromatin remodeler chromodomain helicase DNA-binding 7 (CHD7) gene causes the multiple congenital anomaly CHARGE syndrome. The craniofacial anomalies observed in CHARGE syndrome are caused by dysfunctions of neural crest cells (NCCs), which originate from the neural tube. However, the mechanism by which CHD7 regulates the function of human NCCs (hNCCs) remains unclear. We aimed to characterize the cis-regulatory elements governed by CHD7 in hNCCs by analyzing genome-wide ChIP-Seq data and identifying hNCC-specific CHD7-binding profiles. We compared CHD7-binding regions among cell types, including human induced pluripotent stem cells and human neuroepithelial cells, to determine the comprehensive properties of CHD7-binding in hNCCs. Importantly, analysis of the hNCC-specific CHD7-bound region revealed transcription factor AP-2α as a potential co-factor facilitating the cell type-specific transcriptional program in hNCCs. CHD7 was strongly associated with active enhancer regions, permitting the expression of hNCC-specific genes to sustain the function of hNCCs. Our findings reveal the regulatory mechanisms of CHD7 in hNCCs, thus providing additional information regarding the transcriptional programs in hNCCs.

Human PSCs determine the competency of cerebral organoid differentiation via FGF signaling and epigenetic mechanisms.

Various culture methods have been developed for maintaining human pluripotent stem cells (PSCs). These PSC maintenance methods exhibit biased differentiation; for example, feeder-dependent PSCs efficiently yield cerebral organoids, but it is difficult to generate organoids from feeder-free PSCs. It remains unknown how PSC maintenance conditions affect differentiation. In this study, we identified fibroblast growth factor (FGF) signaling in feeder-free PSC maintenance as a key factor that determines the differentiation toward cerebral organoids. The inhibition of FGF signaling in feeder-free PSCs rescued organoid generation to the same level in feeder-dependent cultures. FGF inhibition induced DNA methylation at the WNT5A locus, and this epigenetic change suppressed the future activation of non-canonical Wnt signaling after differentiation, leading to reliable cerebral organoid generation. This study underscores the importance of PSC culture conditions for directed differentiation into cerebral organoids, and the epigenetic status regulated by FGF signaling is involved in the underlying mechanisms.

Multimodal analyses of a non-human primate model harboring mutant amyloid precursor protein transgenes driven by the human EF1α promoter.

Alzheimer's disease (AD) is the leading cause of dementia which afflicts tens of millions of people worldwide. Despite many scientific progresses to dissect the AD's molecular basis from studies on various mouse models, it has been suffered from evolutionary species differences. Here, we report generation of a non-human primate (NHP), common marmoset model ubiquitously expressing Amyloid-beta precursor protein (APP) transgenes with the Swedish (KM670/671NL) and Indiana (V717F) mutations. The transgene integration of generated two transgenic marmosets (TG1&TG2) was thoroughly investigated by genomic PCR, whole-genome sequencing, and fluorescence in situ hybridization. By reprogramming, we confirmed the validity of transgene expression in induced neurons in vitro. Moreover, we discovered structural changes in specific brain regions of transgenic marmosets by magnetic resonance imaging analysis, including in the entorhinal cortex and hippocampus. In immunohistochemistry, we detected increased Aß plaque-like structures in TG1 brain at 7 years old, although evident neuronal loss or glial inflammation was not observed. Thus, this study summarizes our attempt to establish an NHP AD model. Although the transgenesis approach alone seemed not sufficient to fully recapitulate AD in NHPs, it may be beneficial for drug development and further disease modeling by combination with other genetically engineered models and disease-inducing approaches.

Coupling of angiogenesis and odontogenesis orchestrates tooth mineralization in mice.

The skeletal system consists of bones and teeth, both of which are hardened via mineralization to support daily physical activity and mastication. The precise mechanism for this process, especially how blood vessels contribute to tissue mineralization, remains incompletely understood. Here, we established an imaging technique to visualize the 3D structure of the tooth vasculature at a single-cell level. Using this technique combined with single-cell RNA sequencing, we identified a unique endothelial subtype specialized to dentinogenesis, a process of tooth mineralization, termed periodontal tip-like endothelial cells. These capillaries exhibit high angiogenic activity and plasticity under the control of odontoblasts; in turn, the capillaries trigger odontoblast maturation. Metabolomic analysis demonstrated that the capillaries perform the phosphate delivery required for dentinogenesis. Taken together, our data identified the fundamental cell-to-cell communications that orchestrate tooth formation, angiogenic-odontogenic coupling, a distinct mechanism compared to the angiogenic-osteogenic coupling in bones. This mechanism contributes to our understanding concerning the functional diversity of organotypic vasculature.

Homologous Recombination-Enhancing Factors Identified by Comparative Transcriptomic Analyses of Pluripotent Stem Cell of Human and Common Marmoset.

A previous study assessing the efficiency of the genome editing technology CRISPR-Cas9 for knock-in gene targeting in common marmoset (marmoset; Callithrix jacchus) embryonic stem cells (ESCs) unexpectedly identified innately enhanced homologous recombination activity in marmoset ESCs. Here, we compared gene expression in marmoset and human pluripotent stem cells using transcriptomic and quantitative PCR analyses and found that five HR-related genes (BRCA1, BRCA2, RAD51C, RAD51D, and RAD51) were upregulated in marmoset cells. A total of four of these upregulated genes enhanced HR efficiency with CRISPR-Cas9 in human pluripotent stem cells. Thus, the present study provides a novel insight into species-specific mechanisms for the choice of DNA repair pathways.

SoxE group transcription factor Sox8 promotes astrocytic differentiation of neural stem/precursor cells downstream of Nfia.

The brain consists of three major cell types: neurons and two glial cell types (astrocytes and oligodendrocytes). Although they are generated from common multipotent neural stem/precursor cells (NS/PCs), embryonic NS/PCs cannot generate all of the cell types at the beginning of brain development. NS/PCs first undergo extensive self-renewal to expand their pools, and then acquire the potential to produce neurons, followed by glial cells. Astrocytes are the most frequently found cell type in the central nervous system (CNS), and play important roles in brain development and functions. Although it has been shown that nuclear factor IA (Nfia) is a pivotal transcription factor for conferring gliogenic potential on neurogenic NS/PCs by sequestering DNA methyltransferase 1 (Dnmt1) from astrocyte-specific genes, direct targets of Nfia that participate in astrocytic differentiation have yet to be completely identified. Here we show that SRY-box transcription factor 8 (Sox8) is a direct target gene of Nfia at the initiation of the gliogenic phase. We found that expression of Sox8 augmented leukemia inhibitory factor (LIF)-induced astrocytic differentiation, while Sox8 knockdown inhibited Nfia-enhanced astrocytic differentiation of NS/PCs. In contrast to Nfia, Sox8 did not induce DNA demethylation of an astrocyte-specific marker gene, glial fibrillary acidic protein (Gfap), but instead associated with LIF downstream transcription factor STAT3 through transcriptional coactivator p300, explaining how Sox8 expression further facilitated LIF-induced Gfap expression. Taken together, these results suggest that Sox8 is a crucial Nfia downstream transcription factor for the astrocytic differentiation of NS/PCs in the developing brain.

Non-viral Induction of Transgene-free iPSCs from Somatic Fibroblasts of Multiple Mammalian Species.

Induced pluripotent stem cells (iPSCs) are capable of providing an unlimited source of cells from all three germ layers and germ cells. The derivation and usage of iPSCs from various animal models may facilitate stem cell-based therapy, gene-modified animal production, and evolutionary studies assessing interspecies differences. However, there is a lack of species-wide methods for deriving iPSCs, in particular by means of non-viral and non-transgene-integrating (NTI) approaches. Here, we demonstrate the iPSC derivation from somatic fibroblasts of multiple mammalian species from three different taxonomic orders, including the common marmoset (Callithrix jacchus) in Primates, the dog (Canis lupus familiaris) in Carnivora, and the pig (Sus scrofa) in Cetartiodactyla, by combinatorial usage of chemical compounds and NTI episomal vectors. Interestingly, the fibroblasts temporarily acquired a neural stem cell-like state during the reprogramming. Collectively, our method, robustly applicable to various species, holds a great potential for facilitating stem cell-based research using various animals in Mammalia.

A robust culture system to generate neural progenitors with gliogenic competence from clinically relevant induced pluripotent stem cells for treatment of spinal cord injury.

Cell-based therapy targeting spinal cord injury (SCI) is an attractive approach to promote functional recovery by replacing damaged tissue. We and other groups have reported the effectiveness of transplanting neural stem/progenitor cells (NS/PCs) derived from human induced pluripotent stem cells (hiPSCs) in SCI animal models for neuronal replacement. Glial replacement is an additional approach for tissue repair; however, the lack of robust procedures to drive iPSCs into NS/PCs which can produce glial cells has hindered the development of glial cell transplantation for the restoration of neuronal functions after SCI. Here, we established a method to generate NS/PCs with gliogenic competence (gNS/PCs) optimized for clinical relevance and utilized them as a source of therapeutic NS/PCs for SCI. We could successfully generate gNS/PCs from clinically relevant hiPSCs, which efficiently produced astrocytes and oligodendrocytes in vitro. We also performed comparison between gNS/PCs and neurogenic NS/PCs based on single cell RNA-seq analysis and found that gNS/PCs were distinguished by expression of several transcription factors including HEY2 and NFIB. After gNS/PC transplantation, the graft did not exhibit tumor-like tissue formation, indicating the safety of them as a source of cell therapy. Importantly, the gNS/PCs triggered functional recovery in an SCI animal model, with remyelination of demyelinated axons and improved motor function. Given the inherent safety of gNS/PCs and favorable outcomes observed after their transplantation, cell-based medicine using the gNS/PCs-induction procedure described here together with clinically relevant iPSCs is realistic and would be beneficial for SCI patients.

Single-cell study of neural stem cells derived from human iPSCs reveals distinct progenitor populations with neurogenic and gliogenic potential.

We used single-cell RNA sequencing (seq) on several human induced pluripotent stem (iPS) cell-derived neural stem cell (NSC) lines and one fetal brain-derived NSC line to study inherent cell type heterogeneity at proliferating neural stem cell stage and uncovered predisposed presence of neurogenic and gliogenic progenitors. We observed heterogeneity in neurogenic progenitors that differed between the iPS cell-derived NSC lines and the fetal-derived NSC line, and we also observed differences in spontaneous differentiation potential for inhibitory and excitatory neurons between the iPS cell-derived NSC lines and the fetal-derived NSC line. In addition, using a recently published glia patterning protocol we enriched for gliogenic progenitors and generated glial cells from an iPS cell-derived NSC line.

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.

Chromatin remodeler CHD7 regulates the stem cell identity of human neural progenitors.

Multiple congenital disorders often present complex phenotypes, but how the mutation of individual genetic factors can lead to multiple defects remains poorly understood. In the present study, we used human neuroepithelial (NE) cells and CHARGE patient-derived cells as an in vitro model system to identify the function of chromodomain helicase DNA-binding 7 (CHD7) in NE-neural crest bifurcation, thus revealing an etiological link between the central nervous system (CNS) and craniofacial anomalies observed in CHARGE syndrome. We found that CHD7 is required for epigenetic activation of superenhancers and CNS-specific enhancers, which support the maintenance of the NE and CNS lineage identities. Furthermore, we found that BRN2 and SOX21 are downstream effectors of CHD7, which shapes cellular identities by enhancing a CNS-specific cellular program and indirectly repressing non-CNS-specific cellular programs. Based on our results, CHD7, through its interactions with superenhancer elements, acts as a regulatory hub in the orchestration of the spatiotemporal dynamics of transcription factors to regulate NE and CNS lineage identities.

CHARGE syndrome modeling using patient-iPSCs reveals defective migration of neural crest cells harboring CHD7 mutations.

CHARGE syndrome is caused by heterozygous mutations in the chromatin remodeler, CHD7, and is characterized by a set of malformations that, on clinical grounds, were historically postulated to arise from defects in neural crest formation during embryogenesis. To better delineate neural crest defects in CHARGE syndrome, we generated induced pluripotent stem cells (iPSCs) from two patients with typical syndrome manifestations, and characterized neural crest cells differentiated in vitro from these iPSCs (iPSC-NCCs). We found that expression of genes associated with cell migration was altered in CHARGE iPSC-NCCs compared to control iPSC-NCCs. Consistently, CHARGE iPSC-NCCs showed defective delamination, migration and motility in vitro, and their transplantation in ovo revealed overall defective migratory activity in the chick embryo. These results support the historical inference that CHARGE syndrome patients exhibit defects in neural crest migration, and provide the first successful application of patient-derived iPSCs in modeling craniofacial disorders.

Synergistic induction of astrocytic differentiation by factors secreted from meninges in the mouse developing brain.

Astrocytes, which support diverse neuronal functions, are generated from multipotent neural stem/precursor cells (NS/PCs) during brain development. Although many astrocyte-inducing factors have been identified and studied in vitro, the regions and/or cells that produce these factors in the developing brain remain elusive. Here, we show that meninges-produced factors induce astrocytic differentiation of NS/PCs. Consistent with the timing when astrocytic differentiation of NS/PCs increases, expression of astrocyte-inducing factors is upregulated. Meningeal secretion-mimicking combinatorial treatment of NS/PCs with bone morphogenetic protein 4, retinoic acid and leukemia inhibitory factor synergistically activate the promoter of a typical astrocytic marker, glial fibrillary acidic protein. Taken together, our data suggest that meninges play an important role in astrocytic differentiation of NS/PCs in the developing brain.

Improvement of Foxp3 stability through CNS2 demethylation by TET enzyme induction and activation.

Since induced regulatory T cells (iTregs) can be produced in a large quantity in vitro, these cells are expected to be clinically useful to induce immunological tolerance in various immunological diseases. Foxp3 (Forkhead box P3) expression in iTregs is, however, unstable due to the lack of demethylation of the CpG island in the conserved non-coding sequence 2 (CNS2) of the Foxp3 locus. To facilitate the demethylation of CNS2, we over-expressed the catalytic domain (CD) of the ten-eleven translocation (TET) protein, which catalyzes the steps of the iterative demethylation of 5-methylcytosine. TET-CD over-expression in iTregs resulted in partial demethylation of CNS2 and stable Foxp3 expression. We also discovered that TET expression was enhanced under low oxygen (5%) culture conditions, which facilitated CNS2 DNA demethylation and stabilization of Foxp3 expression in a TET2- and TET3-dependent manner. In combination with vitamin C treatment, which has been reported to enhance TET catalytic activity, iTregs generated under low oxygen conditions retained more stable Foxp3 expression in vitro and in vivo and exhibited stronger suppression activity in a colitis model compared with untreated iTregs. Our data indicate that the induction and activation of TET enzymes in iTregs would be an effective method for Treg-mediated adoptive immunotherapy.

DNA Methylome Analysis Identifies Transcription Factor-Based Epigenomic Signatures of Multilineage Competence in Neural Stem/Progenitor Cells.

Regulation of the epigenome during in vivo specification of brain stem cells is still poorly understood. Here, we report DNA methylome analyses of directly sampled cortical neural stem and progenitor cells (NS/PCs) at different development stages, as well as those of terminally differentiated cortical neurons, astrocytes, and oligodendrocytes. We found that sequential specification of cortical NS/PCs is regulated by two successive waves of demethylation at early and late development stages, which are responsible for the establishment of neuron- and glia-specific low-methylated regions (LMRs), respectively. The regulatory role of demethylation of the gliogenic genes was substantiated by the enrichment of nuclear factor I (NFI)-binding sites. We provide evidence that de novo DNA methylation of neuron-specific LMRs establishes glia-specific epigenotypes, essentially by silencing neuronal genes. Our data highlight the in vivo implications of DNA methylation dynamics in shaping epigenomic features that confer the differentiation potential of NS/PCs sequentially during development.

Whole-Genome DNA Methylation Analyses Revealed Epigenetic Instability in Tumorigenic Human iPS Cell-Derived Neural Stem/Progenitor Cells.

Although human induced pluripotent stem cell (hiPSC) derivatives are considered promising cellular resources for regenerative medicine, their tumorigenicity potentially limits their clinical application in hiPSC technologies. We previously demonstrated that oncogenic hiPSC-derived neural stem/progenitor cells (hiPSC-NS/PCs) produced tumor-like tissues that were distinct from teratomas. To gain insight into the mechanisms underlying the regulation of tumorigenicity in hiPSC-NS/PCs, we performed an integrated analysis using the Infinium HumanMethylation450 BeadChip array and the HumanHT-12 v4.0 Expression BeadChip array to compare the comprehensive DNA methylation and gene expression profiles of tumorigenic hiPSC-NS/PCs (253G1-NS/PCs) and non-tumorigenic cells (201B7-NS/PCs). Although the DNA methylation profiles of 253G1-hiPSCs and 201B7-hiPSCs were similar regardless of passage number, the methylation status of the global DNA methylation profiles of 253G1-NS/PCs and 201B7-NS/PCs differed; the genomic regions surrounding the transcriptional start site of the CAT and PSMD5 genes were hypermethylated in 253G1-NS/PCs but not in 201B7-NS/PCs. Interestingly, the aberrant DNA methylation profile was more pronounced in 253G1-NS/PCs that had been passaged more than 15 times. In addition, we identified aberrations in DNA methylation at the RBP1 gene locus; the DNA methylation frequency in RBP1 changed as 253G1-NS/PCs were sequentially passaged. These results indicate that different NS/PC clones have different DNA methylomes and that DNA methylation patterns are unstable as cells are passaged. Therefore, DNA methylation profiles should be included in the criteria used to evaluate the tumorigenicity of hiPSC-NS/PCs in the clinical setting. Stem Cells 2017;35:1316-1327.

Changeability of the fully methylated status of the 15q11.2 region in induced pluripotent stem cells derived from a patient with Prader-Willi syndrome.

Prader-Will syndrome (PWS) is characterized by hyperphagia, growth hormone deficiency and central hypogonadism caused by the dysfunction of the hypothalamus. Patients with PWS present with methylation abnormalities of the PWS-imprinting control region in chromosome 15q11.2, subject to parent-of-origin-specific methylation and controlling the parent-of-origin-specific expression of other paternally expressed genes flanking the region. In theory, the reversal of hypermethylation in the hypothalamic cells could be a promising strategy for the treatment of PWS patients, since cardinal symptoms of PWS patients are correlated with dysfunction of the hypothalamus. The genome-wide methylation status dramatically changes during the reprograming of somatic cells into induced pluripotent stem cells (iPSCs) and during the in vitro culture of iPSCs. Here, we tested the methylation status of the chromosome 15q11.2 region in iPSCs from a PWS patient using pyrosequencing and a more detailed method of genome-wide DNA methylation profiling to reveal whether iPSCs with a partially unmethylated status for the chromosome 15q11.2 region exhibit global methylation aberrations. As a result, we were able to show that a fully methylated status for chromosome 15q11.2 in a PWS patient could be reversed to a partially unmethylated status in at least some of the PWS-iPSC lines. Genome-wide DNA methylation profiling revealed that the partial unmethylation occurred at differentially methylated regions located in chromosome 15q11.2, but not at other differentially methylated regions associated with genome imprinting. The present data potentially opens a door to cell-based therapy for PWS patients and, possibly, patients with other disorders associated with genomic imprinting.