Conserved and divergent features of DNA methylation in embryonic stem cell-derived neurons

AbstractDNA methylation functions in genome regulation and is implicated in neuronal maturation. Early post-natal accumulation of atypical non-CG methylation (mCH) occurs in neurons of mice and humans, but its precise function remains unknown. Here we investigate mCH deposition in neurons derived from mouse ES-cells in vitro and in cultured primary mouse neurons. We find that both acquire comparable levels of mCH over a similar period as in vivo. In vitro mCH deposition occurs concurrently with a transient increase in Dnmt3a expression, is preceded by expression of the post-mitotic neuronal marker Rbfox3 (NeuN) and is enriched at the nuclear lamina. Despite these similarities, whole genome bisulfite sequencing reveals that mCH patterning in mESC-derived neurons partially differs from in vivo. mESC-derived neurons therefore represent a valuable model system for analyzing the mechanisms and functional consequences of correct and aberrantly deposited CG and non-CG methylation in neuronal maturation.

Download Full-text

220 NONHUMAN PRIMATE EMBRYONIC STEM CELLS SIMILAR TO THE BIOLOGICAL PROPERTIES OF MOUSE EMBRYONIC STEM CELLS

Reproduction Fertility and Development ◽

10.1071/rdv24n1ab220 ◽

2012 ◽

Vol 24 (1) ◽

pp. 222

Author(s):

A. Kusanagi ◽

J. Yamasaki ◽

C. Iwatani ◽

H. Tsuchiya ◽

R. Torii

Keyword(s):

Nonhuman Primate ◽

Cynomolgus Monkey ◽

Es Cells ◽

Embryonic Stem ◽

Biological Properties ◽

Es Cell ◽

Mouse Es Cells ◽

Mouse Es Cell

Human and mouse embryonic stem (ES) cells are derived from the inner cell mass of preimplantation blastocysts and human ES cells were long thought to be equivalent to mouse ES cells, despite clear morphological difference and different signalling pathways to maintain their pluripotency between these two ES cell types. Mouse ES cells depend on leukemia inhibitory factor (LIF) and bone morphogenic protein 4 (BMP4) signalling, whereas their human counterparts rely on basic fibroblast growth factor (bFGF) and activin A signalling. The biggest difference of two ES cells is the ability of chimera formation and mouse ES cells can contribute chimera but primate ES cells fails to do that. Monkey ES cells in primates only can be tested for chimera formation in vivo due to the ethical issue and cynomolgus monkey is the most common nonhuman primate to be used for the safety study of drug discoveries. The objective of this study was to develop novel cynomolgus monkey ES cells that have similar biological properties with mouse ES cell and our ultimate goal is to establish germline competent nonhuman primate ES cells. Ovarian stimulation and oocyte collection were carried out for the derivation of ES cells as previously described by Torii et al. Briefly, GnRH (0.9 mg/head) was administered to cynomolgus monkey and two weeks later, a micro infusion pump (iPRECIO™, Primetech Corp) contains FSH was implanted subcutaneously. Follicular aspiration was then performed 40 h after hCG injection and metaphase II oocytes were fertilized by intracytoplasmic sperm injection (ICSI). Cynomolgus monkey ES cells were then established under mouse ES cell conditions such as LIF/STAT signalling and a dome tree-dimensional (3D) morphology nonhuman primate ES cells were selected. On the other hands, ES cells that were established with the presence of basic FGF showed conventional layer-type morphology. Dome-type ES cells express pluripotent transcriptional factors such as Oct-3/4, Nonog and Sox2 as same as layer-type ES cells and both ES lines were capable of multilineage differentiations in vitro after embryoid body formation. Dome-type nonhuman ES cells can also form teratomas and differentiated into all three germ layers when grafted into immunodeficiency mice. For fluorescent gene delivery to nonhuman primate ES cells, feeder-free condition was applied and CAG-GFP vector was transfected into ES cells using Neon electroporation system (Invitrogen Inc.) for the tracing ES cells in the transplantation study. In this study, we have established dome-type ES cell lines that similar to mouse ES cells in morphology and signalling pathway. Dome-type nonhuman primate ES cells express pluripotent gene markers and prove their pluripotency both of in vitro and in vivo, in addition, these modifications would be important to create germline competent ES cells.

Download Full-text

Disabling de novo DNA methylation in embryonic stem cells allows an illegitimate fate trajectory

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.2109475118 ◽

2021 ◽

Vol 118 (38) ◽

pp. e2109475118

Author(s):

Masaki Kinoshita ◽

Meng Amy Li ◽

Michael Barber ◽

William Mansfield ◽

Sabine Dietmann ◽

...

Keyword(s):

Dna Methylation ◽

Cell Fate ◽

De Novo ◽

Es Cells ◽

Embryonic Stem ◽

Dna Methyltransferases ◽

Bhlh Transcription Factor ◽

Mammalian Development

Genome remethylation is essential for mammalian development but specific reasons are unclear. Here we examined embryonic stem (ES) cell fate in the absence of de novo DNA methyltransferases. We observed that ES cells deficient for both Dnmt3a and Dnmt3b are rapidly eliminated from chimeras. On further investigation we found that in vivo and in vitro the formative pluripotency transition is derailed toward production of trophoblast. This aberrant trajectory is associated with failure to suppress activation of Ascl2. Ascl2 encodes a bHLH transcription factor expressed in the placenta. Misexpression of Ascl2 in ES cells provokes transdifferentiation to trophoblast-like cells. Conversely, Ascl2 deletion rescues formative transition of Dnmt3a/b mutants and improves contribution to chimeric epiblast. Thus, de novo DNA methylation safeguards against ectopic activation of Ascl2. However, Dnmt3a/b-deficient cells remain defective in ongoing embryogenesis. We surmise that multiple developmental transitions may be secured by DNA methylation silencing potentially disruptive genes.

Download Full-text

Brachyury - a gene affecting mouse gastrulation and early organogenesis

Development ◽

10.1242/dev.116.supplement.157 ◽

1992 ◽

Vol 116 (Supplement) ◽

pp. 157-165 ◽

Cited By ~ 20

Author(s):

R. S. P. Beddington ◽

P. Rashbass ◽

V. Wilson

Keyword(s):

Es Cells ◽

Embryonic Stem ◽

Primitive Streak ◽

Coat Colour ◽

Es Cell ◽

Wild Type ◽

Mesoderm Cell ◽

Rostrocaudal Axis

Mouse embryos that are homozygous for the Brachyury (T) deletion die at mid-gestation. They have prominent defects in the notochord, the allantois and the primitive streak. Expression of the T gene commences at the onset of gastrulation and is restricted to the primitive streak, mesoderm emerging from the streak, the head process and the notochord. Genetic evidence has suggested that there may be an increasing demand for T gene function along the rostrocaudal axis. Experiments reported here indicate that this may not be the case. Instead, the gradient in severity of the T defect may be caused by defective mesoderm cell movements, which result in a progressive accumulation of mesoderm cells near the primitive streak. Embryonic stem (ES) cells which are homozygous for the T deletion have been isolated and their differentiation in vitro and in vivo compared with that of heterozygous and wild-type ES cell lines. In +/+ ↔ T/T ES cell chimeras the Brachyury phenotype is not rescued by the presence of wild-type cells and high level chimeras show most of the features characteristic of intact T/T mutants. A few offspring from blastocysts injected with T/T ES cells have been born, several of which had greatly reduced or abnormal tails. However, little or no ES cell contribution was detectable in these animals, either as coat colour pigmentation or by isozyme analysis. Inspection of potential +/+ ↔ T/T ES cell chimeras on the 11th or 12th day of gestation, stages later than that at which intact T/T mutants die, revealed the presence of chimeras with caudal defects. These chimeras displayed a gradient of ES cell colonisation along the rostrocaudal axis with increased colonisation of caudal regions. In addition, the extent of chimerism in ectodermal tissues (which do not invaginate during gastrulation) tended to be higher than that in mesodermal tissues (which are derived from cells invaginating through the primitive streak). These results suggest that nascent mesoderm cells lacking the T gene are compromised in their ability to move away from the primitive streak. This indicates that one function of the T genemay be to regulate cell adhesion or cell motility properties in mesoderm cells. Wild-type cells in +/+ ↔ T/T chimeras appear to move normally to populate trunk and head mesoderm, suggesting that the reduced motility in T/T cells is a cell autonomous defect

Download Full-text

Generation and Characterization of Smac/DIABLO-Deficient Mice

Molecular and Cellular Biology ◽

10.1128/mcb.22.10.3509-3517.2002 ◽

2002 ◽

Vol 22 (10) ◽

pp. 3509-3517 ◽

Cited By ~ 120

Author(s):

Hitoshi Okada ◽

Woong-Kyung Suh ◽

Jianping Jin ◽

Minna Woo ◽

Chunying Du ◽

...

Keyword(s):

Physiological Function ◽

Es Cells ◽

Embryonic Stem ◽

Caspase Activation ◽

Proapoptotic Protein ◽

Apoptosis Proteins ◽

Deficient Mice

ABSTRACT The mitochondrial proapoptotic protein Smac/DIABLO has recently been shown to potentiate apoptosis by counteracting the antiapoptotic function of the inhibitor of apoptosis proteins (IAPs). In response to apoptotic stimuli, Smac is released into the cytosol and promotes caspase activation by binding to IAPs, thereby blocking their function. These observations have suggested that Smac is a new regulator of apoptosis. To better understand the physiological function of Smac in normal cells, we generated Smac-deficient (Smac−/− ) mice by using homologous recombination in embryonic stem (ES) cells. Smac−/− mice were viable, grew, and matured normally and did not show any histological abnormalities. Although the cleavage in vitro of procaspase-3 was inhibited in lysates of Smac−/− cells, all types of cultured Smac−/− cells tested responded normally to all apoptotic stimuli applied. There were also no detectable differences in Fas-mediated apoptosis in the liver in vivo. Our data strongly suggest the existence of a redundant molecule or molecules capable of compensating for a loss of Smac function.

Download Full-text

Induction of Recurrent Break Cluster Genes in Neural Progenitor Cells Differentiated from Embryonic Stem Cells In Culture

10.1101/2019.12.30.891168 ◽

2019 ◽

Author(s):

Aseda Tena ◽

Yuxiang Zhang ◽

Nia Kyritsis ◽

Anne Devorak ◽

Jeffrey Zurita ◽

...

Keyword(s):

Stem Cells ◽

Embryonic Stem Cells ◽

Progenitor Cells ◽

Es Cells ◽

Embryonic Stem ◽

Neural Progenitors ◽

Neural Genes ◽

Genome Wide ◽

Primary Mouse

ABSTRACTMild replication stress enhances appearance of dozens of robust recurrent genomic break clusters, termed RDCs, in cultured primary mouse neural stem and progenitor cells (NSPCs). Robust RDCs occur within genes (“RDC-genes”) that are long and have roles in neural cell communications and/or have been implicated in neuropsychiatric diseases or cancer. We sought to develop an in vitro approach to determine whether specific RDC formation is associated with neural development. For this purpose, we adapted a system to induce neural progenitor cell (NPC) development from mouse embryonic stem cell (ESC) lines deficient for XRCC4 plus p53, a genotype that enhances DNA double-strand break (DSB) persistence to enhance detection. We tested for RDCs by our genome wide DSB identification approach that captures DSBs genome-wide via their ability to join to specific genomic Cas9/sgRNA-generated bait DSBs. In XRCC4/p53-deficient ES cells, we detected 7 RDCs, which were in genes, with two RDCs being robust. In contrast, in NPCs derived from these ES cell lines, we detected 29 RDCs, a large fraction of which were robust and associated with long, transcribed neural genes that were also robust RDC-genes in primary NSPCs. These studies suggest that many RDCs present in NSPCs are developmentally influenced to occur in this cell type and indicate that induced development of NPCs from ES cells provides an approach to rapidly elucidate mechanistic aspects of NPC RDC formation.SIGNIFICANCE STATEMENTWe previously discovered a set of long neural genes susceptible to frequent DNA breaks in primary mouse brain progenitor cells. We termed these genes RDC-genes. RDC-gene breakage during brain development might alter neural gene function and contribute to neurological diseases and brain cancer. To provide an approach to characterize the unknown mechanism of neural RDC-gene breakage, we asked whether RDC-genes appear in neural progenitors differentiated from embryonic stem cells in culture. Indeed, robust RDC-genes appeared in neural progenitors differentiated in culture and many overlapped with robust RDC-genes in primary brain progenitors. These studies indicate that in vitro development of neural progenitors provides a model system for elucidating how RDC-genes are formed.

Download Full-text

Abstract 1457: IGFBP-4-Mediated Wnt Inhibition is Required for Cardiac Myogenesis

Circulation ◽

10.1161/circ.118.suppl_18.s_329-a ◽

2008 ◽

Vol 118 (suppl_18) ◽

Author(s):

Weidong Zhu ◽

Ichiro Shiojima ◽

Li Zhi ◽

Hiroyuki Ikeda ◽

Masashi Yoshida ◽

...

Keyword(s):

Growth Factor ◽

Wnt Signaling ◽

Heart Diseases ◽

Es Cells ◽

Embryonic Stem ◽

Cardiomyocyte Differentiation ◽

Canonical Wnt Signaling ◽

Canonical Wnt

Insulin-like growth factor-binding proteins (IGFBPs) bind to and modulate the actions of insulin-like growth factors (IGFs). Although some of the effects of IGFBPs appear to be independent of IGFs, the precise mechanisms of IGF-independent actions of IGFBPs are largely unknown. In this study we demonstrate that IGFBP-4 is a novel cardiogenic growth factor. IGFBP-4 enhanced cardiomyocyte differentiation of P19CL6 embryonal carcinoma cells and embryonic stem (ES) cells in vitro. Conversely, siRNA-mediated knockdown of IGFBP-4 in P19CL6 cells or ES cells attenuated cardiomyocyte differentiation, and morpholino-mediated knockdown of IGFBP-4 in Xenopus embryos resulted in severe cardiac defects and complete absence of the heart in extreme cases. We also demonstrate that the cardiogenic effect of IGFBP-4 was independent of its IGF-binding activity but was mediated by the inhibitory effect on canonical Wnt signaling. IGFBP-4 physically interacted with a Wnt receptor Frizzled 8 (Frz8) and a Wnt co-receptor low-density lipoprotein receptor-related protein 6 (LRP6), and inhibited the binding of Wnt3A to Frz8 and LRP6. Moreover, the cardiogenic defects induced by IGFBP-4 knockdown both in vitro and in vivo was rescued by simultaneous inhibition of canonical Wnt signaling. Thus, IGFBP-4 is an inhibitor of the canonical Wnt signaling, and Wnt inhibition by IGFBP-4 is required for cardiogenesis. The present study provides a molecular link between IGF signaling and Wnt signaling, and suggests that IGFBP-4 may be a novel therapeutic target for heart diseases.

Download Full-text

Altered imprinted gene methylation and expression in completely ES cell-derived mouse fetuses: association with aberrant phenotypes

Development ◽

10.1242/dev.125.12.2273 ◽

1998 ◽

Vol 125 (12) ◽

pp. 2273-2282 ◽

Cited By ~ 10

Author(s):

W. Dean ◽

L. Bowden ◽

A. Aitchison ◽

J. Klose ◽

T. Moore ◽

...

Keyword(s):

Developmental Stages ◽

Es Cells ◽

Embryonic Stem ◽

Upstream Region ◽

Imprinted Genes ◽

Epigenetic Alterations ◽

Es Cell ◽

Biallelic Expression

In vitro manipulation of preimplantation mammalian embryos can influence differentiation and growth at later stages of development. In the mouse, culture of embryonic stem (ES) cells affects their totipotency and may give rise to fetal abnormalities. To investigate whether this is associated with epigenetic alterations in imprinted genes, we analysed two maternally expressed genes (Igf2r, H19) and two paternally expressed genes (Igf2, U2af1-rs1) in ES cells and in completely ES cell-derived fetuses. Altered allelic methylation patterns were detected in all four genes, and these were consistently associated with allelic changes in gene expression. All the methylation changes that had arisen in the ES cells persisted on in vivo differentiation to fetal stages. Alterations included loss of methylation with biallelic expression of U2af1-rs1, maternal methylation and predominantly maternal expression of Igf2, and biallelic methylation and expression of Igf2r. In many of the ES fetuses, the levels of H19 expression were strongly reduced, and this biallelic repression was associated with biallellic methylation of the H19 upstream region. Surprisingly, biallelic H19 repression was not associated with equal levels of Igf2 expression from both parental chromosomes, but rather with a strong activation of the maternal Igf2 allele. ES fetuses derived from two of the four ES lines appeared developmentally compromised, with polyhydramnios, poor mandible development and interstitial bleeding and, in chimeric fetuses, the degree of chimerism correlated with increased fetal mass. Our study establishes a model for how early embryonic epigenetic alterations in imprinted genes persist to later developmental stages, and are associated with aberrant phenotypes.

Download Full-text

DNA polymerase α interacts with H3-H4 and facilitates the transfer of parental histones to lagging strands

Science Advances ◽

10.1126/sciadv.abb5820 ◽

2020 ◽

Vol 6 (35) ◽

pp. eabb5820 ◽

Cited By ~ 3

Author(s):

Zhiming Li ◽

Xu Hua ◽

Albert Serra-Cardona ◽

Xiaowei Xu ◽

Songlin Gan ◽

...

Keyword(s):

Dna Polymerase ◽

Es Cells ◽

Embryonic Stem ◽

Endogenous Retroviruses ◽

Histone Binding ◽

Dna Polymerase Α ◽

Mouse Es Cells ◽

Dna Strands ◽

Histone Deposition

How parental histones, the carriers of epigenetic modifications, are deposited onto replicating DNA remains poorly understood. Here, we describe the eSPAN method (enrichment and sequencing of protein-associated nascent DNA) in mouse embryonic stem (ES) cells and use it to detect histone deposition onto replicating DNA strands with a relatively small number of cells. We show that DNA polymerase α (Pol α), which synthesizes short primers for DNA synthesis, binds histone H3-H4 preferentially. A Pol α mutant defective in histone binding in vitro impairs the transfer of parental H3-H4 to lagging strands in both yeast and mouse ES cells. Last, dysregulation of both coding genes and noncoding endogenous retroviruses is detected in mutant ES cells defective in parental histone transfer. Together, we report an efficient eSPAN method for analysis of DNA replication–linked processes in mouse ES cells and reveal the mechanism of Pol α in parental histone transfer.

Download Full-text

Multipotent fetal-derived Cdx2 cells from placenta regenerate the heart

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1811827116 ◽

2019 ◽

pp. 201811827 ◽

Cited By ~ 1

Author(s):

Sangeetha Vadakke-Madathil ◽

Gina LaRocca ◽

Koen Raedschelders ◽

Jesse Yoon ◽

Sarah J. Parker ◽

...

Keyword(s):

Fluorescent Protein ◽

Contractile Function ◽

Es Cells ◽

Embryonic Stem ◽

Lineage Tracing ◽

Cardiac Differentiation ◽

Cell Based Therapy ◽

Allogeneic Cell Therapy

The extremely limited regenerative potential of adult mammalian hearts has prompted the need for novel cell-based therapies that can restore contractile function in heart disease. We have previously shown the regenerative potential of mixed fetal cells that were naturally found migrating to the injured maternal heart. Exploiting this intrinsic mechanism led to the current hypothesis that Caudal-type homeobox-2 (Cdx2) cells in placenta may represent a novel cell type for cardiac regeneration. Using a lineage-tracing strategy, we specifically labeled fetal-derived Cdx2 cells with enhanced green fluorescent protein (eGFP). Cdx2-eGFP cells from end-gestation placenta were assayed for cardiac differentiation in vitro and in vivo using a mouse model of myocardial infarction. We observed that these cells differentiated into spontaneously beating cardiomyocytes (CMs) and vascular cells in vitro, indicating multipotentiality. When administered via tail vein to infarcted wild-type male mice, they selectively and robustly homed to the heart and differentiated to CMs and blood vessels, resulting in significant improvement in contractility as noted by MRI. Proteomics and immune transcriptomics studies of Cdx2-eGFP cells compared with embryonic stem (ES) cells reveal that they appear to retain “stem”-related functions of ES cells but exhibit unique signatures supporting roles in homing and survival, with an ability to evade immune surveillance, which is critical for cell-based therapy. Cdx2-eGFP cells may potentially represent a therapeutic advance in allogeneic cell therapy for cardiac repair.

Download Full-text

230 GENERATION OF PUTATIVE RABBIT EMBRYONIC STEM CELLS

Reproduction Fertility and Development ◽

10.1071/rdv19n1ab230 ◽

2007 ◽

Vol 19 (1) ◽

pp. 231

Author(s):

S. Wang ◽

X. Tang ◽

Y. Niu ◽

H. Chen ◽

T. Li ◽

...

Keyword(s):

Stem Cells ◽

Embryonic Stem Cells ◽

High Speed ◽

Es Cells ◽

Research Work ◽

Embryonic Stem ◽

Morphological Characteristics ◽

Mouse Escs

The rabbit, as a laboratory animal model, has several advantages in the study of human physiological disorders. In this study, stable putative pluripotent rabbit embryonic stem cells (rESCs) were derived from in vivo-fertilized and in vitro-cultured blastocysts. The rabbit ICMs were obtained by 0.05% trypsin–0.008% EDTA treatment and mechanical separation; the ES-like cell colonies seen several days later. ICM-derived outgrowths which were treated with 5 mg/mL-1 dispase, followed by 0.05% trypsin–0.008% EDTA, were mechanically disaggregated into small clumps and reseeded on MEFs. The putative ES cell lines maintained expression of pluripotent cells markers and normal XY karyotype for long periods of culture (>1 month). The putative rESCs expressed alkaline phosphatase, transcription factor Oct-4, stage-specific embryonic antigens (SSEA-1, SSEA-3, and SSEA-4), and tumor-related antigens (TRA-1-60 and TRA-1-81). The morphological characteristics of the putative ESCs are closer to those of human ESCs; their high speed of proliferation, however, is closer to that of mouse ESCs. Putative rabbit ESCs were induced to differentiate into many cell types including trophoblast cells, similar to primate ESCs, in vitro, and formed teratomas with derivatives of the 3 major germ layers in vivo when injected into SCID mice. Using RT-PCR measurement, but with some differences in ligands and inhibitors, and comparing with human and mouse ESCs, the putative rabbit ESCs expressed similar genes related to pluripotency (Oct-4, Nanog, SOX2, and UTF-1) and similar genes of FGF, WNT, and TGF signaling pathways related to the proliferation and self-renewal. Our further research work showed that TGF beta and FGF pathways cooperate to maintain pluripotency of rabbit ESCs similar to those of human ES cells.

Download Full-text