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1.
Nat Commun ; 11(1): 5434, 2020 10 28.
Artigo em Inglês | MEDLINE | ID: mdl-33116143

RESUMO

The interfollicular epidermis (IFE) forms a water-tight barrier that is often disrupted in inflammatory skin diseases. During homeostasis, the IFE is replenished by stem cells in the basal layer that differentiate as they migrate toward the skin surface. Conventionally, IFE differentiation is thought to be stepwise as reflected in sharp boundaries between its basal, spinous, granular and cornified layers. The transcription factor GRHL3 regulates IFE differentiation by transcriptionally activating terminal differentiation genes. Here we use single cell RNA-seq to show that murine IFE differentiation is best described as a single step gradualistic process with a large number of transition cells between the basal and spinous layer. RNA-velocity analysis identifies a commitment point that separates the plastic basal and transition cell state from unidirectionally differentiating cells. We also show that in addition to promoting IFE terminal differentiation, GRHL3 is essential for suppressing epidermal stem cell expansion and the emergence of an abnormal stem cell state by suppressing Wnt signaling in stem cells.


Assuntos
Proteínas de Ligação a DNA/metabolismo , Células-Tronco Embrionárias/citologia , Células-Tronco Embrionárias/metabolismo , Células Epidérmicas/citologia , Células Epidérmicas/metabolismo , Fatores de Transcrição/metabolismo , Animais , Animais Recém-Nascidos , Diferenciação Celular , Linhagem da Célula , Proteínas de Ligação a DNA/deficiência , Proteínas de Ligação a DNA/genética , Epiderme/embriologia , Epiderme/metabolismo , Feminino , Perfilação da Expressão Gênica , Idade Gestacional , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Gravidez , Análise de Célula Única , Fatores de Transcrição/deficiência , Fatores de Transcrição/genética
3.
Nat Commun ; 11(1): 4989, 2020 10 05.
Artigo em Inglês | MEDLINE | ID: mdl-33020476

RESUMO

We postulate that exit from pluripotency involves intermediates that retain pluripotency while simultaneously exhibiting lineage-bias. Using a MIXL1 reporter, we explore mesoderm lineage-bias within the human pluripotent stem cell compartment. We identify a substate, which at the single cell level coexpresses pluripotent and mesodermal gene expression programmes. Functionally these cells initiate stem cell cultures and exhibit mesodermal bias in differentiation assays. By promoting mesodermal identity through manipulation of WNT signalling while preventing exit from pluripotency using lysophosphatidic acid, we 'trap' and maintain cells in a lineage-biased stem cell state through multiple passages. These cells correspond to a normal state on the differentiation trajectory, the plasticity of which is evidenced by their reacquisition of an unbiased state upon removal of differentiation cues. The use of 'cross-antagonistic' signalling to trap pluripotent stem cell intermediates with different lineage-bias may have general applicability in the efficient production of cells for regenerative medicine.


Assuntos
Reprogramação Celular , Mesoderma/metabolismo , Células-Tronco Pluripotentes/citologia , Animais , Diferenciação Celular/genética , Linhagem Celular , Linhagem da Célula , Plasticidade Celular/genética , Autorrenovação Celular , Meios de Cultura , Células-Tronco Embrionárias/citologia , Células-Tronco Embrionárias/metabolismo , Humanos , Camundongos , Células-Tronco Pluripotentes/metabolismo , Transdução de Sinais/genética
4.
Nucleic Acids Res ; 48(17): 9505-9520, 2020 09 25.
Artigo em Inglês | MEDLINE | ID: mdl-32870263

RESUMO

Rapid growth of single-cell transcriptomic data provides unprecedented opportunities for close scrutinizing of dynamical cellular processes. Through investigating epithelial-to-mesenchymal transition (EMT), we develop an integrative tool that combines unsupervised learning of single-cell transcriptomic data and multiscale mathematical modeling to analyze transitions during cell fate decision. Our approach allows identification of individual cells making transition between all cell states, and inference of genes that drive transitions. Multiscale extractions of single-cell scale outputs naturally reveal intermediate cell states (ICS) and ICS-regulated transition trajectories, producing emergent population-scale models to be explored for design principles. Testing on the newly designed single-cell gene regulatory network model and applying to twelve published single-cell EMT datasets in cancer and embryogenesis, we uncover the roles of ICS on adaptation, noise attenuation, and transition efficiency in EMT, and reveal their trade-off relations. Overall, our unsupervised learning method is applicable to general single-cell transcriptomic datasets, and our integrative approach at single-cell resolution may be adopted for other cell fate transition systems beyond EMT.


Assuntos
Células-Tronco Embrionárias/patologia , Transição Epitelial-Mesenquimal/fisiologia , Perfilação da Expressão Gênica , Redes Reguladoras de Genes , Modelos Biológicos , Animais , Diferenciação Celular , Células-Tronco Embrionárias/citologia , Células-Tronco Embrionárias/fisiologia , Transição Epitelial-Mesenquimal/genética , Regulação Neoplásica da Expressão Gênica , Neoplasias de Cabeça e Pescoço/genética , Neoplasias de Cabeça e Pescoço/patologia , Humanos , Camundongos , Análise de Célula Única , Neoplasias Cutâneas/genética , Neoplasias Cutâneas/patologia , Carcinoma de Células Escamosas de Cabeça e Pescoço/genética , Carcinoma de Células Escamosas de Cabeça e Pescoço/patologia
5.
PLoS One ; 15(9): e0233072, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32911495

RESUMO

In mammals, the fusion of two gametes, an oocyte and a spermatozoon, during fertilization forms a totipotent zygote. There has been no reported case of adult mammal development by natural parthenogenesis, in which embryos develop from unfertilized oocytes. The genome and epigenetic information of haploid gametes are crucial for mammalian development. Haploid embryonic stem cells (haESCs) can be established from uniparental blastocysts and possess only one set of chromosomes. Previous studies have shown that sperm or oocyte genome can be replaced by haESCs with or without manipulation of genomic imprinting for generation of mice. Recently, these remarkable semi-cloning methods have been applied for screening of key factors of mouse embryonic development. While haESCs have been applied as substitutes of gametic genomes, the fundamental mechanism how haESCs contribute to the genome of totipotent embryos is unclear. Here, we show the generation of fertile semi-cloned mice by injection of parthenogenetic haESCs (phaESCs) into oocytes after deletion of two differentially methylated regions (DMRs), the IG-DMR and H19-DMR. For characterizing the genome of semi-cloned embryos further, we establish ESC lines from semi-cloned blastocysts. We report that polyploid karyotypes are observed in semi-cloned ESCs (scESCs). Our results confirm that mitotically arrested phaESCs yield semi-cloned embryos and mice when the IG-DMR and H19-DMR are deleted. In addition, we highlight the occurrence of polyploidy that needs to be considered for further improving the development of semi-cloned embryos derived by haESC injection.


Assuntos
Clonagem de Organismos/métodos , Desenvolvimento Embrionário , Haploidia , Partenogênese , Poliploidia , Animais , Linhagem Celular , Células-Tronco Embrionárias/citologia , Feminino , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos DBA
6.
Proc Natl Acad Sci U S A ; 117(34): 20625-20635, 2020 08 25.
Artigo em Inglês | MEDLINE | ID: mdl-32788350

RESUMO

Profilin2 (PFN2) is a target of the embryonic stem cell (ESC)-enriched miR-290 family of microRNAs (miRNAs) and an actin/dynamin-binding protein implicated in endocytosis. Here we show that the miR-290-PFN2 pathway regulates many aspects of ESC biology. In the absence of miRNAs, PFN2 is up-regulated in ESCs, with a resulting decrease in endocytosis. Reintroduction of miR-290, knockout of Pfn2, or disruption of the PFN2-dynamin interaction domain in miRNA-deficient cells reverses the endocytosis defect. The reduced endocytosis is associated with impaired extracellular signal-regulated kinase (ERK) signaling, delayed ESC cell cycle progression, and repressed ESC differentiation. Mutagenesis of the single canonical conserved 3' UTR miR-290-binding site of Pfn2 or overexpression of the Pfn2 open reading frame alone in otherwise wild-type cells largely recapitulates these phenotypes. Taken together, these findings define an axis of posttranscriptional control, endocytosis, and signal transduction that is important for ESC proliferation and differentiation.


Assuntos
Células-Tronco Embrionárias/metabolismo , Sistema de Sinalização das MAP Quinases , MicroRNAs/metabolismo , Células-Tronco Pluripotentes/metabolismo , Profilinas/metabolismo , Regiões 3' não Traduzidas , Animais , Diferenciação Celular/genética , Linhagem Celular , Células-Tronco Embrionárias/citologia , Endocitose/fisiologia , Humanos , Camundongos , Camundongos Knockout , MicroRNAs/genética , Células-Tronco Pluripotentes/citologia , Profilinas/genética , Transdução de Sinais/genética
7.
Nat Commun ; 11(1): 4265, 2020 08 26.
Artigo em Inglês | MEDLINE | ID: mdl-32848154

RESUMO

Retinoids regulate a wide spectrum of cellular functions from the embryo throughout adulthood, including cell differentiation, metabolic regulation, and inflammation. These traits make retinoids very attractive molecules for medical purposes. In light of some of the physicochemical limitations of retinoids, the development of drug delivery systems offers several advantages for clinical translation of retinoid-based therapies, including improved solubilization, prolonged circulation, reduced toxicity, sustained release, and improved efficacy. In this Review, we discuss advances in preclinical and clinical tests regarding retinoid formulations, specifically the ones based in natural retinoids, evaluated in the context of regenerative medicine, brain, cancer, skin, and immune diseases. Advantages and limitations of retinoid formulations, as well as prospects to push the field forward, will be presented.


Assuntos
Sistemas de Liberação de Medicamentos/métodos , Medicina Regenerativa/métodos , Retinoides/administração & dosagem , Animais , Encefalopatias/tratamento farmacológico , Diferenciação Celular/efeitos dos fármacos , Ensaios Clínicos como Assunto , Composição de Medicamentos , Sistemas de Liberação de Medicamentos/tendências , Células-Tronco Embrionárias/citologia , Células-Tronco Embrionárias/efeitos dos fármacos , Humanos , Doenças do Sistema Imunitário/tratamento farmacológico , Neoplasias/tratamento farmacológico , Medicina Regenerativa/tendências , Retinoides/química , Retinoides/uso terapêutico , Transdução de Sinais , Dermatopatias/tratamento farmacológico
8.
Mol Cell Biol ; 40(20)2020 09 28.
Artigo em Inglês | MEDLINE | ID: mdl-32817139

RESUMO

Lysine demethylase 6A (KDM6A), also known as UTX, belongs to the KDM6 family of histone H3 lysine 27 (H3K27) demethylases, which also includes UTY and KDM6B (JMJD3). The KDM6A protein contains six tetratricopeptide repeat (TPR) domains and an enzymatic Jumonji C (JmjC) domain that catalyzes the removal of di- and trimethylation on H3K27. KDM6A physically associates with histone H3 lysine 4 monomethyltransferases MLL3 (KMT2C) and MLL4 (KMT2D). Since its identification as an H3K27 demethylase in 2007, studies have reported KDM6A's critical roles in cell differentiation, development, and cancer. KDM6A is important for differentiation of embryonic stem cells and development of various tissues. Mutations of KDM6A cause Kabuki syndrome. KDM6A is frequently mutated in cancers and functions as a tumor suppressor. KDM6A is redundant with UTY and functions largely independently of its demethylase activity. It regulates gene expression, likely through the associated transcription factors and MLL3/4 on enhancers. However, KDM6A enzymatic activity is required in certain cellular contexts. Functional redundancy between H3K27 demethylase activities of KDM6A and KDM6B in vivo has yet to be determined. Further understanding of KDM6A functions and working mechanisms will provide more insights into enhancer regulation and may help generate novel therapeutic approaches to treat KDM6A-related diseases.


Assuntos
Diferenciação Celular/genética , Regulação da Expressão Gênica/genética , Histona Desmetilases/genética , Neoplasias/genética , Domínio Catalítico/genética , Montagem e Desmontagem da Cromatina/genética , Células-Tronco Embrionárias/citologia , Genes Supressores de Tumor , Histona Desmetilases/metabolismo , Humanos , Histona Desmetilases com o Domínio Jumonji/metabolismo , Proteínas Nucleares/metabolismo
9.
Mol Cell Biol ; 40(18)2020 08 28.
Artigo em Inglês | MEDLINE | ID: mdl-32661120

RESUMO

The DNA and protein complex known as chromatin is subject to posttranslational modifications (PTMs) that regulate cellular functions such that PTM dysregulation can lead to disease, including cancer. One critical PTM is acetylation/deacetylation, which is being investigated as a means to develop targeted cancer therapies. The histone acetyltransferase (HAT) family of proteins performs histone acetylation. In humans, MOF (hMOF), a member of the MYST family of HATs, acetylates histone H4 at lysine 16 (H4K16ac). MOF-mediated acetylation plays a critical role in the DNA damage response (DDR) and embryonic stem cell development. Functionally, MOF is found in two distinct complexes: NSL (nonspecific lethal) in humans and MSL (male-specific lethal) in flies. The NSL complex is also able to acetylate additional histone H4 sites. Dysregulation of MOF activity occurs in multiple cancers, including ovarian cancer, medulloblastoma, breast cancer, colorectal cancer, and lung cancer. Bioinformatics analysis of KAT8, the gene encoding hMOF, indicated that it is highly overexpressed in kidney tumors as part of a concerted gene coexpression program that can support high levels of chromosome segregation and cell proliferation. The linkage between MOF and tumor proliferation suggests that there are additional functions of MOF that remain to be discovered.


Assuntos
Dano ao DNA , Células-Tronco Embrionárias/citologia , Histona Acetiltransferases/metabolismo , Acetilação , Carcinogênese/metabolismo , Diferenciação Celular/fisiologia , Núcleo Celular/metabolismo , Proliferação de Células/fisiologia , Transformação Celular Neoplásica/metabolismo , Cromatina/metabolismo , Células-Tronco Embrionárias/metabolismo , Células-Tronco Embrionárias/fisiologia , Histonas/metabolismo , Humanos , Neoplasias Pulmonares/metabolismo , Proteínas Nucleares/metabolismo , Processamento de Proteína Pós-Traducional
10.
PLoS Comput Biol ; 16(7): e1007471, 2020 07.
Artigo em Inglês | MEDLINE | ID: mdl-32716923

RESUMO

Disease development and cell differentiation both involve dynamic changes; therefore, the reconstruction of dynamic gene regulatory networks (DGRNs) is an important but difficult problem in systems biology. With recent technical advances in single-cell RNA sequencing (scRNA-seq), large volumes of scRNA-seq data are being obtained for various processes. However, most current methods of inferring DGRNs from bulk samples may not be suitable for scRNA-seq data. In this work, we present scPADGRN, a novel DGRN inference method using "time-series" scRNA-seq data. scPADGRN combines the preconditioned alternating direction method of multipliers with cell clustering for DGRN reconstruction. It exhibits advantages in accuracy, robustness and fast convergence. Moreover, a quantitative index called Differentiation Genes' Interaction Enrichment (DGIE) is presented to quantify the interaction enrichment of genes related to differentiation. From the DGIE scores of relevant subnetworks, we infer that the functions of embryonic stem (ES) cells are most active initially and may gradually fade over time. The communication strength of known contributing genes that facilitate cell differentiation increases from ES cells to terminally differentiated cells. We also identify several genes responsible for the changes in the DGIE scores occurring during cell differentiation based on three real single-cell datasets. Our results demonstrate that single-cell analyses based on network inference coupled with quantitative computations can reveal key transcriptional regulators involved in cell differentiation and disease development.


Assuntos
Algoritmos , Biologia Computacional/métodos , Redes Reguladoras de Genes/genética , Análise de Sequência de RNA/métodos , Análise de Célula Única/métodos , Animais , Diferenciação Celular/genética , Simulação por Computador , Células-Tronco Embrionárias/citologia , Células-Tronco Embrionárias/fisiologia , Perfilação da Expressão Gênica , Humanos , Camundongos
11.
Mol Cell ; 79(1): 167-179.e11, 2020 07 02.
Artigo em Inglês | MEDLINE | ID: mdl-32497496

RESUMO

The identification of microRNA (miRNA) targets by Ago2 crosslinking-immunoprecipitation (CLIP) methods has provided major insights into the biology of this important class of non-coding RNAs. However, these methods are technically challenging and not easily applicable to an in vivo setting. To overcome these limitations and facilitate the investigation of miRNA functions in vivo, we have developed a method based on a genetically engineered mouse harboring a conditional Halo-Ago2 allele expressed from the endogenous Ago2 locus. By using a resin conjugated to the HaloTag ligand, Ago2-miRNA-mRNA complexes can be purified from cells and tissues expressing the endogenous Halo-Ago2 allele. We demonstrate the reproducibility and sensitivity of this method in mouse embryonic stem cells, developing embryos, adult tissues, and autochthonous mouse models of human brain and lung cancers. This method and the datasets we have generated will facilitate the characterization of miRNA-mRNA networks in vivo under physiological and pathological conditions.


Assuntos
Proteínas Argonauta/fisiologia , Células-Tronco Embrionárias/metabolismo , Glioma/metabolismo , MicroRNAs/metabolismo , RNA Mensageiro/metabolismo , Proteínas Recombinantes de Fusão/metabolismo , Animais , Células-Tronco Embrionárias/citologia , Feminino , Regulação da Expressão Gênica , Glioma/genética , Glioma/patologia , Sequenciamento de Nucleotídeos em Larga Escala , Hidrolases/genética , Camundongos , Camundongos Knockout , MicroRNAs/genética , Ligação Proteica , RNA Mensageiro/genética , Proteínas Recombinantes de Fusão/genética
12.
Proc Natl Acad Sci U S A ; 117(27): 15702-15711, 2020 07 07.
Artigo em Inglês | MEDLINE | ID: mdl-32576691

RESUMO

Mammalian cells contain two isoforms of RNA polymerase III (Pol III) that differ in only a single subunit, with POLR3G in one form (Pol IIIα) and the related POLR3GL in the other form (Pol IIIß). Previous research indicates that POLR3G and POLR3GL are differentially expressed, with POLR3G expression being highly enriched in embryonic stem cells (ESCs) and tumor cells relative to the ubiquitously expressed POLR3GL. To date, the functional differences between these two subunits remain largely unexplored, especially in vivo. Here, we show that POLR3G and POLR3GL containing Pol III complexes bind the same target genes and assume the same functions both in vitro and in vivo and, to a significant degree, can compensate for each other in vivo. Notably, an observed defect in the differentiation ability of POLR3G knockout ESCs can be rescued by exogenous expression of POLR3GL. Moreover, whereas POLR3G knockout mice die at a very early embryonic stage, POLR3GL knockout mice complete embryonic development without noticeable defects but die at about 3 wk after birth with signs of both general growth defects and potential cerebellum-related neuronal defects. The different phenotypes of the knockout mice likely reflect differential expression levels of POLR3G and POLR3GL across developmental stages and between tissues and insufficient amounts of total Pol III in vivo.


Assuntos
Cerebelo/crescimento & desenvolvimento , Desenvolvimento Embrionário/genética , Neurônios/metabolismo , RNA Polimerase III/genética , Animais , Sítios de Ligação/genética , Diferenciação Celular/genética , Cerebelo/patologia , Células-Tronco Embrionárias/citologia , Células-Tronco Embrionárias/metabolismo , Regulação da Expressão Gênica no Desenvolvimento/genética , Humanos , Camundongos , Camundongos Knockout , Neurônios/patologia , Ligação Proteica/genética , Isoformas de Proteínas/genética , Subunidades Proteicas/genética
13.
Proc Natl Acad Sci U S A ; 117(22): 12182-12191, 2020 06 02.
Artigo em Inglês | MEDLINE | ID: mdl-32414917

RESUMO

In multicellular organisms, paralogs from gene duplication survive purifying selection by evolving tissue-specific expression and function. Whether this genetic redundancy is also selected for within a single cell type is unclear for multimember paralogs, as exemplified by the four obligatory Lef/Tcf transcription factors of canonical Wnt signaling, mainly due to the complex genetics involved. Using the developing mouse lung as a model system, we generate two quadruple conditional knockouts, four triple mutants, and various combinations of double mutants, showing that the four Lef/Tcf genes function redundantly in the presence of at least two Lef/Tcf paralogs, but additively upon losing additional paralogs to specify and maintain lung epithelial progenitors. Prelung-specification, pan-epithelial double knockouts have no lung phenotype; triple knockouts have varying phenotypes, including defective branching and tracheoesophageal fistulas; and the quadruple knockout barely forms a lung, resembling the Ctnnb1 mutant. Postlung-specification deletion of all four Lef/Tcf genes leads to branching defects, down-regulation of progenitor genes, premature alveolar differentiation, and derepression of gastrointestinal genes, again phenocopying the corresponding Ctnnb1 mutant. Our study supports a monotonic, positive signaling relationship between CTNNB1 and Lef/Tcf in lung epithelial progenitors as opposed to reported repressor functions of Lef/Tcf, and represents a thorough in vivo analysis of cell-type-specific genetic redundancy among the four Lef/Tcf paralogs.


Assuntos
Embrião de Mamíferos/metabolismo , Células-Tronco Embrionárias/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Pulmão/metabolismo , Fator 1 de Ligação ao Facilitador Linfoide/fisiologia , Células-Tronco/metabolismo , beta Catenina/metabolismo , Animais , Diferenciação Celular , Embrião de Mamíferos/citologia , Células-Tronco Embrionárias/citologia , Feminino , Fator 1-alfa Nuclear de Hepatócito/fisiologia , Pulmão/citologia , Camundongos , Camundongos Knockout , Análise de Célula Única , Células-Tronco/citologia , Proteína 1 Semelhante ao Fator 7 de Transcrição/fisiologia , Proteína 2 Semelhante ao Fator 7 de Transcrição/fisiologia , Proteínas Wnt/genética , Proteínas Wnt/metabolismo , beta Catenina/genética
14.
PLoS One ; 15(5): e0233057, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32396545

RESUMO

Poor survival of human pluripotent stem cells (hPSCs) following freezing, thawing, or passaging hinders the maintenance and differentiation of stem cells. Rho-associated kinases (ROCKs) play a crucial role in hPSC survival. To date, a typical ROCK inhibitor, Y-27632, has been the primary agent used in hPSC research. Here, we report that another ROCK inhibitor, fasudil, can be used as an alternative and is cheaper than Y-27632. It increased hPSC growth following thawing and passaging, like Y-27632, and did not affect pluripotency, differentiation ability, and chromosome integrity. Furthermore, fasudil promoted retinal pigment epithelium (RPE) differentiation and the survival of neural crest cells (NCCs) during differentiation. It was also useful for single-cell passaging of hPSCs and during aggregation. These findings suggest that fasudil can replace Y-27632 for use in stem research.


Assuntos
1-(5-Isoquinolinasulfonil)-2-Metilpiperazina/análogos & derivados , Células-Tronco Pluripotentes/citologia , Células-Tronco Pluripotentes/efeitos dos fármacos , Quinases Associadas a rho/antagonistas & inibidores , 1-(5-Isoquinolinasulfonil)-2-Metilpiperazina/farmacologia , Amidas/farmacologia , Diferenciação Celular/efeitos dos fármacos , Linhagem Celular , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Células-Tronco Embrionárias/citologia , Células-Tronco Embrionárias/efeitos dos fármacos , Humanos , Células-Tronco Pluripotentes Induzidas/citologia , Células-Tronco Pluripotentes Induzidas/efeitos dos fármacos , Crista Neural/citologia , Crista Neural/efeitos dos fármacos , Inibidores de Proteínas Quinases/farmacologia , Piridinas/farmacologia , Epitélio Pigmentado da Retina/citologia , Epitélio Pigmentado da Retina/efeitos dos fármacos , Pesquisa com Células-Tronco
15.
Nat Cell Biol ; 22(6): 651-662, 2020 06.
Artigo em Inglês | MEDLINE | ID: mdl-32393886

RESUMO

BMP4 regulates a plethora of developmental processes, including the dorsal-ventral axis and neural patterning. Here, we report that BMP4 reconfigures the nuclear architecture during the primed-to-naive transition (PNT). We first established a BMP4-driven PNT and show that BMP4 orchestrates the chromatin accessibility dynamics during PNT. Among the loci opened early by BMP4, we identified Zbtb7a and Zbtb7b (Zbtb7a/b) as targets that drive PNT. ZBTB7A/B in turn facilitate the opening of naive pluripotent chromatin loci and the activation of nearby genes. Mechanistically, ZBTB7A not only binds to chromatin loci near to the genes that are activated, but also strategically occupies those that are silenced, consistent with a role of BMP4 in both activating and suppressing gene expression during PNT at the chromatin level. Our results reveal a previously unknown function of BMP4 in regulating nuclear architecture and link its targets ZBTB7A/B to chromatin remodelling and pluripotent fate control.


Assuntos
Proteína Morfogenética Óssea 4/metabolismo , Cromatina/metabolismo , Proteínas de Ligação a DNA/metabolismo , Células-Tronco Embrionárias/citologia , Camadas Germinativas/citologia , Células-Tronco Pluripotentes/citologia , Fatores de Transcrição/metabolismo , Animais , Blastocisto/citologia , Blastocisto/metabolismo , Proteína Morfogenética Óssea 4/genética , Diferenciação Celular , Células Cultivadas , Cromatina/genética , Proteínas de Ligação a DNA/genética , Células-Tronco Embrionárias/metabolismo , Feminino , Regulação da Expressão Gênica no Desenvolvimento , Camadas Germinativas/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos CBA , Células-Tronco Pluripotentes/metabolismo , Transdução de Sinais , Fatores de Transcrição/genética
16.
Life Sci ; 255: 117817, 2020 Aug 15.
Artigo em Inglês | MEDLINE | ID: mdl-32446845

RESUMO

Glucocorticoids can promote cardiomyocyte maturation. However, the mechanism underlying glucocorticoid-mediated cardiomyocyte maturation is still unclear. Mitophagy plays a key role in cardiomyocyte maturation. Based on current knowledge, our study evaluated the effects of the glucocorticoid dexamethasone (100 nM) on the maturation of mouse embryonic stem cell-derived cardiomyocytes and the role of mitophagy in this maturation. The results showed that dexamethasone can promote embryonic stem cell-derived cardiomyocyte maturation, inhibit cardiomyocyte proliferation, and promote myocardial fiber arrangement. However, dexamethasone did not affect mitochondrial morphology in cardiomyocytes. Glucocorticoid receptor inhibitors (RU486, 1 nM) can inhibit dexamethasone-mediated cardiomyocyte maturation. Additionally, dexamethasone can promote mitophagy in embryonic stem cell-derived cardiomyocytes and induce LC3 and lysosomal aggregation in mitochondria. The inhibition of mitophagy can inhibit the cardiomyocyte maturation effect of dexamethasone. Furthermore, our research found that dexamethasone may mediate the occurrence of mitophagy in cardiomyocytes through Parkin. The siRNA-mediated inhibition of Parkin expression can inhibit mitochondrial autophagy caused by dexamethasone, thus inhibiting cardiomyocyte maturation. Overall, our study found that dexamethasone can promote embryonic stem cell-derived cardiomyocyte maturation through Parkin-mediated mitophagy.


Assuntos
Dexametasona/farmacologia , Glucocorticoides/farmacologia , Mitofagia/efeitos dos fármacos , Miócitos Cardíacos/efeitos dos fármacos , Ubiquitina-Proteína Ligases/metabolismo , Animais , Autofagia/efeitos dos fármacos , Linhagem Celular , Células-Tronco Embrionárias/citologia , Células-Tronco Embrionárias/efeitos dos fármacos , Camundongos , Mitocôndrias/efeitos dos fármacos , Miócitos Cardíacos/citologia , RNA Interferente Pequeno/administração & dosagem , Receptores de Glucocorticoides/metabolismo
17.
Nat Commun ; 11(1): 2434, 2020 05 15.
Artigo em Inglês | MEDLINE | ID: mdl-32415072

RESUMO

Cell replacement is a long-standing and realistic goal for the treatment of Parkinson's disease (PD). Cells for transplantation can be obtained from fetal brain tissue or from stem cells. However, after transplantation, dopamine (DA) neurons are seen to be a minor component of grafts, and it has remained difficult to determine the identity of other cell types. Here, we report analysis by single-cell RNA sequencing (scRNA-seq) combined with comprehensive histological analyses to characterize intracerebral grafts from human embryonic stem cells (hESCs) and fetal tissue after functional maturation in a pre-clinical rat PD model. We show that neurons and astrocytes are major components in both fetal and stem cell-derived grafts. Additionally, we identify a cell type closely resembling a class of recently identified perivascular-like cells in stem cell-derived grafts. Thus, this study uncovers previously unknown cellular diversity in a clinically relevant cell replacement PD model.


Assuntos
Neurônios Dopaminérgicos/citologia , Doença de Parkinson/terapia , Transplante de Células-Tronco , Células-Tronco/citologia , Animais , Encéfalo/metabolismo , Diferenciação Celular , Corpo Estriado , Modelos Animais de Doenças , Dopamina/metabolismo , Células-Tronco Embrionárias/citologia , Feminino , Sobrevivência de Enxerto , Humanos , Família Multigênica , RNA-Seq , Ratos , Ratos Nus , Regeneração , Análise de Célula Única , Transcriptoma
18.
Nat Commun ; 11(1): 2420, 2020 05 15.
Artigo em Inglês | MEDLINE | ID: mdl-32415101

RESUMO

Archetypal human pluripotent stem cells (hPSC) are widely considered to be equivalent in developmental status to mouse epiblast stem cells, which correspond to pluripotent cells at a late post-implantation stage of embryogenesis. Heterogeneity within hPSC cultures complicates this interspecies comparison. Here we show that a subpopulation of archetypal hPSC enriched for high self-renewal capacity (ESR) has distinct properties relative to the bulk of the population, including a cell cycle with a very low G1 fraction and a metabolomic profile that reflects a combination of oxidative phosphorylation and glycolysis. ESR cells are pluripotent and capable of differentiation into primordial germ cell-like cells. Global DNA methylation levels in the ESR subpopulation are lower than those in mouse epiblast stem cells. Chromatin accessibility analysis revealed a unique set of open chromatin sites in ESR cells. RNA-seq at the subpopulation and single cell levels shows that, unlike mouse epiblast stem cells, the ESR subset of hPSC displays no lineage priming, and that it can be clearly distinguished from gastrulating and extraembryonic cell populations in the primate embryo. ESR hPSC correspond to an earlier stage of post-implantation development than mouse epiblast stem cells.


Assuntos
Células-Tronco Embrionárias/citologia , Camadas Germinativas/citologia , Células-Tronco Pluripotentes/citologia , Animais , Diferenciação Celular , Cromatina/metabolismo , Metilação de DNA , Epigenoma , Citometria de Fluxo , Técnica Indireta de Fluorescência para Anticorpo , Fase G1 , Camadas Germinativas/metabolismo , Glicólise , Humanos , Sistema de Sinalização das MAP Quinases , Metabolômica , Camundongos , Mitocôndrias/metabolismo , Fosforilação Oxidativa , RNA-Seq , Transdução de Sinais
19.
Sci Rep ; 10(1): 6785, 2020 04 22.
Artigo em Inglês | MEDLINE | ID: mdl-32321995

RESUMO

The adult human central nervous system (CNS) has very limited regenerative capability, and injury at the cellular and molecular level cannot be studied in vivo. Modelling neural damage in human systems is crucial to identifying species-specific responses to injury and potentially neurotoxic compounds leading to development of more effective neuroprotective agents. Hence we developed human neural stem cell (hNSC) 3-dimensional (3D) cultures and tested their potential for modelling neural insults, including hypoxic-ischaemic and Ca2+-dependent injury. Standard 3D conditions for rodent cells support neuroblastoma lines used as human CNS models, but not hNSCs, but in all cases changes in culture architecture alter gene expression. Importantly, response to damage differs in 2D and 3D cultures and this is not due to reduced drug accessibility. Together, this study highlights the impact of culture cytoarchitecture on hNSC phenotype and damage response, indicating that 3D models may be better predictors of in vivo response to damage and compound toxicity.


Assuntos
Técnicas de Cultura de Células/métodos , Sistema Nervoso Central/fisiologia , Células-Tronco Embrionárias/fisiologia , Células-Tronco Neurais/fisiologia , Neurônios/fisiologia , Diferenciação Celular/efeitos dos fármacos , Diferenciação Celular/genética , Diferenciação Celular/fisiologia , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Proliferação de Células/genética , Proliferação de Células/fisiologia , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/genética , Células Cultivadas , Sistema Nervoso Central/citologia , Sistema Nervoso Central/embriologia , Células-Tronco Embrionárias/citologia , Células-Tronco Embrionárias/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Glucose/metabolismo , Glucose/farmacologia , Humanos , Células-Tronco Neurais/citologia , Células-Tronco Neurais/metabolismo , Neuroblastoma/genética , Neuroblastoma/metabolismo , Neuroblastoma/patologia , Neurônios/citologia , Neurônios/metabolismo , Oxigênio/metabolismo , Oxigênio/farmacologia , Tapsigargina/farmacologia , Traumatismos do Sistema Nervoso/genética , Traumatismos do Sistema Nervoso/metabolismo , Traumatismos do Sistema Nervoso/patologia
20.
Biochem Pharmacol ; 177: 113984, 2020 07.
Artigo em Inglês | MEDLINE | ID: mdl-32311348

RESUMO

Pluripotent stem cells are have therapeutic applications in regenerative medicine and drug discovery. However, the differentiation of stem cells in vitro hinders their large-scale production and clinical applications. The maintenance of cell pluripotency relies on a complex network of transcription factors; of these, octamer-binding transcription factor-4 (Oct4) plays a key role. This study aimed to construct an Oct4 gene promoter-driven firefly luciferase reporter and screen small-molecule compounds could maintain cell self-renewal and pluripotency. The results showed that ethyl-p-methoxycinnamate (EPMC) enhance the promoter activity of the Oct4 gene, increased the expression of Oct4 at both mRNA and protein levels, and significantly promoted the colony formation of P19 cells. These findings suggesting that EPMC could reinforce the self-renewal capacity of P19 cells. The pluripotency markers Oct4, SRY-related high-mobility-group-box protein-2, and Nanog were expressed at higher levels in EPMC-induced colonies. EPMC could promote teratoma formation and differentiation potential of P19 cells in vivo. It also enhanced self-renewal and pluripotency of human umbilical cord mesenchymal stem cells and mouse embryonic stem cells. Moreover, it significantly activated the nuclear factor kappa B (NF-κB) signaling pathway via the myeloid differentiation factor 88-dependent pathway. The expression level of Oct4 decreased after blocking the NF-κB signaling pathway, suggesting that EPMC promoted the expression of Oct4 partially through the NF-κB signaling pathway. This study indicated that EPMC could maintain self-renewal and pluripotency of stem cells.


Assuntos
Autorrenovação Celular/efeitos dos fármacos , Cinamatos/farmacologia , NF-kappa B/genética , Fator 3 de Transcrição de Octâmero/genética , Células-Tronco Pluripotentes/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacos , Animais , Diferenciação Celular/efeitos dos fármacos , Autorrenovação Celular/genética , Células-Tronco Embrionárias/citologia , Células-Tronco Embrionárias/efeitos dos fármacos , Células-Tronco Embrionárias/metabolismo , Fibroblastos/citologia , Fibroblastos/efeitos dos fármacos , Fibroblastos/metabolismo , Regulação da Expressão Gênica , Genes Reporter , Humanos , Luciferases/genética , Luciferases/metabolismo , Células-Tronco Mesenquimais/citologia , Células-Tronco Mesenquimais/efeitos dos fármacos , Células-Tronco Mesenquimais/metabolismo , Camundongos , Fator 88 de Diferenciação Mieloide/genética , Fator 88 de Diferenciação Mieloide/metabolismo , Inibidor de NF-kappaB alfa/genética , Inibidor de NF-kappaB alfa/metabolismo , NF-kappa B/agonistas , NF-kappa B/metabolismo , Proteína Homeobox Nanog/genética , Proteína Homeobox Nanog/metabolismo , Fator 3 de Transcrição de Octâmero/agonistas , Fator 3 de Transcrição de Octâmero/metabolismo , Células-Tronco Pluripotentes/citologia , Células-Tronco Pluripotentes/metabolismo , Regiões Promotoras Genéticas , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Fatores de Transcrição SOXB1/genética , Fatores de Transcrição SOXB1/metabolismo , Transdução de Sinais/genética
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