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2.
Mol Biol (Mosk) ; 53(5): 725-740, 2019.
Artigo em Russo | MEDLINE | ID: mdl-31661474

RESUMO

Human pluripotent stem cells, which include embryonic stem cells and induced pluripotent cells (iPSCs), are capable of unlimited division and differentiation into all cells of the body. These cells are considered as a potential source of various types of cells for transplantations. The use of autologous iPSCs is not potentially associated with immune rejection and does not require immunosuppression required for allogeneic grafts. However, the high cost of this technology and the duration of obtaining iPSCs and differentiated cells may limit the use of autologous iPSCs in clinical practice. In addition, full equivalence and immunological compatibility of autologous iPSCs and their derivatives have been repeatedly questioned. One approach to solving the problem of the immunological compatibility of allogeneic derivatives of iPSCs can be the establishment of cell lines with reduced immunogenicity. Differentiated derivatives of such iPSCs may be suitable for transplantation to any patient. This review discusses the strategies for evading immune surveillance in normal and tumor processes that can be used to establish stem cell lines with reduced immunogenicity.


Assuntos
Linhagem Celular/citologia , Linhagem Celular/imunologia , Células-Tronco Pluripotentes Induzidas/citologia , Células-Tronco Pluripotentes Induzidas/imunologia , Diferenciação Celular , Células-Tronco Embrionárias/citologia , Células-Tronco Embrionárias/imunologia , Humanos
3.
Nat Cell Biol ; 21(9): 1060-1067, 2019 09.
Artigo em Inglês | MEDLINE | ID: mdl-31481793

RESUMO

Cyclins, cyclin-dependent kinases and other components of the core cell cycle machinery drive cell division. Growing evidence indicates that this machinery operates in a distinct fashion in some mammalian stem cell types, such as pluripotent embryonic stem cells. In this Review, we discuss our current knowledge of how cell cycle proteins mechanistically link cell proliferation, pluripotency and cell fate specification. We focus on embryonic stem cells, induced pluripotent stem cells and embryonic neural stem/progenitor cells.


Assuntos
Ciclo Celular/fisiologia , Diferenciação Celular/fisiologia , Células-Tronco Embrionárias/citologia , Células-Tronco Pluripotentes/citologia , Animais , Proliferação de Células/fisiologia , Humanos , Células-Tronco Pluripotentes Induzidas/citologia
4.
Adv Exp Med Biol ; 1169: 81-93, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31487020

RESUMO

Identification of thyroid stem cells in the past few years has made important contributions to our understanding of the cellular and molecular mechanisms that induce tissue regeneration and repair. Embryonic stem (ES) cells and induced-pluripotent stem cells have been used to establish reliable protocols to obtain mature thyrocytes and functional follicles for the treatment of thyroid diseases in mice. In addition, the discovery of resident thyroid progenitor cells, along with other sources of stem cells, has defined in detail the mechanisms responsible for tissue repair upon moderate or severe organ injury.In this chapter, we highlight in detail the current state of research on thyroid stem cells by focusing on (1) the description of the first experiments performed to obtain thyroid follicles from embryonic stem cells, (2) the identification of resident stem cells in the thyroid gland, and (3) the definition of the current translational in vivo and in vitro models used for thyroid tissue repair and regeneration.


Assuntos
Células-Tronco Embrionárias , Glândula Tireoide , Animais , Diferenciação Celular , Células-Tronco Embrionárias/citologia , Humanos , Camundongos , Pesquisa/tendências , Glândula Tireoide/citologia
5.
Adv Exp Med Biol ; 1169: 213-223, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31487026

RESUMO

Every organ in the body is thought to harbor two populations of stem cells, including the quiescent and the actively dividing, that leads to heterogeneity among them. It is generally believed that the ovary harbors a fixed number of follicles at birth that differentiate during fetal development from the primordial germ cells. The numbers of follicles decrease by age, leading to menopause. However, in 2004, it was suggested that ovary may harbor stem cells that are possibly involved in the formation of new follicles throughout reproductive life. Research over little more than a decade shows that ovarian stem cells include a quiescent population of very small embryonic-like stem cells (VSELs) and slightly bigger, actively dividing ovarian stem cells (OSCs). This heterogeneity among ovarian stem cells is similar to the presence of VSELs along with spermatogonial stem cells (SSCs) in the testis or hematopoietic stem cells (HSCs) in the hematopoietic system. VSELs express embryonic markers, including nuclear OCT-4, and are lodged in the ovary surface epithelium (OSE). Ovarian VSELs undergo asymmetric cell division to self-renew and give rise to OSCs that in turn undergo symmetric cell divisions and clonal expansion (germ cell nest) followed by meiosis to form an oocyte that gets assembled as a primordial follicle. Both VSELs and OSCs also express receptors for follicle-stimulating hormone (FSHR) and are directly activated by FSH to undergo neo-oogenesis and primordial follicle assembly. Whether stimulation of ovaries by FSH in Infertility Clinics activates the stem cells leading to the formation of multiple follicles needs further investigation. Epithelial cells lining the surface of ovary provide a niche to the stem cells under normal circumstances and undergo epithelial-mesenchymal transition (EMT) to form granulosa cells for primordial follicle assembly. Compromised function of the epithelial cells with age possibly leads to inability of stem cells to form follicles, leading to menopause. More than 90% of ovarian cancers arise in the OSE, possibly due to excessive self-renewal of VSELs. Altered biology of the OSE cells results in the formation of myofibroblasts by EMT and may provide a cancerous niche that supports excessive expansion of the stem cells lodged in the OSE, leading to ovarian cancer. Ovarian cancer cells express markers like OCT-4 and FSHR, which are also expressed by the VSELs lodged in the OSE, whereas the epithelial cells are distinctly negative for the same. Lot more research is required in the field to gain further understanding of ovarian stem cell biology.


Assuntos
Células-Tronco Embrionárias , Folículo Ovariano , Células-Tronco Embrionárias/citologia , Feminino , Humanos , Masculino , Oogênese , Folículo Ovariano/citologia
6.
Cell Physiol Biochem ; 53(2): 337-354, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31373783

RESUMO

BACKGROUND/AIMS: The availability of truly maturated cardiomyocytic subtypes is a major prerequisite for cardiovascular cell replacement therapies. Pluripotent stem cells provide a suitable source for the development of new strategies to overcome enormous hurdles such as yield, purity and safety of in vitro generated cells. METHODS: To address these issues, we have refined existing forward programming protocols by combining forced exogenous overexpression of the early cardiovascular transcription factor Nkx2.5 with a αMHC-promoter-based antibiotic selection step. Additionally, we applied small molecules such as ascorbic acid to enhance cardiomyogenic differentiation efficiency. Subsequently, we evaluated the cell fate of the resulting cardiomyocytes on the mRNA as well as protein levels. The latter was performed using high-resolution confocal microscopy. Furthermore, we examined the response of the cells` beating activities to pharmacological substance administration. RESULTS: Our results reveal an apparent influence of Nkx2.5 on the cell fate of ESC-derived cardiomyocytes. Resulting single cells exhibit characteristics of early ventricular cardiomyocytes, such as sarcomeric marker expression, spontaneous beating frequency, and distinct L-type calcium channel occurrence. CONCLUSION: Therefore, we demonstrate cardiovascular subtype forward programming of ESCs using a combination of transcription factors along with small molecule administration. However, our findings also underline current assumptions, that a terminal maturation of PSC derived cardiomyocytes in vitro is still an unsolved problem which urgently needs to be addressed in the field.


Assuntos
Reprogramação Celular , Células-Tronco Embrionárias/metabolismo , Proteína Homeobox Nkx-2.5/metabolismo , Miócitos Cardíacos/metabolismo , Animais , Ácido Ascórbico/farmacologia , Diferenciação Celular/efeitos dos fármacos , Células-Tronco Embrionárias/citologia , Proteína Homeobox Nkx-2.5/antagonistas & inibidores , Proteína Homeobox Nkx-2.5/genética , Camundongos , Microscopia Confocal , Miócitos Cardíacos/citologia , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Verapamil/farmacologia
7.
Cancer Sci ; 110(10): 3027-3037, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31348591

RESUMO

We previously established a method to generate myeloid cells with a proliferative capability from pluripotent stem cells and designated them iPS-ML. Human iPS-ML cells share features with physiological macrophages including the capability to infiltrate into cancer tissues. We observed therapeutic effects of human iPS-ML cells expressing interferon ß (iPS-ML/interferon (IFN)-ß) in xenograft cancer models. However, assessment of host immune system-mediated therapeutic and adverse effects of this therapy is impossible by xenograft models. We currently evaluated the therapeutic effects of a mouse equivalent of human iPS-ML/IFN, a mouse embryonic stem (ES) cell-derived myeloid cell line producing IFN (ES-ML/IFN). The ES-MLs producing IFN-ß (ß-ML) and IFN-γ (γ-ML) and originating from E14 ES cells derived from the 129 mouse strain (H-2b ) were generated, and the MHC (H-2Kb , Db , and I-Ab ) genes of the ES-ML/IFN were disrupted using the clustered regularly interspaced short palindromic repeats (CRISPR)/CAS9 method. We used the ES-ML/IFN to treat allogeneic BALB/c mice (H-2d ) transplanted with Colon26 cancer cells. Treatment with ß-ML but not with γ-ML cells repressed the growth of colon cancer in the peritoneal cavity and liver. The transferred ES-ML/IFN infiltrated into cancer tissues and enhanced infiltration of T cells into cancer tissues. ES-ML/IFN therapy increased the number of immune cells in the lymphoid organs. Sensitization of both cancer antigen-specific CD8+ T cells and natural killer (NK) cells were enhanced by the therapy, and CD8+ T cells were essential for the therapeutic effect, implying that donor MHC-deficient ß-ML exhibited a therapeutic effect through the activation of host immune cells derived from allogeneic recipient mice. The results suggested the usefulness of HLA-deficient human iPS-ML/IFN-ß cells for therapy of HLA-mismatched allogeneic cancer patients.


Assuntos
Neoplasias do Colo/terapia , Células-Tronco Embrionárias/citologia , Antígenos de Histocompatibilidade/genética , Interferon beta/metabolismo , Células Mieloides/transplante , Animais , Linfócitos T CD8-Positivos/metabolismo , Linhagem Celular Tumoral , Neoplasias do Colo/imunologia , Células-Tronco Embrionárias/metabolismo , Feminino , Humanos , Células-Tronco Pluripotentes Induzidas/citologia , Células-Tronco Pluripotentes Induzidas/metabolismo , Células Matadoras Naturais/metabolismo , Linfócitos do Interstício Tumoral/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Células Mieloides/citologia , Células Mieloides/metabolismo , Transplante Homólogo , Ensaios Antitumorais Modelo de Xenoenxerto
8.
Neuron ; 103(4): 617-626.e6, 2019 08 21.
Artigo em Inglês | MEDLINE | ID: mdl-31257103

RESUMO

The autism-associated synaptic-adhesion gene Neuroligin-4 (NLGN4) is poorly conserved evolutionarily, limiting conclusions from Nlgn4 mouse models for human cells. Here, we show that the cellular and subcellular expression of human and murine Neuroligin-4 differ, with human Neuroligin-4 primarily expressed in cerebral cortex and localized to excitatory synapses. Overexpression of NLGN4 in human embryonic stem cell-derived neurons resulted in an increase in excitatory synapse numbers but a remarkable decrease in synaptic strength. Human neurons carrying the syndromic autism mutation NLGN4-R704C also formed more excitatory synapses but with increased functional synaptic transmission due to a postsynaptic mechanism, while genetic loss of NLGN4 did not significantly affect synapses in the human neurons analyzed. Thus, the NLGN4-R704C mutation represents a change-of-function mutation. Our work reveals contrasting roles of NLGN4 in human and mouse neurons, suggesting that human evolution has impacted even fundamental cell biological processes generally assumed to be highly conserved.


Assuntos
Moléculas de Adesão Celular Neuronais/fisiologia , Transmissão Sináptica/fisiologia , Animais , Transtorno Autístico/genética , Moléculas de Adesão Celular Neuronais/genética , Células Cultivadas , Córtex Cerebral/fisiologia , Células-Tronco Embrionárias/citologia , Potenciais Pós-Sinápticos Excitadores/fisiologia , Genes Reporter , Ácido Glutâmico/fisiologia , Humanos , Camundongos , Potenciais Pós-Sinápticos em Miniatura/fisiologia , Mutação de Sentido Incorreto , Neurogênese , Neurônios/fisiologia , Fenótipo , Receptores de Glutamato/fisiologia , Especificidade da Espécie , Sinapses/química
9.
Methods Mol Biol ; 2018: 115-130, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31228154

RESUMO

Transgenic technology in rats is increasingly important for the design and implementation of biological and physiological studies in the fields of neuroscience, pharmacology, and toxicology. Pluripotent embryonic stem cells (ESCs) are a useful tool for generation of gene-modified rats. During the last decade, not only foreign DNA introduction but also endogenous DNA modification has been successfully achieved with rat ESCs. Detailed protocols for establishment of bona fide rat ESCs and their use for production of gene-modified rats are described in this chapter.


Assuntos
Blastocisto/citologia , Células-Tronco Embrionárias/citologia , Edição de Genes/veterinária , Animais , Animais Geneticamente Modificados , Células Cultivadas , Células Alimentadoras/citologia , Feminino , Masculino , Camundongos , Ratos , Ratos Transgênicos
10.
Nat Cell Biol ; 21(7): 824-834, 2019 07.
Artigo em Inglês | MEDLINE | ID: mdl-31235934

RESUMO

How pluripotent stem cells differentiate into the main germ layers is a key question of developmental biology. Here, we show that the chromatin-related factor Whsc1 (also known as Nsd2 and MMSET) has a dual role in pluripotency exit and germ layer specification of embryonic stem cells. On induction of differentiation, a proportion of Whsc1-depleted embryonic stem cells remain entrapped in a pluripotent state and fail to form mesendoderm, although they are still capable of generating neuroectoderm. These functions of Whsc1 are independent of its methyltransferase activity. Whsc1 binds to enhancers of the mesendodermal regulators Gata4, T (Brachyury), Gata6 and Foxa2, together with Brd4, and activates the expression of these genes. Depleting each of these regulators also delays pluripotency exit, suggesting that they mediate the effects observed with Whsc1. Our data indicate that Whsc1 links silencing of the pluripotency regulatory network with activation of mesendoderm lineages.


Assuntos
Diferenciação Celular/fisiologia , Endoderma/citologia , Histona-Lisina N-Metiltransferase/metabolismo , Células-Tronco Pluripotentes/citologia , Animais , Diferenciação Celular/genética , Linhagem da Célula , Células-Tronco Embrionárias/citologia , Camadas Germinativas/citologia , Camundongos , Placa Neural/citologia , Proteínas Nucleares/metabolismo , Fatores de Transcrição/metabolismo
11.
Nat Cell Biol ; 21(6): 687-699, 2019 06.
Artigo em Inglês | MEDLINE | ID: mdl-31160711

RESUMO

We recently derived mouse expanded potential stem cells (EPSCs) from individual blastomeres by inhibiting the critical molecular pathways that predispose their differentiation. EPSCs had enriched molecular signatures of blastomeres and possessed developmental potency for all embryonic and extra-embryonic cell lineages. Here, we report the derivation of porcine EPSCs, which express key pluripotency genes, are genetically stable, permit genome editing, differentiate to derivatives of the three germ layers in chimeras and produce primordial germ cell-like cells in vitro. Under similar conditions, human embryonic stem cells and induced pluripotent stem cells can be converted, or somatic cells directly reprogrammed, to EPSCs that display the molecular and functional attributes reminiscent of porcine EPSCs. Importantly, trophoblast stem-cell-like cells can be generated from both human and porcine EPSCs. Our pathway-inhibition paradigm thus opens an avenue for generating mammalian pluripotent stem cells, and EPSCs present a unique cellular platform for translational research in biotechnology and regenerative medicine.


Assuntos
Diferenciação Celular/genética , Reprogramação Celular/genética , Células-Tronco Pluripotentes Induzidas/citologia , Células-Tronco Pluripotentes/citologia , Animais , Blastômeros/citologia , Blastômeros/metabolismo , Linhagem da Célula/genética , Células-Tronco Embrionárias/citologia , Camadas Germinativas/crescimento & desenvolvimento , Camadas Germinativas/metabolismo , Humanos , Camundongos , Medicina Regenerativa , Transdução de Sinais/genética , Suínos , Trofoblastos/citologia , Trofoblastos/metabolismo
12.
Genes Dev ; 33(13-14): 782-798, 2019 07 01.
Artigo em Inglês | MEDLINE | ID: mdl-31171699

RESUMO

Mouse embryonic stem cell (ESC) cultures contain a rare cell population of "2C-like" cells resembling two-cell embryos, the key stage of zygotic genome activation (ZGA). Little is known about positive regulators of the 2C-like state and two-cell stage embryos. Here we show that GADD45 (growth arrest and DNA damage 45) proteins, regulators of TET (TET methylcytosine dioxygenase)-mediated DNA demethylation, promote both states. Methylome analysis of Gadd45a,b,g triple-knockout (TKO) ESCs reveal locus-specific DNA hypermethylation of ∼7000 sites, which are enriched for enhancers and loci undergoing TET-TDG (thymine DNA glycosylase)-mediated demethylation. Gene expression is misregulated in TKOs, notably upon differentiation, and displays signatures of DNMT (DNA methyltransferase) and TET targets. TKOs manifest impaired transition into the 2C-like state and exhibit DNA hypermethylation and down-regulation of 2C-like state-specific genes. Gadd45a,b double-mutant mouse embryos display embryonic sublethality, deregulated ZGA gene expression, and developmental arrest. Our study reveals an unexpected role of GADD45 proteins in embryonic two-cell stage regulation.


Assuntos
Antígenos de Diferenciação/genética , Antígenos de Diferenciação/metabolismo , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Desmetilação do DNA , Células-Tronco Embrionárias/citologia , Regulação da Expressão Gênica no Desenvolvimento , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Animais , Células Cultivadas , Técnicas de Inativação de Genes , Camundongos
13.
Int J Mol Sci ; 20(11)2019 May 30.
Artigo em Inglês | MEDLINE | ID: mdl-31151253

RESUMO

Ubiquitination regulates nearly every aspect of cellular events in eukaryotes. It modifies intracellular proteins with 76-amino acid polypeptide ubiquitin (Ub) and destines them for proteolysis or activity alteration. Ubiquitination is generally achieved by a tri-enzyme machinery involving ubiquitin activating enzymes (E1), ubiquitin conjugating enzymes (E2) and ubiquitin ligases (E3). E1 activates Ub and transfers it to the active cysteine site of E2 via a transesterification reaction. E3 coordinates with E2 to mediate isopeptide bond formation between Ub and substrate protein. The E1-E2-E3 cascade can create diverse types of Ub modifications, hence effecting distinct outcomes on the substrate proteins. Dysregulation of ubiquitination results in severe consequences and human diseases. There include cancers, developmental defects and immune disorders. In this review, we provide an overview of the ubiquitination machinery and discuss the recent progresses in the ubiquitination-mediated regulation of embryonic stem cell maintenance and cancer biology.


Assuntos
Autorrenovação Celular , Transformação Celular Neoplásica/metabolismo , Células-Tronco Embrionárias/metabolismo , Neoplasias/etiologia , Neoplasias/metabolismo , Animais , Biomarcadores , Diferenciação Celular , Autorrenovação Celular/genética , Transformação Celular Neoplásica/genética , Células-Tronco Embrionárias/citologia , Epigênese Genética , Regulação da Expressão Gênica , Humanos , Neoplasias/patologia , Transdução de Sinais , Ubiquitinação
14.
Nat Genet ; 51(6): 941-946, 2019 06.
Artigo em Inglês | MEDLINE | ID: mdl-31152160

RESUMO

The histone variant H3.3 is enriched at enhancers and active genes, as well as repeat regions such as telomeres and retroelements, in mouse embryonic stem cells (mESCs)1-3. Although recent studies demonstrate a role for H3.3 and its chaperones in establishing heterochromatin at repeat regions4-8, the function of H3.3 in transcription regulation has been less clear9-16. Here, we find that H3.3-specific phosphorylation17-19 stimulates activity of the acetyltransferase p300 in trans, suggesting that H3.3 acts as a nucleosomal cofactor for p300. Depletion of H3.3 from mESCs reduces acetylation on histone H3 at lysine 27 (H3K27ac) at enhancers. Compared with wild-type cells, those lacking H3.3 demonstrate reduced capacity to acetylate enhancers that are activated upon differentiation, along with reduced ability to reprogram cell fate. Our study demonstrates that a single amino acid in a histone variant can integrate signaling information and impact genome regulation globally, which may help to better understand how mutations in these proteins contribute to human cancers20,21.


Assuntos
Elementos Facilitadores Genéticos , Regulação da Expressão Gênica , Histonas/metabolismo , Serina/metabolismo , Fatores de Transcrição de p300-CBP/metabolismo , Acetilação , Animais , Diferenciação Celular , Células-Tronco Embrionárias/citologia , Células-Tronco Embrionárias/metabolismo , Camundongos , Fosforilação , Processamento de Proteína Pós-Traducional
15.
Nature ; 570(7759): 77-82, 2019 06.
Artigo em Inglês | MEDLINE | ID: mdl-31086336

RESUMO

Ontogeny describes the emergence of complex multicellular organisms from single totipotent cells. This field is particularly challenging in mammals, owing to the indeterminate relationship between self-renewal and differentiation, variation in progenitor field sizes, and internal gestation in these animals. Here we present a flexible, high-information, multi-channel molecular recorder with a single-cell readout and apply it as an evolving lineage tracer to assemble mouse cell-fate maps from fertilization through gastrulation. By combining lineage information with single-cell RNA sequencing profiles, we recapitulate canonical developmental relationships between different tissue types and reveal the nearly complete transcriptional convergence of endodermal cells of extra-embryonic and embryonic origins. Finally, we apply our cell-fate maps to estimate the number of embryonic progenitor cells and their degree of asymmetric partitioning during specification. Our approach enables massively parallel, high-resolution recording of lineage and other information in mammalian systems, which will facilitate the construction of a quantitative framework for understanding developmental processes.


Assuntos
Embrião de Mamíferos/embriologia , Embrião de Mamíferos/metabolismo , Desenvolvimento Embrionário/genética , Animais , Diferenciação Celular/genética , Linhagem da Célula/genética , Embrião de Mamíferos/citologia , Células-Tronco Embrionárias/citologia , Células-Tronco Embrionárias/metabolismo , Endoderma/embriologia , Endoderma/metabolismo , Feminino , Fertilização , Gastrulação , Regulação da Expressão Gênica no Desenvolvimento/genética , Masculino , Camundongos , Especificidade de Órgãos/genética , Fenótipo , Análise de Sequência de RNA , Análise de Célula Única
16.
Int J Mol Sci ; 20(9)2019 May 07.
Artigo em Inglês | MEDLINE | ID: mdl-31067653

RESUMO

In recent years, great interest has been devoted to finding alternative sources for human stem cells which can be easily isolated, ideally without raising ethical objections. These stem cells should furthermore have a high proliferation rate and the ability to differentiate into all three germ layers. Amniotic fluid, ordinarily discarded as medical waste, is potentially such a novel source of stem cells, and these amniotic fluid derived stem cells are currently gaining a lot of attention. However, further information will be required about the properties of these cells before they can be used for therapeutic purposes. For example, the risk of tumor formation after cell transplantation needs to be explored. The tumor suppressor protein p53, well known for its activity in controlling Cell Prolif.eration and cell death in differentiated cells, has more recently been found to be also active in amniotic fluid stem cells. In this review, we summarize the major findings about human amniotic fluid stem cells since their discovery, followed by a brief overview of the important role played by p53 in embryonic and adult stem cells. In addition, we explore what is known about p53 in amniotic fluid stem cells to date, and emphasize the need to investigate its role, particularly in the context of cell tumorigenicity.


Assuntos
Líquido Amniótico/citologia , Células-Tronco Embrionárias/metabolismo , Proteína Supressora de Tumor p53/metabolismo , Líquido Amniótico/metabolismo , Animais , Diferenciação Celular , Células-Tronco Embrionárias/citologia , Humanos , Proteína Supressora de Tumor p53/genética
17.
Nat Genet ; 51(6): 1024-1034, 2019 06.
Artigo em Inglês | MEDLINE | ID: mdl-31133748

RESUMO

The mouse X-inactivation center (Xic) locus represents a powerful model for understanding the links between genome architecture and gene regulation, with the non-coding genes Xist and Tsix showing opposite developmental expression patterns while being organized as an overlapping sense/antisense unit. The Xic is organized into two topologically associating domains (TADs) but the role of this architecture in orchestrating cis-regulatory information remains elusive. To explore this, we generated genomic inversions that swap the Xist/Tsix transcriptional unit and place their promoters in each other's TAD. We found that this led to a switch in their expression dynamics: Xist became precociously and ectopically upregulated, both in male and female pluripotent cells, while Tsix expression aberrantly persisted during differentiation. The topological partitioning of the Xic is thus critical to ensure proper developmental timing of X inactivation. Our study illustrates how the genomic architecture of cis-regulatory landscapes can affect the regulation of mammalian developmental processes.


Assuntos
Regulação da Expressão Gênica no Desenvolvimento , RNA Longo não Codificante/genética , Inativação do Cromossomo X , Animais , Diferenciação Celular/genética , Expressão Ectópica do Gene , Células-Tronco Embrionárias/citologia , Células-Tronco Embrionárias/metabolismo , Feminino , Inativação Gênica , Loci Gênicos , Masculino , Camundongos , Modelos Biológicos , Regiões Promotoras Genéticas , Inversão de Sequência , Transcrição Genética
18.
Nat Methods ; 16(6): 489-492, 2019 06.
Artigo em Inglês | MEDLINE | ID: mdl-31133759

RESUMO

Modular domains of long non-coding RNAs can serve as scaffolds to bring distant regions of the linear genome into spatial proximity. Here, we present HiChIRP, a method leveraging bio-orthogonal chemistry and optimized chromosome conformation capture conditions, which enables interrogation of chromatin architecture focused around a specific RNA of interest down to approximately ten copies per cell. HiChIRP of three nuclear RNAs reveals insights into promoter interactions (7SK), telomere biology (telomerase RNA component) and inflammatory gene regulation (lincRNA-EPS).


Assuntos
Cromatina/química , Cromatina/genética , Células-Tronco Embrionárias/metabolismo , Regulação da Expressão Gênica , RNA Longo não Codificante/genética , RNA/química , Telomerase/química , Animais , Células Cultivadas , Cromossomos , Células-Tronco Embrionárias/citologia , Genoma , Camundongos , Regiões Promotoras Genéticas , RNA/genética , Telomerase/genética
19.
Methods Mol Biol ; 1965: 1-6, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31069664

RESUMO

Developmental toxicity associated with exposure to exogenous compounds such as drugs and environmental chemicals can be assessed using a variety of different in vitro models, each with their own advantages and disadvantages. These models include cultured cells (Chapters 3 - 6 ), organ and tissue cultures (Chapters 7 and 8 ), and whole embryo cultures (Chapters 13 - 15 ) and typically support the guiding principles of the three Rs: replace, reduce, and refine. These models can be used in early chemical screens and have helped further our understanding into the mechanisms associated with developmental toxicity following exposure to many chemicals.


Assuntos
Técnicas de Cultura de Células/métodos , Células-Tronco Embrionárias/citologia , Animais , Células Cultivadas , Técnicas de Cultura Embrionária , Células-Tronco Embrionárias/efeitos dos fármacos , Humanos , Modelos Biológicos , Técnicas de Cultura de Tecidos , Testes de Toxicidade
20.
PLoS Genet ; 15(4): e1008058, 2019 04.
Artigo em Inglês | MEDLINE | ID: mdl-30933982

RESUMO

In the skin and gill epidermis of fish, ionocytes develop alongside keratinocytes and maintain body fluid ionic homeostasis that is essential for adaptation to environmental fluctuations. It is known that ionocyte progenitors in zebrafish embryos are specified from p63+ epidermal stem cells through a patterning process involving DeltaC (Dlc)-Notch-mediated lateral inhibition, which selects scattered dlc+ cells into the ionocyte progenitor fate. However, mechanisms by which the ionocyte progenitor population is modulated remain unclear. Krüppel-like factor 4 (Klf4) transcription factor was previously implicated in the terminal differentiation of mammalian skin epidermis and is known for its bifunctional regulation of cell proliferation in a tissue context-dependent manner. Here, we report novel roles for zebrafish Klf4 in the ventral ectoderm during embryonic skin development. We found that Klf4 was expressed in p63+ epidermal stem cells of the ventral ectoderm from 90% epiboly onward. Knockdown or knockout of klf4 expression reduced the proliferation rate of p63+ stem cells, resulting in decreased numbers of p63+ stem cells, dlc-p63+ keratinocyte progenitors and dlc+ p63+ ionocyte progenitor cells. These reductions subsequently led to diminished keratinocyte and ionocyte densities and resulted from upregulation of the well-known cell cycle regulators, p53 and cdkn1a/p21. Moreover, mutation analyses of the KLF motif in the dlc promoter, combined with VP16-klf4 or engrailed-klf4 mRNA overexpression analyses, showed that Klf4 can bind the dlc promoter and modulate lateral inhibition by directly repressing dlc expression. This idea was further supported by observing the lateral inhibition outcomes in klf4-overexpressing or knockdown embryos. Overall, our experiments delineate novel roles for zebrafish Klf4 in regulating the ionocyte progenitor population throughout early stem cell stage to initiation of terminal differentiation, which is dependent on Dlc-Notch-mediated lateral inhibition.


Assuntos
Células-Tronco Embrionárias/citologia , Células-Tronco Embrionárias/metabolismo , Células Epidérmicas/citologia , Células Epidérmicas/metabolismo , Fatores de Transcrição Kruppel-Like/metabolismo , Proteínas de Peixe-Zebra/metabolismo , Peixe-Zebra/embriologia , Peixe-Zebra/metabolismo , Sequência de Aminoácidos , Animais , Animais Geneticamente Modificados , Padronização Corporal , Diferenciação Celular , Proliferação de Células , Inibidor de Quinase Dependente de Ciclina p21/genética , Inibidor de Quinase Dependente de Ciclina p21/metabolismo , Ectoderma/citologia , Ectoderma/embriologia , Ectoderma/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Brânquias/citologia , Brânquias/embriologia , Brânquias/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/genética , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Transporte de Íons , Fatores de Transcrição Kruppel-Like/deficiência , Fatores de Transcrição Kruppel-Like/genética , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Fosfoproteínas/genética , Fosfoproteínas/metabolismo , Regiões Promotoras Genéticas , Receptores Notch/genética , Receptores Notch/metabolismo , Transativadores/genética , Transativadores/metabolismo , Proteína Supressora de Tumor p53/genética , Proteína Supressora de Tumor p53/metabolismo , Peixe-Zebra/genética , Proteínas de Peixe-Zebra/deficiência , Proteínas de Peixe-Zebra/genética
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