Dynamic Change of R-Loop Implicates in the Regulation of Zygotic Genome Activation in Mouse.

In mice, zygotic genome activation (ZGA) occurs in two steps: minor ZGA at the one-cell stage and major ZGA at the two-cell stage. Regarding the regulation of gene transcription, minor ZGA is known to have unique features, including a transcriptionally permissive state of chromatin and insufficient splicing processes. The molecular characteristics may originate from extremely open chromatin states in the one-cell stage zygotes, yet the precise underlying mechanism has not been well studied. Recently, the R-loop, a triple-stranded nucleic acid structure of the DNA/RNA hybrid, has been implicated in gene transcription and DNA replication. Therefore, in the present study, we examined the changes in R-loop dynamics during mouse zygotic development, and its roles in zygotic transcription or DNA replication. Our analysis revealed that R-loops persist in the genome of metaphase II oocytes and preimplantation embryos from the zygote to the blastocyst stage. In particular, zygotic R-loop levels dynamically change as development proceeds, showing that R-loop levels decrease as pronucleus maturation occurs. Mechanistically, R-loop dynamics are likely linked to ZGA, as inhibition of either DNA replication or transcription at the time of minor ZGA decreases R-loop levels in the pronuclei of zygotes. However, the induction of DNA damage by treatment with anticancer agents, including cisplatin or doxorubicin, does not elicit genome-wide changes in zygotic R-loop levels. Therefore, our study suggests that R-loop formation is mechanistically associated with the regulation of mouse ZGA, especially minor ZGA, by modulating gene transcription and DNA replication.

INO80 Is Required for the Cell Cycle Control, Survival, and Differentiation of Mouse ESCs by Transcriptional Regulation.

Precise regulation of the cell cycle of embryonic stem cells (ESCs) is critical for their self-maintenance and differentiation. The cell cycle of ESCs differs from that of somatic cells and is different depending on the cell culture conditions. However, the cell cycle regulation in ESCs via epigenetic mechanisms remains unclear. Here, we showed that the ATP-dependent chromatin remodeler Ino80 regulates the cell cycle genes in ESCs under primed conditions. Ino80 loss led to a significantly extended length of the G1-phase in ESCs grown under primed culture conditions. Ino80 directly bound to the transcription start site and regulated the expression of cell cycle-related genes. Furthermore, Ino80 loss induced cell apoptosis. However, the regulatory mechanism of Ino80 in differentiating ESC cycle slightly differed; an extended S-phase was detected in differentiating inducible Ino80 knockout ESCs. RNA-seq analysis of differentiating ESCs revealed that the expression of genes associated with organ development cell cycle is persistently altered in Ino80 knockout cells, suggesting that cell cycle regulation by Ino80 is not limited to undifferentiated ESCs. Therefore, our study establishes the function of Ino80 in ESC cycle via transcriptional regulation, at least partly. Moreover, this Ino80 function may be universal to other cell types.

Efficient Generation of Neural Stem Cells from Embryonic Stem Cells Using a Three-Dimensional Differentiation System.

Mouse embryonic stem cells (ESCs) are useful tools for studying early embryonic development and tissue formation in mammals. Since neural lineage differentiation is a major subject of organogenesis, the development of efficient techniques to induce neural stem cells (NSCs) from pluripotent stem cells must be preceded. However, the currently available NSC differentiation methods are complicated and time consuming. This study aimed to propose an efficient method for the derivation of NSCs from mouse ESCs; early neural lineage commitment was achieved using a three-dimensional (3D) culture system, followed by a two-dimensional (2D) NSC derivation. To select early neural lineage cell types during differentiation, Sox1-GFP transgenic ESCs were used. They were differentiated into early neural lineage, forming spherical aggregates, which were subsequently picked for the establishment of 2D NSCs. The latter showed a morphology similar to that of brain-derived NSCs and expressed NSC markers, Musashi, Nestin, N-cadherin, and Sox2. Moreover, the NSCs could differentiate into three subtypes of neural lineages, neurons, astrocytes, and oligodendrocytes. The results together suggested that ESCs could efficiently differentiate into tripotent NSCs through specification in 3D culture (for approximately 10 days) followed by 2D culture (for seven days).

Insights from the Applications of Single-Cell Transcriptomic Analysis in Germ Cell Development and Reproductive Medicine.

Mechanistic understanding of germ cell formation at a genome-scale level can aid in developing novel therapeutic strategies for infertility. Germ cell formation is a complex process that is regulated by various mechanisms, including epigenetic regulation, germ cell-specific gene transcription, and meiosis. Gonads contain a limited number of germ cells at various stages of differentiation. Hence, genome-scale analysis of germ cells at the single-cell level is challenging. Conventional genome-scale approaches cannot delineate the landscape of genomic, transcriptomic, and epigenomic diversity or heterogeneity in the differentiating germ cells of gonads. Recent advances in single-cell genomic techniques along with single-cell isolation methods, such as microfluidics and fluorescence-activated cell sorting, have helped elucidate the mechanisms underlying germ cell development and reproductive disorders in humans. In this review, the history of single-cell transcriptomic analysis and their technical advantages over the conventional methods have been discussed. Additionally, recent applications of single-cell transcriptomic analysis for analyzing germ cells have been summarized.

Anisotropic Platinum Nanoparticle-Induced Cytotoxicity, Apoptosis, Inflammatory Response, and Transcriptomic and Molecular Pathways in Human Acute Monocytic Leukemia Cells.

The thermoplasmonic properties of platinum nanoparticles (PtNPs) render them desirable for use in diagnosis, detection, therapy, and surgery. However, their toxicological effects and impact at the molecular level remain obscure. Nanotoxicology is mainly focused on the interactions of nanostructures with biological systems, particularly with an emphasis on elucidating the relationship between the physical and chemical properties such as size and shape. Therefore, we hypothesized whether these unique anisotropic nanoparticles could induce cytotoxicity similar to that of spherical nanoparticles and the mechanism involved. Thus, we synthesized unique and distinct anisotropic PtNPs using lycopene as a biological template and investigated their biological activities in model human acute monocytic leukemia (THP-1) macrophages. Exposure to PtNPs for 24 h dose-dependently decreased cell viability and proliferation. Levels of the cytotoxic markers lactate dehydrogenase and intracellular protease significantly and dose-dependently increased with PtNP concentration. Furthermore, cells incubated with PtNPs dose-dependently produced oxidative stress markers including reactive oxygen species (ROS), malondialdehyde, nitric oxide, and carbonylated protein. An imbalance in pro-oxidants and antioxidants was confirmed by significant decreases in reduced glutathione, thioredoxin, superoxide dismutase, and catalase levels against oxidative stress. The cell death mechanism was confirmed by mitochondrial dysfunction and decreased ATP levels, mitochondrial copy numbers, and PGC-1α expression. To further substantiate the mechanism of cell death mediated by endoplasmic reticulum stress (ERS), we determined the expression of the inositol-requiring enzyme (IRE1), (PKR-like ER kinase) PERK, activating transcription factor 6 (ATF6), and activating transcription factor 4 ATF4, the apoptotic markers p53, Bax, and caspase 3, and the anti-apoptotic marker Bcl-2. PtNPs could activate ERS and apoptosis mediated by mitochondria. A proinflammatory response to PtNPs was confirmed by significant upregulation of interleukin-1-beta (IL-1ß), interferon γ (IFNγ), tumor necrosis factor alpha (TNFα), and interleukin (IL-6). Transcriptomic and molecular pathway analyses of THP-1 cells incubated with the half maximal inhibitory concentration (IC50) of PtNPs revealed the altered expression of genes involved in protein misfolding, mitochondrial function, protein synthesis, inflammatory responses, and transcription regulation. We applied transcriptomic analyses to investigate anisotropic PtNP-induced toxicity for further mechanistic studies. Isotropic nanoparticles are specifically used to inhibit non-specific cellular uptake, leading to enhanced in vivo bio-distribution and increased targeting capabilities due to the higher radius of curvature. These characteristics of anisotropic nanoparticles could enable the technology as an attractive platform for nanomedicine in biomedical applications.

DOT1-like histone lysine methyltransferase is critical for adult vessel maintenance and functions.

OBJECTIVE: Disruptor of telomeric silencing 1-like (DOT1L) is the only known histone H3K79 methyltransferase essential for the development of the embryonic cardiovascular system, including the heart, blood vessels, and lymphatic vessels, through transcriptional regulation. Our previous study demonstrated that Dot1l deletion results in aberrant lymphatic development and function. However, its precise function in the postnatal cardiovascular system remains unknown. METHODS: Using conditional and inducible Dot1l knockout (KO) mice, along with a reporter strain carrying the Geo gene at the Dot1l locus, DOT1L expression and its function in the vascular system during postnatal life were investigated. To assess vessel morphology and vascular permeability, we administered Latex or Evans blue dye to KO mice. In addition, in vitro tube formation and cell migration assays were performed using DOT1L-depleted human umbilical vein endothelial cells (HUVECs). Changes in the expression of vascular genes in HUVECs were measured by quantitative polymerase chain reaction. RESULTS: Our findings demonstrate that conditional Dot1l knockout in the Tg (Tie2-cre) strain results in abnormal blood vessel formation and lymphatic anomalies in the intestine. In a mouse model of Rosa26-creER-mediated inducible Dot1l knockout, we observed vascular phenotypes, including increased vascular permeability and brain hemorrhage, when DOT1L was deleted in adulthood. Additionally, DOT1L depletion in cultured HUVECs led to impaired cell migration and tube formation, likely due to altered gene transcription. These findings highlight the essential role of DOT1L in maintaining vascular integrity and function during embryonic development and postnatal life. CONCLUSION: Our study revealed that DOT1L is required for the maintenance of adult vascular function through the regulation of gene expression.

RNA helicase DEAD-box-5 is involved in R-loop dynamics of preimplantation embryos.

OBJECTIVE: R-loops are DNA:RNA triplex hybrids, and their metabolism is tightly regulated by transcriptional regulation, DNA damage response, and chromatin structure dynamics. R-loop homeostasis is dynamically regulated and closely associated with gene transcription in mouse zygotes. However, the factors responsible for regulating these dynamic changes in the R-loops of fertilized mouse eggs have not yet been investigated. This study examined the functions of candidate factors that interact with R-loops during zygotic gene activation. METHODS: In this study, we used publicly available next-generation sequencing datasets, including low-input ribosome profiling analysis and polymerase II chromatin immunoprecipitation-sequencing (ChIP-seq), to identify potential regulators of R-loop dynamics in zygotes. These datasets were downloaded, reanalyzed, and compared with mass spectrometry data to identify candidate factors involved in regulating R-loop dynamics. To validate the functions of these candidate factors, we treated mouse zygotes with chemical inhibitors using in vitro fertilization. Immunofluorescence with an anti-R-loop antibody was then performed to quantify changes in R-loop metabolism. RESULTS: We identified DEAD-box-5 (DDX5) and histone deacetylase-2 (HDAC2) as candidates that potentially regulate R-loop metabolism in oocytes, zygotes and two-cell embryos based on change of their gene translation. Our analysis revealed that the DDX5 inhibition of activity led to decreased R-loop accumulation in pronuclei, indicating its involvement in regulating R-loop dynamics. However, the inhibition of histone deacetylase-2 activity did not significantly affect R-loop levels in pronuclei. CONCLUSION: These findings suggest that dynamic changes in R-loops during mouse zygote development are likely regulated by RNA helicases, particularly DDX5, in conjunction with transcriptional processes. Our study provides compelling evidence for the involvement of these factors in regulating R-loop dynamics during early embryonic development.

Common and distinct functions of mouse Dot1l in the regulation of endothelial transcriptome.

Epigenetic mechanisms are mandatory for endothelial called lymphangioblasts during cardiovascular development. Dot1l-mediated gene transcription in mice is essential for the development and function of lymphatic ECs (LECs). The role of Dot1l in the development and function of blood ECs blood endothelial cells is unclear. RNA-seq datasets from Dot1l-depleted or -overexpressing BECs and LECs were used to comprehensively analyze regulatory networks of gene transcription and pathways. Dot1l depletion in BECs changed the expression of genes involved in cell-to-cell adhesion and immunity-related biological processes. Dot1l overexpression modified the expression of genes involved in different types of cell-to-cell adhesion and angiogenesis-related biological processes. Genes involved in specific tissue development-related biological pathways were altered in Dot1l-depleted BECs and LECs. Dot1l overexpression altered ion transportation-related genes in BECs and immune response regulation-related genes in LECs. Importantly, Dot1l overexpression in BECs led to the expression of genes related to the angiogenesis and increased expression of MAPK signaling pathways related was found in both Dot1l-overexpressing BECs and LECs. Therefore, our integrated analyses of transcriptomics in Dot1l-depleted and Dot1l-overexpressed ECs demonstrate the unique transcriptomic program of ECs and the differential functions of Dot1l in the regulation of gene transcription in BECs and LECs.

INO80 function is required for mouse mammary gland development, but mutation alone may be insufficient for breast cancer.

The aberrant function of ATP-dependent chromatin remodeler INO80 has been implicated in multiple types of cancers by altering chromatin architecture and gene expression; however, the underlying mechanism of the functional involvement of INO80 mutation in cancer etiology, especially in breast cancer, remains unclear. In the present study, we have performed a weighted gene co-expression network analysis (WCGNA) to investigate links between INO80 expression and breast cancer sub-classification and progression. Our analysis revealed that INO80 repression is associated with differential responsiveness of estrogen receptors (ERs) depending upon breast cancer subtype, ER networks, and increased risk of breast carcinogenesis. To determine whether INO80 loss induces breast tumors, a conditional INO80-knockout (INO80 cKO) mouse model was generated using the Cre-loxP system. Phenotypic characterization revealed that INO80 cKO led to reduced branching and length of the mammary ducts at all stages. However, the INO80 cKO mouse model had unaltered lumen morphology and failed to spontaneously induce tumorigenesis in mammary gland tissue. Therefore, our study suggests that the aberrant function of INO80 is potentially associated with breast cancer by modulating gene expression. INO80 mutation alone is insufficient for breast tumorigenesis.

Production of Highly Uniform Midbrain Organoids from Human Pluripotent Stem Cells.

Brain organoids have been considered as an advanced platform for in vitro disease modeling and drug screening, but numerous roadblocks exist, such as lack of large-scale production technology and lengthy protocols with multiple manipulation steps, impeding the industrial translation of brain organoid technology. Here, we describe the high-speed and large-scale production of midbrain organoids using a high-throughput screening-compatible platform within 30 days. Micro midbrain organoids (µMOs) exhibit a highly uniform morphology and gene expression pattern with minimal variability. Notably, µMOs show dramatically accelerated maturation, resulting in the generation of functional µMOs within only 30 days of differentiation. Furthermore, individual µMOs display highly consistent responsiveness to neurotoxin, suggesting their usefulness as an in vitro high-throughput drug toxicity screening platform. Collectively, our data indicate that µMO technology could represent an advanced and robust platform for in vitro disease modeling and drug screening for human neuronal diseases.

CFP1 governs uterine epigenetic landscapes to intervene in progesterone responses for uterine physiology and suppression of endometriosis.

Progesterone (P4) is required for the preparation of the endometrium for a successful pregnancy. P4 resistance is a leading cause of the pathogenesis of endometrial disorders like endometriosis, often leading to infertility; however, the underlying epigenetic cause remains unclear. Here we demonstrate that CFP1, a regulator of H3K4me3, is required for maintaining epigenetic landscapes of P4-progesterone receptor (PGR) signaling networks in the mouse uterus. Cfp1f/f;Pgr-Cre (Cfp1d/d) mice showed impaired P4 responses, leading to complete failure of embryo implantation. mRNA and chromatin immunoprecipitation sequencing analyses showed that CFP1 regulates uterine mRNA profiles not only in H3K4me3-dependent but also in H3K4me3-independent manners. CFP1 directly regulates important P4 response genes, including Gata2, Sox17, and Ihh, which activate smoothened signaling pathway in the uterus. In a mouse model of endometriosis, Cfp1d/d ectopic lesions showed P4 resistance, which was rescued by a smoothened agonist. In human endometriosis, CFP1 was significantly downregulated, and expression levels between CFP1 and these P4 targets are positively related regardless of PGR levels. In brief, our study provides that CFP1 intervenes in the P4-epigenome-transcriptome networks for uterine receptivity for embryo implantation and the pathogenesis of endometriosis.

Epigenetic Factors as Etiological Agents, Diagnostic Markers, and Therapeutic Targets for Luminal Breast Cancer.

Luminal breast cancer, an etiologically heterogeneous disease, is characterized by high steroid hormone receptor activity and aberrant gene expression profiles. Endocrine therapy and chemotherapy are promising therapeutic approaches to mitigate breast cancer proliferation and recurrence. However, the treatment of therapy-resistant breast cancer is a major challenge. Recent studies on breast cancer etiology have revealed the critical roles of epigenetic factors in luminal breast cancer tumorigenesis and drug resistance. Tumorigenic epigenetic factor-induced aberrant chromatin dynamics dysregulate the onset of gene expression and consequently promote tumorigenesis and metastasis. Epigenetic dysregulation, a type of somatic mutation, is a high-risk factor for breast cancer progression and therapy resistance. Therefore, epigenetic modulators alone or in combination with other therapies are potential therapeutic agents for breast cancer. Several clinical trials have analyzed the therapeutic efficacy of potential epi-drugs for breast cancer and reported beneficial clinical outcomes, including inhibition of tumor cell adhesion and invasiveness and mitigation of endocrine therapy resistance. This review focuses on recent findings on the mechanisms of epigenetic factors in the progression of luminal breast cancer. Additionally, recent findings on the potential of epigenetic factors as diagnostic biomarkers and therapeutic targets for breast cancer are discussed.

Developmental Potency and Metabolic Traits of Extended Pluripotency Are Faithfully Transferred to Somatic Cells via Cell Fusion-Induced Reprogramming.

As a novel cell type from eight-cell-stage embryos, extended pluripotent stem cells (EPSCs) are known for diverse differentiation potency in both extraembryonic and embryonic lineages, suggesting new possibilities as a developmental research model. Although various features of EPSCs have been defined, their ability to directly transfer extended pluripotency to differentiated somatic cells by cell fusion remains to be elucidated. Here, we derived EPSCs from eight-cell mouse embryos and confirmed their extended pluripotency at the molecular level and extraembryonic differentiation ability. Then, they were fused with OG2+/- ROSA+/- neural stem cells (NSCs) by the polyethylene-glycol (PEG)-mediated method and further analyzed. The resulting fused hybrid cells exhibited pluripotential markers with upregulated EPSC-specific gene expression. Furthermore, the hybrid cells contributed to the extraembryonic and embryonic lineages in vivo and in vitro. RNA sequencing analysis confirmed that the hybrid cells showed distinct global expression patterns resembling EPSCs without parental expression of NSC markers, indicating the complete acquisition of extended pluripotency and the erasure of the somatic memory of NSCs. Furthermore, ultrastructural observation and metabolic analysis confirmed that the hybrid cells rearranged the mitochondrial morphology and bivalent metabolic profile to those of EPSCs. In conclusion, the extended pluripotency of EPSCs could be transferred to somatic cells through fusion-induced reprogramming.

Role of Transcriptional and Epigenetic Regulation in Lymphatic Endothelial Cell Development.

The lymphatic system is critical for maintaining the homeostasis of lipids and interstitial fluid and regulating the immune cell development and functions. Developmental anomaly-induced lymphatic dysfunction is associated with various pathological conditions, including lymphedema, inflammation, and cancer. Most lymphatic endothelial cells (LECs) are derived from a subset of endothelial cells in the cardinal vein. However, recent studies have reported that the developmental origin of LECs is heterogeneous. Multiple regulatory mechanisms, including those mediated by signaling pathways, transcription factors, and epigenetic pathways, are involved in lymphatic development and functions. Recent studies have demonstrated that the epigenetic regulation of transcription is critical for embryonic LEC development and functions. In addition to the chromatin structures, epigenetic modifications may modulate transcriptional signatures during the development or differentiation of LECs. Therefore, the understanding of the epigenetic mechanisms involved in the development and function of the lymphatic system can aid in the management of various congenital or acquired lymphatic disorders. Future studies must determine the role of other epigenetic factors and changes in mammalian lymphatic development and function. Here, the recent findings on key factors involved in the development of the lymphatic system and their epigenetic regulation, LEC origins from different organs, and lymphatic diseases are reviewed.

Epigenetic regulator Cfp1 safeguards male meiotic progression by regulating meiotic gene expression.

Meiosis occurs specifically in germ cells to produce sperm and oocytes that are competent for sexual reproduction. Multiple factors are required for successful meiotic entry, progression, and termination. Among them, trimethylation of histone H3 on lysine 4 (H3K4me3), a mark of active transcription, has been implicated in spermatogenesis by forming double-strand breaks (DSBs). However, the role of H3K4me in transcriptional regulation during meiosis remains poorly understood. Here, we reveal that mouse CXXC finger protein 1 (Cfp1), a component of the H3K4 methyltransferase Setd1a/b, is dynamically expressed in differentiating male germ cells and safeguards meiosis by controlling gene expression. Genetic ablation of mouse CFP1 in male germ cells caused complete infertility with failure in prophase I of the 1st meiosis. Mechanistically, CFP1 binds to genes essential for spermatogenesis, and its loss leads to a reduction in H3K4me3 levels and gene expression. Importantly, CFP1 is highly enriched within the promoter/TSS of target genes to elevate H3K4me3 levels and gene expression at the pachytene stage of meiotic prophase I. The most enriched genes were associated with meiosis and homologous recombination during the differentiation of spermatocytes to round spermatids. Therefore, our study establishes a mechanistic link between CFP1-mediated transcriptional control and meiotic progression and might provide an unprecedented genetic basis for understanding human sterility.

Genome stabilization by RAD51-stimulatory compound 1 enhances efficiency of somatic cell nuclear transfer-mediated reprogramming and full-term development of cloned mouse embryos.

OBJECTIVES: The genetic instability and DNA damage arise during transcription factor-mediated reprogramming of somatic cells, and its efficiency may be reduced due to abnormal chromatin remodelling. The efficiency in somatic cell nuclear transfer (SCNT)-mediated reprogramming is also very low, and it is caused by development arrest of most reconstituted embryos. MATERIALS AND METHODS: Whether the repair of genetic instability or double-strand breaks (DSBs) during SCNT reprogramming may play an important role in embryonic development, we observed and analysed the effect of Rad 51, a key modulator of DNA damage response (DDR) in SCNT-derived embryos. RESULTS: Here, we observed that the activity of Rad 51 is lower in SCNT eggs than in conventional IVF and found a significantly lower level of DSBs in SCNT embryos during reprogramming. To address this difference, supplementation with RS-1, an activator of Rad51, during the activation of SCNT embryos can increase RAD51 expression and DSB foci and thereby increased the efficiency of SCNT reprogramming. Through subsequent single-cell RNA-seq analysis, we observed the reactivation of a large number of genes that were not expressed in SCNT-2-cell embryos by the upregulation of DDR, which may be related to overcoming the developmental block. Additionally, there may be an independent pathway involving histone demethylase that can reduce reprograming-resistance regions. CONCLUSIONS: This technology can contribute to the production of comparable cell sources for regenerative medicine.

ATP-Dependent Chromatin Remodeler CHD9 Controls the Proliferation of Embryonic Stem Cells in a Cell Culture Condition-Dependent Manner.

Emerging evidence suggests that chromodomain-helicase-DNA-binding (CHD) proteins are involved in stem cell maintenance and differentiation via the coordination of chromatin structure and gene expression. However, the molecular function of some CHD proteins in stem cell regulation is still poorly understood. Herein, we show that Chd9 knockdown (KD) in mouse embryonic stem cells (ESCs) cultured in normal serum media, not in 2i-leukemia inhibitory factor (LIF) media, causes rapid cell proliferation. This is caused by transcriptional regulation related to the cell cycle and the response to growth factors. Our analysis showed that, unlike the serum cultured-Chd9 KD ESCs, the 2i-LIF-cultured-Chd9 KO ESCs displayed elevated levels of critical G1 phase regulators such as p21 and p27. Consistently, the DNA binding sites of CHD9 overlap with some transcription factor DNA motifs that are associated with genes regulating the cell cycle and growth pathways. These transcription factors include the cycle gene homology region (CHR), Arid5a, and LIN54. Collectively, our results provide new insights into CHD9-mediated gene transcription for controlling the cell cycle of ESCs.

Role of mitochondrial fission-related genes in mitochondrial morphology and energy metabolism in mouse embryonic stem cells.

Mitochondria, the major organelles that produce energy for cell survival and function, dynamically change their morphology via fusion and fission, a process called mitochondrial dynamics. The details of the underlying mechanism of mitochondrial dynamics have not yet been elucidated. Here, we aimed to investigate the function of mitochondrial fission genes in embryonic stem cells (ESCs). To this end, we generated homozygous knockout ESC lines, namely, Fis1-/-, Mff-/-, and Dnm1l-/- ESCs, using the CRISPR-Cas9 system. Interestingly, the Fis1-/-, Mff-/-, and Dnm1l-/- ESCs showed normal morphology, self-renewal, and the ability to differentiate into all three germ layers in vitro. However, transmission electron microscopy showed a significant increase in the cytoplasm to nucleus ratio and mitochondrial elongation in Dnm1l-/- ESCs, which was due to incomplete fission. To assess the change in metabolic energy, we analyzed oxidative phosphorylation (OXPHOS), glycolysis, and the intracellular ATP concentration. The ESC knockout lines showed an increase in OXPHOS, decrease in glycolysis, and an increase in intracellular ATP concentration, which was related to mitochondrial elongation. In particular, the Dnm1l knockout most significantly affected mitochondrial morphology, energy metabolism, and ATP production in ESCs. Furthermore, RNA sequencing and gene ontology analysis showed that the differentially expressed genes in Mff-/- ESCs were distinct from those in Dnm1l-/- or Fis1-/- ESCs. In total, five metabolism-related genes, namely, Aass, Cdo1, Cyp2b23, Nt5e, and Pck2, were expressed in all three knockout ESC lines, and three of them were associated with regulation of ATP generation.

Robust and Reproducible Generation of Induced Neural Stem Cells from Human Somatic Cells by Defined Factors.

BACKGROUND AND OBJECTIVES: Recent studies have described direct reprogramming of mouse and human somatic cells into induced neural stem cells (iNSCs) using various combinations of transcription factors. Although iNSC technology holds a great potential for clinical applications, the low conversion efficiency and limited reproducibility of iNSC generation hinder its further translation into the clinic, strongly suggesting the necessity of highly reproducible method for human iNSCs (hiNSCs). Thus, in orderto develop a highly efficient and reproducible protocol for hiNSC generation, we revisited the reprogramming potentials of previously reported hiNSC reprogramming cocktails by comparing the reprogramming efficiency of distinct factor combinations including ours. METHODS: We introduced distinct factor combinations, OSKM (OCT4+SOX2+KLF4+C-MYC), OCT4 alone, SOX2 alone, SOX2+HMGA2, BRN4+SKM+SV40LT (BSKMLT), SKLT, SMLT, and SKMLT and performed comparative analysis of reprogramming potentials of distinct factor combinations in hiNSC generation. RESULTS: Here we show that ectopic expression of five reprogramming factors, BSKMLT leads the robust hiNSC generation (>80 folds enhanced efficiency) from human somatic cells compared with previously described factor combinations. With our combination, we were able to observe hiNSC conversion within 7 days of transduction. Throughout further optimization steps, we found that both BRN4 and KLF4 are not essential for hiNSC conversion. CONCLUSIONS: Our factor combination could robustly and reproducibly generate hiNSCs from human somatic cells with distinct origins. Therefore, our novel reprogramming strategy might serve as a useful tool for hiNSC-based clinical application.

Comparative analysis of the mitochondrial morphology, energy metabolism, and gene expression signatures in three types of blastocyst-derived stem cells.

Pre-implantation mouse blastocyst-derived stem cells, namely embryonic stem cells (ESCs), trophoblast stem cells (TSCs), and extraembryonic endoderm (XEN) cells, have their own characteristics and lineage specificity. So far, several studies have attempted to identify these three stem cell types based on genetic markers, morphologies, and factors involved in maintaining cell self-renewal. In this study, we focused on characterizing the three stem cell types derived from mouse blastocysts by observing cellular organelles, especially the mitochondria, and analyzing how mitochondrial dynamics relates to the energy metabolism in each cell type. Our study revealed that XEN cells have distinct mitochondrial morphology and energy metabolism compared with that in ESCs and TSCs. In addition, by analyzing the energy metabolism (oxygen consumption and extracellular acidification rates), we demonstrated that differences in the mitochondria affect the cellular metabolism in the stem cells. RNA sequencing analysis showed that although ESCs are developmentally closer to XEN cells in origin, their gene expression pattern is relatively closer to that of TSCs. Notably, mitochondria-, mitochondrial metabolism-, transport/secretory action-associated genes were differentially expressed in XEN cells compared with that in ESCs and TSCs, and this feature corresponds with the morphology of the cells.