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1.
Development ; 148(17)2021 09 01.
Artigo em Inglês | MEDLINE | ID: mdl-34373891

RESUMO

The Krüppel-like factor (Klf) gene family encodes transcription factors that play an important role in the regulation of stem cell proliferation, cell differentiation and development in bilaterians. Although Klf genes have been shown to specify functionally various cell types in non-bilaterian animals, their role in early-diverging animal lineages has not been assessed. Thus, the ancestral activity of these transcription factors in animal development is not well understood. The ctenophore Mnemiopsis leidyi has emerged as an important non-bilaterian model system for understanding early animal evolution. Here, we characterize the expression and functional role of Klf genes during M. leidyi embryogenesis. Zygotic Klf gene function was assessed with both CRISPR/Cas9-mediated genome editing and splice-blocking morpholino oligonucleotide knockdown approaches. Abrogation of zygotic Klf expression during M. leidyi embryogenesis resulted in abnormal development of several organs, including the pharynx, tentacle bulbs and apical organ. Our data suggest an ancient role for Klf genes in regulating endodermal patterning, possibly through regulation of cell proliferation.


Assuntos
Ctenóforos/embriologia , Fatores de Transcrição Kruppel-Like/metabolismo , Animais , Padronização Corporal , Sistemas CRISPR-Cas , Ctenóforos/citologia , Ctenóforos/genética , Ctenóforos/metabolismo , Desenvolvimento Embrionário , Endoderma/citologia , Endoderma/embriologia , Edição de Genes , Expressão Gênica , Fatores de Transcrição Kruppel-Like/genética
2.
Cells ; 10(8)2021 08 08.
Artigo em Inglês | MEDLINE | ID: mdl-34440795

RESUMO

During liver organogenesis, cellular transcriptional profiles are constantly reshaped by the action of hepatic transcriptional regulators, including FoxA1-3, GATA4/6, HNF1α/ß, HNF4α, HNF6, OC-2, C/EBPα/ß, Hex, and Prox1. These factors are crucial for the activation of hepatic genes that, in the context of compact chromatin, cannot access their targets. The initial opening of highly condensed chromatin is executed by a special class of transcription factors known as pioneer factors. They bind and destabilize highly condensed chromatin and facilitate access to other "non-pioneer" factors. The association of target genes with pioneer and non-pioneer transcription factors takes place long before gene activation. In this way, the underlying gene regulatory regions are marked for future activation. The process is called "bookmarking", which confers transcriptional competence on target genes. Developmental bookmarking is accompanied by a dynamic maturation process, which prepares the genomic loci for stable and efficient transcription. Stable hepatic expression profiles are maintained during development and adulthood by the constant availability of the main regulators. This is achieved by a self-sustaining regulatory network that is established by complex cross-regulatory interactions between the major regulators. This network gradually grows during liver development and provides an epigenetic memory mechanism for safeguarding the optimal expression of the regulators.


Assuntos
Regulação da Expressão Gênica no Desenvolvimento , Fígado/metabolismo , Organogênese/genética , Fatores de Transcrição/genética , Animais , Diferenciação Celular/genética , Endoderma/citologia , Endoderma/embriologia , Endoderma/metabolismo , Hepatócitos/citologia , Hepatócitos/metabolismo , Humanos , Fígado/citologia , Fígado/embriologia , Células-Tronco/citologia , Células-Tronco/metabolismo , Fatores de Transcrição/metabolismo
3.
Development ; 148(19)2021 10 01.
Artigo em Inglês | MEDLINE | ID: mdl-34463740

RESUMO

Using scRNA-seq coupled with computational approaches, we studied transcriptional changes in cell states of sea urchin embryos during development to the larval stage. Eighteen closely spaced time points were taken during the first 24 h of development of Lytechinus variegatus (Lv). Developmental trajectories were constructed using Waddington-OT, a computational approach to 'stitch' together developmental time points. Skeletogenic and primordial germ cell trajectories diverged early in cleavage. Ectodermal progenitors were distinct from other lineages by the 6th cleavage, although a small percentage of ectoderm cells briefly co-expressed endoderm markers that indicated an early ecto-endoderm cell state, likely in cells originating from the equatorial region of the egg. Endomesoderm cells also originated at the 6th cleavage and this state persisted for more than two cleavages, then diverged into distinct endoderm and mesoderm fates asynchronously, with some cells retaining an intermediate specification status until gastrulation. Seventy-nine out of 80 genes (99%) examined, and included in published developmental gene regulatory networks (dGRNs), are present in the Lv-scRNA-seq dataset and are expressed in the correct lineages in which the dGRN circuits operate.


Assuntos
Genômica/métodos , Lytechinus/genética , RNA-Seq/métodos , Análise de Célula Única/métodos , Transcriptoma , Animais , Linhagem da Célula , Endoderma/citologia , Mesoderma/citologia
4.
PLoS One ; 16(7): e0254373, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34252142

RESUMO

Human induced pluripotent stem cells (hiPSCs) are considered a promising source of pancreatic ß-cells for the treatment of diabetes. However, this approach is limited by issues such as low efficiency and high cost. Here, we have developed a new protocol to induce insulin-producing cells. To reduce costs, we decreased the number of reagents and replaced protein reagents with chemical compounds. In this method, we increased induction efficiency with ascorbic acid (vitamin C) and an ALK5 inhibitor, RepSox. In 2D culture, the majority of cells were immature ß-cells with low glucose-stimulated insulin secretion. Transferring to 3D culture immediately after endocrine progenitor cell differentiation, however, improved glucose-stimulated insulin secretion. This simplified method will contribute to realizing transplantation therapy of ß-cells using iPSCs.


Assuntos
Ácido Ascórbico/farmacologia , Técnicas de Cultura de Células/métodos , Diferenciação Celular , Células-Tronco Pluripotentes Induzidas/citologia , Células Secretoras de Insulina/citologia , Pirazóis/farmacologia , Piridinas/farmacologia , Animais , Contagem de Células , Endoderma/citologia , Glucose/farmacologia , Humanos , Secreção de Insulina/efeitos dos fármacos , Camundongos
5.
PLoS One ; 16(7): e0254024, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34234366

RESUMO

During embryonic development, cells differentiate into a variety of distinct cell types and subtypes with diverse transcriptional profiles. To date, transcriptomic signatures of different cell lineages that arise during development have been only partially characterized. Here we used single-cell RNA-seq to perform transcriptomic analysis of over 20,000 cells disaggregated from the trunk region of zebrafish embryos at the 30 hpf stage. Transcriptional signatures of 27 different cell types and subtypes were identified and annotated during this analysis. This dataset will be a useful resource for many researchers in the fields of developmental and cellular biology and facilitate the understanding of molecular mechanisms that regulate cell lineage choices during development.


Assuntos
Embrião não Mamífero/metabolismo , Perfilação da Expressão Gênica , Análise de Célula Única , Tronco/embriologia , Peixe-Zebra/embriologia , Peixe-Zebra/genética , Animais , Linhagem da Célula/genética , Ectoderma/citologia , Ectoderma/embriologia , Endoderma/citologia , Endoderma/embriologia , Endotélio Vascular/citologia , Endotélio Vascular/embriologia , Eritrócitos/metabolismo , Fibroblastos/citologia , Regulação da Expressão Gênica no Desenvolvimento , Mesoderma/citologia , Mesoderma/embriologia , Músculo Esquelético/embriologia , Músculo Esquelético/metabolismo , Proteínas de Peixe-Zebra/genética , Proteínas de Peixe-Zebra/metabolismo
6.
PLoS One ; 16(7): e0254763, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34320001

RESUMO

Understanding the processes by which the mammalian embryo implants in the maternal uterus is a long-standing challenge in embryology. New insights into this morphogenetic event could be of great importance in helping, for example, to reduce human infertility. During implantation the blastocyst, composed of epiblast, trophectoderm and primitive endoderm, undergoes significant remodelling from an oval ball to an egg cylinder. A main feature of this transformation is symmetry breaking and reshaping of the epiblast into a "cup". Based on previous studies, we hypothesise that this event is the result of mechanical constraints originating from the trophectoderm, which is also significantly transformed during this process. In order to investigate this hypothesis we propose MG# (MechanoGenetic Sharp), an original computational model of biomechanics able to reproduce key cell shape changes and tissue level behaviours in silico. With this model, we simulate epiblast and trophectoderm morphogenesis during implantation. First, our results uphold experimental findings that repulsion at the apical surface of the epiblast is essential to drive lumenogenesis. Then, we provide new theoretical evidence that trophectoderm morphogenesis indeed can dictate the cup shape of the epiblast and fosters its movement towards the uterine tissue. Our results offer novel mechanical insights into mouse peri-implantation and highlight the usefulness of agent-based modelling methods in the study of embryogenesis.


Assuntos
Endoderma/citologia , Camadas Germinativas/citologia , Modelos Biológicos , Animais , Proliferação de Células , Implantação do Embrião , Embrião de Mamíferos/citologia , Embrião de Mamíferos/metabolismo , Desenvolvimento Embrionário , Endoderma/metabolismo , Camadas Germinativas/metabolismo , Camundongos
7.
Nat Commun ; 12(1): 3277, 2021 06 02.
Artigo em Inglês | MEDLINE | ID: mdl-34078907

RESUMO

Generating properly differentiated embryonic structures in vitro from pluripotent stem cells remains a challenge. Here we show that instruction of aggregates of mouse embryonic stem cells with an experimentally engineered morphogen signalling centre, that functions as an organizer, results in the development of embryo-like entities (embryoids). In situ hybridization, immunolabelling, cell tracking and transcriptomic analyses show that these embryoids form the three germ layers through a gastrulation process and that they exhibit a wide range of developmental structures, highly similar to neurula-stage mouse embryos. Embryoids are organized around an axial chordamesoderm, with a dorsal neural plate that displays histological properties similar to the murine embryo neuroepithelium and that folds into a neural tube patterned antero-posteriorly from the posterior midbrain to the tip of the tail. Lateral to the chordamesoderm, embryoids display somitic and intermediate mesoderm, with beating cardiac tissue anteriorly and formation of a vasculature network. Ventrally, embryoids differentiate a primitive gut tube, which is patterned both antero-posteriorly and dorso-ventrally. Altogether, embryoids provide an in vitro model of mammalian embryo that displays extensive development of germ layer derivatives and that promises to be a powerful tool for in vitro studies and disease modelling.


Assuntos
Padronização Corporal/genética , Corpos Embrioides/metabolismo , Desenvolvimento Embrionário/genética , Células-Tronco Embrionárias Murinas/metabolismo , Transdução de Sinais/genética , Animais , Ectoderma/citologia , Ectoderma/crescimento & desenvolvimento , Ectoderma/metabolismo , Embrião de Mamíferos , Corpos Embrioides/citologia , Endoderma/citologia , Endoderma/crescimento & desenvolvimento , Endoderma/metabolismo , Fator de Transcrição GATA6/genética , Fator de Transcrição GATA6/metabolismo , Gástrula/citologia , Gástrula/crescimento & desenvolvimento , Gástrula/metabolismo , Gastrulação/genética , Regulação da Expressão Gênica no Desenvolvimento , Proteínas HMGB/genética , Proteínas HMGB/metabolismo , Camundongos , Células-Tronco Embrionárias Murinas/citologia , Proteína Homeobox Nanog/genética , Proteína Homeobox Nanog/metabolismo , Tubo Neural/citologia , Tubo Neural/crescimento & desenvolvimento , Tubo Neural/metabolismo , Notocorda/citologia , Notocorda/crescimento & desenvolvimento , Notocorda/metabolismo , Fatores de Transcrição SOXF/genética , Fatores de Transcrição SOXF/metabolismo
8.
Proc Natl Acad Sci U S A ; 118(23)2021 06 08.
Artigo em Inglês | MEDLINE | ID: mdl-34083443

RESUMO

Markers for the endoderm and mesoderm germ layers are commonly expressed together in the early embryo, potentially reflecting cells' ability to explore potential fates before fully committing. It remains unclear when commitment to a single-germ layer is reached and how it is impacted by external signals. Here, we address this important question in Drosophila, a convenient model system in which mesodermal and endodermal fates are associated with distinct cellular movements during gastrulation. Systematically applying endoderm-inducing extracellular signal-regulated kinase (ERK) signals to the ventral medial embryo-which normally only receives a mesoderm-inducing cue-reveals a critical time window during which mesodermal cell movements and gene expression are suppressed by proendoderm signaling. We identify the ERK target gene huckebein (hkb) as the main cause of the ventral furrow suppression and use computational modeling to show that Hkb repression of the mesoderm-associated gene snail is sufficient to account for a broad range of transcriptional and morphogenetic effects. Our approach, pairing precise signaling perturbations with observation of transcriptional dynamics and cell movements, provides a general framework for dissecting the complexities of combinatorial tissue patterning.


Assuntos
Gástrula/metabolismo , Gastrulação/fisiologia , Sistema de Sinalização das MAP Quinases , Modelos Biológicos , Animais , Proteínas de Ligação a DNA/metabolismo , Proteínas de Drosophila/metabolismo , Drosophila melanogaster , Endoderma/citologia , Endoderma/embriologia , Gástrula/citologia , Mesoderma/citologia , Mesoderma/embriologia
9.
Nat Commun ; 12(1): 3780, 2021 06 18.
Artigo em Inglês | MEDLINE | ID: mdl-34145242

RESUMO

RNA N6-methyladenosine (m6A), the most abundant internal modification of mRNAs, plays key roles in human development and health. Post-translational methylation of proteins is often critical for the dynamic regulation of enzymatic activity. However, the role of methylation of the core methyltransferase METTL3/METTL14 in m6A regulation remains elusive. We find by mass spectrometry that METTL14 arginine 255 (R255) is methylated (R255me). Global mRNA m6A levels are greatly decreased in METTL14 R255K mutant mouse embryonic stem cells (mESCs). We further find that R255me greatly enhances the interaction of METTL3/METTL14 with WTAP and promotes the binding of the complex to substrate RNA. We show that protein arginine N-methyltransferases 1 (PRMT1) interacts with and methylates METTL14 at R255, and consistent with this, loss of PRMT1 reduces mRNA m6A modification globally. Lastly, we find that loss of R255me preferentially affects endoderm differentiation in mESCs. Collectively, our findings show that arginine methylation of METTL14 stabilizes the binding of the m6A methyltransferase complex to its substrate RNA, thereby promoting global m6A modification and mESC endoderm differentiation. This work highlights the crosstalk between protein methylation and RNA methylation in gene expression.


Assuntos
Adenosina/análogos & derivados , Arginina/metabolismo , Endoderma/citologia , Metiltransferases/metabolismo , Células-Tronco Embrionárias Murinas/citologia , Adenosina/genética , Adenosina/metabolismo , Animais , Diferenciação Celular/genética , Regulação da Expressão Gênica/genética , Células HEK293 , Células HeLa , Humanos , Metilação , Metiltransferases/genética , Camundongos , Processamento de Proteína Pós-Traducional/genética , Proteína-Arginina N-Metiltransferases/genética , Proteína-Arginina N-Metiltransferases/metabolismo , Interferência de RNA , RNA Interferente Pequeno/genética
10.
Commun Biol ; 4(1): 788, 2021 06 25.
Artigo em Inglês | MEDLINE | ID: mdl-34172827

RESUMO

Successful specification of the two mouse blastocyst inner cell mass (ICM) lineages (the primitive endoderm (PrE) and epiblast) is a prerequisite for continued development and requires active fibroblast growth factor 4 (FGF4) signaling. Previously, we identified a role for p38 mitogen-activated protein kinases (p38-MAPKs) during PrE differentiation, but the underlying mechanisms have remained unresolved. Here, we report an early blastocyst window of p38-MAPK activity that is required to regulate ribosome-related gene expression, rRNA precursor processing, polysome formation and protein translation. We show that p38-MAPK inhibition-induced PrE phenotypes can be partially rescued by activating the translational regulator mTOR. However, similar PrE phenotypes associated with extracellular signal-regulated kinase (ERK) pathway inhibition targeting active FGF4 signaling are not affected by mTOR activation. These data indicate a specific role for p38-MAPKs in providing a permissive translational environment during mouse blastocyst PrE differentiation that is distinct from classically reported FGF4-based mechanisms.


Assuntos
Blastocisto/fisiologia , Endoderma/citologia , Biossíntese de Proteínas , Proteínas Quinases p38 Ativadas por Mitógeno/fisiologia , Animais , Diferenciação Celular , Linhagem da Célula , Proteínas de Ligação a DNA/fisiologia , Desenvolvimento Embrionário , Camundongos , Proteínas de Ligação a RNA/fisiologia , Serina-Treonina Quinases TOR/fisiologia , Fatores de Transcrição/fisiologia , Proteínas Quinases p38 Ativadas por Mitógeno/antagonistas & inibidores
11.
Nat Cell Biol ; 23(7): 692-703, 2021 07.
Artigo em Inglês | MEDLINE | ID: mdl-34168324

RESUMO

It is generally accepted that epiblast cells ingress into the primitive streak by epithelial-to-mesenchymal transition (EMT) to give rise to the mesoderm; however, it is less clear how the endoderm acquires an epithelial fate. Here, we used embryonic stem cell and mouse embryo knock-in reporter systems to combine time-resolved lineage labelling with high-resolution single-cell transcriptomics. This allowed us to resolve the morphogenetic programs that segregate the mesoderm from the endoderm germ layer. Strikingly, while the mesoderm is formed by classical EMT, the endoderm is formed independent of the key EMT transcription factor Snail1 by mechanisms of epithelial cell plasticity. Importantly, forkhead box transcription factor A2 (Foxa2) acts as an epithelial gatekeeper and EMT suppressor to shield the endoderm from undergoing a mesenchymal transition. Altogether, these results not only establish the morphogenetic details of germ layer formation, but also have broader implications for stem cell differentiation and cancer metastasis.


Assuntos
Blastocisto/fisiologia , Plasticidade Celular , Endoderma/fisiologia , Células Epiteliais/fisiologia , Transição Epitelial-Mesenquimal , Gastrulação , Células-Tronco Embrionárias Murinas/fisiologia , Animais , Blastocisto/citologia , Blastocisto/metabolismo , Diferenciação Celular , Linhagem Celular , Endoderma/citologia , Endoderma/metabolismo , Células Epiteliais/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Idade Gestacional , Fator 3-beta Nuclear de Hepatócito/genética , Fator 3-beta Nuclear de Hepatócito/metabolismo , Camundongos , Camundongos Transgênicos , Células-Tronco Embrionárias Murinas/metabolismo , Fenótipo , Fatores de Transcrição da Família Snail/genética , Fatores de Transcrição da Família Snail/metabolismo , Fatores de Tempo
12.
J Vis Exp ; (170)2021 04 12.
Artigo em Inglês | MEDLINE | ID: mdl-33900299

RESUMO

Human lung development and disease has been difficult to study due to the lack of biologically relevant in vitro model systems. Human induced pluripotent stem cells (hiPSCs) can be differentiated stepwise into 3D multicellular lung organoids, made of both epithelial and mesenchymal cell populations. We recapitulate embryonic developmental cues by temporally introducing a variety of growth factors and small molecules to efficiently generate definitive endoderm, anterior foregut endoderm, and subsequently lung progenitor cells. These cells are then embedded in growth factor reduced (GFR)-basement membrane matrix medium, allowing them to spontaneously develop into 3D lung organoids in response to external growth factors. These whole lung organoids (WLO) undergo early lung developmental stages including branching morphogenesis and maturation after exposure to dexamethasone, cyclic AMP and isobutylxanthine. WLOs possess airway epithelial cells expressing the markers KRT5 (basal), SCGB3A2 (club) and MUC5AC (goblet) as well as alveolar epithelial cells expressing HOPX (alveolar type I) and SP-C (alveolar type II). Mesenchymal cells are also present, including smooth muscle actin (SMA), and platelet-derived growth factor receptor A (PDGFRα). iPSC derived WLOs can be maintained in 3D culture conditions for many months and can be sorted for surface markers to purify a specific cell population. iPSC derived WLOs can also be utilized to study human lung development, including signaling between the lung epithelium and mesenchyme, to model genetic mutations on human lung cell function and development, and to determine the cytotoxicity of infective agents.


Assuntos
Células Epiteliais Alveolares/citologia , Endoderma/citologia , Células-Tronco Pluripotentes Induzidas/citologia , Pulmão/citologia , Organogênese , Organoides/citologia , Engenharia Tecidual/métodos , Células Epiteliais Alveolares/metabolismo , Técnicas de Cultura de Células , Diferenciação Celular , Biologia do Desenvolvimento , Endoderma/metabolismo , Humanos , Células-Tronco Pluripotentes Induzidas/metabolismo , Pulmão/metabolismo , Organoides/metabolismo
13.
Cells ; 10(4)2021 04 06.
Artigo em Inglês | MEDLINE | ID: mdl-33917333

RESUMO

The endoderm, differentiated from human induced pluripotent stem cells (iPSCs), can differentiate into the small intestine and liver, which are vital for drug absorption and metabolism. The development of human iPSC-derived enterocytes (HiEnts) and hepatocytes (HiHeps) has been reported. However, pharmacokinetic function-deficiency of these cells remains to be elucidated. Here, we aimed to develop an efficient differentiation method to induce endoderm formation from human iPSCs. Cells treated with activin A for 168 h expressed higher levels of endodermal genes than those treated for 72 h. Using activin A (days 0-7), CHIR99021 and PI-103 (days 0-2), and FGF2 (days 3-7), the hiPSC-derived endoderm (HiEnd) showed 97.97% CD-117 and CD-184 double-positive cells. Moreover, HiEnts derived from the human iPSC line Windy had similar or higher expression of small intestine-specific genes than adult human small intestine. Activities of the drug transporter P-glycoprotein and drug-metabolizing enzyme cytochrome P450 (CYP) 3A4/5 were confirmed. Additionally, Windy-derived HiHeps expressed higher levels of hepatocyte- and pharmacokinetics-related genes and proteins and showed higher CYP3A4/5 activity than those derived through the conventional differentiation method. Thus, using this novel method, the differentiated HiEnts and HiHeps with pharmacokinetic functions could be used for drug development.


Assuntos
Técnicas de Cultura de Células/métodos , Diferenciação Celular , Endoderma/citologia , Enterócitos/citologia , Hepatócitos/citologia , Células-Tronco Pluripotentes Induzidas/citologia , Ativinas/farmacologia , Proteína Morfogenética Óssea 4/farmacologia , Diferenciação Celular/efeitos dos fármacos , Linhagem Celular , Células Cultivadas , Dimetil Sulfóxido/farmacologia , Enterócitos/efeitos dos fármacos , Células Alimentadoras/citologia , Fator 2 de Crescimento de Fibroblastos/farmacologia , Furanos/farmacologia , Hepatócitos/efeitos dos fármacos , Humanos , Células-Tronco Pluripotentes Induzidas/efeitos dos fármacos , Intestino Delgado/citologia , Linha Primitiva/citologia , Piridinas/farmacologia , Pirimidinas/farmacologia , Reprodutibilidade dos Testes
14.
Cells ; 10(4)2021 03 30.
Artigo em Inglês | MEDLINE | ID: mdl-33808472

RESUMO

The cells present in the stromal compartment of many tissues are a heterogeneous population containing stem cells, progenitor cells, fibroblasts, and other stromal cells. A SSEA3(+) cell subpopulation isolated from human stromal compartments showed stem cell properties. These cells, known as multilineage-differentiating stress-enduring (MUSE) cells, are capable of resisting stress and possess an excellent ability to repair DNA damage. We isolated MUSE cells from different mouse stromal compartments, such as those present in bone marrow, subcutaneous white adipose tissue, and ear connective tissue. These cells showed overlapping in vitro biological properties. The mouse MUSE cells were positive for stemness markers such as SOX2, OCT3/4, and NANOG. They also expressed TERT, the catalytic telomerase subunit. The mouse MUSE cells showed spontaneous commitment to differentiation in meso/ecto/endodermal derivatives. The demonstration that multilineage stem cells can be isolated from an animal model, such as the mouse, could offer a valid alternative to the use of other stem cells for disease studies and envisage of cellular therapies.


Assuntos
Tecido Adiposo/citologia , Células da Medula Óssea/citologia , Compartimento Celular , Separação Celular , Células do Tecido Conjuntivo/citologia , Orelha/anatomia & histologia , Células-Tronco/citologia , Animais , Biomarcadores/metabolismo , Ciclo Celular , Diferenciação Celular , Ectoderma/citologia , Endoderma/citologia , Mesoderma/citologia , Camundongos Endogâmicos C57BL , Células Estromais/citologia
15.
Development ; 148(6)2021 03 29.
Artigo em Inglês | MEDLINE | ID: mdl-33782045

RESUMO

The esophagus is derived from the anterior portion of the foregut endoderm, which also gives rise to the respiratory system. As it develops, the esophageal lining is transformed from a simple columnar epithelium into a stratified squamous cell layer, accompanied by the replacement of unspecified mesenchyme with layers of muscle cells. Studies in animal models have provided significant insights into the roles of various signaling pathways in esophageal development. More recent studies using human pluripotent stem cells (hPSCs) further demonstrate that some of these signaling pathways are conserved in human esophageal development. In addition, a combination of mouse genetics and hPSC differentiation approaches have uncovered new players that control esophageal morphogenesis. In this Review, we summarize these new findings and discuss how the esophagus is established and matures throughout different stages, including its initial specification, respiratory-esophageal separation, epithelial morphogenesis and maintenance. We also discuss esophageal muscular development and enteric nervous system innervation, which are essential for esophageal structure and function.


Assuntos
Esôfago/citologia , Células-Tronco Pluripotentes/metabolismo , Animais , Diferenciação Celular , Endoderma/citologia , Endoderma/metabolismo , Sistema Nervoso Entérico/citologia , Sistema Nervoso Entérico/crescimento & desenvolvimento , Sistema Nervoso Entérico/metabolismo , Esôfago/metabolismo , Proteínas Hedgehog/metabolismo , Humanos , Células-Tronco Pluripotentes/citologia , Transdução de Sinais , Fatores de Transcrição/antagonistas & inibidores , Fatores de Transcrição/metabolismo
16.
Nat Protoc ; 16(4): 1802-1829, 2021 04.
Artigo em Inglês | MEDLINE | ID: mdl-33649566

RESUMO

Lung and airway epithelial cells generated in vitro from human pluripotent stem cells (hPSCs) have applications in regenerative medicine, modeling of lung disease, drug screening and studies of human lung development. Here, we describe a strategy for directed differentiation of hPSCs into mature lung and airway epithelial cells obtained through maturation of NKX2.1+ hPSC-derived lung progenitors in a 3D matrix of collagen I in the absence of glycogen synthase kinase 3 inhibition. This protocol is an extension of our previously published protocol on the directed differentiation of lung and airway epithelium from hPSCs that modifies the technique and offers additional applications. This protocol is conducted in defined media conditions, has a duration of 50-80 d, does not require reporter lines and results in cultures containing mature alveolar type II and I cells as well as airway basal, ciliated, club and neuroendocrine cells. We also present a flow cytometry strategy to assess maturation in the cultures. Several of these populations, including mature NGFR+ basal cells, can be prospectively isolated by cell sorting and expanded for further investigation.


Assuntos
Técnicas de Cultura de Células/métodos , Diferenciação Celular , Linhagem da Célula , Imageamento Tridimensional , Pulmão/citologia , Células-Tronco Pluripotentes/citologia , Animais , Biomarcadores/metabolismo , Células Cultivadas , Endoderma/citologia , Células-Tronco Embrionárias Humanas/citologia , Humanos , Camundongos , Vírus da Parainfluenza 3 Humana/fisiologia
17.
Sci Rep ; 11(1): 6137, 2021 03 17.
Artigo em Inglês | MEDLINE | ID: mdl-33731744

RESUMO

Low differentiation propensity towards a targeted lineage can significantly hamper the utility of individual human pluripotent stem cell (hPSC) lines in biomedical applications. Here, we use monolayer and micropatterned cell cultures, as well as transcriptomic profiling, to investigate how variability in signalling pathway activity between human embryonic stem cell lines affects their differentiation efficiency towards definitive endoderm (DE). We show that endogenous suppression of WNT signalling in hPSCs at the onset of differentiation prevents the switch from self-renewal to DE specification. Gene expression profiling reveals that this inefficient switch is reflected in NANOG expression dynamics. Importantly, we demonstrate that higher WNT stimulation or inhibition of the PI3K/AKT signalling can overcome the DE commitment blockage. Our findings highlight that redirection of the activity of Activin/NODAL pathway by WNT signalling towards mediating DE fate specification is a vulnerable spot, as disruption of this process can result in poor hPSC specification towards DE.


Assuntos
Endoderma , Regulação da Expressão Gênica no Desenvolvimento , Células-Tronco Embrionárias Humanas , Via de Sinalização Wnt , Diferenciação Celular , Linhagem Celular , Endoderma/citologia , Endoderma/metabolismo , Células-Tronco Embrionárias Humanas/citologia , Células-Tronco Embrionárias Humanas/metabolismo , Humanos
18.
Biochem Biophys Res Commun ; 552: 142-149, 2021 05 07.
Artigo em Inglês | MEDLINE | ID: mdl-33744762

RESUMO

Fully understanding the regulatory network under the pluripotency of embryonic stem cells (ESC) is a prerequisite for their safe application. Here, we addressed the characteristics of metastasis-associated (MTA) family members in human ESCs and found that knockdown of the expression of MTA2 and MTA3, but not MTA1, would induce differentiation. High-throughput sequence and quantitative real-time PCR showed that the decreased MTA2 or MTA3 gene transcript mainly led to the emergence of mesendoderm associated markers. Finally, based on the chemical small molecule library screening, we observed that addition of ID8, a specific inhibitor of the dual-specificity tyrosine phosphorylation-regulated kinases (DYRKs), was able to impair the differentiation phenotype induced by MTA2 and MTA3 reduction. Functional assay showed that ID8 could mediate differentiation caused by MTA2 or MTA3 knockdown mainly through inhibition of DYRK4 activity. Therefore, our finding provides the evidence that the functions of MTA family genes in human ESCs are different. Revealing the function of MTA in ESCs with different pluripotency states will help us better understand and apply stem cells.


Assuntos
Diferenciação Celular/genética , Endoderma/metabolismo , Histona Desacetilases/genética , Células-Tronco Embrionárias Humanas/metabolismo , Mesoderma/metabolismo , Proteínas de Neoplasias/genética , Proteínas Repressoras/genética , Animais , Linhagem Celular , Endoderma/citologia , Inibidores Enzimáticos/farmacologia , Perfilação da Expressão Gênica/métodos , Regulação da Expressão Gênica , Histona Desacetilases/metabolismo , Células-Tronco Embrionárias Humanas/citologia , Humanos , Mesoderma/citologia , Proteínas de Neoplasias/antagonistas & inibidores , Proteínas de Neoplasias/metabolismo , /genética , Proteínas Tirosina Quinases/antagonistas & inibidores , Proteínas Tirosina Quinases/genética , Proteínas Tirosina Quinases/metabolismo , Interferência de RNA , Proteínas Repressoras/antagonistas & inibidores , Proteínas Repressoras/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa
19.
J Biosci ; 462021.
Artigo em Inglês | MEDLINE | ID: mdl-33737502

RESUMO

Autophagy is a vacuolar pathway for the regulated degradation and recycling of cellular components. Beclin1, a Bcl2-interacting protein, is a well-studied autophagy regulator. Homozygous loss of Beclin1 in mice leads to early embryonic lethality. However, the role of Beclin1 in regulating the pluripotency of embryonic stem cells and their differentiation remains poorly explored. To study this, we generated Beclin1-Knockout (KO) mouse embryonic stem cells (mESCs) using the CRISPR-Cas9 genome-editing tool. Interestingly, Beclin1-KO mESCs did not show any change in the expression of pluripotency marker genes. Beclin1-KO mESCs also displayed active autophagy, suggesting the presence of Beclin1-independent autophagy in mESCs. However, loss of Beclin1 resulted in compromised differentiation of mESCs in vitro and in vivo due to misregulated expression of transcription factors. Our results suggest that Beclin1 may play an autophagy-independent role in regulating the differentiation of mESCs.


Assuntos
Proteína Beclina-1/fisiologia , Células-Tronco Embrionárias Murinas/citologia , Células-Tronco Embrionárias Murinas/fisiologia , Animais , Autofagia/genética , Proteína Beclina-1/genética , Sistemas CRISPR-Cas , Diferenciação Celular/genética , Endoderma/citologia , Mesoderma/citologia , Camundongos , Camundongos Knockout , Camundongos SCID
20.
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
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