Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 20 de 1.735
Filtrar
1.
Mol Cell ; 81(8): 1732-1748.e8, 2021 04 15.
Artigo em Inglês | MEDLINE | ID: mdl-33730542

RESUMO

During self-renewal, cell-type-defining features are drastically perturbed in mitosis and must be faithfully reestablished upon G1 entry, a process that remains largely elusive. Here, we characterized at a genome-wide scale the dynamic transcriptional and architectural resetting of mouse pluripotent stem cells (PSCs) upon mitotic exit. We captured distinct waves of transcriptional reactivation with rapid induction of stem cell genes and transient activation of lineage-specific genes. Topological reorganization at different hierarchical levels also occurred in an asynchronous manner and showed partial coordination with transcriptional resetting. Globally, rapid transcriptional and architectural resetting associated with mitotic retention of H3K27 acetylation, supporting a bookmarking function. Indeed, mitotic depletion of H3K27ac impaired the early reactivation of bookmarked, stem-cell-associated genes. However, 3D chromatin reorganization remained largely unaffected, suggesting that these processes are driven by distinct forces upon mitotic exit. This study uncovers principles and mediators of PSC molecular resetting during self-renewal.


Assuntos
Cromatina/genética , Código das Histonas/genética , Histonas/genética , Mitose/genética , Células-Tronco Pluripotentes/fisiologia , Acetilação , Animais , Linhagem Celular , Drosophila/genética , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Transcrição Genética/genética , Ativação Transcricional/genética
2.
Science ; 371(6529)2021 02 05.
Artigo em Inglês | MEDLINE | ID: mdl-33542111

RESUMO

During development, cells progress from a pluripotent state to a more restricted fate within a particular germ layer. However, cranial neural crest cells (CNCCs), a transient cell population that generates most of the craniofacial skeleton, have much broader differentiation potential than their ectodermal lineage of origin. Here, we identify a neuroepithelial precursor population characterized by expression of canonical pluripotency transcription factors that gives rise to CNCCs and is essential for craniofacial development. Pluripotency factor Oct4 is transiently reactivated in CNCCs and is required for the subsequent formation of ectomesenchyme. Furthermore, open chromatin landscapes of Oct4+ CNCC precursors resemble those of epiblast stem cells, with additional features suggestive of priming for mesenchymal programs. We propose that CNCCs expand their developmental potential through a transient reacquisition of molecular signatures of pluripotency.


Assuntos
Crista Neural/embriologia , Células-Tronco Pluripotentes/fisiologia , Animais , Diferenciação Celular/genética , Movimento Celular , Embrião de Mamíferos , Camadas Germinativas/citologia , Camundongos , Crista Neural/citologia , Crista Neural/metabolismo , Fator 3 de Transcrição de Octâmero/genética , Fator 3 de Transcrição de Octâmero/metabolismo , Células-Tronco Pluripotentes/citologia , RNA-Seq , Transcrição Genética , Transcriptoma
3.
Nat Protoc ; 16(3): 1581-1599, 2021 03.
Artigo em Inglês | MEDLINE | ID: mdl-33580232

RESUMO

The endodermal germ layer gives rise to respiratory epithelium, hepatocytes, pancreatic cells and intestinal lineages, among other cell types. These lineages can be differentiated from human pluripotent stem cells (hPSCs) via a common definitive endoderm (DE) intermediate that is characterized by the co-expression of the cell surface markers CXCR4, c-KIT and EPCAM and the transcription factors SOX17 and FOXA2. Here we provide a detailed protocol for mass production of DE from hPSCs in scalable and easy-to-handle suspension culture using a rotating Erlenmeyer flask or a sophisticated, fully controllable, 150-ml stirred tank bioreactor. This protocol uses two different media formulations that are chemically defined and xeno free and therefore good manufacturing practice ready. Our protocol allows for efficient hPSC-derived DE specification in multicellular aggregates within 3 days and generates up to 1 × 108 DE cells with >92% purity in one differentiation batch when using the bioreactor. The hPSC-derived DE cells that are generated can be cryopreserved for later downstream differentiation into various endodermal lineages. This protocol should facilitate the flexible production of mature DE derivatives for physiologically relevant disease models, high-throughput drug screening, toxicology testing and cellular therapies.


Assuntos
Técnicas de Cultura de Células/métodos , Endoderma/citologia , Células-Tronco Pluripotentes/citologia , Diferenciação Celular/fisiologia , Linhagem Celular , Células Cultivadas , Criopreservação/métodos , Meios de Cultura , Endoderma/crescimento & desenvolvimento , Endoderma/metabolismo , Hepatócitos/citologia , Humanos , Células-Tronco Pluripotentes/metabolismo , Células-Tronco Pluripotentes/fisiologia
4.
Methods Mol Biol ; 2258: 73-92, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33340355

RESUMO

Biophysical cues synergize with biochemical cues to drive differentiation of pluripotent stem cells through specific phenotypic trajectory. Tools to manipulate the cell biophysical environment and identify the influence of specific environment perturbation in the presence of combinatorial inputs will be critical to control the development trajectory. Here we describe the procedure to perturb biophysical environment of pluripotent stem cells while maintaining them in 3D culture configuration. We also discuss a high-throughput platform for combinatorial perturbation of the cell microenvironment, and detail a statistical procedure to extract dominant environmental influences.


Assuntos
Diferenciação Celular , Linhagem da Célula , Endoderma/fisiologia , Imunofluorescência , Mecanotransdução Celular , Microscopia de Fluorescência , Células-Tronco Pluripotentes/fisiologia , Nicho de Células-Tronco , Engenharia Tecidual , Alginatos/química , Técnicas de Cultura de Células , Células Cultivadas , Endoderma/citologia , Regulação da Expressão Gênica no Desenvolvimento , Humanos , Modelos Estatísticos , Fenótipo , Fatores de Tempo
5.
Methods Mol Biol ; 2258: 105-116, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33340357

RESUMO

Pluripotent stem cells (PSCs) possess the ability to self-organize into complex tissue-like structures; however, the genetic mechanisms and multicellular dynamics that direct such patterning are difficult to control. Here, we pair live imaging with controlled induction of gene knockdown by CRISPR interference (CRISPRi) to generate changes within subpopulations of human PSCs, allowing for control over organization and analysis of emergent behaviors. Specifically, we use forced aggregation of mixtures of cells with and without an inducible CRISPRi system to knockdown molecular regulators of tissue symmetry. We then track the resulting multicellular organization through fluorescence live imaging concurrent with the induction of knockdown. Overall, this technique allows for controlled initiation of symmetry breaking by CRISPRi to produce changes in cellular behavior that can be tracked over time within high-density pluripotent stem cell colonies.


Assuntos
Padronização Corporal , Sistemas CRISPR-Cas , Edição de Genes , Células-Tronco Pluripotentes/fisiologia , Proteínas Associadas a CRISPR/genética , Proteínas Associadas a CRISPR/metabolismo , Células Cultivadas , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , Regulação da Expressão Gênica no Desenvolvimento , Microscopia de Fluorescência , Microscopia de Vídeo , RNA Guia/genética , RNA Guia/metabolismo , Transdução de Sinais , Fatores de Tempo , Imagem com Lapso de Tempo
6.
Methods Mol Biol ; 2258: 171-192, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33340361

RESUMO

Our understanding in the inherent properties of human pluripotent stem cells (hPSCs) have made possible the development of differentiation procedures to generate three-dimensional tissue-like cultures, so-called organoids. Here we detail a stepwise methodology to generate kidney organoids from hPSCs. This is achieved through direct differentiation of hPSCs in two-dimensional monolayer culture toward the posterior primitive streak fate, followed by induction of intermediate mesoderm-committed cells, which are further aggregated and cultured in three-dimensions to generate kidney organoids containing segmented nephron-like structures in a process that lasts 20 days. We also provide a concise description on how to assess renal commitment during the time course of kidney organoid generation. This includes the use of flow cytometry and immunocytochemistry analyses for the detection of specific renal differentiation markers.


Assuntos
Diferenciação Celular , Rim/fisiologia , Células-Tronco Pluripotentes/fisiologia , Engenharia Tecidual , Técnicas de Cultura de Células , Células Cultivadas , Citometria de Fluxo , Imunofluorescência , Regulação da Expressão Gênica no Desenvolvimento , Humanos , Rim/citologia , Microscopia de Fluorescência , Morfogênese , Organoides , Transdução de Sinais , Fatores de Tempo
7.
Elife ; 92020 12 17.
Artigo em Inglês | MEDLINE | ID: mdl-33331818

RESUMO

WNT proteins are secreted symmetry breaking signals that interact with cell surface receptors of the FZD family to regulate a multitude of developmental processes. Studying selectivity between WNTs and FZDs has been hampered by the paucity of purified WNT proteins and by their apparent non-selective interactions with the FZD receptors. Here, we describe an engineered protein, called F7L6, comprised of antibody-derived single-chain variable fragments, that selectively binds to human FZD7 and the co-receptor LRP6. F7L6 potently activates WNT/ß-catenin signaling in a manner similar to Wnt3a. In contrast to Wnt3a, F7L6 engages only FZD7 and none of the other FZD proteins. Treatment of human pluripotent stem (hPS) cells with F7L6 initiates transcriptional programs similar to those observed during primitive streak formation and subsequent gastrulation in the mammalian embryo. This demonstrates that selective engagement and activation of FZD7 signaling is sufficient to promote mesendodermal differentiation of hPS cells.


Assuntos
Diferenciação Celular/fisiologia , Receptores Frizzled/fisiologia , Mesoderma/embriologia , Células-Tronco Pluripotentes/fisiologia , Western Blotting , Regulação da Expressão Gênica , Humanos , Mesoderma/citologia , Mesoderma/crescimento & desenvolvimento , Reação em Cadeia da Polimerase em Tempo Real , Proteínas Recombinantes , Via de Sinalização Wnt/fisiologia
8.
Biochem Biophys Res Commun ; 533(4): 1477-1483, 2020 12 17.
Artigo em Inglês | MEDLINE | ID: mdl-33333713

RESUMO

Development of the mammalian central nervous system is an important process, which is accomplished through precise regulations of many different genes. Zinc finger protein 179 (Znf179) is one of the essential genes that plays a critical role in neuronal differentiation. In our previous study, Znf179 knockout mice displayed brain malformation and impaired brain functions. We have also previously shown that Znf179 involves in cell cycle regulation, but the regulatory mechanism of Znf179 expression is not yet fully characterized. Herein, we identified that Purα is an essential factor for the promotor activity of Znf179. We also showed concurrent expression of Znf179 and Purα during neuronal differentiation. We also found that overexpression of Purα increased Znf179 expression in neuronal differentiated P19 cells. Through its direct binding to Znf179, as shown using DAPA, Purα upregulates Znf179 expression, suggesting that Purα is important for the regulation of Znf179 expression during neuronal differentiation. Our data indicated that Purα is involved in the transcriptional regulation of Znf179 gene during neuronal differentiation, and is indispensable during the brain development.


Assuntos
Diferenciação Celular/genética , Proteínas de Ligação a DNA/genética , Proteínas do Tecido Nervoso/genética , Neurônios/fisiologia , Animais , Proteínas de Ligação a DNA/metabolismo , Luciferases/genética , Camundongos , Proteínas do Tecido Nervoso/metabolismo , Neurônios/citologia , Células-Tronco Pluripotentes/citologia , Células-Tronco Pluripotentes/fisiologia , Regiões Promotoras Genéticas , Transcrição Genética
9.
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
10.
Nat Genet ; 52(9): 931-938, 2020 09.
Artigo em Inglês | MEDLINE | ID: mdl-32632336

RESUMO

Many chromatin-binding proteins and protein complexes that regulate transcription also bind RNA. One of these, Polycomb repressive complex 2 (PRC2), deposits the H3K27me3 mark of facultative heterochromatin and is required for stem cell differentiation. PRC2 binds RNAs broadly in vivo and in vitro. Yet, the biological importance of this RNA binding remains unsettled. Here, we tackle this question in human induced pluripotent stem cells by using multiple complementary approaches. Perturbation of RNA-PRC2 interaction by RNase A, by a chemical inhibitor of transcription or by an RNA-binding-defective mutant all disrupted PRC2 chromatin occupancy and localization genome wide. The physiological relevance of PRC2-RNA interactions is further underscored by a cardiomyocyte differentiation defect upon genetic disruption. We conclude that PRC2 requires RNA binding for chromatin localization in human pluripotent stem cells and in turn for defining cellular state.


Assuntos
Cromatina/genética , Células-Tronco Pluripotentes Induzidas/fisiologia , Células-Tronco Pluripotentes/fisiologia , Complexo Repressor Polycomb 2/genética , RNA/genética , Sítios de Ligação/genética , Proteínas de Transporte , Diferenciação Celular/genética , Genoma/genética , Histonas/genética , Humanos , Ligação Proteica/genética
11.
Sci Rep ; 10(1): 9464, 2020 06 11.
Artigo em Inglês | MEDLINE | ID: mdl-32528170

RESUMO

The autonomic nervous system (ANS) regulates tissue homeostasis and remodelling through antagonistic effects of noradrenergic sympathetic and cholinergic parasympathetic signalling. Despite numerous reports on the induction of sympathetic neurons from human pluripotent stem cells (hPSCs), no induction methods have effectively derived cholinergic parasympathetic neurons from hPSCs. Considering the antagonistic effects of noradrenergic and cholinergic inputs on target organs, both sympathetic and parasympathetic neurons are expected to be induced. This study aimed to develop a stepwise chemical induction method to induce sympathetic-like and parasympathetic-like ANS neurons. Autonomic specification was achieved through restricting signals inducing sensory or enteric neurogenesis and activating bone morphogenetic protein (BMP) signals. Global mRNA expression analyses after stepwise induction, including single-cell RNA-seq analysis of induced neurons and functional assays revealed that each induced sympathetic-like or parasympathetic-like neuron acquired pharmacological and electrophysiological functional properties with distinct marker expression. Further, we identified selective induction methods using appropriate seeding cell densities and neurotrophic factor concentrations. Neurons were individually induced, facilitating the regulation of the beating rates of hiPSC-derived cardiomyocytes in an antagonistic manner. The induction methods yield specific neuron types, and their influence on various tissues can be studied by co-cultured assays.


Assuntos
Frequência Cardíaca/fisiologia , Miócitos Cardíacos/fisiologia , Neurônios/fisiologia , Sistema Nervoso Parassimpático/fisiologia , Sistema Nervoso Simpático/fisiologia , Vias Autônomas/metabolismo , Vias Autônomas/fisiologia , Proteínas Morfogenéticas Ósseas/metabolismo , Células Cultivadas , Humanos , Interneurônios/metabolismo , Interneurônios/fisiologia , Masculino , Miócitos Cardíacos/metabolismo , Neurônios/metabolismo , Sistema Nervoso Parassimpático/metabolismo , Células-Tronco Pluripotentes/metabolismo , Células-Tronco Pluripotentes/fisiologia , RNA Mensageiro/metabolismo , Transdução de Sinais/fisiologia , Sistema Nervoso Simpático/metabolismo
12.
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
13.
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
14.
J Biomed Sci ; 27(1): 67, 2020 May 22.
Artigo em Inglês | MEDLINE | ID: mdl-32443983

RESUMO

Over the last 4 decades, cell culture techniques have evolved towards the creation of in vitro multicellular entities that incorporate the three-dimensional complexity of in vivo tissues and organs. As a result, stem cells and adult progenitor cells have been used to derive self-organized 3D cell aggregates that mimic the morphological and functional traits of organs in vitro. These so-called organoids were first generated from primary animal and human tissues, then human pluripotent stem cells (hPSCs) arose as a new tool for organoid generation. Due to their self-renewal capacity and differentiation potential, hPSCs are an unlimited source of cells used for organoids. Today, hPSC-derived small intestinal, kidney, brain, liver, and pancreas organoids, among others, have been produced and are promising in vitro human models for diverse applications, including fundamental research, drug development and regenerative medicine. However, achieving in vivo-like organ complexity and maturation in vitro remains a challenge. Current hPSC-derived organoids are often limited in size and developmental state, resembling embryonic or fetal organs rather than adult organs. The use of endothelial cells to vascularize hPSC-derived organoids may represent a key to ensuring oxygen and nutrient distribution in large organoids, thus contributing to the maturation of adult-like organoids through paracrine signaling.Here, we review the current state of the art regarding vascularized hPSC-derived organoids (vhPSC-Orgs). We analyze the progress achieved in the generation of organoids derived from the three primary germ layers (endoderm, mesoderm and ectoderm) exemplified by the pancreas, liver, kidneys and brain. Special attention will be given to the role of the endothelium in the organogenesis of the aforementioned organs, the sources of endothelial cells employed in vhPSC-Org protocols and the remaining challenges preventing the creation of ex vivo functional and vascularized organs.


Assuntos
Endotélio/fisiologia , Organogênese , Organoides/crescimento & desenvolvimento , Células-Tronco Pluripotentes/fisiologia , Células Endoteliais/fisiologia , Humanos , Organoides/citologia
15.
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
16.
Circ Res ; 126(8): 1086-1106, 2020 04 10.
Artigo em Inglês | MEDLINE | ID: mdl-32271675

RESUMO

Maturation is the last phase of heart development that prepares the organ for strong, efficient, and persistent pumping throughout the mammal's lifespan. This process is characterized by structural, gene expression, metabolic, and functional specializations in cardiomyocytes as the heart transits from fetal to adult states. Cardiomyocyte maturation gained increased attention recently due to the maturation defects in pluripotent stem cell-derived cardiomyocyte, its antagonistic effect on myocardial regeneration, and its potential contribution to cardiac disease. Here, we review the major hallmarks of ventricular cardiomyocyte maturation and summarize key regulatory mechanisms that promote and coordinate these cellular events. With advances in the technical platforms used for cardiomyocyte maturation research, we expect significant progress in the future that will deepen our understanding of this process and lead to better maturation of pluripotent stem cell-derived cardiomyocyte and novel therapeutic strategies for heart disease.


Assuntos
Diferenciação Celular/fisiologia , Miócitos Cardíacos/fisiologia , Animais , Cardiopatias/patologia , Cardiopatias/fisiopatologia , Humanos , Células-Tronco Pluripotentes/fisiologia
17.
J Toxicol Sci ; 45(4): 187-199, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32238694

RESUMO

In vitro human induced pluripotent stem (iPS) cells testing (iPST) to assess developmental toxicity, e.g., the induction of malformation or dysfunction, was developed by modifying a mouse embryonic stem cell test (EST), a promising animal-free approach. The iPST evaluates the potential risks and types of drugs-induced developmental toxicity in humans by assessing three endpoints: the inhibitory effects of the drug on the cardiac differentiation of iPS cells and on the proliferation/survival of iPS cells and human fibroblasts. In the present study, the potential developmental toxicity of drugs was divided into three classes (1: non-developmentally toxic, 2: weakly developmentally toxic and 3: strongly developmentally toxic) according to the EST criteria. In addition, the type of developmental toxicity of drugs was grouped into three types (1: non-effective, 2: embryotoxic [inducing growth retardation/dysfunction]/deadly or 3: teratogenic [inducing malformation]/deadly) by comparing the three endpoints. The present study was intended to validate the clinical predictability of the iPST. The traditionally developmentally toxic drugs of aminopterin, methotrexate, all-trans-retinoic acid, thalidomide, tetracycline, lithium, phenytoin, 5-fluorouracil, warfarin and valproate were designated as class 2 or 3 according to the EST criteria, and their developmental toxicity was type 3. The non-developmentally toxic drugs of ascorbic acid, saccharin, isoniazid and penicillin G were designated as class 1, and ascorbic acid, saccharin and isoniazid were grouped as type 1 while penicillin G was type 2 but not teratogenic. These results suggest that the iPST is useful for predicting the human developmental toxicity of drug candidates in a preclinical setting.


Assuntos
Avaliação Pré-Clínica de Medicamentos/métodos , Células-Tronco Pluripotentes/efeitos dos fármacos , Células-Tronco Pluripotentes/fisiologia , Testes de Toxicidade/métodos , Aminopterina/toxicidade , Animais , Ácido Ascórbico/toxicidade , Diferenciação Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Células Cultivadas , Fibroblastos/efeitos dos fármacos , Humanos , Isoniazida/toxicidade , Metotrexato , Camundongos , Sacarina/toxicidade , Teratogênese/efeitos dos fármacos , Tretinoína/toxicidade
18.
Nat Commun ; 11(1): 1528, 2020 03 23.
Artigo em Inglês | MEDLINE | ID: mdl-32251294

RESUMO

The occurrence of repetitive genomic changes that provide a selective growth advantage in pluripotent stem cells is of concern for their clinical application. However, the effect of different culture conditions on the underlying mutation rate is unknown. Here we show that the mutation rate in two human embryonic stem cell lines derived and banked for clinical application is low and not substantially affected by culture with Rho Kinase inhibitor, commonly used in their routine maintenance. However, the mutation rate is reduced by >50% in cells cultured under 5% oxygen, when we also found alterations in imprint methylation and reversible DNA hypomethylation. Mutations are evenly distributed across the chromosomes, except for a slight increase on the X-chromosome, and an elevation in intergenic regions suggesting that chromatin structure may affect mutation rate. Overall the results suggest that pluripotent stem cells are not subject to unusually high rates of genetic or epigenetic alterations.


Assuntos
Técnicas de Cultura de Células/métodos , Cromossomos Humanos X/genética , DNA Intergênico/genética , Taxa de Mutação , Células-Tronco Pluripotentes/fisiologia , Linhagem Celular , Meios de Cultura/farmacologia , Metilação de DNA , Análise Mutacional de DNA , Epigênese Genética , Humanos , Estresse Oxidativo/efeitos dos fármacos , Estresse Oxidativo/genética , Oxigênio/química , Oxigênio/farmacologia , Análise de Sequência de RNA , Sequenciamento Completo do Genoma
19.
Nat Immunol ; 21(5): 578-587, 2020 05.
Artigo em Inglês | MEDLINE | ID: mdl-32231298

RESUMO

The pool of beta cell-specific CD8+ T cells in type 1 diabetes (T1D) sustains an autoreactive potential despite having access to a constant source of antigen. To investigate the long-lived nature of these cells, we established a DNA methylation-based T cell 'multipotency index' and found that beta cell-specific CD8+ T cells retained a stem-like epigenetic multipotency score. Single-cell assay for transposase-accessible chromatin using sequencing confirmed the coexistence of naive and effector-associated epigenetic programs in individual beta cell-specific CD8+ T cells. Assessment of beta cell-specific CD8+ T cell anatomical distribution and the establishment of stem-associated epigenetic programs revealed that self-reactive CD8+ T cells isolated from murine lymphoid tissue retained developmentally plastic phenotypic and epigenetic profiles relative to the same cells isolated from the pancreas. Collectively, these data provide new insight into the longevity of beta cell-specific CD8+ T cell responses and document the use of this methylation-based multipotency index for investigating human and mouse CD8+ T cell differentiation.


Assuntos
Linfócitos T CD8-Positivos/fisiologia , Diabetes Mellitus Tipo 1/imunologia , Células Secretoras de Insulina/imunologia , Células-Tronco Pluripotentes/fisiologia , Adolescente , Adulto , Animais , Autoantígenos/imunologia , Plasticidade Celular , Células Cultivadas , Metilação de DNA , Epigênese Genética , Feminino , Citometria de Fluxo , Humanos , Memória Imunológica , Masculino , Camundongos , Análise de Célula Única , Adulto Jovem
20.
Int J Mol Sci ; 21(6)2020 Mar 12.
Artigo em Inglês | MEDLINE | ID: mdl-32178385

RESUMO

Heart disease is the most common cause of death in developed countries, but the medical treatments for heart failure remain limited. In this context, the development of cardiac regeneration therapy for severe heart failure is important. Owing to their unique characteristics, including multiple differentiation and infinitive self-renewal, pluripotent stem cells can be considered as a novel source for regenerative medicine. Janus kinase/signal transducer and activator of transcription 3 (JAK/STAT3) signaling plays critical roles in the induction, maintenance, and differentiation of pluripotent stem cells. In the heart, JAK/STAT3 signaling has diverse cellular functions, including myocardial differentiation, cell cycle re-entry of matured myocyte after injury, and anti-apoptosis in pathological conditions. Therefore, regulating STAT3 activity has great potential as a strategy of cardiac regeneration therapy. In this review, we summarize the current understanding of STAT3, focusing on stem cell biology and pathophysiology, as they contribute to cardiac regeneration therapy. We also introduce a recently reported therapeutic strategy for myocardial regeneration that uses engineered artificial receptors that trigger endogenous STAT3 signal activation.


Assuntos
Coração/fisiopatologia , Células-Tronco Pluripotentes/metabolismo , Células-Tronco Pluripotentes/fisiologia , Regeneração/fisiologia , Medicina Regenerativa/métodos , Fator de Transcrição STAT3/metabolismo , Animais , Bioengenharia/métodos , Diferenciação Celular/fisiologia , Humanos , Miocárdio/metabolismo
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA
...