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1.
Nat Commun ; 11(1): 382, 2020 01 20.
Artigo em Inglês | MEDLINE | ID: mdl-31959746

RESUMO

Neurogenesis, a highly orchestrated process, entails the transition from a pluripotent to neural state and involves neural progenitor cells (NPCs) and neuronal/glial subtypes. However, the precise epigenetic mechanisms underlying fate decision remain poorly understood. Here, we delete KDM6s (JMJD3 and/or UTX), the H3K27me3 demethylases, in human embryonic stem cells (hESCs) and show that their deletion does not impede NPC generation from hESCs. However, KDM6-deficient NPCs exhibit poor proliferation and a failure to differentiate into neurons and glia. Mechanistically, both JMJD3 and UTX are found to be enriched in gene loci essential for neural development in hNPCs, and KDM6 impairment leads to H3K27me3 accumulation and blockade of DNA accessibility at these genes. Interestingly, forced expression of neuron-specific chromatin remodelling BAF (nBAF) rescues the neuron/glia defect in KDM6-deficient NPCs despite H3K27me3 accumulation. Our findings uncover the differential requirement of KDM6s in specifying NPCs and neurons/glia and highlight the contribution of individual epigenetic regulators in fate decisions in a human development model.


Assuntos
Diferenciação Celular/genética , Regulação da Expressão Gênica no Desenvolvimento , Histona Desmetilases/metabolismo , Histona Desmetilases com o Domínio Jumonji/metabolismo , Células-Tronco Neurais/fisiologia , Linhagem Celular , Proliferação de Células/genética , Metilação de DNA , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Células-Tronco Embrionárias/fisiologia , Epigênese Genética/fisiologia , Técnicas de Introdução de Genes , Técnicas de Inativação de Genes , Histona Desmetilases/genética , Histonas/genética , Histonas/metabolismo , Humanos , Histona Desmetilases com o Domínio Jumonji/genética , Neurogênese/genética , RNA-Seq
2.
EMBO J ; 38(24): e101196, 2019 12 16.
Artigo em Inglês | MEDLINE | ID: mdl-31750563

RESUMO

Parkinson's disease (PD) is neurodegenerative movement disorder characterized by degeneration of midbrain-type dopamine (mDA) neurons in the substantia nigra (SN). The RNA-binding protein Lin28 plays a role in neuronal stem cell development and neuronal differentiation. In this study, we reveal that Lin28 conditional knockout (cKO) mice show degeneration of mDA neurons in the SN, as well as PD-related behavioral deficits. We identify a loss-of-function variant of LIN28A (R192G substitution) in two early-onset PD patients. Using an isogenic human embryonic stem cell (hESC)/human induced pluripotent stem cell (hiPSC)-based disease model, we find that the Lin28 R192G variant leads to developmental defects and PD-related phenotypes in mDA neuronal cells that can be rescued by expression of wild-type Lin28A. Cell transplantation experiments in PD model rats show that correction of the LIN28A variant in the donor patient (pt)-hiPSCs leads to improved behavioral phenotypes. Our data link LIN28A to PD pathogenesis and suggest future personalized medicine targeting this variant in patients.


Assuntos
Doença de Parkinson/metabolismo , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/fisiologia , Substância Negra/metabolismo , Animais , Comportamento Animal , Transplante de Células , Modelos Animais de Doenças , Dopamina/metabolismo , Neurônios Dopaminérgicos/fisiologia , Células-Tronco Embrionárias/fisiologia , Edição de Genes , Predisposição Genética para Doença , Humanos , Células-Tronco Pluripotentes Induzidas/fisiologia , Células-Tronco Pluripotentes Induzidas/transplante , Camundongos , Camundongos Knockout , Mutação , Células-Tronco Neurais/fisiologia , Células-Tronco Neurais/transplante , Doença de Parkinson/genética , Ratos , Transplante de Células-Tronco
3.
Elife ; 82019 10 10.
Artigo em Inglês | MEDLINE | ID: mdl-31599722

RESUMO

The access of Transcription Factors (TFs) to their cognate DNA binding motifs requires a precise control over nucleosome positioning. This is especially important following DNA replication and during mitosis, both resulting in profound changes in nucleosome organization over TF binding regions. Using mouse Embryonic Stem (ES) cells, we show that the TF CTCF displaces nucleosomes from its binding site and locally organizes large and phased nucleosomal arrays, not only in interphase steady-state but also immediately after replication and during mitosis. Correlative analyses suggest this is associated with fast gene reactivation following replication and mitosis. While regions bound by other TFs (Oct4/Sox2), display major rearrangement, the post-replication and mitotic nucleosome positioning activity of CTCF is not unique: Esrrb binding regions are also characterized by persistent nucleosome positioning. Therefore, selected TFs such as CTCF and Esrrb act as resilient TFs governing the inheritance of nucleosome positioning at regulatory regions throughout the cell-cycle.


Assuntos
Fator de Ligação a CCCTC/metabolismo , Replicação do DNA , Células-Tronco Embrionárias/fisiologia , Mitose , Nucleossomos/metabolismo , Animais , Células Cultivadas , Regulação da Expressão Gênica , Camundongos , Ativação Transcricional
4.
Elife ; 82019 09 30.
Artigo em Inglês | MEDLINE | ID: mdl-31566562

RESUMO

Base excision repair (BER) functions not only in the maintenance of genomic integrity but also in active DNA demethylation and epigenetic gene regulation. This dual role raises the question if phenotypic abnormalities resulting from deficiency of BER factors are due to DNA damage or impaired DNA demethylation. Here we investigate the bifunctional DNA glycosylases/lyases NEIL1 and NEIL2, which act in repair of oxidative lesions and in epigenetic demethylation. Neil-deficiency in Xenopus embryos and differentiating mouse embryonic stem cells (mESCs) leads to a surprisingly restricted defect in cranial neural crest cell (cNCC) development. Neil-deficiency elicits an oxidative stress-induced TP53-dependent DNA damage response, which impairs early cNCC specification. Epistasis experiments with Tdg-deficient mESCs show no involvement of epigenetic DNA demethylation. Instead, Neil-deficiency results in oxidative damage specific to mitochondrial DNA, which triggers a TP53-mediated intrinsic apoptosis. Thus, NEIL1 and NEIL2 DNA glycosylases protect mitochondrial DNA against oxidative damage during neural crest differentiation.


Assuntos
Dano ao DNA , DNA Glicosilases/metabolismo , Células-Tronco Embrionárias/fisiologia , Mitocôndrias/metabolismo , Crista Neural/embriologia , Estresse Oxidativo , Animais , Diferenciação Celular , Linhagem Celular , Reparo do DNA , Camundongos , Xenopus
5.
PLoS Biol ; 17(9): e3000453, 2019 09.
Artigo em Inglês | MEDLINE | ID: mdl-31557150

RESUMO

The link between single-cell variation and population-level fate choices lacks a mechanistic explanation despite extensive observations of gene expression and epigenetic variation among individual cells. Here, we found that single human embryonic stem cells (hESCs) have different and biased differentiation potentials toward either neuroectoderm or mesendoderm depending on their G1 lengths before the onset of differentiation. Single-cell variation in G1 length operates in a dynamic equilibrium that establishes a G1 length probability distribution for a population of hESCs and predicts differentiation outcome toward neuroectoderm or mesendoderm lineages. Although sister stem cells generally share G1 lengths, a variable proportion of cells have asymmetric G1 lengths, which maintains the population dispersion. Environmental Wingless-INT (WNT) levels can control the G1 length distribution, apparently as a means of priming the fate of hESC populations once they undergo differentiation. As a downstream mechanism, global 5-hydroxymethylcytosine levels are regulated by G1 length and thereby link G1 length to differentiation outcomes of hESCs. Overall, our findings suggest that intrapopulation heterogeneity in G1 length underlies the pluripotent differentiation potential of stem cell populations.


Assuntos
Diferenciação Celular , Células-Tronco Embrionárias/fisiologia , Fase G1 , Proteínas Wnt/fisiologia , Linhagem Celular , Humanos
6.
Nat Biotechnol ; 37(10): 1198-1208, 2019 10.
Artigo em Inglês | MEDLINE | ID: mdl-31501559

RESUMO

Harnessing the potential of human embryonic stem cells to mimic normal and aberrant development with standardized models is a pressing challenge. Here we use micropattern technology to recapitulate early human neurulation in large numbers of nearly identical structures called neuruloids. Dual-SMAD inhibition followed by bone morphogenic protein 4 stimulation induced self-organization of neuruloids harboring neural progenitors, neural crest, sensory placode and epidermis. Single-cell transcriptomics unveiled the precise identities and timing of fate specification. Investigation of the molecular mechanism of neuruloid self-organization revealed a pulse of pSMAD1 at the edge that induced epidermis, whose juxtaposition to central neural fates specifies neural crest and placodes, modulated by fibroblast growth factor and Wnt. Neuruloids provide a unique opportunity to study the developmental aspects of human diseases. Using isogenic Huntington's disease human embryonic stem cells and deep neural network analysis, we show how specific phenotypic signatures arise in our model of early human development as a consequence of mutant huntingtin protein, outlining an approach for phenotypic drug screening.


Assuntos
Ectoderma/fisiologia , Células-Tronco Embrionárias/fisiologia , Doença de Huntington , Neurulação/fisiologia , Telencéfalo/crescimento & desenvolvimento , Técnicas de Cultura de Células , Diferenciação Celular/fisiologia , Linhagem Celular , Humanos , Neurogênese , Telencéfalo/fisiologia
7.
Methods Mol Biol ; 2029: 257-271, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31273748

RESUMO

Calcium imaging in an ex-vivo setup is used to understand the calcium status of isolated cells or tissue. In this chapter we explain the use of the ratiometric chemical indicator Fura-2 which can be loaded into isolated cells in the form of lipophilic acetomethyl (AM) esters. Fura-2 is a combination of calcium chelator and fluorophore, and can be used with dual wavelength excitation (340/380 nm) for quantitative calcium concentrations. The cells can then be viewed using a fluorescence microscope and captured by a CCD camera. We specifically discuss the technique involved in understanding the endoplasmic reticulum (ER)-driven store-operated calcium entry (SOCE) in human neural precursors (NPCs) and spontaneously differentiated neurons derived from a pluripotent human embryonic stem cell (hESC) line. The derivation of neural precursors from stem cells and their subsequent spontaneous neural differentiation is also explained. The method can be used for various non-excitable and excitable cell types including neurons, be it freshly isolated, from frozen vials, or derived from different stem cell lines.


Assuntos
Cálcio/metabolismo , Diferenciação Celular/fisiologia , Neurônios/metabolismo , Neurônios/fisiologia , Canais de Cálcio/metabolismo , Canais de Cálcio/fisiologia , Sinalização do Cálcio/fisiologia , Linhagem Celular , Células-Tronco Embrionárias/metabolismo , Células-Tronco Embrionárias/fisiologia , Retículo Endoplasmático/metabolismo , Retículo Endoplasmático/fisiologia , Corantes Fluorescentes/metabolismo , Fura-2/metabolismo , Humanos
8.
Science ; 364(6444): 948-951, 2019 Jun 07.
Artigo em Inglês | MEDLINE | ID: mdl-31171690

RESUMO

Embryonic development is orchestrated by robust and complex regulatory mechanisms acting at different scales of organization. In vivo studies are particularly challenging for mammals after implantation, owing to the small size and inaccessibility of the embryo. The generation of stem cell models of the embryo represents a powerful system with which to dissect this complexity. Control of geometry, modulation of the physical environment, and priming with chemical signals reveal the intrinsic capacity of embryonic stem cells to make patterns. Adding the stem cells for the extraembryonic lineages generates three-dimensional models that are more autonomous from the environment and recapitulate many features of the pre- and postimplantation mouse embryo, including gastrulation. Here, we review the principles of self-organization and how they set cells in motion to create an embryo.


Assuntos
Padronização Corporal , Desenvolvimento Embrionário , Células-Tronco Embrionárias/fisiologia , Animais , Humanos , Camundongos , Modelos Biológicos
9.
Theriogenology ; 135: 164-168, 2019 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-31216507

RESUMO

Though blastocyst production in vitro has been successful in several animal species, a culture system to produce viable and normal canine blastocysts in vitro remains to be established. In this study, we report the development of an in vitro culture system for canine preimplantation embryos produced via parthenogenetic activation (PA) and somatic cell nucleus transfer (SCNT). Our results show that the medium developed by us, named "Qingdao Agricultural University's (QAU)-4 medium", successfully breaks the developmental arrest observed at the eight-cell stage in canine embryos grown in other culture systems. The blastocysts produced in QAU-4 displayed normal blastocyst structures, including a clear inner cell mass and blastocyst cavity. We also found that blastocyst formation in PA embryos cultured in QAU-4 medium was quite high, though this was not so in the case of SCNT embryos. However, supplementation of QAU-4 medium with 100 nM of scriptaid caused a sharp increase in blastocyst formation in SCNT embryos. After culture, hatched blastocysts were also observed to successfully adhere to collagen-coated dishes, where further growth and differentiation occurred. To our knowledge, this is the first in vitro canine preimplantation embryo culture system that can successfully produce canine blastocysts.


Assuntos
Blastocisto/fisiologia , Cães/embriologia , Técnicas de Cultura Embrionária/veterinária , Técnicas de Transferência Nuclear/veterinária , Animais , Biomarcadores , Sobrevivência Celular , Células-Tronco Embrionárias/fisiologia , Feminino , Masculino , Partenogênese
10.
Elife ; 82019 05 24.
Artigo em Inglês | MEDLINE | ID: mdl-31124784

RESUMO

Enhancers are important regulatory elements that can control gene activity across vast genetic distances. However, the underlying nature of this regulation remains obscured because it has been difficult to observe in living cells. Here, we visualize the spatial organization and transcriptional output of the key pluripotency regulator Sox2 and its essential enhancer Sox2 Control Region (SCR) in living embryonic stem cells (ESCs). We find that Sox2 and SCR show no evidence of enhanced spatial proximity and that spatial dynamics of this pair is limited over tens of minutes. Sox2 transcription occurs in short, intermittent bursts in ESCs and, intriguingly, we find this activity demonstrates no association with enhancer proximity, suggesting that direct enhancer-promoter contacts do not drive contemporaneous Sox2 transcription. Our study establishes a framework for interrogation of enhancer function in living cells and supports an unexpected mechanism for enhancer control of Sox2 expression that uncouples transcription from enhancer proximity.


Assuntos
Células-Tronco Embrionárias/fisiologia , Elementos Facilitadores Genéticos , Regulação da Expressão Gênica , Fatores de Transcrição SOXB1/biossíntese , Transcrição Genética , Animais , Camundongos , Fatores de Transcrição SOXB1/genética
11.
Circ Res ; 125(1): 14-25, 2019 06 21.
Artigo em Inglês | MEDLINE | ID: mdl-30964391

RESUMO

RATIONALE: Embryonic heart is characterized of rapidly dividing cardiomyocytes required to build a working myocardium. Cardiomyocytes retain some proliferative capacity in the neonates but lose it in adulthood. Consequently, a number of signaling hubs including microRNAs are altered during cardiac development that adversely impacts regenerative potential of cardiac tissue. Embryonic stem cell cycle miRs are a class of microRNAs exclusively expressed during developmental stages; however, their effect on cardiomyocyte proliferation and heart function in adult myocardium has not been studied previously. OBJECTIVE: To determine whether transient reintroduction of embryonic stem cell cycle miR-294 promotes cardiomyocyte cell cycle reentry enhancing cardiac repair after myocardial injury. METHODS AND RESULTS: miR-294 is expressed in the heart during development, prenatal stages, lost in the neonate, and adult heart confirmed by qRT-PCR and in situ hybridization. Neonatal ventricular myocytes treated with miR-294 showed elevated expression of Ki67, p-histone H3, and Aurora B confirmed by immunocytochemistry compared with control cells. miR-294 enhanced oxidative phosphorylation and glycolysis in Neonatal ventricular myocytes measured by seahorse assay. Mechanistically, miR-294 represses Wee1 leading to increased activity of the cyclin B1/CDK1 complex confirmed by qRT-PCR and immunoblot analysis. Next, a doxycycline-inducible AAV9-miR-294 vector was delivered to mice for activating miR-294 in myocytes for 14 days continuously after myocardial infarction. miR-294-treated mice significantly improved left ventricular functions together with decreased infarct size and apoptosis 8 weeks after MI. Myocyte cell cycle reentry increased in miR-294 hearts analyzed by Ki67, pH3, and AurB (Aurora B kinase) expression parallel to increased small myocyte number in the heart. Isolated adult myocytes from miR-294 hearts showed increased 5-ethynyl-2'-deoxyuridine+ cells and upregulation of cell cycle markers and miR-294 targets 8 weeks after MI. CONCLUSIONS: Ectopic transient expression of miR-294 recapitulates developmental signaling and phenotype in cardiomyocytes promoting cell cycle reentry that leads to augmented cardiac function in mice after myocardial infarction.


Assuntos
Ciclo Celular/fisiologia , Células-Tronco Embrionárias/fisiologia , MicroRNAs/metabolismo , Infarto do Miocárdio/metabolismo , Miócitos Cardíacos/fisiologia , Animais , Animais Recém-Nascidos , Células Cultivadas , Feminino , Masculino , Camundongos , Camundongos Endogâmicos C57BL , MicroRNAs/genética , Infarto do Miocárdio/genética , Gravidez , Ratos
12.
Comput Biol Chem ; 80: 111-120, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-30947069

RESUMO

Single cell technology is a powerful tool to reveal intercellular heterogeneity and discover cellular developmental processes. When analyzing the complexity of cellular dynamics and variability, it is important to construct a pseudo-time trajectory using single-cell expression data to reflect the process of cellular development. Although a number of computational and statistical methods have been developed recently for single-cell analysis, more effective and efficient methods are still strongly needed. In this work we propose a new method named SCOUT for the inference of single-cell pseudo-time ordering with bifurcation trajectories. We first propose to use the fixed-radius near neighbors algorithms based on cell densities to find landmarks to represent the cell states, and employ the minimum spanning tree (MST) to determine the developmental branches. We then propose to use the projection of Apollonian circle or a weighted distance to determine the pseudo-time trajectories of single cells. The proposed algorithm is applied to one synthetic and two realistic single-cell datasets (including single-branching and multi-branching trajectories) and the cellular developmental dynamics is recovered successfully. Compared with other popular methods, numerical results show that our proposed method is able to generate more robust and accurate pseudo-time trajectories. The code of the method is implemented in Python and available at https://github.com/statway/SCOUT.


Assuntos
Células-Tronco Embrionárias/fisiologia , Análise de Célula Única/métodos , Algoritmos , Animais , Diferenciação Celular/genética , Diferenciação Celular/fisiologia , Bases de Dados Factuais/estatística & dados numéricos , Expressão Gênica , Humanos , Fatores de Tempo
13.
Elife ; 82019 04 23.
Artigo em Inglês | MEDLINE | ID: mdl-31012843

RESUMO

Transposable elements (TEs) are thought to have helped establish gene regulatory networks. Both the embryonic and extraembryonic lineages of the early mouse embryo have seemingly co-opted TEs as enhancers, but there is little evidence that they play significant roles in gene regulation. Here we tested a set of long terminal repeat TE families for roles as enhancers in mouse embryonic and trophoblast stem cells. Epigenomic and transcriptomic data suggested that a large number of TEs helped to establish tissue-specific gene expression programmes. Genetic editing of individual TEs confirmed a subset of these regulatory relationships. However, a wider survey via CRISPR interference of RLTR13D6 elements in embryonic stem cells revealed that only a minority play significant roles in gene regulation. Our results suggest that a subset of TEs are important for gene regulation in early mouse development, and highlight the importance of functional experiments when evaluating gene regulatory roles of TEs.


Assuntos
Elementos de DNA Transponíveis , Células-Tronco Embrionárias/fisiologia , Regulação da Expressão Gênica no Desenvolvimento , Trofoblastos/fisiologia , Animais , Camundongos
14.
Elife ; 82019 04 16.
Artigo em Inglês | MEDLINE | ID: mdl-30990414

RESUMO

During early mammalian development, the chromatin landscape undergoes profound transitions. The Zdbf2 gene-involved in growth control-provides a valuable model to study this window: upon exit from naïve pluripotency and prior to tissue differentiation, it undergoes a switch from a distal to a proximal promoter usage, accompanied by a switch from polycomb to DNA methylation occupancy. Using a mouse embryonic stem cell (ESC) system to mimic this period, we show here that four enhancers contribute to the Zdbf2 promoter switch, concomitantly with dynamic changes in chromatin architecture. In ESCs, the locus is partitioned to facilitate enhancer contacts with the distal Zdbf2 promoter. Relieving the partition enhances proximal Zdbf2 promoter activity, as observed during differentiation or with genetic mutants. Importantly, we show that 3D regulation occurs upstream of the polycomb and DNA methylation pathways. Our study reveals the importance of multi-layered regulatory frameworks to ensure proper spatio-temporal activation of developmentally important genes.


Assuntos
Diferenciação Celular , Células-Tronco Embrionárias/fisiologia , Regulação da Expressão Gênica no Desenvolvimento , Animais , Cromatina/metabolismo , Proteínas de Ligação a DNA/biossíntese , Camundongos , Regiões Promotoras Genéticas
15.
Bioessays ; 41(3): e1800222, 2019 03.
Artigo em Inglês | MEDLINE | ID: mdl-30793782

RESUMO

Targeted transitions in chromatin states at thousands of genes are essential drivers of eukaryotic development. Therefore, understanding the in vivo dynamics of epigenetic regulators is crucial for deciphering the mechanisms underpinning cell fate decisions. This review illustrates how, in addition to its cell memory function, the Polycomb group of transcriptional regulators orchestrates temporal, cell and tissue-specific expression of master genes during development. These highly sophisticated developmental transitions are dependent on the context- and tissue-specific assembly of the different types of Polycomb Group (PcG) complexes, which regulates their targeting and/or activities on chromatin. Here, an overview is provided of how PcG complexes function at multiple scales to regulate transcription, local chromatin environment, and higher order structures that support normal differentiation and are perturbed in tumorigenesis.


Assuntos
Cromatina/genética , Epigênese Genética , Genoma , Proteínas do Grupo Polycomb/genética , Animais , Carcinogênese/genética , Diferenciação Celular , Montagem e Desmontagem da Cromatina/genética , Drosophila , Proteínas de Drosophila/genética , Células-Tronco Embrionárias/fisiologia , Inativação Gênica , Humanos , Camundongos , Transcrição Genética
16.
Development ; 146(4)2019 02 18.
Artigo em Inglês | MEDLINE | ID: mdl-30777863

RESUMO

In the adult rodent brain, neural stem cells (NSCs) persist in the ventricular-subventricular zone (V-SVZ) and the subgranular zone (SGZ), which are specialized niches in which young neurons for the olfactory bulb (OB) and hippocampus, respectively, are generated. Recent studies have significantly modified earlier views on the mechanisms of NSC self-renewal and neurogenesis in the adult brain. Here, we discuss the molecular control, heterogeneity, regional specification and cell division modes of V-SVZ NSCs, and draw comparisons with NSCs in the SGZ. We highlight how V-SVZ NSCs are regulated by local signals from their immediate neighbors, as well as by neurotransmitters and factors that are secreted by distant neurons, the choroid plexus and vasculature. We also review recent advances in single cell RNA analyses that reveal the complexity of adult neurogenesis. These findings set the stage for a better understanding of adult neurogenesis, a process that one day may inspire new approaches to brain repair.


Assuntos
Células-Tronco Adultas/fisiologia , Hipocampo/fisiologia , Ventrículos Laterais/fisiologia , Células-Tronco Neurais/fisiologia , Neurogênese/fisiologia , Bulbo Olfatório/fisiologia , Animais , Comunicação Celular , Diferenciação Celular , Linhagem da Célula , Células-Tronco Embrionárias/fisiologia , Hipocampo/embriologia , Humanos , Interneurônios/fisiologia , Ventrículos Laterais/embriologia , Camundongos , Neurônios/fisiologia , Bulbo Olfatório/embriologia , Análise de Sequência de RNA , Transdução de Sinais , Análise de Célula Única , Transcriptoma
17.
Nat Cell Biol ; 21(2): 263-274, 2019 02.
Artigo em Inglês | MEDLINE | ID: mdl-30710150

RESUMO

Despite advances in the differentiation of insulin-producing cells from human embryonic stem cells, the generation of mature functional ß cells in vitro has remained elusive. To accomplish this goal, we have developed cell culture conditions to closely mimic events occurring during pancreatic islet organogenesis and ß cell maturation. In particular, we have focused on recapitulating endocrine cell clustering by isolating and reaggregating immature ß-like cells to form islet-sized enriched ß-clusters (eBCs). eBCs display physiological properties analogous to primary human ß cells, including robust dynamic insulin secretion, increased calcium signalling in response to secretagogues, and improved mitochondrial energization. Notably, endocrine cell clustering induces metabolic maturation by driving mitochondrial oxidative respiration, a process central to stimulus-secretion coupling in mature ß cells. eBCs display glucose-stimulated insulin secretion as early as three days after transplantation in mice. In summary, replicating aspects of endocrine cell clustering permits the generation of stem-cell-derived ß cells that resemble their endogenous counterparts.


Assuntos
Diferenciação Celular , Células-Tronco Embrionárias/citologia , Células Endócrinas/citologia , Fibroblastos/citologia , Células-Tronco Embrionárias Humanas/citologia , Células Secretoras de Insulina/citologia , Animais , Células Cultivadas , Células-Tronco Embrionárias/fisiologia , Células Endócrinas/fisiologia , Fibroblastos/fisiologia , Glucose/farmacologia , Células-Tronco Embrionárias Humanas/fisiologia , Humanos , Secreção de Insulina/efeitos dos fármacos , Células Secretoras de Insulina/fisiologia , Ilhotas Pancreáticas/citologia , Camundongos , Mitocôndrias/metabolismo
18.
Stem Cell Reports ; 12(2): 305-318, 2019 02 12.
Artigo em Inglês | MEDLINE | ID: mdl-30713040

RESUMO

Although pluripotent stem cells can generate various types of differentiated cells, it is unclear why lineage-committed stem/progenitor cells derived from pluripotent stem cells are decelerated and why the differentiation-resistant propensity of embryonic stem cell (ESC)/induced pluripotent stem cell (iPSC)-derived cells is predominant compared with the in vivo equivalents derived from embryonic/adult tissues. In this study, we demonstrated that iPSCs reprogrammed and maintained with three chemical inhibitors of the fibroblast growth factor 4-mitogen-activated protein kinase cascade and GSK3ß (3i) could be differentiated into all three germ layers more efficiently than the iPSCs reprogrammed without the 3i chemicals, even though they were maintained with 3i chemicals once they were reprogrammed. Although the iPSCs reprogrammed with 3i had increased numbers of Zscan4-positive cells, the Zscan4-positive cells among iPSCs that were reprogrammed without 3i did not have an accelerated differentiation ability. These observations suggest that 3i exposure during the reprogramming period determines the accelerated differentiation/maturation potentials of iPSCs that are stably maintained at the distinct state.


Assuntos
Biomarcadores/metabolismo , Diferenciação Celular/fisiologia , Reprogramação Celular/fisiologia , Células-Tronco Pluripotentes Induzidas/metabolismo , Células-Tronco Pluripotentes Induzidas/fisiologia , Animais , Células Cultivadas , Células-Tronco Embrionárias/metabolismo , Células-Tronco Embrionárias/fisiologia , Fator 4 de Crescimento de Fibroblastos/metabolismo , Camadas Germinativas/metabolismo , Camadas Germinativas/fisiologia , Glicogênio Sintase Quinase 3 beta/metabolismo , Camundongos
19.
BMC Genomics ; 20(1): 131, 2019 Feb 12.
Artigo em Inglês | MEDLINE | ID: mdl-30755158

RESUMO

BACKGROUND: Genetically engineered mice (GEM) are essential tools for understanding gene function and disease modeling. Historically, gene targeting was first done in embryonic stem cells (ESCs) derived from the 129 family of inbred strains, leading to a mixed background or congenic mice when crossed with C57BL/6 mice. Depending on the number of backcrosses and breeding strategies, genomic segments from 129-derived ESCs can be introgressed into the C57BL/6 genome, establishing a unique genetic makeup that needs characterization in order to obtain valid conclusions from experiments using GEM lines. Currently, SNP genotyping is used to detect the extent of 129-derived ESC genome introgression into C57BL/6 recipients; however, it fails to detect novel/rare variants. RESULTS: Here, we present a computational pipeline implemented in the Galaxy platform and in BASH/R script to determine genetic introgression of GEM using next generation sequencing data (NGS), such as whole genome sequencing (WGS), whole exome sequencing (WES) and RNA-Seq. The pipeline includes strategies to uncover variants linked to a targeted locus, genome-wide variant visualization, and the identification of potential modifier genes. Although these methods apply to congenic mice, they can also be used to describe variants fixed by genetic drift. As a proof of principle, we analyzed publicly available RNA-Seq data from five congenic knockout (KO) lines and our own RNA-Seq data from the Sall2 KO line. Additionally, we performed target validation using several genetics approaches. CONCLUSIONS: We revealed the impact of the 129-derived ESC genome introgression on gene expression, predicted potential modifier genes, and identified potential phenotypic interference in KO lines. Our results demonstrate that our new approach is an effective method to determine genetic introgression of GEM.


Assuntos
Animais Geneticamente Modificados/genética , Patrimônio Genético , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Animais , Biologia Computacional , Inibidor de Quinase Dependente de Ciclina p21/genética , Proteínas de Ligação a DNA , Células-Tronco Embrionárias/fisiologia , Regulação da Expressão Gênica , Genes Modificadores , Peptídeos e Proteínas de Sinalização Intracelular/genética , Camundongos , Camundongos da Linhagem 129 , Camundongos Congênicos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Polimorfismo de Nucleotídeo Único , Locos de Características Quantitativas/genética , Análise de Sequência de RNA , Sequenciamento Completo do Exoma
20.
Nat Genet ; 51(3): 529-540, 2019 03.
Artigo em Inglês | MEDLINE | ID: mdl-30804559

RESUMO

Here, we report a single-cell DNA replication sequencing method, scRepli-seq, a genome-wide methodology that measures copy number differences between replicated and unreplicated DNA. Using scRepli-seq, we demonstrate that replication-domain organization is conserved among individual mouse embryonic stem cells (mESCs). Differentiated mESCs exhibited distinct profiles, which were also conserved among cells. Haplotype-resolved scRepli-seq revealed similar replication profiles of homologous autosomes, while the inactive X chromosome was clearly replicated later than its active counterpart. However, a small degree of cell-to-cell replication-timing heterogeneity was present, which was smallest at the beginning and the end of S phase. In addition, developmentally regulated domains were found to deviate from others and showed a higher degree of heterogeneity, thus suggesting a link to developmental plasticity. Moreover, allelic expression imbalance was found to strongly associate with replication-timing asynchrony. Our results form a foundation for single-cell-level understanding of DNA replication regulation and provide insights into three-dimensional genome organization.


Assuntos
Replicação do DNA/genética , DNA/genética , Mamíferos/genética , Animais , Diferenciação Celular/genética , Linhagem Celular , Variações do Número de Cópias de DNA/genética , Período de Replicação do DNA/genética , Células-Tronco Embrionárias/fisiologia , Genoma/genética , Estudo de Associação Genômica Ampla/métodos , Instabilidade Genômica/genética , Humanos , Camundongos , Células-Tronco Embrionárias Murinas/fisiologia , Fase S/genética , Cromossomo X/genética
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