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1.
Adv Exp Med Biol ; 1186: 121-140, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31654388

RESUMO

Human pluripotent stem cell (hPSC) technology has revolutionized the field of biology through the unprecedented ability to study the differentiation of human cells in vitro. In the past decade, hPSCs have been applied to study development, model disease, develop drugs, and devise cell replacement therapies for numerous biological systems. Of particular interest is the application of this technology to study and treat optic neuropathies such as glaucoma. Retinal ganglion cells (RGCs) are the primary cell type affected in these diseases, and once lost, they are unable to regenerate in adulthood. This necessitates the development of strategies to study the mechanisms of degeneration as well as develop translational therapeutic approaches to treat early- and late-stage disease progression. Numerous protocols have been established to derive RGCs from hPSCs, with the ability to generate large populations of human RGCs for translational applications. In this review, the key applications of hPSCs within the retinal field are described, including the use of these cells as developmental models, disease models, drug development, and finally, cell replacement therapies. In greater detail, the current report focuses on the differentiation of hPSC-derived RGCs and the many unique characteristics associated with these cells in vitro including their genetic identifiers, their electrophysiological activity, and their morphological maturation. Also described is the current progress in the use of patient-specific hPSCs to study optic neuropathies affecting RGCs, with emphasis on the use of these RGCs for studying disease mechanisms and pathogenesis, drug screening, and cell replacement therapies in future studies.


Assuntos
Diferenciação Celular , Células-Tronco Pluripotentes , Células Ganglionares da Retina , Glaucoma/terapia , Humanos , Neuropatologia/tendências , Doenças do Nervo Óptico/terapia , Células-Tronco Pluripotentes/citologia , Retina/citologia , Retina/patologia , Células Ganglionares da Retina/citologia
2.
Nat Cell Biol ; 21(9): 1060-1067, 2019 09.
Artigo em Inglês | MEDLINE | ID: mdl-31481793

RESUMO

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


Assuntos
Ciclo Celular/fisiologia , Diferenciação Celular/fisiologia , Células-Tronco Embrionárias/citologia , Células-Tronco Pluripotentes/citologia , Animais , Proliferação de Células/fisiologia , Humanos , Células-Tronco Pluripotentes Induzidas/citologia
3.
Biomed Khim ; 65(4): 339-346, 2019 Jun.
Artigo em Russo | MEDLINE | ID: mdl-31436176

RESUMO

Secretion of 21 cytokines, chemokines and growth factors (LIF, SCF, SDF-1a, SCGF-b, M-CSF, MCP-3, MIF, MIG, TRAIL, GRO-a; IL-1a, IL-2ra, IL-3, IL-12(p40), IL-16, IL-18, HGF, TNF-b, b-NGF, IFN-a2, CTACK) has been studied in vitro in the culture of human adipose-derived multipotent mesenchymal stromal cells (hAMMSCs) in conditions of its osteogenic differentiation caused by 14-day contact with calcium phosphate (CP) surface with different roughness. Bilateral X-ray amorphous CP coatings were prepared on the samples of commercially pure titanium in the anodal regime using a micro-arc method. An aqueous solution prepared from 20 wt% phosphoric acid, 6 wt% dissolved hydrohyapatite nanopowder (particle diameter 10-30 nm with single agglomerates up to 100 nm), and 9 wt% dissolved calcium carbonate was used to obtain CP coating. hAMMSCs isolated from lipoaspirate were co-cultured after 4 passages with the CP-coated samples at final concentration of 1.5´105 viable karyocytes per 1.5 mL of standard nutrition medium (without osteogenic stimulators) for 14 days (a determination of [CD45,34,14,20], CD73, CD90 и CD105 cell immunophenotype; an analysis of secretory activity) and 21 days (alizarin red S staining of culture) with medium replacement every 3-4 days. Under conditions of in vitro contact with rough CP coating hAMMSCs differentiated into osteoblasts synthesizing the mineralized bone matrix; this was accompanied by 2-3-fold increasing ratio of [CD45,34,14,20]+ hemopoietic cells. The following humoral factors of hemopoietic niches acted as the signal molecules escalating in vitro the hemopoietic base in 14 days of differentiating three-dimensional culture of hAMMSCs: either leukemia inhibitory factor (LIF) and stem cell factor (SCF) cytokines under mean index of CP roughness Ra=2.4-2.6 mm or stromal derived factor-1 (SDF-1a, CXCL12 chemokine) under Ra=3.1-4.4 mm.


Assuntos
Fosfatos de Cálcio/farmacologia , Diferenciação Celular , Células-Tronco Mesenquimais/citologia , Osteogênese , Células-Tronco Pluripotentes/citologia , Tecido Adiposo/química , Células Cultivadas , Quimiocinas/metabolismo , Citocinas/metabolismo , Humanos , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Células Estromais/citologia
4.
Brain Nerve ; 71(8): 895-900, 2019 Aug.
Artigo em Japonês | MEDLINE | ID: mdl-31346146

RESUMO

Muse cells are non-tumorigenic reparative endogenous stem cells identified by SSEA-3+. They are pluripotent and are stably mobilized from the bone marrow to the peripheral blood and distribute to organ connective tissue, where they contribute to daily minute repair of damaged/lost cells by spontaneous differentiation into tissue-constituent cells. Muse cells specifically home to damaged site to repair the tissue by simultaneous differentiation into multiple tissue-constituent cells. When the number of endogenous Muse cells is not sufficient, administration of exogenous Muse cells delivers robust functional recovery. Muse cells do not need to be "induced" or genetically manipulated. Intravenous drip is the main method of administration, making surgical operation unnecessary. Because Muse cells have an immunomodulatory system similar to the placenta, donor-derived Muse cells can be directly administered to patients without HLA-matching or immunosuppression therapy. Allogeneic Muse cells remain in the host tissue as differentiated cells for more than half a year. Clinical trials for the treatment of myocardial infarction, stroke and epidermolysis bullosa with intravenous injection of donor-derived Muse cells are currently conducted by the Life Science Institute Inc. Muse cells may safely provide clinically relevant effects compatible with the 'body's natural repair systems' by a simple cost-effective strategy.


Assuntos
Infusões Intravenosas , Células-Tronco Pluripotentes/citologia , Medicina Regenerativa , Transplante de Células-Tronco , Diferenciação Celular , Humanos
5.
Nat Methods ; 16(7): 640-648, 2019 07.
Artigo em Inglês | MEDLINE | ID: mdl-31249412

RESUMO

Signaling centers, localized groups of cells that secrete morphogens, play a key role in early development and organogenesis by orchestrating spatial cell fate patterning. Here we present a microfluidic approach that exposes human pluripotent stem cell (hPSC) colonies to spatiotemporally controlled morphogen gradients generated from artificial signaling centers. In response to a localized source of bone morphogenetic protein 4 (BMP4), hPSC colonies reproducibly break their intrinsic radial symmetry to produce distinct, axially arranged differentiation domains. Counteracting sources of the BMP antagonist NOGGIN enhance this spatial control of cell fate patterning. We also show how morphogen concentration and cell density affect the BMP response and germ layer patterning. These results demonstrate that the intrinsic capacity of stem cells for self-organization can be extrinsically controlled through the use of engineered signaling centers.


Assuntos
Células-Tronco Pluripotentes/citologia , Padronização Corporal , Proteína Morfogenética Óssea 4/farmacologia , Contagem de Células , Diferenciação Celular , Humanos , Dispositivos Lab-On-A-Chip
6.
Nat Cell Biol ; 21(6): 687-699, 2019 06.
Artigo em Inglês | MEDLINE | ID: mdl-31160711

RESUMO

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


Assuntos
Diferenciação Celular/genética , Reprogramação Celular/genética , Células-Tronco Pluripotentes Induzidas/citologia , Células-Tronco Pluripotentes/citologia , Animais , Blastômeros/citologia , Blastômeros/metabolismo , Linhagem da Célula/genética , Células-Tronco Embrionárias/citologia , Camadas Germinativas/crescimento & desenvolvimento , Camadas Germinativas/metabolismo , Humanos , Camundongos , Medicina Regenerativa , Transdução de Sinais/genética , Suínos , Trofoblastos/citologia , Trofoblastos/metabolismo
7.
Int J Mol Sci ; 20(11)2019 Jun 02.
Artigo em Inglês | MEDLINE | ID: mdl-31159483

RESUMO

The repair of damaged articular cartilage using currently available implantation techniques is not sufficient for the full recovery of patients. Pluripotent stem cells (iPSC)-based therapies could bring new perspectives in the treatment of joint diseases. A number of protocols of in vitro differentiation of iPSC in chondrocytes for regenerative purposes have been recently described. However, in order to use these cells in clinics, the elimination of animal serum and feeder cells is essential. In our study, a strictly defined and controllable protocol was designed for the differentiation of pluripotent stem cells (BG01V, ND 41658*H, GPCCi001-A) in chondrocyte-like cells in serum- and a feeder cell-free system, using the embryoid bodies step. The extension of the protocol and culture conditions (monolayer versus 3D culture) was also tested after the initial 21 days of chondrogenic differentiation. Promotion of the chondrogenic differentiation in 3D culture via the elevated expression of genes related to chondrogenesis was achieved. Using immunofluorescence and immunohistochemistry staining techniques, the increased deposition of the specific extracellular matrix was indicated. As a result, chondrocyte-like cells in the early stages of their differentiation using pellet culture under fully controlled and defined conditions were obtained.


Assuntos
Diferenciação Celular , Condrócitos/citologia , Condrogênese , Células-Tronco Pluripotentes/citologia , Técnicas de Cultura de Células/métodos , Linhagem Celular , Corpos Embrioides/citologia , Células-Tronco Embrionárias Humanas/citologia , Humanos , Células-Tronco Pluripotentes Induzidas/citologia
8.
Biochemistry (Mosc) ; 84(3): 321-328, 2019 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-31221070

RESUMO

Organoids are three-dimensional (3D) cell cultures that replicate some of the key features of morphology, spatial architecture, and functions of a particular organ. Organoids can be generated from both adult and pluripotent stem cells (PSCs), and complex organoids can also be obtained by combining different types of cells, including differentiated cells. The ability of pluripotent cells to self-organize into organotypic structures containing several cell subtypes specific for a particular organ was used for creating organoids of the brain, eye, kidney, intestine, and other organs. Despite the advantages of using PSCs for obtaining organoids, an essential shortcoming that prevents their widespread use has been a low yield when they are obtained from a PSC monolayer culture and a large variation in size. This leads to great heterogeneity on further differentiation. In this article, we describe our own protocol for generating standardized organoids, with emphasis on a method for generating brain organoids, which allows scaling-up experiments and makes their cultivation less expensive and easier.


Assuntos
Invenções , Organoides/citologia , Diferenciação Celular , Células Cultivadas , Humanos , Tamanho da Partícula , Células-Tronco Pluripotentes/citologia , Propriedades de Superfície
9.
Nat Cell Biol ; 21(7): 835-844, 2019 07.
Artigo em Inglês | MEDLINE | ID: mdl-31209294

RESUMO

Totipotency refers to the ability of a cell to generate all of the cell types of an organism. Unlike pluripotency, the establishment of totipotency is poorly understood. In mouse embryonic stem cells, Dux drives a small percentage of cells into a totipotent state by expressing 2-cell-embryo-specific transcripts. To understand how this transition takes place, we performed single-cell RNA-seq, which revealed a two-step transcriptional reprogramming process characterized by downregulation of pluripotent genes in the first step and upregulation of the 2-cell-embryo-specific elements in the second step. To identify factors controlling the transition, we performed a CRISPR-Cas9-mediated screen, which revealed Myc and Dnmt1 as two factors preventing the transition. Mechanistic studies demonstrate that Myc prevents downregulation of pluripotent genes in the first step, while Dnmt1 impedes 2-cell-embryo-specific gene activation in the second step. Collectively, the findings of our study reveal insights into the establishment and regulation of the totipotent state in mouse embryonic stem cells.


Assuntos
Reprogramação Celular/genética , DNA (Citosina-5-)-Metiltransferase 1/genética , Células-Tronco Embrionárias/metabolismo , Genes myc/genética , Animais , Diferenciação Celular/genética , Epigênese Genética/genética , Camundongos , Células-Tronco Embrionárias Murinas , Células-Tronco Pluripotentes/citologia , Células-Tronco Totipotentes/citologia
10.
Nat Cell Biol ; 21(7): 824-834, 2019 07.
Artigo em Inglês | MEDLINE | ID: mdl-31235934

RESUMO

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


Assuntos
Diferenciação Celular/fisiologia , Endoderma/citologia , Histona-Lisina N-Metiltransferase/metabolismo , Células-Tronco Pluripotentes/citologia , Animais , Diferenciação Celular/genética , Linhagem da Célula , Células-Tronco Embrionárias/citologia , Camadas Germinativas/citologia , Camundongos , Placa Neural/citologia , Proteínas Nucleares/metabolismo , Fatores de Transcrição/metabolismo
11.
Cell Physiol Biochem ; 52(6): 1309-1324, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31050280

RESUMO

BACKGROUND/AIMS: Different approaches have been considered to improve heart reconstructive medicine and direct delivery of pluripotent stem cell-derived cardiomyocytes (PSC-CMs) appears to be highly promising in this context. However, low cell persistence post-transplantation remains a bottleneck hindering the approach. Here, we present a novel strategy to overcome the low engraftment of PSC-CMs during the early post-transplantation phase into the myocardium of both healthy and cryoinjured syngeneic mice. METHODS: Adult murine bone marrow mesenchymal stem cells (MSCs) and PSC-CMs were co-cultured on thermo-responsive polymers and later detached through temperature reduction, resulting in the protease-free generation of cell clusters (micro-tissues) composed of both cells types. Micro-tissues were transplanted into healthy and cryo-injured murine hearts. Short term cell retention was quantified by real-time-PCR. Longitudinal cell tracking was performed by bioluminescence imaging for four weeks. Transplanted cells were further detected by immunofluorescence staining of tissue sections. RESULTS: We demonstrated that in vitro grown micro-tissues consisting of PSC-CMs and MSCs can increase cardiomyocyte retention by >10fold one day post-transplantation, but could not fully rescue a further cell loss between day 1 and day 2. Neutrophil infiltration into the transplanted area was detected in healthy hearts and could be attributed to the cellular implantation rather than tissue damage exerted by the transplantation cannula. Injected PSC-CMs were tracked and successfully detected for up to four weeks by bioluminescence imaging. CONCLUSION: This approach demonstrated that in vitro grown micro-tissues might contribute to the development of cardiac cell replacement therapies.


Assuntos
Transplante de Células-Tronco Mesenquimais , Células-Tronco Mesenquimais/citologia , Miocárdio/patologia , Miócitos Cardíacos/transplante , Animais , Células da Medula Óssea/citologia , Linhagem Celular , Rastreamento de Células , Técnicas de Cocultura , Imunidade Inata , Masculino , Células-Tronco Mesenquimais/metabolismo , Camundongos , Microscopia de Fluorescência , Infarto do Miocárdio/patologia , Infarto do Miocárdio/terapia , Miocárdio/imunologia , Miócitos Cardíacos/citologia , Miócitos Cardíacos/metabolismo , Infiltração de Neutrófilos , Imagem Óptica , Células-Tronco Pluripotentes/citologia , Polímeros/química
12.
Tissue Cell ; 58: 51-60, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-31133246

RESUMO

Tissue-specific adult stem cells (ASC) are heterogeneous and characterized by a mix of progenitor cells that produce cells at various stages of differentiation, and ultimately different terminally differentiated cells. Understanding the heterogeneity of ASCs may lead to the development of improved protocols of cell isolation and optimized cell therapy clinical protocols. Using a combination of enzymatic and explant culture protocols, we obtained pADSC population, which is composed by two distinct morphologies: fibroblast-like cells (FLCs) and endothelial-like cells (ELCs). Both cell sub-types efficiently formed colonies, expressed CD90+/CD105+/CD44+, and differentially expressed such markers such as Nestin, Vimentin, Fibronectin, Cytokeratin, Connexin 43, CD31, CD34 and CD146 as well as the pluripotent stem cell markers Oct-4, Nanog and Sox2. Mixed populations of pADSCs did not lose their multipotentiality and the cells were able to undergo osteogenic, chondrogenic, adipogenic and myogenic differentiation. Furthermore, the mixed population spontaneously formed capillary tube structures. Our findings suggest that different subpopulations can be isolated from adipose tissue and that the ADSCs need to be better evaluated using a wide panel of different markers related to cell differentiation, which is important for stem cell therapy and regenerative medicine, particularly for advanced stem cells therapies - products that are currently under investigation or even use.


Assuntos
Tecido Adiposo/metabolismo , Células-Tronco Adultas/metabolismo , Antígenos de Diferenciação/metabolismo , Separação Celular , Células-Tronco Pluripotentes/metabolismo , Tecido Adiposo/citologia , Células-Tronco Adultas/citologia , Animais , Células-Tronco Pluripotentes/citologia , Suínos
13.
Methods Mol Biol ; 1975: 53-77, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31062305

RESUMO

Human pluripotent stem cells are defined by their potential to give rise to all of the lineages of an embryo proper. Guiding the differentiation of embryonic stem cells or induced pluripotent stem cells can be achieved by exposing them to a succession of signaling conditions meant to mimic developmental milieus. However, achieving a quantitative understanding of the relationship between proliferation, cell death, and commitment has been difficult due to the inherent heterogeneity of pluripotent stem cells and their differentiation. Here, we describe a computational modeling approach to track the dynamics of germ layer commitment of human embryonic stem cells. We demonstrate that simulations using this model yield specific hypotheses regarding proliferation, cell death, and commitment and that these predictions are consistent with experimental measurements.


Assuntos
Diferenciação Celular , Linhagem da Célula , Endoderma/citologia , Células-Tronco Embrionárias Humanas/citologia , Modelos Teóricos , Células-Tronco Pluripotentes/citologia , Ativinas/metabolismo , Endoderma/metabolismo , Células-Tronco Embrionárias Humanas/metabolismo , Humanos , Células-Tronco Pluripotentes/metabolismo
14.
Methods Mol Biol ; 1975: 79-105, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31062306

RESUMO

The Reasoning Engine for Interaction Networks (RE:IN) is a tool that was developed initially for the study of pluripotency in mouse embryonic stem cells. A set of critical factors that regulate the pluripotent state had been identified experimentally, but it was not known how these genes interacted to stabilize self-renewal or commit the cell to differentiation. The methodology encapsulated in RE:IN enabled the exploration of a space of possible network interaction models, allowing for uncertainty in whether individual interactions exist between the pluripotency factors. This concept of an "abstract" network was combined with automated reasoning that allows the user to eliminate models that are inconsistent with experimental observations. The tool generalizes beyond the study of stem cell decision-making, allowing for the study of interaction networks more broadly across biology.


Assuntos
Diferenciação Celular , Linhagem da Célula , Biologia Computacional/métodos , Células-Tronco Embrionárias Murinas/citologia , Células-Tronco Pluripotentes/citologia , Animais , Perfilação da Expressão Gênica , Redes Reguladoras de Genes , Camundongos , Células-Tronco Pluripotentes/metabolismo
15.
Methods Mol Biol ; 1975: 107-129, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31062307

RESUMO

Studying cell fate dynamics is complicated by the fact that direct in vivo observation of individual cell fate outcomes is usually not possible and only multicellular data of cell clones can be obtained. In this situation, experimental data alone is not sufficient to validate biological models because the hypotheses and the data cannot be directly compared and thus standard statistical tests cannot be leveraged. On the other hand, mathematical modelling can bridge the scales between a hypothesis and measured data via quantitative predictions from a mathematical model. Here, we describe how to implement the rules behind a hypothesis (cell fate outcomes) one-to-one as a stochastic model, how to evaluate such a rule-based model mathematically via analytical calculation or stochastic simulations of the model's Master equation, and to predict the outcomes of clonal statistics for respective hypotheses. We also illustrate two approaches to compare these predictions directly with the clonal data to assess the models.


Assuntos
Diferenciação Celular , Linhagem da Célula , Endoderma/citologia , Modelos Teóricos , Células-Tronco Embrionárias Murinas/citologia , Células-Tronco Pluripotentes/citologia , Animais , Células Clonais , Endoderma/metabolismo , Camundongos , Células-Tronco Embrionárias Murinas/metabolismo , Células-Tronco Pluripotentes/metabolismo
16.
Methods Mol Biol ; 1975: 305-320, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31062316

RESUMO

Stem cell metabolism is intrinsically tied to stem cell pluripotency and function. Yet, understanding metabolic rewiring in stem cells has been challenging due to the complex and highly interconnected nature of the metabolic network. Genome-scale metabolic network models are increasingly used to holistically model the metabolic behavior of various cells and tissues using transcriptomics data. However, these powerful approaches that model steady-state behavior have limited utility for studying dynamic stem cell state transitions. To address this complexity, we recently developed the dynamic flux activity (DFA) approach; DFA is a genome-scale modeling approach that uses time-course metabolic data to predict metabolic flux rewiring. This protocol outlines the steps for modeling steady-state and dynamic metabolic behavior using transcriptomics and time-course metabolomics data, respectively. Using data from naive and primed pluripotent stem cells, we demonstrate how we can use genome-scale modeling and DFA to comprehensively characterize the metabolic differences between these states.


Assuntos
Diferenciação Celular , Linhagem da Célula , Biologia Computacional/métodos , Redes Reguladoras de Genes , Metaboloma , Células-Tronco Pluripotentes/citologia , Células-Tronco Pluripotentes/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Humanos , Transcriptoma
17.
Methods Mol Biol ; 1975: 407-426, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31062320

RESUMO

Due to their unique cellular features, pluripotent stem cells (PSCs) acquire chromosomal aberrations at a rather high frequency during their growth in culture. Analysis of chromosomal integrity should be routinely performed and usually is done at the DNA level of the cells. RNA sequencing (RNA-Seq) has recently become the basic tool for transcriptional studies. Therefore, methods that utilize this already available data to inspect the genomic integrity are very valuable. In this chapter, we provide a practical guide to implement methods of detection of chromosomal aberrations, which are based on RNA-Seq data. The expression-based karyotyping (e-Karyotyping) method is based on global gene expression analysis, while the expressed-SNP-karyotyping (eSNP-Karyotyping) method is based on changes in the ratio between alleles.


Assuntos
Aneuploidia , Biologia Computacional/métodos , Regulação da Expressão Gênica , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Cariotipagem/métodos , Células-Tronco Pluripotentes/citologia , Polimorfismo de Nucleotídeo Único , Humanos
18.
Int J Mol Sci ; 20(9)2019 May 07.
Artigo em Inglês | MEDLINE | ID: mdl-31067778

RESUMO

Pluripotent stem cells (PSCs), including embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs), hold a huge promise for regenerative medicine, drug development, and disease modeling. PSCs have unique metabolic features that are akin to those of cancer cells, in which glycolysis predominates to produce energy as well as building blocks for cellular components. Recent studies indicate that the unique metabolism in PSCs is not a mere consequence of their preference for a low oxygen environment, but is an active process for maintaining self-renewal and pluripotency, possibly in preparation for rapid response to the metabolic demands of differentiation. Understanding the regulatory mechanisms of this unique metabolism in PSCs is essential for proper derivation, generation, and maintenance of PSCs. In this review, we discuss the metabolic features of PSCs and describe the current understanding of the mechanisms of the metabolic shift during reprogramming from somatic cells to iPSCs, in which the metabolism switches from oxidative phosphorylation (OxPhos) to glycolysis.


Assuntos
Reprogramação Celular , Metabolismo Energético , Células-Tronco Pluripotentes/metabolismo , Animais , Humanos , Mitocôndrias/metabolismo , Células-Tronco Pluripotentes/citologia
19.
Methods Mol Biol ; 1965: 35-48, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31069667

RESUMO

Cranial neural crest cells (NCCs) migrate to the branchial arches and give rise to the majority of cranial mesenchyme that eventually differentiates into odontoblasts, cartilage, craniofacial bone, and connective tissue; a subset of these cells differentiate into cranial ganglia. Here we present a protocol that describes directed differentiation method of human pluripotent stem cells into cranial NCC-like cells and a cytotoxicity assay using hPSC-derived cranial NCC-like cells. This cell-based assay system allows for high-sensitive cytotoxicity detection of test chemicals. These methods can be applied to predict drug/chemical toxicity effect on early craniofacial development.


Assuntos
Encéfalo/citologia , Crista Neural/citologia , Células-Tronco Pluripotentes/citologia , Animais , Técnicas de Cultura de Células , Diferenciação Celular , Células Cultivadas , Humanos , Modelos Biológicos , Testes de Toxicidade
20.
Nat Genet ; 51(6): 999-1010, 2019 06.
Artigo em Inglês | MEDLINE | ID: mdl-31110351

RESUMO

Human embryonic stem cells (ESCs) and human induced pluripotent stem cells hold great promise for cell-based therapies and drug discovery. However, homogeneous differentiation remains a major challenge, highlighting the need for understanding developmental mechanisms. We performed genome-scale CRISPR screens to uncover regulators of definitive endoderm (DE) differentiation, which unexpectedly uncovered five Jun N-terminal kinase (JNK)-JUN family genes as key barriers of DE differentiation. The JNK-JUN pathway does not act through directly inhibiting the DE enhancers. Instead, JUN co-occupies ESC enhancers with OCT4, NANOG, SMAD2 and SMAD3, and specifically inhibits the exit from the pluripotent state by impeding the decommissioning of ESC enhancers and inhibiting the reconfiguration of SMAD2 and SMAD3 chromatin binding from ESC to DE enhancers. Therefore, the JNK-JUN pathway safeguards pluripotency from precocious DE differentiation. Direct pharmacological inhibition of JNK significantly improves the efficiencies of generating DE and DE-derived pancreatic and lung progenitor cells, highlighting the potential of harnessing the knowledge from developmental studies for regenerative medicine.


Assuntos
Diferenciação Celular/genética , Endoderma/embriologia , Endoderma/metabolismo , Genoma , Genômica , Sistema de Sinalização das MAP Quinases , Células-Tronco Pluripotentes/citologia , Células-Tronco Pluripotentes/metabolismo , Linhagem Celular , Cromatina/genética , Cromatina/metabolismo , Montagem e Desmontagem da Cromatina , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , Expressão Gênica , Técnicas de Inativação de Genes , Genes Reporter , Genômica/métodos , Humanos , Células-Tronco Pluripotentes Induzidas , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Modelos Biológicos , Reprodutibilidade dos Testes , Proteínas Smad
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