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1.
Nature ; 574(7776): 112-116, 2019 10.
Artigo em Inglês | MEDLINE | ID: mdl-31554966

RESUMO

Organogenesis is a complex and interconnected process that is orchestrated by multiple boundary tissue interactions1-7. However, it remains unclear how individual, neighbouring components coordinate to establish an integral multi-organ structure. Here we report the continuous patterning and dynamic morphogenesis of hepatic, biliary and pancreatic structures, invaginating from a three-dimensional culture of human pluripotent stem cells. The boundary interactions between anterior and posterior gut spheroids differentiated from human pluripotent stem cells enables retinoic acid-dependent emergence of hepato-biliary-pancreatic organ domains specified at the foregut-midgut boundary organoids in the absence of extrinsic factors. Whereas transplant-derived tissues are dominated by midgut derivatives, long-term-cultured microdissected hepato-biliary-pancreatic organoids develop into segregated multi-organ anlages, which then recapitulate early morphogenetic events including the invagination and branching of three different and interconnected organ structures, reminiscent of tissues derived from mouse explanted foregut-midgut culture. Mis-segregation of multi-organ domains caused by a genetic mutation in HES1 abolishes the biliary specification potential in culture, as seen in vivo8,9. In sum, we demonstrate that the experimental multi-organ integrated model can be established by the juxtapositioning of foregut and midgut tissues, and potentially serves as a tractable, manipulatable and easily accessible model for the study of complex human endoderm organogenesis.


Assuntos
Sistema Biliar/embriologia , Intestinos/embriologia , Fígado/embriologia , Modelos Biológicos , Morfogênese , Pâncreas/embriologia , Animais , Sistema Biliar/citologia , Biomarcadores/análise , Biomarcadores/metabolismo , Padronização Corporal , Endoderma/citologia , Endoderma/embriologia , Humanos , Células-Tronco Pluripotentes Induzidas/citologia , Células-Tronco Pluripotentes Induzidas/metabolismo , Intestinos/citologia , Fígado/citologia , Masculino , Camundongos , Organoides/citologia , Organoides/embriologia , Pâncreas/citologia , Esferoides Celulares/citologia , Esferoides Celulares/metabolismo , Esferoides Celulares/transplante , Fatores de Transcrição HES-1/análise , Fatores de Transcrição HES-1/metabolismo
2.
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
3.
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
4.
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
5.
Nat Commun ; 10(1): 1089, 2019 03 06.
Artigo em Inglês | MEDLINE | ID: mdl-30842446

RESUMO

The essential roles played by Nodal and Bmp signalling during early mouse development have been extensively documented. Here we use conditional deletion strategies to investigate functional contributions made by Nodal, Bmp and Smad downstream effectors during primordial germ cell (PGC) development. We demonstrate that Nodal and its target gene Eomes provide early instructions during formation of the PGC lineage. We discover that Smad2 inactivation in the visceral endoderm results in increased numbers of PGCs due to an expansion of the PGC niche. Smad1 is required for specification, whereas in contrast Smad4 controls the maintenance and migration of PGCs. Additionally we find that beside Blimp1, down-regulated phospho-Smad159 levels also distinguishes PGCs from their somatic neighbours so that emerging PGCs become refractory to Bmp signalling that otherwise promotes mesodermal development in the posterior epiblast. Thus balanced Nodal/Bmp signalling cues regulate germ cell versus somatic cell fate decisions in the early posterior epiblast.


Assuntos
Proteínas Morfogenéticas Ósseas/metabolismo , Diferenciação Celular/fisiologia , Regulação da Expressão Gênica no Desenvolvimento/fisiologia , Células Germinativas/fisiologia , Proteína Nodal/metabolismo , Animais , Proteínas Morfogenéticas Ósseas/genética , Linhagem Celular , Movimento Celular/fisiologia , Embrião de Mamíferos , Endoderma/citologia , Endoderma/fisiologia , Feminino , Técnicas de Inativação de Genes , Masculino , Camundongos , Camundongos Knockout , Células-Tronco Embrionárias Murinas , Proteína Nodal/genética , Transdução de Sinais/genética , Proteínas Smad/genética , Proteínas Smad/metabolismo
6.
Cell Prolif ; 52(3): e12591, 2019 May.
Artigo em Inglês | MEDLINE | ID: mdl-30896067

RESUMO

OBJECTIVES: To date, many efforts have been made to establish porcine embryonic stem (pES) cells without success. Extraembryonic endoderm (XEN) cells can self-renew and differentiate into the visceral endoderm and parietal endoderm. XEN cells are derived from the primitive endoderm of the inner cell mass of blastocysts and may be an intermediate state in cell reprogramming. MATERIALS AND METHODS: Porcine XEN cells (pXENCs) were generated from porcine pluripotent stem cells (pPSCs) and were characterized by RNA sequencing and immunofluorescence analyses. The developmental potential of pXENCs was investigated in chimeric mouse embryos. RESULTS: Porcine XEN cells derived from porcine pPSCs were successfully expanded in N2B27 medium supplemented with bFGF for least 30 passages. RNA sequencing and immunofluorescence analyses showed that pXENCs expressed the murine and canine XEN markers Gata6, Gata4, Sox17 and Pdgfra but not the pluripotent markers Oct4, Sox2 and TE marker Cdx2. Moreover, these cells contributed to the XEN when injected into four-cell stage mouse embryos. Supplementation with Chir99021 and SB431542 promoted the pluripotency of the pXENCs. CONCLUSIONS: We successfully derived pXENCs and showed that supplementation with Chir99021 and SB431542 confer them with pluripotency. Our results provide a new resource for investigating the reprogramming mechanism of porcine-induced pluripotent stem cells.


Assuntos
Endoderma/citologia , Endoderma/embriologia , Suínos/embriologia , Animais , Técnicas de Cultura de Células , Diferenciação Celular , Linhagem Celular , Técnicas de Cocultura , Cães , Células-Tronco Embrionárias/citologia , Células-Tronco Embrionárias/metabolismo , Endoderma/metabolismo , Expressão Gênica , Camundongos , Células-Tronco Pluripotentes/citologia , Células-Tronco Pluripotentes/metabolismo , Análise de Sequência de RNA , Transdução de Sinais , Suínos/genética , Suínos/metabolismo , Quimeras de Transplante
7.
Nature ; 566(7745): 490-495, 2019 02.
Artigo em Inglês | MEDLINE | ID: mdl-30787436

RESUMO

Across the animal kingdom, gastrulation represents a key developmental event during which embryonic pluripotent cells diversify into lineage-specific precursors that will generate the adult organism. Here we report the transcriptional profiles of 116,312 single cells from mouse embryos collected at nine sequential time points ranging from 6.5 to 8.5 days post-fertilization. We construct a molecular map of cellular differentiation from pluripotency towards all major embryonic lineages, and explore the complex events involved in the convergence of visceral and primitive streak-derived endoderm. Furthermore, we use single-cell profiling to show that Tal1-/- chimeric embryos display defects in early mesoderm diversification, and we thus demonstrate how combining temporal and transcriptional information can illuminate gene function. Together, this comprehensive delineation of mammalian cell differentiation trajectories in vivo represents a baseline for understanding the effects of gene mutations during development, as well as a roadmap for the optimization of in vitro differentiation protocols for regenerative medicine.


Assuntos
Diferenciação Celular/genética , Embrião de Mamíferos/citologia , Embrião de Mamíferos/metabolismo , Gastrulação , Organogênese , Análise de Célula Única , Animais , Linhagem da Célula/genética , Quimera/embriologia , Quimera/genética , Quimera/metabolismo , Endoderma/citologia , Endoderma/embriologia , Endoderma/metabolismo , Endotélio/citologia , Endotélio/embriologia , Endotélio/metabolismo , Feminino , Gastrulação/genética , Perfilação da Expressão Gênica , Regulação da Expressão Gênica no Desenvolvimento/genética , Hematopoese/genética , Masculino , Mesoderma/citologia , Mesoderma/embriologia , Camundongos , Mutação/genética , Células Mieloides/citologia , Organogênese/genética , Células-Tronco Pluripotentes/citologia , Células-Tronco Pluripotentes/metabolismo , Linha Primitiva/citologia , Linha Primitiva/embriologia , Proteína 1 de Leucemia Linfocítica Aguda de Células T/deficiência , Proteína 1 de Leucemia Linfocítica Aguda de Células T/genética
8.
PLoS One ; 14(2): e0212109, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30735538

RESUMO

During preimplantation mouse development stages, emerging pluripotent epiblast (Epi) and extraembryonic primitive endoderm (PrE) cells are first distributed in the blastocyst in a "salt-and-pepper" manner before they segregate into separate layers. As a result of segregation, PrE cells become localised on the surface of the inner cell mass (ICM), and the Epi is enclosed by the PrE on one side and by the trophectoderm on the other. During later development, a subpopulation of PrE cells migrates away from the ICM and forms the parietal endoderm (PE), while cells remaining in contact with the Epi form the visceral endoderm (VE). Here, we asked: what are the mechanisms mediating Epi and PrE cell segregation and the subsequent VE vs PE specification? Differences in cell adhesion have been proposed; however, we demonstrate that the levels of plasma membrane-bound E-cadherin (CDH1, cadherin 1) in Epi and PrE cells only differ after the segregation of these lineages within the ICM. Moreover, manipulating E-cadherin levels did not affect lineage specification or segregation, thus failing to confirm its role during these processes. Rather, we report changes in E-cadherin localisation during later PrE-to-PE transition which are accompanied by the presence of Vimentin and Twist, supporting the hypothesis that an epithelial-to-mesenchymal transition process occurs in the mouse peri-implantation blastocyst.


Assuntos
Blastocisto/citologia , Blastocisto/metabolismo , Caderinas/metabolismo , Endoderma/citologia , Células-Tronco Pluripotentes/citologia , Animais , Morte Celular , Linhagem da Célula , Membrana Celular/metabolismo , Implantação do Embrião , Transição Epitelial-Mesenquimal , Feminino , Camundongos , Transporte Proteico
9.
Dev Cell ; 48(3): 361-370.e3, 2019 02 11.
Artigo em Inglês | MEDLINE | ID: mdl-30753836

RESUMO

The Erk mitogen-activated protein kinase plays diverse roles in animal development. Its widespread reuse raises a conundrum: when a single kinase like Erk is activated, how does a developing cell know which fate to adopt? We combine optogenetic control with genetic perturbations to dissect Erk-dependent fates in the early Drosophila embryo. We find that Erk activity is sufficient to "posteriorize" 88% of the embryo, inducing gut endoderm-like gene expression and morphogenetic movements in all cells within this region. Gut endoderm fate adoption requires at least 1 h of signaling, whereas a 30-min Erk pulse specifies a distinct ectodermal cell type, intermediate neuroblasts. We find that the endoderm-ectoderm cell fate switch is controlled by the cumulative load of Erk activity, not the duration of a single pulse. The fly embryo thus harbors a classic example of dynamic control, where the temporal profile of Erk signaling selects between distinct physiological outcomes.


Assuntos
Ectoderma/citologia , Embrião de Mamíferos/metabolismo , Endoderma/citologia , Regulação da Expressão Gênica no Desenvolvimento , Animais , Diferenciação Celular/genética , Diferenciação Celular/fisiologia , Drosophila/embriologia , Ectoderma/embriologia , Embrião não Mamífero/metabolismo , Endoderma/embriologia , Endoderma/metabolismo , Morfogênese/genética
10.
Dev Comp Immunol ; 95: 89-95, 2019 06.
Artigo em Inglês | MEDLINE | ID: mdl-30753854

RESUMO

The chicken yolk sac (YS) plays an important role in nutrient absorption and immune function for the developing embryo. The avian ß-defensins (AvBD) are cationic peptides that are important members of the innate immune system. The objective of this study was to profile AvBD mRNA expression patterns and distribution of cells expressing AvBD mRNA in the chicken YS. Expression of AvBD1, 2, 7, and 10 mRNA was low at embryonic day 7 (e7), increased to e9 through e13 and then declined to e19. Using in situ hybridization, AvBD10 mRNA was found to be expressed in endodermal epithelial cells, while AvBD1, 2, and 7 mRNA were expressed in heterophils. The developmental expression pattern and distribution of AvBD mRNA in the YS reveals the importance of these genes to protection of the developing chick embryo.


Assuntos
Proteínas Aviárias/genética , Desenvolvimento Embrionário/imunologia , Regulação da Expressão Gênica no Desenvolvimento/imunologia , Saco Vitelino/imunologia , beta-Defensinas/genética , Animais , Proteínas Aviárias/imunologia , Embrião de Galinha , Galinhas , Endoderma/citologia , Endoderma/imunologia , Endoderma/metabolismo , Células Epiteliais/imunologia , Células Epiteliais/metabolismo , RNA Mensageiro/metabolismo , Saco Vitelino/crescimento & desenvolvimento , Saco Vitelino/metabolismo , beta-Defensinas/imunologia
11.
Curr Stem Cell Res Ther ; 14(3): 278-289, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30674265

RESUMO

The discovery of small non-coding RNAs began an interesting era in cellular and molecular biology. To date, miRNAs are the best recognized non-coding RNAs for maintenance and differentiation of pluripotent stem cells including embryonic stem cells (ES), induced pluripotent stem cells (iPSC), and cancer stem cells. ES cells are defined by their ability to self-renew, teratoma formation, and to produce numerous types of differentiated cells. Dual capacity of ES cells for self-renewal and differentiation is controlled by specific interaction with the neighboring cells and intrinsic signaling pathways from the level of transcription to translation. The ES cells have been the suitable model for evaluating the function of non-coding RNAs and in specific miRNAs. So far, the general function of the miRNAs in ES cells has been assessed in mammalian and non-mammalian stem cells. Nowadays, the evolution of sequencing technology led to the discovery of numerous miRNAs in human and mouse ES cells that their expression levels significantly changes during proliferation and differentiation. Several miRNAs have been identified in ectoderm, mesoderm, and endoderm cells, as well. This review would focus on recent knowledge about the expression and functional roles of miRNAs in embryonic and lineage-specific stem cells. It also describes that miRNAs might have essential roles in orchestrating the Waddington's landscape structure during development.


Assuntos
Linhagem da Célula/genética , Células-Tronco Embrionárias/metabolismo , Células-Tronco Pluripotentes Induzidas/metabolismo , MicroRNAs/genética , Células-Tronco Neoplásicas/metabolismo , Células-Tronco Neurais/metabolismo , Animais , Comunicação Celular , Diferenciação Celular , Proliferação de Células , Ectoderma/citologia , Ectoderma/metabolismo , Células-Tronco Embrionárias/citologia , Endoderma/citologia , Endoderma/metabolismo , Regulação da Expressão Gênica , Humanos , Células-Tronco Pluripotentes Induzidas/citologia , Mesoderma/citologia , Mesoderma/metabolismo , MicroRNAs/classificação , MicroRNAs/metabolismo , Células-Tronco Neoplásicas/patologia , Células-Tronco Neurais/citologia , Transdução de Sinais
12.
Nature ; 565(7740): 480-484, 2019 01.
Artigo em Inglês | MEDLINE | ID: mdl-30651642

RESUMO

The embryonic gut tube is a cylindrical structure from which the respiratory and gastrointestinal tracts develop1. Although the early emergence of the endoderm as an epithelial sheet2,3 and later morphogenesis of the definitive digestive and respiratory organs4-6 have been investigated, the intervening process of gut tube formation remains relatively understudied7,8. Here we investigate the molecular control of macroscopic forces underlying early morphogenesis of the gut tube in the chick embryo. The gut tube has been described as forming from two endodermal invaginations-the anterior intestinal portal (AIP) towards the rostral end of the embryo and the caudal intestinal portal (CIP) at the caudal end-that migrate towards one another, internalizing the endoderm until they meet at the yolk stalk (umbilicus in mammals)1,6. Migration of the AIP to form foregut has been descriptively characterized8,9, but the hindgut is likely to form by a distinct mechanism that has not been fully explained10. We find that the hindgut is formed by collective cell movements through a stationary CIP, rather than by movement of the CIP itself. Further, combining in vivo imaging, biophysics and mathematical modelling with molecular and embryological approaches, we identify a contractile force gradient that drives cell movements in the hindgut-forming endoderm, enabling tissue-scale posterior extension of the forming hindgut tube. The force gradient, in turn, is established in response to a morphogenic gradient of fibroblast growth factor signalling. As a result, we propose that an important positive feedback arises, whereby contracting cells draw passive cells from low to high fibroblast growth factor levels, recruiting them to contract and pull more cells into the elongating hindgut. In addition to providing insight into the early gut development, these findings illustrate how large-scale tissue level forces can be traced to developmental signals during vertebrate morphogenesis.


Assuntos
Trato Gastrointestinal/embriologia , Morfogênese , Animais , Padronização Corporal , Movimento Celular , Embrião de Galinha , Endoderma/citologia , Endoderma/embriologia , Endoderma/metabolismo , Fator 8 de Crescimento de Fibroblasto/metabolismo , Trato Gastrointestinal/citologia , Trato Gastrointestinal/metabolismo , Transdução de Sinais
13.
Nat Protoc ; 14(2): 350-378, 2019 02.
Artigo em Inglês | MEDLINE | ID: mdl-30617351

RESUMO

Molecular and embryology studies have demonstrated that mouse pre-implantation embryo development is a process of progressive cell fate determination. At the time of implantation, three cell lineages are present in the developing blastocyst: the trophectoderm (TE), the epiblast (Epi) and the primitive endoderm (PrE). From these early embryo cells, trophoblast stem (TS) cells, embryonic stem (ES) cells and extra-embryonic endoderm stem (XEN) cells can be derived. Recently, we derived stem cells with blastomere-like features from mouse cleavage-stage embryos, which we named expanded-potential stem cells (EPSCs). Here, we provide detailed protocols that describe how to establish EPSCs from single eight-cell-stage blastomeres or whole eight-cell pre-implantation mouse embryos, or by conversion of mouse ES cells or induced pluripotent stem (iPS) cells reprogrammed from fibroblasts. It takes 2-3 weeks to derive EPSCs from each cell source. The EPSCs derived from these protocols can differentiate into all embryonic and extra-embryonic lineages when implanted into chimeras. Furthermore, bona fide TS and XEN cell lines can be derived from EPSCs in vitro.


Assuntos
Técnicas de Cultura de Células , Linhagem da Célula , Desenvolvimento Embrionário , Células-Tronco Pluripotentes Induzidas/citologia , Células-Tronco Embrionárias Murinas/citologia , Trofoblastos/citologia , Animais , Biomarcadores/metabolismo , Blastômeros/citologia , Blastômeros/metabolismo , Fator de Transcrição CDX2/genética , Fator de Transcrição CDX2/metabolismo , Diferenciação Celular , Proliferação de Células , Reprogramação Celular , Endoderma/citologia , Endoderma/metabolismo , Feminino , Fibroblastos/citologia , Fibroblastos/metabolismo , Expressão Gênica , Camadas Germinativas/citologia , Camadas Germinativas/metabolismo , Células-Tronco Pluripotentes Induzidas/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Células-Tronco Embrionárias Murinas/metabolismo , Fator 3 de Transcrição de Octâmero/genética , Fator 3 de Transcrição de Octâmero/metabolismo , Cultura Primária de Células , Trofoblastos/metabolismo
14.
Dev Biol ; 447(1): 14-23, 2019 03 01.
Artigo em Inglês | MEDLINE | ID: mdl-29653088

RESUMO

The secreted TGF-ß superfamily signals Nodal and BMP coordinate the patterning of vertebrate embryos. Nodal specifies endoderm and mesoderm during germ layer formation, and BMP specifies ventral fates and patterns the dorsal/ventral axis. Five major models have been proposed to explain how the correct distributions of Nodal and BMP are achieved within tissues to orchestrate embryogenesis: source/sink, transcriptional determination, relay, self-regulation, and shuttling. Here, we discuss recent experiments probing these signal dispersal models, focusing on early zebrafish development.


Assuntos
Proteínas Morfogenéticas Ósseas/metabolismo , Desenvolvimento Embrionário/fisiologia , Modelos Biológicos , Ligantes da Sinalização Nodal/metabolismo , Proteínas de Peixe-Zebra/metabolismo , Peixe-Zebra/embriologia , Animais , Proteínas Morfogenéticas Ósseas/genética , Endoderma/citologia , Endoderma/embriologia , Mesoderma/citologia , Mesoderma/embriologia , Ligantes da Sinalização Nodal/genética , Peixe-Zebra/genética , Proteínas de Peixe-Zebra/genética
15.
Dev Biol ; 445(2): 256-270, 2019 01 15.
Artigo em Inglês | MEDLINE | ID: mdl-30472119

RESUMO

The enteric nervous system is thought to originate solely from the neural crest. Transgenic lineage tracing revealed a novel population of clonal pancreatic duodenal homeobox-1 (Pdx1)-Cre lineage progenitor cells in the tunica muscularis of the gut that produced pancreatic descendants as well as neurons upon differentiation in vitro. Additionally, an in vivo subpopulation of endoderm lineage enteric neurons, but not glial cells, was seen especially in the proximal gut. Analysis of early transgenic embryos revealed Pdx1-Cre progeny (as well as Sox-17-Cre and Foxa2-Cre progeny) migrating from the developing pancreas and duodenum at E11.5 and contributing to the enteric nervous system. These results show that the mammalian enteric nervous system arises from both the neural crest and the endoderm. Moreover, in adult mice there are separate Wnt1-Cre neural crest stem cells and Pdx1-Cre pancreatic progenitors within the muscle layer of the gut.


Assuntos
Sistema Nervoso Entérico/embriologia , Animais , Linhagem da Célula/genética , Duodeno/embriologia , Duodeno/inervação , Duodeno/metabolismo , Endoderma/citologia , Endoderma/embriologia , Endoderma/metabolismo , Sistema Nervoso Entérico/citologia , Sistema Nervoso Entérico/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Proteínas HMGB/genética , Proteínas HMGB/metabolismo , Fator 3-beta Nuclear de Hepatócito/genética , Fator 3-beta Nuclear de Hepatócito/metabolismo , Proteínas de Homeodomínio/genética , Proteínas de Homeodomínio/metabolismo , Camundongos , Camundongos Transgênicos , Crista Neural/citologia , Crista Neural/embriologia , Crista Neural/metabolismo , Células-Tronco Neurais/citologia , Células-Tronco Neurais/metabolismo , Pâncreas/embriologia , Pâncreas/inervação , Pâncreas/metabolismo , Fatores de Transcrição SOXF/genética , Fatores de Transcrição SOXF/metabolismo , Transativadores/genética , Transativadores/metabolismo , Proteína Wnt1/genética , Proteína Wnt1/metabolismo
16.
Tissue Eng Part A ; 25(5-6): 457-467, 2019 03.
Artigo em Inglês | MEDLINE | ID: mdl-30141379

RESUMO

IMPACT STATEMENT: Although oxygen is a vital nutrient for the hepatocytes in vitro, few reports have focused on its effect during hepatic differentiation of induced pluripotent stem cells (iPSCs). In this report, we performed the hepatic differentiation of human iPSCs (hiPSCs) under different atmospheric oxygen concentrations and oxygen supply fluxes to investigate the effects of oxygen in terms of both the concentration and the supply flux. Results demonstrate that direct oxygenation through a polydimethylsiloxane (PDMS) membrane enhances the maturation and efficient production of hiPSC-derived hepatocyte-like cells (iHeps). Thus, direct oxygenation through a PDMS membrane is a better alternative culture method over conventional tissue culture-treated polystyrene (TCPS) plates for the maturation of hiPSC-derived hepatocytes in vitro.


Assuntos
Diferenciação Celular/efeitos dos fármacos , Permeabilidade da Membrana Celular , Células-Tronco Pluripotentes Induzidas/citologia , Fígado/citologia , Oxigênio/farmacologia , Albuminas/genética , Albuminas/metabolismo , Fosfatase Alcalina/genética , Fosfatase Alcalina/metabolismo , Permeabilidade da Membrana Celular/efeitos dos fármacos , Respiração Celular/efeitos dos fármacos , DNA/metabolismo , Dimetilpolisiloxanos/farmacologia , Endoderma/citologia , Fator de Transcrição GATA4/metabolismo , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Células-Tronco Pluripotentes Induzidas/efeitos dos fármacos , Consumo de Oxigênio/efeitos dos fármacos
17.
Nat Protoc ; 14(1): 28-50, 2019 01.
Artigo em Inglês | MEDLINE | ID: mdl-30470820

RESUMO

The human stomach contains two primary domains: the corpus, which contains the fundic epithelium, and the antrum. Each of these domains has distinct cell types and functions, and therefore each presents with unique disease pathologies. Here, we detail two protocols to differentiate human pluripotent stem cells (hPSCs) into human gastric organoids (hGOs) that recapitulate both domains. Both protocols begin with the differentiation of hPSCs into definitive endoderm (DE) using activin A, followed by the generation of free-floating 3D posterior foregut spheroids using FGF4, Wnt pathway agonist CHIR99021 (CHIR), BMP pathway antagonist Noggin, and retinoic acid. Embedding spheroids in Matrigel and continuing 3D growth in epidermal growth factor (EGF)-containing medium for 4 weeks results in antral hGOs (hAGOs). To obtain fundic hGOs (hFGOs), spheroids are additionally treated with CHIR and FGF10. Induced differentiation of acid-secreting parietal cells in hFGOs requires temporal treatment of BMP4 and the MEK inhibitor PD0325901 for 48 h on protocol day 30. In total, it takes ~34 d to generate hGOs from hPSCs. To date, this is the only approach that generates functional human differentiated gastric cells de novo from hPSCs.


Assuntos
Técnicas de Cultura de Células , Endoderma/citologia , Células Epiteliais/citologia , Fundo Gástrico/citologia , Organoides/citologia , Células-Tronco Pluripotentes/citologia , Antro Pilórico/citologia , Ativinas/farmacologia , Benzamidas/farmacologia , Proteínas de Transporte/farmacologia , Diferenciação Celular , Colágeno/química , Meios de Cultura/química , Meios de Cultura/farmacologia , Difenilamina/análogos & derivados , Difenilamina/farmacologia , Combinação de Medicamentos , Endoderma/efeitos dos fármacos , Endoderma/metabolismo , Fator de Crescimento Epidérmico/farmacologia , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/metabolismo , Fator 10 de Crescimento de Fibroblastos/farmacologia , Fator 4 de Crescimento de Fibroblastos/farmacologia , Fundo Gástrico/metabolismo , Humanos , Laminina/química , Especificidade de Órgãos , Organoides/efeitos dos fármacos , Organoides/metabolismo , Células-Tronco Pluripotentes/efeitos dos fármacos , Células-Tronco Pluripotentes/metabolismo , Proteoglicanas/química , Antro Pilórico/metabolismo , Piridinas/farmacologia , Pirimidinas/farmacologia , Tretinoína/farmacologia , Via de Sinalização Wnt/efeitos dos fármacos
18.
Nature ; 564(7735): 219-224, 2018 12.
Artigo em Inglês | MEDLINE | ID: mdl-30518857

RESUMO

Direct lineage reprogramming involves the conversion of cellular identity. Single-cell technologies are useful for deconstructing the considerable heterogeneity that emerges during lineage conversion. However, lineage relationships are typically lost during cell processing, complicating trajectory reconstruction. Here we present 'CellTagging', a combinatorial cell-indexing methodology that enables parallel capture of clonal history and cell identity, in which sequential rounds of cell labelling enable the construction of multi-level lineage trees. CellTagging and longitudinal tracking of fibroblast to induced endoderm progenitor reprogramming reveals two distinct trajectories: one leading to successfully reprogrammed cells, and one leading to a 'dead-end' state, paths determined in the earliest stages of lineage conversion. We find that expression of a putative methyltransferase, Mettl7a1, is associated with the successful reprogramming trajectory; adding Mettl7a1 to the reprogramming cocktail increases the yield of induced endoderm progenitors. Together, these results demonstrate the utility of our lineage-tracing method for revealing the dynamics of direct reprogramming.


Assuntos
Linhagem da Célula , Rastreamento de Células/métodos , Reprogramação Celular , Células Clonais/citologia , Análise de Célula Única/métodos , Animais , Linhagem da Célula/efeitos dos fármacos , Separação Celular , Reprogramação Celular/efeitos dos fármacos , Células Clonais/efeitos dos fármacos , Endoderma/citologia , Endoderma/efeitos dos fármacos , Fibroblastos/citologia , Fibroblastos/efeitos dos fármacos , Células HEK293 , Humanos , Metiltransferases/metabolismo , Camundongos , Células-Tronco/citologia , Células-Tronco/efeitos dos fármacos , Fatores de Tempo
19.
Stem Cell Reports ; 11(6): 1357-1364, 2018 12 11.
Artigo em Inglês | MEDLINE | ID: mdl-30449705

RESUMO

Specifying the primitive streak (PS) guides stem cell differentiation in vitro; however, much remains to be learned about the transcription networks that direct anterior and posterior PS cells (APS and PPS, respectively) to differentiate to distinct mesendodermal subpopulations. Here, we show that APS genes are predominantly induced in YAP1-/- human embryonic stem cells (hESCs) in response to ACTIVIN. This finding establishes the Hippo effector YAP1 as a master regulator of PS specification, functioning to repress ACTIVIN-regulated APS genes in hESCs. Moreover, transient exposure of wild-type hESCs to dasatinib, a potent C-SRC/YAP1 inhibitor, enables differentiation to APS-derived endoderm and cardiac mesoderm in response to ACTIVIN. Importantly, these cells can differentiate efficiently to normal beating cardiomyocytes without the cytoskeletal defect seen in YAP1-/- hESC-derived cardiomyocytes. Overall, we uncovered an induction mechanism to generate APS cells using a cocktail of ACTIVIN and YAP1i molecules that holds practical implications for hESC and induced pluripotent stem cell differentiation into distinct mesendodermal lineages.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Fosfoproteínas/metabolismo , Células-Tronco Pluripotentes/metabolismo , Linha Primitiva/metabolismo , Ativinas/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/antagonistas & inibidores , Diferenciação Celular/efeitos dos fármacos , Linhagem da Célula/efeitos dos fármacos , Reprogramação Celular/efeitos dos fármacos , Dasatinibe/farmacologia , Endoderma/citologia , Células-Tronco Embrionárias Humanas/citologia , Células-Tronco Embrionárias Humanas/efeitos dos fármacos , Células-Tronco Embrionárias Humanas/metabolismo , Humanos , Miocárdio/citologia , Miócitos Cardíacos/citologia , Miócitos Cardíacos/efeitos dos fármacos , Miócitos Cardíacos/metabolismo , Fosfoproteínas/antagonistas & inibidores , Células-Tronco Pluripotentes/citologia , Células-Tronco Pluripotentes/efeitos dos fármacos , Fatores de Transcrição
20.
PLoS One ; 13(11): e0207211, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-30440053

RESUMO

Standard methods for seeding monolayer cell cultures in a multiwell plate or dish do not uniformly distribute cells on the surface. With traditional methods, users find aggregation around the circumference, in the centre, or a combination of the two. This variation is introduced due to the macro scale flow of the cell seeding suspension, and movement of the dish before cells can settle and attach to the surface. Reproducibility between labs, users, and experiments is hampered by this variability in cell seeding. We present a simple method for uniform and user-independent the cell seeding using an easily produced uniform cell seeder (UCS) device. This allows precise control of cell density in a reproducible manner. By containing the cell seeding suspension in a defined volume above the culture surface with the UCS, fluctuations in cell density are minimised. Seeding accuracy, as defined by the actual cell density versus the target seeding density is improved dramatically across users with various levels of expertise. We go on to demonstrate the impact of local variation in cell density on the lineage commitment of human embryonic stem cells (hESCs) towards pancreatic endoderm (PE). Variations in the differentiation profile of cells across a culture well closely mirror variations in cell density introduced by seeding method-with the UCS correcting variations in differentiation efficiency. The UCS device provides a simple and reproducible method for uniform seeding across multiple culture systems.


Assuntos
Técnicas de Cultura de Células/instrumentação , Técnicas de Cultura de Células/métodos , Contagem de Células , Diferenciação Celular , Células Cultivadas , Células-Tronco Embrionárias/citologia , Células-Tronco Embrionárias/fisiologia , Endoderma/citologia , Endoderma/fisiologia , Humanos , Pâncreas/citologia , Pâncreas/embriologia , Pâncreas/fisiologia , Impressão Tridimensional , Melhoria de Qualidade
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