The naïve state of pluripotency is actively being explored by a number of labs. There is some controversy in the field as to the true identity of naïve human pluripotent cells as they are not exact ...mirrors of the mouse. The various reports published, although in basic agreement, present discrepancies in the characterization of the various lines, which likely reflect the etiology of these lines. The primary lesson learned from these contributions is that a human naïve state reflecting the preimplantation human is likely to exist. The essential factors that will universally maintain the naïve state in human cells in vitro are not yet fully understood. These first need to be identified in order to describe the definitive characteristics of this state. Comparisons of naïve and primed human pluripotent cells have also highlighted consistencies between states and broadened our understanding of embryonic metabolism, epigenetic change required for development, embryonic DNA repair strategies and embryonic expression dynamics. Stem Cells 2017;35:35–41
Single-cell Hi-C (scHi-C) interrogates genome-wide chromatin interaction in individual cells, allowing us to gain insights into 3D genome organization. However, the extremely sparse nature of scHi-C ...data poses a significant barrier to analysis, limiting our ability to tease out hidden biological information. In this work, we approach this problem by applying topic modeling to scHi-C data. Topic modeling is well-suited for discovering latent topics in a collection of discrete data. For our analysis, we generate nine different single-cell combinatorial indexed Hi-C (sci-Hi-C) libraries from five human cell lines (GM12878, H1Esc, HFF, IMR90, and HAP1), consisting over 19,000 cells. We demonstrate that topic modeling is able to successfully capture cell type differences from sci-Hi-C data in the form of "chromatin topics." We further show enrichment of particular compartment structures associated with locus pairs in these topics.
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Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Endogenous retroviruses (ERVs) are remnants of ancient retroviral infections, and comprise nearly 8% of the human genome. The most recently acquired human ERV is HERVK(HML-2), which repeatedly ...infected the primate lineage both before and after the divergence of the human and chimpanzee common ancestor. Unlike most other human ERVs, HERVK retained multiple copies of intact open reading frames encoding retroviral proteins. However, HERVK is transcriptionally silenced by the host, with the exception of in certain pathological contexts such as germ-cell tumours, melanoma or human immunodeficiency virus (HIV) infection. Here we demonstrate that DNA hypomethylation at long terminal repeat elements representing the most recent genomic integrations, together with transactivation by OCT4 (also known as POU5F1), synergistically facilitate HERVK expression. Consequently, HERVK is transcribed during normal human embryogenesis, beginning with embryonic genome activation at the eight-cell stage, continuing through the emergence of epiblast cells in preimplantation blastocysts, and ceasing during human embryonic stem cell derivation from blastocyst outgrowths. Remarkably, we detected HERVK viral-like particles and Gag proteins in human blastocysts, indicating that early human development proceeds in the presence of retroviral products. We further show that overexpression of one such product, the HERVK accessory protein Rec, in a pluripotent cell line is sufficient to increase IFITM1 levels on the cell surface and inhibit viral infection, suggesting at least one mechanism through which HERVK can induce viral restriction pathways in early embryonic cells. Moreover, Rec directly binds a subset of cellular RNAs and modulates their ribosome occupancy, indicating that complex interactions between retroviral proteins and host factors can fine-tune pathways of early human development.
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DOBA, IJS, IZUM, KILJ, KISLJ, NUK, PILJ, PNG, SAZU, SBMB, SIK, UILJ, UKNU, UL, UM, UPUK
For nearly a century developmental biologists have recognized that cells from embryos can differ in their potential to differentiate into distinct cell types. Recently, it has been recognized that ...embryonic stem cells derived from both mice and humans exhibit two stable yet epigenetically distinct states of pluripotency: naive and primed. We now show that nicotinamide N-methyltransferase (NNMT) and the metabolic state regulate pluripotency in human embryonic stem cells (hESCs). Specifically, in naive hESCs, NNMT and its enzymatic product 1-methylnicotinamide are highly upregulated, and NNMT is required for low S-adenosyl methionine (SAM) levels and the H3K27me3 repressive state. NNMT consumes SAM in naive cells, making it unavailable for histone methylation that represses Wnt and activates the HIF pathway in primed hESCs. These data support the hypothesis that the metabolome regulates the epigenetic landscape of the earliest steps in human development.
Derivation of naïve human embryonic stem cells Ware, Carol B.; Nelson, Angelique M.; Mecham, Brigham ...
Proceedings of the National Academy of Sciences - PNAS,
03/2014, Letnik:
111, Številka:
12
Journal Article
Recenzirano
Odprti dostop
The naïve pluripotent state has been shown in mice to lead to broad and more robust developmental potential relative to primed mouse epiblast cells. The human naïve ES cell state has eluded ...derivation without the use of transgenes, and forced expression of OCT4, KLF4, and KLF2 allows maintenance of human cells in a naïve state Hanna J, et al. (2010) Proc Natl Acad Sci USA 107 (20):9222—9227. We describe two routes to generate nontransgenic naïve human ES cells (hESCs). The first is by reverse toggling of preexisting primed hESC lines by preculture in the histone deacetylase inhibitors butyrate and suberoylanilide hydroxamic acid, followed by culture in MEK/ERK and GSK3 inhibitors (2i) with FGF2. The second route is by direct derivation from a human embryo in 2i with FGF2. We show that human naïve cells meet mouse criteria for the naïve state by growth characteristics, antibody labeling profile, gene expression, X-inactivation profile, mitochondrial morphology, microRNA profile and development in the context of teratomas. hESCs can exist in a naïve state without the need for transgenes. Direct derivation is an elusive, but attainable, process, leading to cells at the earliest stage of in vitro pluripotency described for humans. Reverse toggling of primed cells to naïve is efficient and reproducible.
High-throughput methods based on chromosome conformation capture have greatly advanced our understanding of the three-dimensional (3D) organization of genomes but are limited in resolution by their ...reliance on restriction enzymes. Here we describe a method called DNase Hi-C for comprehensively mapping global chromatin contacts. DNase Hi-C uses DNase I for chromatin fragmentation, leading to greatly improved efficiency and resolution over that of Hi-C. Coupling this method with DNA-capture technology provides a high-throughput approach for targeted mapping of fine-scale chromatin architecture. We applied targeted DNase Hi-C to characterize the 3D organization of 998 large intergenic noncoding RNA (lincRNA) promoters in two human cell lines. Our results revealed that expression of lincRNAs is tightly controlled by complex mechanisms involving both super-enhancers and the Polycomb repressive complex. Our results provide the first glimpse of the cell type-specific 3D organization of lincRNA genes.
The retina is subject to degenerative conditions, leading to blindness. Although retinal regeneration is robust in lower vertebrates, regeneration does not occur in the adult mammalian retina. Thus, ...we have developed efficient methods for deriving retinal neurons from human embryonic stem (hES) cells. Under appropriate culture conditions, up to 80% of the H1 line can be directed to the retinal progenitor fate, and express a gene expression profile similar to progenitors derived from human fetal retina. The hES cellderived progenitors differentiate primarily into inner retinal neurons (ganglion and amacrine cells), with functional glutamate receptors. Upon coculture with retinas derived from a mouse model of retinal degeneration, the hES cell derived retinal progenitors integrate with the degenerated mouse retina and increase in their expression of photoreceptor-specific markers. These results demonstrate that human ES cells can be selectively directed to a neural retinal cell fate and thus may be useful in the treatment of retinal degenerations.
Each unit of the D4Z4 macrosatellite repeat contains a retrotransposed gene encoding the DUX4 double-homeobox transcription factor. Facioscapulohumeral dystrophy (FSHD) is caused by deletion of a ...subset of the D4Z4 units in the subtelomeric region of chromosome 4. Although it has been reported that the deletion of D4Z4 units induces the pathological expression of DUX4 mRNA, the association of DUX4 mRNA expression with FSHD has not been rigorously investigated, nor has any human tissue been identified that normally expresses DUX4 mRNA or protein. We show that FSHD muscle expresses a different splice form of DUX4 mRNA compared to control muscle. Control muscle produces low amounts of a splice form of DUX4 encoding only the amino-terminal portion of DUX4. FSHD muscle produces low amounts of a DUX4 mRNA that encodes the full-length DUX4 protein. The low abundance of full-length DUX4 mRNA in FSHD muscle cells represents a small subset of nuclei producing a relatively high abundance of DUX4 mRNA and protein. In contrast to control skeletal muscle and most other somatic tissues, full-length DUX4 transcript and protein is expressed at relatively abundant levels in human testis, most likely in the germ-line cells. Induced pluripotent (iPS) cells also express full-length DUX4 and differentiation of control iPS cells to embryoid bodies suppresses expression of full-length DUX4, whereas expression of full-length DUX4 persists in differentiated FSHD iPS cells. Together, these findings indicate that full-length DUX4 is normally expressed at specific developmental stages and is suppressed in most somatic tissues. The contraction of the D4Z4 repeat in FSHD results in a less efficient suppression of the full-length DUX4 mRNA in skeletal muscle cells. Therefore, FSHD represents the first human disease to be associated with the incomplete developmental silencing of a retrogene array normally expressed early in development.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
ABSTRACT
Embryonic stem (ES) cells are promising for cardiac repair’ but directing their differentiation toward cardiomyocytes remains challenging. We investigated whether the heart guides ES cells ...toward cardiomyocytes in vivo and whether allogeneic ES cells were immunologically tolerated. Undifferentiated mouse ES cells consistently formed cardiac teratomas in nude or immunocompetent syngeneic mice. Cardiac teratomas contained no more cardiomyocytes than hind‐limb teratomas’ suggesting lack of guided differentiation. ES cells also formed teratomas in infarcted hearts’ indicating injury‐related signals did not direct cardiac differentiation. Allogeneic ES cells also caused cardiac teratomas’ but these were immunologically rejected after several weeks’ in association with increased inflammation and up‐regulation of class I and II histocompatibility antigens. Fusion between ES cells and cardiomyocytes occurred in vivo’ but was rare. Infarct autofluorescence was identified as an artifact that might be mistaken for enhanced GFP expression and true regeneration. Hence’ undifferentiated ES cells were not guided toward a cardiomyocyte fate in either normal or infarcted hearts’ and there was no evidence for allogeneic immune tolerance of ES cell derivatives. Successful cardiac repair strategies involving ES cells will need to control cardiac differentiation’ avoid introducing undifferentiated cells’ and will likely require immune modulation to avoid rejection.—Nussbaum, J., Minami, E., Laflamme, M. A., Virag, J. A. I., Ware, C. B., Masino, A., Muskheli, V., Pabon, L., Reinecke, H., Murry, C. E. Transplantation of undifferentiated mu‐rine embryonic stem cells in the heart: teratoma formation and immune response. FASEB J. 21, 1345–1357 (2007)
Regulation of embryonic diapause, dormancy that interrupts the tight connection between developmental stage and time, is still poorly understood. Here, we characterize the transcriptional and ...metabolite profiles of mouse diapause embryos and identify unique gene expression and metabolic signatures with activated lipolysis, glycolysis, and metabolic pathways regulated by AMPK. Lipolysis is increased due to mTORC2 repression, increasing fatty acids to support cell survival. We further show that starvation in pre-implantation ICM-derived mouse ESCs induces a reversible dormant state, transcriptionally mimicking the in vivo diapause stage. During starvation, Lkb1, an upstream kinase of AMPK, represses mTOR, which induces a reversible glycolytic and epigenetically H4K16Ac-negative, diapause-like state. Diapause furthermore activates expression of glutamine transporters SLC38A1/2. We show by genetic and small molecule inhibitors that glutamine transporters are essential for the H4K16Ac-negative, diapause state. These data suggest that mTORC1/2 inhibition, regulated by amino acid levels, is causal for diapause metabolism and epigenetic state.
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•Diapause is associated with increased lipolysis and glutamine transporter expression•Upregulation of lipolysis in diapause is associated with downregulation of mTORC2•Starvation results in a diapause-like state, enriched in glutamine transporters•Inhibition of glutamine transporters leads to exit from the diapause epigenetic state
Hussein et al. report that, during starvation, mTOR is repressed through LKB1-AMPK, inducing a reversible metabolically active but epigenetically silenced embryonic diapause-like state that upregulates expression of the glutamine transporters SLC38A1/2. These transporters are required for the H4K16ac-negative, diapause state.
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