吉原 正仁

BACKGROUND: Primary cilia emanate from most human cell types, including neurons. Cilia are important for communicating with the cell's immediate environment: signal reception and transduction to/from the ciliated cell. Deregulation of ciliary signaling can lead to ciliopathies and certain neurodevelopmental disorders. In the developing brain cilia play well-documented roles for the expansion of the neural progenitor cell pool, while information about the roles of cilia during post-mitotic neuron differentiation and maturation is scarce. RESULTS: We employed ciliated Lund Human Mesencephalic (LUHMES) cells in time course experiments to assess the impact of ciliary signaling on neuron differentiation. By comparing ciliated and non-ciliated neuronal precursor cells and neurons in wild type and in RFX2 -/- mutant neurons with altered cilia, we discovered an early-differentiation "ciliary time window" during which transient cilia promote axon outgrowth, branching and arborization. Experiments in neurons with IFT88 and IFT172 ciliary gene knockdowns, leading to shorter cilia, confirm these results. Cilia promote neuron differentiation by tipping WNT signaling toward the non-canonical pathway, in turn activating WNT pathway output genes implicated in cyto-architectural changes. CONCLUSIONS: We provide a mechanistic entry point into when and how ciliary signaling coordinates, promotes and translates into anatomical changes. We hypothesize that ciliary alterations causing neuron differentiation defects may result in "mild" impairments of brain development, possibly underpinning certain aspects of neurodevelopmental disorders.

Embryonic genome activation (EGA) is a critical step in embryonic development. However, while EGA has been studied in mice using mouse 2-cell-like cells, human EGA remains incompletely elucidated due to the lack of an in vitro cell model recapitulating the early blastomere stage in humans. Recently, five groups independently reported human 8-cell-like cells (8CLCs, also called induced blastomere-like cells) developed from pluripotent stem cells and used single-cell RNA sequencing (scRNA-seq) to specify their cellular identities. Here we summarize the methods developed to produce the 8CLCs and compare their transcriptomic profiles by integrating them with the scRNA-seq datasets of human embryos. These observations will allow comparison and validation of the models, stimulate further in-depth research to characterize the genes involved in human EGA and pre-implantation development, and facilitate studies on human embryogenesis.

Induced pluripotent stem cell (iPSC) reprogramming is inefficient and understanding the molecular mechanisms underlying this inefficiency holds the key to successfully control cellular identity. Here, we report 24 reprogramming roadblock genes identified by CRISPR/Cas9-mediated genome-wide knockout (KO) screening. Of these, depletion of the predicted KRAB zinc finger protein (KRAB-ZFP) Zfp266 strongly and consistently enhances murine iPSC generation in several reprogramming settings, emerging as the most robust roadblock. We show that ZFP266 binds Short Interspersed Nuclear Elements (SINEs) adjacent to binding sites of pioneering factors, OCT4 (POU5F1), SOX2, and KLF4, and impedes chromatin opening. Replacing the KRAB co-suppressor with co-activator domains converts ZFP266 from an inhibitor to a potent facilitator of iPSC reprogramming. We propose that the SINE-KRAB-ZFP interaction is a critical regulator of chromatin accessibility at regulatory elements required for efficient cellular identity changes. In addition, this work serves as a resource to further illuminate molecular mechanisms hindering reprogramming.

Ovaries are central to development, fertility, and reproduction in women. A particularly interesting feature of ovaries is their accelerated ageing compared to other tissues, leading to loss of function far before other organs senesce. The limited pool of ovarian follicles is generated before birth and once exhausted, menopause will inevitably commence around the age of 50 years marking the end of fertility. Yet, there are reports suggesting the presence of germline stem cells and neo-oogenesis in adult human ovaries. These observations have fueled a long debate, created experimental fertility treatments, and opened business opportunities. Our recent analysis of cell types in ovarian cortex of women in fertile age could not find evidence of germline stem cells. Like before, our work has been met with critique suggesting methodological shortcomings. We agree that excellence starts with methods, and welcome discussion on pros and cons of different protocols. In this commentary, we discuss the recent re-interpretation of our work.

DUX4 has recently been recognized as a key regulator in human embryonic genome activation (EGA). The exact role of DUX4 in human embryo is still elusive, partly due to the cytotoxicity of persistent <italic>DUX4</italic> expression in cellular models. We report here that a transient <italic>DUX4</italic> expression in human embryonic stem cells (hESCs) retains cell viability while inducing an EGA-like expression program in a subpopulation of the cells. These cells showed resemblance to 8-cell stage blastomeres and were thus named induced blastomere-like (iBM) cells. Trajectory inference from the single-cell RNA-seq data suggested that the expression profile of these cells progressed in a manner similar to the morula to blastocyst transition in human embryo. Finally, viable iBM cells could be enriched using an antibody against NaPi2b (SLC34A2), paving the way for further experimental approaches. The iBM cells can become a powerful tool to model transcriptional dynamics and regulation during early human embryogenesis.

Conventional reprogramming methods rely on the ectopic expression of transcription factors to reprogram somatic cells into induced pluripotent stem cells (iPSCs). The forced expression of transcription factors may lead to off-target gene activation and heterogeneous reprogramming, resulting in the emergence of alternative cell types and aberrant iPSCs. Activation of endogenous pluripotency factors by CRISPR activation (CRISPRa) can reduce this heterogeneity. Here, we describe a high-efficiency reprogramming of human somatic cells into iPSCs using optimized CRISPRa. Efficient reprogramming was dependent on the additional targeting of the embryo genome activation-enriched Alu-motif and the miR-302/367 locus. Single-cell transcriptome analysis revealed that the optimized CRISPRa reprogrammed cells more directly and specifically into the pluripotent state when compared to the conventional reprogramming method. These findings support the use of CRISPRa for high-quality pluripotent reprogramming of human cells.

Many human cell types are ciliated, including neural progenitors and differentiated neurons. Ciliopathies are characterized by defective cilia and comprise various disease states, including brain phenotypes, where the underlying biological pathways are largely unknown. Our understanding of neuronal cilia is rudimentary, and an easy-to-maintain, ciliated human neuronal cell model is absent. The Lund human mesencephalic (LUHMES) cell line is a ciliated neuronal cell line derived from human fetal mesencephalon. LUHMES cells can easily be maintained and differentiated into mature, functional neurons within one week. They have a single primary cilium as proliferating progenitor cells and as postmitotic, differentiating neurons. These developmental stages are completely separable within one day of culture condition change. The sonic hedgehog (SHH) signaling pathway is active in differentiating LUHMES neurons. RNA-sequencing timecourse analyses reveal molecular pathways and gene-regulatory networks critical for ciliogenesis and axon outgrowth at the interface between progenitor cell proliferation, polarization and neuronal differentiation. Gene expression dynamics of cultured LUHMES neurons faithfully mimic the corresponding in vivo dynamics of human fetal midbrain. In LUHMES cells, neuronal cilia biology can be investigated from proliferation through differentiation to mature neurons.

Developmental dyslexia (DD) is a neurodevelopmental condition with complex genetic mechanisms. A number of candidate genes have been identified, some of which are linked to neuronal development and migration and to ciliary functions. However, expression and regulation of these genes in human brain development and neuronal differentiation remain uncharted. Here, we used human long-term self-renewing neuroepithelial stem (lt-NES, here termed NES) cells derived from human induced pluripotent stem cells to study neuronal differentiation in vitro. We characterized gene expression changes during differentiation by using RNA sequencing and validated dynamics for selected genes by qRT-PCR. Interestingly, we found that genes related to cilia were significantly enriched among upregulated genes during differentiation, including genes linked to ciliopathies with neurodevelopmental phenotypes. We confirmed the presence of primary cilia throughout neuronal differentiation. Focusing on dyslexia candidate genes, 33 out of 50 DD candidate genes were detected in NES cells by RNA sequencing, and seven candidate genes were upregulated during differentiation to neurons, including DYX1C1 (DNAAF4), a highly replicated DD candidate gene. Our results suggest a role of ciliary genes in differentiating neuronal cells and show that NES cells provide a relevant human neuronal model to study ciliary and DD candidate genes.

The human ovary orchestrates sex hormone production and undergoes monthly structural changes to release mature oocytes. The outer lining of the ovary (cortex) has a key role in defining fertility in women as it harbors the ovarian reserve. It has been postulated that putative oogonial stem cells exist in the ovarian cortex and that these can be captured by DDX4 antibody isolation. Here, we report single-cell transcriptomes and cell surface antigen profiles of over 24,000 cells from high quality ovarian cortex samples from 21 patients. Our data identify transcriptional profiles of six main cell types; oocytes, granulosa cells, immune cells, endothelial cells, perivascular cells, and stromal cells. Cells captured by DDX4 antibody are perivascular cells, not oogonial stem cells. Our data do not support the existence of germline stem cells in adult human ovaries, thereby reinforcing the dogma of a limited ovarian reserve.

Corneal endothelial cells (CECs) are essential for maintaining the clarity of the cornea. Because CECs have limited proliferative ability, interest is growing in their potentially therapeutic regeneration from pluripotent stem cells. However, the molecular mechanisms of human CEC differentiation remain largely unknown. To determine the key regulators of CEC characteristics, here we generated a comprehensive promoter-level expression profile of human CECs, using cap analysis of gene expression (CAGE) with a single molecule sequencer. Integration with the FANTOM5 promoter-level expression atlas, which includes transcriptome profiles of various human tissues and cells, enabled us to identify 45 promoters at 28 gene loci that are specifically expressed in CECs. We further discovered that the expression of transcription factor POU class 6 homeobox 2 (POU6F2) is restricted to CECs, and upregulated during human CEC differentiation, suggesting that POU6F2 is pivotal to terminal differentiation of CECs. These CEC-specific promoters would be useful for the assessment of fully differentiated CECs derived from pluripotent stem cells. These findings promote the development of corneal regenerative medicine.

Induced pluripotent stem cells (iPSCs) are generated by direct reprogramming of somatic cells and hold great promise for novel therapies. However, several studies have reported genetic variations in iPSC genomes. Here, we investigated point mutations identified by whole-genome sequencing in mouse and human iPSCs in the context of epigenetic status. In contrast to disease-causing single-nucleotide polymorphisms, de novo point mutations introduced during reprogramming were underrepresented in protein-coding genes and in open chromatin regions, including transcription factor binding sites. Instead, these mutations occurred preferentially in structurally condensed lamina-associated heterochromatic domains, suggesting that chromatin organization is a factor that can bias the regional mutation rate in iPSC genomes. Mutation signature analysis implicated oxidative stress associated with reprogramming as a likely cause of point mutations. Altogether, our study provides deeper understanding of the mutational landscape of iPSC genomes, paving an important way toward the translation of iPSC-based cell therapy.

An in vitro model of corneal epithelial cells (CECs) has been developed to study and treat corneal disorders. Nevertheless, conventional CEC culture supplemented with epidermal growth factor (EGF) results in a loss of CEC characteristics. It has recently been reported that limbal epithelial cells (LECs) cultured with keratinocyte growth factor (KGF) and the rho kinase inhibitor Y-27632 could maintain the expression of several CEC-specific markers. However, the molecular mechanism underlying the effect of culture media on LECs remains to be elucidated. To elucidate this mechanism, we performed comprehensive gene expression analysis of human LECs cultured with EGF or KGF/Y-27632, by cap analysis of gene expression (CAGE). Here, we found that LECs cultured with KGF and Y-27632 presented a gene expression profile highly similar to that of CECs in vivo. In contrast, LECs cultured with EGF lost the characteristic CEC gene expression profile. We further discovered that CEC-specific PAX6 promoters are highly activated in LECs cultured with KGF and Y-27632. Our results provide strong evidence that LECs cultured with KGF and Y-27632 would be an improved in vitro model in the context of gene expression. These findings will accelerate basic studies of CECs and clinical applications in regenerative medicine.

Induced pluripotent stem cells (iPSCs) are a type of pluripotent stem cells generated directly from mature cells through the introduction of key transcription factors. iPSCs can be propagated and differentiated into many cell types in the human body, holding enormous potential in the field of regenerative medicine. However, genomic instability of iPSCs has been reported with the advent of high-throughput technologies such as next-generation sequencing. The presence of genetic variations in iPSCs has raised serious safety concerns, hampering the advancement of iPSC-based novel therapies. Here we summarize our current knowledge on genomic instability of iPSCs, with a particular focus on types of genetic variations and their origins. Importantly, it remains elusive whether genetic variations in iPSCs can be an actual risk factor for adverse effects including malignant outgrowth. Furthermore, we discuss novel approaches to generate iPSCs with fewer genetic variations. Lastly, we outline the safety issues and monitoring strategies of iPSCs in clinical settings.

Enhancers are distal cis-regulatory DNA elements that increase the expression of target genes. Various experimental and computational approaches including chromatin signature profiling have been developed to predict enhancers on a genome-wide scale, although each method has its advantages and disadvantages. Here we overview an emerging method to identify transcribed enhancers at exceedingly high nucleotide resolution based on enhancer RNA transcripts captured by Cap Analysis of Gene Expression (CAGE) technology. We further argue that disease-causative regulatory mutations at enhancers are increasingly recognized, emphasizing the importance of enhancer identification in functional and clinical genomics including, but not limited to, genome-wide association studies (GWASs) and cancer genomics studies.

PURPOSE: To investigate the corneal topography and visual function of patients with Mooren ulcer using 3-dimensional anterior segment optical coherence tomography (3-D AS-OCT). METHODS: Fourteen eyes of 9 patients with Mooren ulcer were studied. Pachymetric and axial power maps were obtained by 3-D AS-OCT. The axial power maps were classified into 3 patterns by visual inspection. The distribution of the corneal dioptric power was analyzed by Fourier harmonic expansion. The magnitudes of the spherical component, asymmetry, regular astigmatism, higher-order irregularity, and radial distance from the corneal vertex to the thinnest point of the lesion were determined. RESULTS: The axial power maps of 9 eyes were classified into arcuate patterns, 4 into crab-claw patterns, and 1 eye into an intermediate pattern. The radial distance from the corneal vertex to the thinnest point of the lesion was significantly shorter in the crab-claw pattern group than in the arcuate pattern group (P = 0.007). The magnitudes of asymmetry, regular astigmatism, and higher-order irregularity of the crab-claw pattern group were significantly greater than those of the arcuate pattern group (P = 0.017, P = 0.011, and P = 0.030, respectively). CONCLUSIONS: Three-dimensional AS-OCT is able to evaluate the corneal topography of opacified peripheral lesions in eyes with Mooren ulcer, and the results showed that irregular astigmatism is higher when the lesion is closer to the center of the cornea.

The corneal endothelium is a monolayer of hexagonal corneal endothelial cells (CECs) on the inner surface of the cornea. CECs are critical in maintaining corneal transparency through their barrier and pump functions. CECs in vivo have a limited capacity in proliferation, and loss of a significant number of CECs results in corneal edema called bullous keratopathy which can lead to severe visual loss. Corneal transplantation is the most effective method to treat corneal endothelial dysfunction, where it suffers from donor shortage. Therefore, regeneration of CECs from other cell types attracts increasing interests, and specific markers of CECs are crucial to identify actual CECs. However, the currently used markers are far from satisfactory because of their non-specific expression in other cell types. Here, we explored molecular markers to discriminate CECs from other cell types in the human body by integrating the published RNA-seq data of CECs and the FANTOM5 atlas representing diverse range of cell types based on expression patterns. We identified five genes, CLRN1, MRGPRX3, HTR1D, GRIP1 and ZP4 as novel markers of CECs, and the specificities of these genes were successfully confirmed by independent experiments at both the RNA and protein levels. Notably none of them have been documented in the context of CEC function. These markers could be useful for the purification of actual CECs, and also available for the evaluation of the products derived from other cell types. Our results demonstrate an effective approach to identify molecular markers for CECs and open the door for the regeneration of CECs in vitro.