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1.
Int J Mol Sci ; 22(9)2021 Apr 28.
Artigo em Inglês | MEDLINE | ID: mdl-33924833

RESUMO

Glaucoma, the second leading cause of blindness worldwide, is an incurable neurodegenerative disorder due to the dysfunction of retinal ganglion cells (RGCs). RGCs function as the only output neurons conveying the detected light information from the retina to the brain, which is a bottleneck of vision formation. RGCs in mammals cannot regenerate if injured, and RGC subtypes differ dramatically in their ability to survive and regenerate after injury. Recently, novel RGC subtypes and markers have been uncovered in succession. Meanwhile, apart from great advances in RGC axon regeneration, some degree of experimental RGC regeneration has been achieved by the in vitro differentiation of embryonic stem cells and induced pluripotent stem cells or in vivo somatic cell reprogramming, which provides insights into the future therapy of myriad neurodegenerative disorders. Further approaches to the combination of different factors will be necessary to develop efficacious future therapeutic strategies to promote ultimate axon and RGC regeneration and functional vision recovery following injury.


Assuntos
Regeneração Nervosa , Células Ganglionares da Retina/fisiologia , Animais , Axônios/fisiologia , Diferenciação Celular , Células-Tronco Embrionárias/fisiologia , Humanos , Células-Tronco Pluripotentes Induzidas/fisiologia , Nervo Óptico/fisiologia
3.
Nat Genet ; 53(2): 215-229, 2021 02.
Artigo em Inglês | MEDLINE | ID: mdl-33526924

RESUMO

Naive epiblast and embryonic stem cells (ESCs) give rise to all cells of adults. Such developmental plasticity is associated with genome hypomethylation. Here, we show that LIF-Stat3 signaling induces genomic hypomethylation via metabolic reconfiguration. Stat3-/- ESCs show decreased α-ketoglutarate production from glutamine, leading to increased Dnmt3a and Dnmt3b expression and DNA methylation. Notably, genome methylation is dynamically controlled through modulation of α-ketoglutarate availability or Stat3 activation in mitochondria. Alpha-ketoglutarate links metabolism to the epigenome by reducing the expression of Otx2 and its targets Dnmt3a and Dnmt3b. Genetic inactivation of Otx2 or Dnmt3a and Dnmt3b results in genomic hypomethylation even in the absence of active LIF-Stat3. Stat3-/- ESCs show increased methylation at imprinting control regions and altered expression of cognate transcripts. Single-cell analyses of Stat3-/- embryos confirmed the dysregulated expression of Otx2, Dnmt3a and Dnmt3b as well as imprinted genes. Several cancers display Stat3 overactivation and abnormal DNA methylation; therefore, the molecular module that we describe might be exploited under pathological conditions.


Assuntos
Blastocisto/fisiologia , Metilação de DNA/fisiologia , Células-Tronco Embrionárias/metabolismo , Fator de Transcrição STAT3/metabolismo , Animais , Diferenciação Celular , Células Cultivadas , DNA (Citosina-5-)-Metiltransferases/genética , DNA (Citosina-5-)-Metiltransferases/metabolismo , Células-Tronco Embrionárias/fisiologia , Regulação da Expressão Gênica , Histonas/metabolismo , Ácidos Cetoglutáricos/metabolismo , Fator Inibidor de Leucemia/metabolismo , Camundongos Knockout , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo , Fatores de Transcrição Otx/genética , Fatores de Transcrição Otx/metabolismo , Células-Tronco Pluripotentes/metabolismo , Regiões Promotoras Genéticas , Fator de Transcrição STAT3/genética
4.
Nat Genet ; 53(2): 230-242, 2021 02.
Artigo em Inglês | MEDLINE | ID: mdl-33526923

RESUMO

Noncoding RNAs are exquisitely titrated by the cellular RNA surveillance machinery for regulating diverse biological processes. The RNA exosome, the predominant 3' RNA exoribonuclease in mammalian cells, is composed of nine core and two catalytic subunits. Here, we developed a mouse model with a conditional allele to study the RNA exosome catalytic subunit DIS3. In DIS3-deficient B cells, integrity of the immunoglobulin heavy chain (Igh) locus in its topologically associating domain is affected, with accumulation of DNA-associated RNAs flanking CTCF-binding elements, decreased CTCF binding to CTCF-binding elements and disorganized cohesin localization. DIS3-deficient B cells also accumulate activation-induced cytidine deaminase-mediated asymmetric nicks, altering somatic hypermutation patterns and increasing microhomology-mediated end-joining DNA repair. Altered mutation patterns and Igh architectural defects in DIS3-deficient B cells lead to decreased class-switch recombination but increased chromosomal translocations. Our observations of DIS3-mediated architectural regulation at the Igh locus are reflected genome wide, thus providing evidence that noncoding RNA processing is an important mechanism for controlling genome organization.


Assuntos
Linfócitos B/fisiologia , Complexo Multienzimático de Ribonucleases do Exossomo/genética , RNA não Traduzido/genética , Hipermutação Somática de Imunoglobulina/fisiologia , Animais , Linfócitos B/efeitos dos fármacos , Fator de Ligação a CCCTC/genética , Fator de Ligação a CCCTC/metabolismo , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Proteínas Cromossômicas não Histona/metabolismo , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Células-Tronco Embrionárias/fisiologia , Complexo Multienzimático de Ribonucleases do Exossomo/metabolismo , Exossomos/genética , Proteínas de Fluorescência Verde/genética , Camundongos Knockout , Camundongos Transgênicos , Mutação , Processamento Pós-Transcricional do RNA , Recombinação Genética , Tamoxifeno/farmacologia
5.
Nat Genet ; 53(3): 379-391, 2021 03.
Artigo em Inglês | MEDLINE | ID: mdl-33603234

RESUMO

Rapid cellular responses to environmental stimuli are fundamental for development and maturation. Immediate early genes can be transcriptionally induced within minutes in response to a variety of signals. How their induction levels are regulated and their untimely activation by spurious signals prevented during development is poorly understood. We found that in developing sensory neurons, before perinatal sensory-activity-dependent induction, immediate early genes are embedded into a unique bipartite Polycomb chromatin signature, carrying active H3K27ac on promoters but repressive Ezh2-dependent H3K27me3 on gene bodies. This bipartite signature is widely present in developing cell types, including embryonic stem cells. Polycomb marking of gene bodies inhibits mRNA elongation, dampening productive transcription, while still allowing for fast stimulus-dependent mark removal and bipartite gene induction. We reveal a developmental epigenetic mechanism regulating the rapidity and amplitude of the transcriptional response to relevant stimuli, while preventing inappropriate activation of stimulus-response genes.


Assuntos
Cromatina/genética , Regulação da Expressão Gênica no Desenvolvimento , Genes Precoces , Proteínas do Grupo Polycomb/genética , Animais , Cromatina/metabolismo , Células-Tronco Embrionárias/fisiologia , Proteína Potenciadora do Homólogo 2 de Zeste/genética , Proteína Potenciadora do Homólogo 2 de Zeste/metabolismo , Epigênese Genética , Histonas/metabolismo , Camundongos Transgênicos , Mutação , Proteínas do Grupo Polycomb/metabolismo , Regiões Promotoras Genéticas , RNA Polimerase II/genética , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Rombencéfalo/efeitos dos fármacos , Rombencéfalo/embriologia , Células Receptoras Sensoriais/fisiologia
6.
Nat Commun ; 11(1): 5095, 2020 10 09.
Artigo em Inglês | MEDLINE | ID: mdl-33037201

RESUMO

Nucleosome turnover concomitant with incorporation of the replication-independent histone variant H3.3 is a hallmark of regulatory regions in the animal genome. Nucleosome turnover is known to be universally linked to DNA accessibility and histone acetylation. In mouse embryonic stem cells, H3.3 is also highly enriched at interstitial heterochromatin, most prominently at intracisternal A-particle endogenous retroviral elements. Interstitial heterochromatin is established over confined domains by the TRIM28-KAP1/SETDB1 corepressor complex and has stereotypical features of repressive chromatin, such as H3K9me3 and recruitment of all HP1 isoforms. Here, we demonstrate that fast histone turnover and H3.3 incorporation is compatible with these hallmarks of heterochromatin. Further, we find that Smarcad1 chromatin remodeler evicts nucleosomes generating accessible DNA. Free DNA is repackaged via DAXX-mediated nucleosome assembly with histone variant H3.3 in this dynamic heterochromatin state. Loss of H3.3 in mouse embryonic stem cells elicits a highly specific opening of interstitial heterochromatin with minimal effects on other silent or active regions of the genome.


Assuntos
Células-Tronco Embrionárias/fisiologia , Heterocromatina/metabolismo , Histonas/metabolismo , Animais , Células Cultivadas , Imunoprecipitação da Cromatina , DNA/metabolismo , DNA Helicases/metabolismo , Heterocromatina/genética , Histonas/genética , Camundongos Knockout , Nucleossomos/genética , Nucleossomos/metabolismo , Células-Tronco Pluripotentes/fisiologia , Retroelementos/genética
7.
Phys Rev Lett ; 125(8): 088101, 2020 Aug 21.
Artigo em Inglês | MEDLINE | ID: mdl-32909810

RESUMO

Characterization of the differences between biological and random networks can reveal the design principles that enable the robust realization of crucial biological functions including the establishment of different cell types. Previous studies, focusing on identifying topological features that are present in biological networks but not in random networks, have, however, provided few functional insights. We use a Boolean modeling framework and ideas from the spin glass literature to identify functional differences between five real biological networks and random networks with similar topological features. We show that minimal frustration is a fundamental property that allows biological networks to robustly establish cell types and regulate cell fate choice, and that this property can emerge in complex networks via Darwinian evolution. The study also provides clues regarding how the regulation of cell fate choice can go awry in a disease like cancer and lead to the emergence of aberrant cell types.


Assuntos
Fenômenos Fisiológicos Celulares , Modelos Biológicos , Diferenciação Celular/fisiologia , Células-Tronco Embrionárias/fisiologia , Humanos
8.
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
9.
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.
Science ; 369(6502): 397-403, 2020 07 24.
Artigo em Inglês | MEDLINE | ID: mdl-32703873

RESUMO

Embryonic stem cells can propagate indefinitely in a pluripotent state, able to differentiate into all types of specialized cells when restored to the embryo. What sustains their pluripotency during propagation remains unclear. Here, we show that core pluripotency factors OCT4 and SOX2 suppress chaperone-mediated autophagy (CMA), a selective form of autophagy, until the initiation of differentiation. Low CMA activity promotes embryonic stem cell self-renewal, whereas its up-regulation enhances differentiation. CMA degrades isocitrate dehydrogenases IDH1 and IDH2 and reduces levels of intracellular α-ketoglutarate, an obligatory cofactor for various histone and DNA demethylases involved in pluripotency. These findings suggest that CMA mediates the effect of core pluripotency factors on metabolism, shaping the epigenetic landscape of stem cells and governing the balance between self-renewal and differentiation.


Assuntos
Diferenciação Celular , Autofagia Mediada por Chaperonas , Células-Tronco Embrionárias/fisiologia , Animais , Linhagem Celular , Epigênese Genética , Histonas/fisiologia , Ácidos Cetoglutáricos/metabolismo , Camundongos , Fator 3 de Transcrição de Octâmero/fisiologia , Fatores de Transcrição SOXB1/fisiologia
12.
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
13.
Nat Rev Endocrinol ; 16(9): 506-518, 2020 09.
Artigo em Inglês | MEDLINE | ID: mdl-32587391

RESUMO

Diabetes mellitus, which affects more than 463 million people globally, is caused by the autoimmune ablation or functional loss of insulin-producing ß-cells, and prevalence is projected to continue rising over the next decades. Generating ß-cells to mitigate the aberrant glucose homeostasis manifested in the disease has remained elusive. Substantial advances have been made in producing mature ß-cells from human pluripotent stem cells that respond appropriately to dynamic changes in glucose concentrations in vitro and rapidly function in vivo following transplantation in mice. Other potential avenues to produce functional ß-cells include: transdifferentiation of closely related cell types (for example, other pancreatic islet cells such as α-cells, or other cells derived from endoderm); the engineering of non-ß-cells that are capable of modulating blood sugar; and the construction of synthetic 'cells' or particles mimicking functional aspects of ß-cells. This Review focuses on the current status of generating ß-cells via these diverse routes, highlighting the unique advantages and challenges of each approach. Given the remarkable progress in this field, scalable bioengineering processes are also discussed for the realization of the therapeutic potential of derived ß-cells.


Assuntos
Diferenciação Celular , Diabetes Mellitus/terapia , Células Secretoras de Insulina/fisiologia , Células-Tronco Pluripotentes/fisiologia , Células-Tronco/fisiologia , Animais , Reatores Biológicos , Blastocisto/citologia , Células-Tronco Embrionárias/fisiologia , Humanos , Imunossupressores , Lactente , Recém-Nascido , Ilhotas Pancreáticas/fisiologia , Camundongos , Transplante de Células-Tronco
14.
Proc Natl Acad Sci U S A ; 117(26): 15209-15220, 2020 06 30.
Artigo em Inglês | MEDLINE | ID: mdl-32541058

RESUMO

Preclinical assessment of the therapeutic potential of dopamine (DA) neuron replacement in Parkinson's disease (PD) has primarily been performed in the 6-hydroxydopamine toxin model. While this is a good model to assess graft function, it does not reflect the pathological features or progressive nature of the disease. In this study, we establish a humanized transplantation model of PD that better recapitulates the main disease features, obtained by coinjection of preformed human α-synuclein (α-syn) fibrils and adeno-associated virus (AAV) expressing human wild-type α-syn unilaterally into the rat substantia nigra (SN). This model gives rise to DA neuron dysfunction and progressive loss of DA neurons from the SN and terminals in the striatum, accompanied by extensive α-syn pathology and a prominent inflammatory response, making it an interesting and relevant model in which to examine long-term function and integrity of transplanted neurons in a PD-like brain. We transplanted DA neurons derived from human embryonic stem cells (hESCs) into the striatum and assessed their survival, growth, and function over 6 to 18 wk. We show that the transplanted cells, even in the presence of ongoing pathology, are capable of innervating the DA-depleted striatum. However, on closer examination of the grafts, we found evidence of α-syn pathology in the form of inclusions of phosphorylated α-syn in a small fraction of the grafted DA neurons, indicating host-to-graft transfer of α-syn pathology, a phenomenon that has previously been observed in PD patients receiving fetal tissue grafts but has not been possible to demonstrate and study in toxin-based animal models.


Assuntos
Células-Tronco Embrionárias/fisiologia , Transplante de Células-Tronco , Sinucleinopatias , alfa-Sinucleína/metabolismo , Animais , Sobrevivência Celular , Neurônios Dopaminérgicos/metabolismo , Regulação para Baixo , Feminino , Humanos , Inflamação , Degeneração Neural , Ratos , Ratos Sprague-Dawley , Substância Negra/citologia
15.
Nat Genet ; 52(8): 819-827, 2020 08.
Artigo em Inglês | MEDLINE | ID: mdl-32514123

RESUMO

Mammalian cells stably maintain high levels of DNA methylation despite expressing both positive (DNMT3A/B) and negative (TET1-3) regulators. Here, we analyzed the independent and combined effects of these regulators on the DNA methylation landscape using a panel of knockout human embryonic stem cell (ESC) lines. The greatest impact on global methylation levels was observed in DNMT3-deficient cells, including reproducible focal demethylation at thousands of normally methylated loci. Demethylation depends on TET expression and occurs only when both DNMT3s are absent. Dynamic loci are enriched for hydroxymethylcytosine and overlap with subsets of putative somatic enhancers that are methylated in ESCs and can be activated upon differentiation. We observe similar dynamics in mouse ESCs that were less frequent in epiblast stem cells (EpiSCs) and scarce in somatic tissues, suggesting a conserved pluripotency-linked mechanism. Taken together, our data reveal tightly regulated competition between DNMT3s and TETs at thousands of somatic regulatory sequences within pluripotent cells.


Assuntos
DNA (Citosina-5-)-Metiltransferases/genética , Metilação de DNA/genética , Elementos Facilitadores Genéticos/genética , Oxigenases de Função Mista/genética , Células-Tronco Pluripotentes/fisiologia , Proteínas Proto-Oncogênicas/genética , Animais , Diferenciação Celular/genética , Linhagem Celular , Células-Tronco Embrionárias/fisiologia , Epigênese Genética/genética , Regulação da Expressão Gênica no Desenvolvimento/genética , Camadas Germinativas/fisiologia , Humanos , Camundongos , Camundongos Knockout
16.
Mol Cell ; 78(3): 493-505.e8, 2020 05 07.
Artigo em Inglês | MEDLINE | ID: mdl-32353257

RESUMO

The promyelocytic leukemia (PML) body is a phase-separated nuclear structure physically associated with chromatin, implying its crucial roles in genome functions. However, its role in transcriptional regulation is largely unknown. We developed APEX-mediated chromatin labeling and purification (ALaP) to identify the genomic regions proximal to PML bodies. We found that PML bodies associate with active regulatory regions across the genome and with ∼300 kb of the short arm of the Y chromosome (YS300) in mouse embryonic stem cells. The PML body association with YS300 is essential for the transcriptional activity of the neighboring Y-linked clustered genes. Mechanistically, PML bodies provide specific nuclear spaces that the de novo DNA methyltransferase DNMT3A cannot access, resulting in the steady maintenance of a hypo-methylated state at Y-linked gene promoters. Our study underscores a new mechanism for gene regulation in the 3D nuclear space and provides insights into the functional properties of nuclear structures for genome function.


Assuntos
DNA (Citosina-5-)-Metiltransferases/metabolismo , Regulação da Expressão Gênica , Corpos de Inclusão Intranuclear/genética , Cromossomo Y/genética , Animais , Linhagem Celular , Cromatina/genética , Cromatina/metabolismo , RNA Helicases DEAD-box/genética , DNA (Citosina-5-)-Metiltransferases/genética , Metilação de DNA , DNA Liase (Sítios Apurínicos ou Apirimidínicos)/genética , Células-Tronco Embrionárias/fisiologia , Endonucleases/genética , Sequenciamento de Nucleotídeos em Larga Escala , Corpos de Inclusão Intranuclear/metabolismo , Camundongos Knockout , Antígenos de Histocompatibilidade Menor/genética , Enzimas Multifuncionais/genética , Família Multigênica , Estresse Oxidativo , Proteína da Leucemia Promielocítica/genética , Proteína da Leucemia Promielocítica/metabolismo , Proteínas/genética , Fatores de Transcrição/genética , Cromossomo Y/metabolismo
17.
Nat Cell Biol ; 22(5): 534-545, 2020 05.
Artigo em Inglês | MEDLINE | ID: mdl-32367046

RESUMO

Following implantation, the naive pluripotent epiblast of the mouse blastocyst generates a rosette, undergoes lumenogenesis and forms the primed pluripotent egg cylinder, which is able to generate the embryonic tissues. How pluripotency progression and morphogenesis are linked and whether intermediate pluripotent states exist remain controversial. We identify here a rosette pluripotent state defined by the co-expression of naive factors with the transcription factor OTX2. Downregulation of blastocyst WNT signals drives the transition into rosette pluripotency by inducing OTX2. The rosette then activates MEK signals that induce lumenogenesis and drive progression to primed pluripotency. Consequently, combined WNT and MEK inhibition supports rosette-like stem cells, a self-renewing naive-primed intermediate. Rosette-like stem cells erase constitutive heterochromatin marks and display a primed chromatin landscape, with bivalently marked primed pluripotency genes. Nonetheless, WNT induces reversion to naive pluripotency. The rosette is therefore a reversible pluripotent intermediate whereby control over both pluripotency progression and morphogenesis pivots from WNT to MEK signals.


Assuntos
Células-Tronco Embrionárias/fisiologia , Células-Tronco Pluripotentes/fisiologia , Animais , Blastocisto/metabolismo , Blastocisto/fisiologia , Diferenciação Celular/fisiologia , Cromatina/metabolismo , Células-Tronco Embrionárias/metabolismo , Feminino , Regulação da Expressão Gênica no Desenvolvimento/fisiologia , Camadas Germinativas/metabolismo , Camadas Germinativas/fisiologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Morfogênese/fisiologia , Fatores de Transcrição Otx/metabolismo , Células-Tronco Pluripotentes/metabolismo
18.
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
19.
Curr Pharm Biotechnol ; 21(12): 1154-1164, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32297579

RESUMO

BACKGROUND: Stem cells are of two types: embryonic and adult stem cells and they act as a repair system by replenishing body tissue. Stem cells differentiate into different types of cells, such as neural, hematopoietic, adipose, etc. and are used for the treatment of various conditions like myocardial infarction, spinal cord injury, Parkinson's disease and diabetes. METHODS: This article focuses on recent research development that addresses the viability issues of stem cells. The efficiency of transplanted stem cells reduces due to conditions like hypoxia, inflammation, nutrient deprivation, immunogenicity, extracellular matrix loss on delivery and mechanical stress. RESULTS: To increase the viability of stem cells, techniques like scaffolds of stem cells with hydrogel or alginate, pre-conditioning, different routes of administration and encapsulation, are implemented. CONCLUSION: For the protection of stem cells against apoptosis, different pathways, namely Phosphoinositide 3-Kinase (PI3K/AKT), Hypoxia-Inducible Factor (HIF1), Mitogen-Activated Protein Kinases (MAPK) and Hippo, are discussed. DISCUSSION: Activation of the PI3K/AKT pathway decreases the concentration of apoptotic factors, while the HIF pathway protects stem cells against the micro-environment of tissue (hypoxia).


Assuntos
Células-Tronco Adultas/fisiologia , Técnicas de Cultura de Células/métodos , Células-Tronco Embrionárias/fisiologia , Células-Tronco Mesenquimais/fisiologia , Células-Tronco Adultas/efeitos dos fármacos , Células-Tronco Adultas/metabolismo , Animais , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/fisiologia , Criopreservação , Células-Tronco Embrionárias/efeitos dos fármacos , Células-Tronco Embrionárias/metabolismo , Células-Tronco Mesenquimais/efeitos dos fármacos , Células-Tronco Mesenquimais/metabolismo , Substâncias Protetoras/farmacologia , Ratos , Transplante de Células-Tronco
20.
PLoS Biol ; 18(4): e3000684, 2020 04.
Artigo em Inglês | MEDLINE | ID: mdl-32275660

RESUMO

5-hydroxymethylcytosine (5hmC) is the most prevalent intermediate on the oxidative DNA demethylation pathway and is implicated in regulation of embryogenesis, neurological processes, and cancerogenesis. Profiling of this relatively scarce genomic modification in clinical samples requires cost-effective high-resolution techniques that avoid harsh chemical treatment. Here, we present a bisulfite-free approach for 5hmC profiling at single-nucleotide resolution, named hmTOP-seq (5hmC-specific tethered oligonucleotide-primed sequencing), which is based on direct sequence readout primed at covalently labeled 5hmC sites from an in situ tethered DNA oligonucleotide. Examination of distinct conjugation chemistries suggested a structural model for the tether-directed nonhomologous polymerase priming enabling theoretical evaluation of suitable tethers at the design stage. The hmTOP-seq procedure was optimized and validated on a small model genome and mouse embryonic stem cells, which allowed construction of single-nucleotide 5hmC maps reflecting subtle differences in strand-specific CG hydroxymethylation. Collectively, hmTOP-seq provides a new valuable tool for cost-effective and precise identification of 5hmC in characterizing its biological role and epigenetic changes associated with human disease.


Assuntos
5-Metilcitosina/análogos & derivados , Análise de Sequência de DNA/métodos , 5-Metilcitosina/química , Acetilação , Animais , Bacteriófago lambda/genética , Linhagem Celular , Metilação de DNA , Células-Tronco Embrionárias/fisiologia , Genoma , Histonas/metabolismo , Lisina/metabolismo , Camundongos , Oligonucleotídeos , Reprodutibilidade dos Testes , Sulfitos
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