Tatsuyuki Takada

PRDM14 promotes active DNA demethylation through the Ten-eleven translocation (TET)-mediated base excision repair pathway in embryonic stem cells

Ten-eleven translocation (TET) proteins oxidize 5-methylcytosine (5mC) to 5-hydroxymethylcytosine (5hmC), 5-formylcytosine (5fC) and 5-carboxylcytosine (5caC). 5fC and 5caC can be excised and repaired by the base excision repair (BER) pathway, implicating 5mC oxidation in active DNA demethylation. Genome-wide DNA methylation is erased in the transition from metastable states to the ground state of embryonic stem cells (ESCs) and in migrating primordial germ cells (PGCs), although some resistant regions become demethylated only in gonadal PGCs. Understanding the mechanisms underlying global hypomethylation in naive ESCs and developing PGCs will be useful for realizing cellular pluripotency and totipotency. In this study, we found that PRDM14, the PR domain-containing transcriptional regulator, accelerates the TET-BER cycle, resulting in the promotion of active DNA demethylation in ESCs. Induction of Prdm14 expression transiently elevated 5hmC, followed by the reduction of 5mC at pluripotency-associated genes, germline-specific genes and imprinted loci, but not across the entire genome, which resembles the second wave of DNA demethylation observed in gonadal PGCs. PRDM14 physically interacts with TET1 and TET2 and enhances the recruitment of TET1 and TET2 at target loci. Knockdown of TET1 and TET2 impaired transcriptional regulation and DNA demethylation by PRDM14. The repression of the BER pathway by administration of pharmacological inhibitors of APE1 and PARP1 and the knockdown of thymine DNA glycosylase (TDG) also impaired DNA demethylation by PRDM14. Furthermore, DNA demethylation induced by PRDM14 takes place normally in the presence of aphidicolin, which is an inhibitor of G1/S progression. Together, our analysis provides mechanistic insight into DNA demethylation in naive pluripotent stem cells and developing PGCs.

Monkey embryonic stem cell lines expressing green fluorescent protein.

The major limitation of nonhuman primate (NHP) embryonic stem (ES) cell research is inefficient genetic modification and limited knowledge of differentiation mechanisms. A genetically modified NHP-ES cell with biomarkers, such as green fluorescent protein (GFP), that allow noninvasive monitoring of transgenic cells, is a useful tool to study cell differentiation control during preimplantation and fetal development, which also plays a crucial role in the development of cell transplantation medicine. Here we report the establishment of transgenic NHP-ES cell lines that express GFP without jeopardizing their pluripotency, which was confirmed by in vitro and in vivo differentiation. These GFP-expressing ES cells reproducibly differentiated into embryoid bodies, neural cells, and cardiac myocytes. They formed teratoma composed of tissues derived from the three embryonic germ layers when transplanted into severe combined immunodeficient disease (SCID) mice. GFP expression was maintained in these differentiated cells, suggesting that these cells were useful for cell transplantation experiments. Furthermore, we showed that these ES cells have the ability to form chimeric blastocysts by introducing into the early preimplantation stage NHP embryo.

Transient suppression of PPARγ directed ES cells into an osteoblastic lineage

Osteoblasts and adipocytes are believed to share a common progenitor. Peroxisome proliferator‐activated receptor γ (PPARγ) plays a key role in the switching of these two cell lineages. Here, we demonstrated the differentiation of ES cells into an osteoblastic lineage using siRNA against PPARγ without the addition of any osteogenic factors. We found that PPARγ‐siRNA downregulated the expression of aP2 mRNA and lipid accumulation, whereas it upregulated the expression of osteocalcin and calcium deposition. These results suggested that ES cells were directed into an osteoblastic lineage. Therefore, transient suppression using PPARγ‐siRNA may be a novel tool to induce differentiation of ES cells into osteoblasts.

Bisphenol A modulates germ cell differentiation and retinoic acid signaling in mouse ES cells.

Bisphenol A (BPA) has been reported to have an adverse effect on mammalian reproduction and recognized as an endocrine disruptor. However, the molecular mechanism that causes impaired development of germ cells remains elusive. In this study, we investigated the effect of BPA using in vitro differentiation of embryonic stem (ES) cells focusing on the expression of germ cell marker genes. We found that the BPA-treated embryoid bodies (EBs), exhibited the most prominent up-regulation of meiotic entry gene Stra8 and induction mechanism appeared to be different from that of retinoic acid. Localization of aggregated Sycp3 signal in nuclei, characteristic to leptotene of meiosis, was also detected. In addition, up-regulation of ovarian markers (Foxl2 and Wnt4) and suppression of testicular markers (Sox9 and Fgf9) were observed. These results suggest that BPA might affect testicular and ovarian development as well as germ cell differentiation, and appears to induce genes responsible for ovary development.

BMP4 Induces Primitive Endoderm But Not Trophectoderm in Monkey Embryonic Stem Cells

Monkey embryonic stem (ES) cells share similar characteristics to human ES cells and provide a primate model of allotransplantation, which allows to validate efficacy and safety of cell transplantation therapy in regenerative medicine. Bone morphogenetic protein 4 (BMP4) is known to promote trophoblast differentiation in human ES cells in contrast to mouse ES cells where BMP4 synergistically maintains self-renewal with leukemia inhibitory factor (LIF), which represents a significant difference in signal transduction of self-renewal and differentiation between murine and human ES cells. As the similarity of the differentiation mechanism between monkey and human ES cells is of critical importance for their use as a primate model system, we investigated whether BMP4 induces trophoblast differentiation in monkey ES cells. Interestingly, BMP4 did not induce trophoblast differentiation, but instead induced primitive endoderm differentiation. Prominent downregulation of Sox2, which plays a pivotal role not only in pluripotency but also placenta development, was observed in cells treated with BMP4. In addition, upregulation of Hand1, Cdx2, and chorionic gonadotropin beta (CG-beta), which are markers of trophoblast, was not observed. In contrast, BMP4 induced significant upregulation of Gata6, Gata4, and LamininB1, suggesting differentiation into the primitive endoderm, visceral endoderm, and parietal endoderm, respectively. The threshold of BMP4 activity was estimated as about 10 ng/mL. These findings suggest that BMP4 induced differentiation into the primitive endoderm lineage but not into trophoblast in monkey ES cells.

DNA methylation dynamics in mouse preimplantation embryos revealed by mass spectrometry

Following fertilization in mammals, paternal genomic 5-methyl-2′-deoxycytidine (5 mC) content is thought to decrease via oxidation to 5-hydroxymethyl-2′-deoxycytidine (5 hmC). This reciprocal model of demethylation and hydroxymethylation is inferred from indirect, non-quantitative methods. We here report direct quantification of genomic 5 mC and 5 hmC in mouse embryos by small scale liquid chromatographic tandem mass spectrometry (SMM). Profiles of absolute 5 mC levels in embryos produced by in vitro fertilization (IVF) and intracytoplasmic sperm injection (ICSI) were almost identical. By 10 h after fertilization, 5 mC levels had declined by ~40%, consistent with active genomic DNA demethylation. Levels of 5 mC in androgenotes (containing only a paternal genome) and parthenogenotes (containing only a maternal genome) underwent active 5 mC loss in the first 6 h, showing that both parental genomes can undergo demethylation independently. We found no evidence for net loss of 5 mC 10–48 h after fertilization, implying that any passive ‘demethylation’ following DNA replication was balanced by active 5 mC maintenance methylation. However, levels of 5 mC declined during development after 48 h, to 1% (measured as a fraction of G-residues) in blastocysts (~96 h). 5 hmC levels were consistently low (<0.2% of G-residues) throughout development in normal diploid embryos. This work directly quantifies the dynamics of global genomic DNA modification in mouse preimplantation embryos, suggesting that SMM will be applicable to other biomedical situations with limiting sample sizes.

Molecular cloning of BNP from heart and its immunohistochemical localization in the hypothalamus of monkey.

Previous physiological studies have suggested central roles of brain natriuretic peptide (BNP). However, little information is available about the localization of BNP in the brain. In this study, we determined cDNA sequence encoding the entire coding region of prepro-BNP of Japanese and cynomologus monkeys, and then examined the immunohistochemical localization of BNP in the monkey hypothalamus. Japanese and cynomologus monkey prepro-BNP consisted of 132 amino acid residues with biologically active C-terminal 32 amino acids. Comparisons of deduced amino acid sequences among different species revealed high homology between monkey and human (91% in prerpro-BNP and 97% in the mature region). Immunohistochemical examination showed that BNP immunoreactive dots were observed in the paraventricular, periventricular, and supraoptic nuclei of the monkey hypothalamus. The present result suggests the central role of BNP in the neuroendocrine system in the hypothalamus.

Evaluation of heterologous insulator function with regard to chromosomal position effect in the mouse blastocyst and fetus

Insulators are located at the boundaries of differentially regulated genes and delimit their interactions by establishing independent chromatin structures. Recently, an insulator sequence has been found in the 5′‐flanking region of arylsulfatase (ARS) gene from sea urchin. To investigate functional conservation of this ARS insulator in mice, we performed blastocyst assays to evaluate the effect of this insulator on the chromosomal position effect, quantitatively. We constructed transgenes that have a luciferase gene under the control of the CMV‐IE enhancer and the human elongation factor 1 α promoter in the presence or absence of the ARS insulator in both flanking regions. These transgenes were microinjected into 1‐cell mouse embryos and luciferase activity was measured at the blastocyst stage. We found that the presence of ARS insulator sequence doubled the number of luciferase‐expressing blastocysts, and that the proportion of the blastocysts with high‐level expression (≥ 1 × 104 relative light units (RLU)) was increased more than tenfold. In the case of transgenic fetuses, however, the presence of ARS insulator did not seem to improve transgene expression. These results suggest that the sea urchin ARS insulator confers position‐independent expression driven by the human elongation factor 1 α promoter, at least in the blastocyst stage of the mouse. Mol. Reprod. Dev. 57:232–237, 2000.

Magnetic resonance imaging using hemagglutinating virus of Japan-envelope vector successfully detects localization of intra-cardially administered microglia in normal mouse brain

Although the therapeutic use of microglia has received some attention for the treatment of brain diseases, few non-invasive techniques exist for monitoring the cells after administration. Here, we present a technique using magnetic resonance imaging (MRI) to track microglia injected intra-cardially. We labeled microglia expressing enhanced green fluorescent protein with superparamagnetic iron oxide (Resovist) using the hemagglutinating virus of Japan-envelope vector. We injected labeled microglia into the left ventricle of the heart of mice. After monitoring exogenously administered microglia in the mouse brain in vivo using T(2)*-weighted MRI at a magnetic field of 7T, we compared the MR images with histochemical localization of exogenous microglia in vitro. MRI revealed clear signal changes attributable to Resovist-containing microglia in the mouse brain. Histochemistry demonstrated the presence of exogenous microglia in the brain at the same locations shown by MRI. This study demonstrates the usefulness of MRI for non-invasive monitoring of exogenous microglia, and suggests a promising future for microglia/macrophages as therapeutic tools for brain disease.

Immunohistochemical mapping of natriuretic peptide receptor-A in the brainstem of Macaca fascicularis

Natriuretic peptide receptor-A (NPR-A) mediates the biological effects of atrial natriuretic peptide (ANP) and brain natriuretic peptide (BNP), and is involved in maintaining cardiovascular homeostasis. In this immunohistochemical study we examined the distribution of NPR-A in the brainstem of the cynomolgus monkey. NPR-A immunoreactivity was localized to neurons in specific brainstem regions. NPR-A-immunoreactive perikarya were found in the red nucleus and the oculomotor nucleus in the midbrain, the parabrachial nucleus and the locus coeruleus in the pons, and the dorsal motor nucleus of the vagus, the hypoglossal nucleus, the cuneate nucleus, the gracile nucleus, the nucleus ambiguus, the lateral reticular nucleus, the reticular formation, and the inferior olivary nucleus in the medulla oblongata. Extensive networks of immunoreactive fibers were apparent in the red nucleus, the oculomotor nucleus, the principal sensory trigeminal nucleus, and the parabrachial nucleus. Double immunostaining revealed NPR-A immunoreactivity in cholinergic neurons of the parabrachial nucleus, the dorsal motor nucleus of vagus, the hypoglossal nucleus, and the nucleus ambiguus. However, there was no colocalization of NPR-A and tyrosine hydroxylase in the locus coeruleus. The wide anatomical distribution of NPR-A-immunoreactive structures suggests that natriuretic peptides, besides having a role in the central regulation of endocrine and cardiovascular homeostasis, may also mediate diverse physiological functions.