RESUMO
BACKGROUND: Transcriptional reconfiguration is central to heart failure, the most common cause of which is dilated cardiomyopathy (DCM). The effect of 3-dimensional chromatin topology on transcriptional dysregulation and pathogenesis in human DCM remains elusive. METHODS: We generated a compendium of 3-dimensional epigenome and transcriptome maps from 101 biobanked human DCM and nonfailing heart tissues through highly integrative chromatin immunoprecipitation (H3K27ac [acetylation of lysine 27 on histone H3]), in situ high-throughput chromosome conformation capture, chromatin immunoprecipitation sequencing, assay for transposase-accessible chromatin using sequencing, and RNA sequencing. We used human induced pluripotent stem cell-derived cardiomyocytes and mouse models to interrogate the key transcription factor implicated in 3-dimensional chromatin organization and transcriptional regulation in DCM pathogenesis. RESULTS: We discovered that the active regulatory elements (H3K27ac peaks) and their connectome (H3K27ac loops) were extensively reprogrammed in DCM hearts and contributed to transcriptional dysregulation implicated in DCM development. For example, we identified that nontranscribing NPPA-AS1 (natriuretic peptide A antisense RNA 1) promoter functions as an enhancer and physically interacts with the NPPA (natriuretic peptide A) and NPPB (natriuretic peptide B) promoters, leading to the cotranscription of NPPA and NPPB in DCM hearts. We revealed that DCM-enriched H3K27ac loops largely resided in conserved high-order chromatin architectures (compartments, topologically associating domains) and their anchors unexpectedly had equivalent chromatin accessibility. We discovered that the DCM-enriched H3K27ac loop anchors exhibited a strong enrichment for HAND1 (heart and neural crest derivatives expressed 1), a key transcription factor involved in early cardiogenesis. In line with this, its protein expression was upregulated in human DCM and mouse failing hearts. To further validate whether HAND1 is a causal driver for the reprogramming of enhancer-promoter connectome in DCM hearts, we performed comprehensive 3-dimensional epigenome mappings in human induced pluripotent stem cell-derived cardiomyocytes. We found that forced overexpression of HAND1 in human induced pluripotent stem cell-derived cardiomyocytes induced a distinct gain of enhancer-promoter connectivity and correspondingly increased the expression of their connected genes implicated in DCM pathogenesis, thus recapitulating the transcriptional signature in human DCM hearts. Electrophysiology analysis demonstrated that forced overexpression of HAND1 in human induced pluripotent stem cell-derived cardiomyocytes induced abnormal calcium handling. Furthermore, cardiomyocyte-specific overexpression of Hand1 in the mouse hearts resulted in dilated cardiac remodeling with impaired contractility/Ca2+ handling in cardiomyocytes, increased ratio of heart weight/body weight, and compromised cardiac function, which were ascribed to recapitulation of transcriptional reprogramming in DCM. CONCLUSIONS: This study provided novel chromatin topology insights into DCM pathogenesis and illustrated a model whereby a single transcription factor (HAND1) reprograms the genome-wide enhancer-promoter connectome to drive DCM pathogenesis.
Assuntos
Cardiomiopatia Dilatada , Células-Tronco Pluripotentes Induzidas , Animais , Cardiomiopatia Dilatada/metabolismo , Cromatina/genética , Cromatina/metabolismo , Histonas/metabolismo , Humanos , Células-Tronco Pluripotentes Induzidas/metabolismo , Camundongos , Fatores de Transcrição/genéticaRESUMO
Stem cells can self-renew and differentiate into multiple cell types. These characteristics are maintained by the combination of specific signaling pathways and transcription factors that cooperate to establish a unique epigenetic state. Despite the broad interest of these mechanisms, the precise molecular controls by which extracellular signals organize epigenetic marks to confer multipotency remain to be uncovered. Here, we use human embryonic stem cells (hESCs) to show that the Activin-SMAD2/3 signaling pathway cooperates with the core pluripotency factor NANOG to recruit the DPY30-COMPASS histone modifiers onto key developmental genes. Functional studies demonstrate the importance of these interactions for correct histone 3 Lys4 trimethylation and also self-renewal and differentiation. Finally, genetic studies in mice show that Dpy30 is also necessary to maintain pluripotency in the pregastrulation embryo, thereby confirming the existence of similar regulations in vivo during early embryonic development. Our results reveal the mechanisms by which extracellular factors coordinate chromatin status and cell fate decisions in hESCs.
Assuntos
Ativinas/metabolismo , Diferenciação Celular/genética , Cromatina/genética , Histonas/genética , Proteínas de Homeodomínio/metabolismo , Proteína Nodal/metabolismo , Transdução de Sinais , Animais , Células Cultivadas , Cromatina/metabolismo , Embrião de Mamíferos , Células-Tronco Embrionárias , Epigênese Genética/genética , Histona Metiltransferases , Histona-Lisina N-Metiltransferase/metabolismo , Humanos , Camundongos , Proteína Homeobox Nanog , Proteína Smad2/metabolismo , Proteína Smad3/metabolismoRESUMO
The epicardium has emerged as a multipotent cardiovascular progenitor source with therapeutic potential for coronary smooth muscle cell, cardiac fibroblast (CF) and cardiomyocyte regeneration, owing to its fundamental role in heart development and its potential ability to initiate myocardial repair in injured adult tissues. Here, we describe a chemically defined method for generating epicardium and epicardium-derived smooth muscle cells (EPI-SMCs) and CFs from human pluripotent stem cells (HPSCs) through an intermediate lateral plate mesoderm (LM) stage. HPSCs were initially differentiated to LM in the presence of FGF2 and high levels of BMP4. The LM was robustly differentiated to an epicardial lineage by activation of WNT, BMP and retinoic acid signalling pathways. HPSC-derived epicardium displayed enhanced expression of epithelial- and epicardium-specific markers, exhibited morphological features comparable with human foetal epicardial explants and engrafted in the subepicardial space in vivo. The in vitro-derived epicardial cells underwent an epithelial-to-mesenchymal transition when treated with PDGF-BB and TGFß1, resulting in vascular SMCs that displayed contractile ability in response to vasoconstrictors. Furthermore, the EPI-SMCs displayed low density lipoprotein uptake and effective lowering of lipoprotein levels upon treatment with statins, similar to primary human coronary artery SMCs. Cumulatively, these findings suggest that HPSC-derived epicardium and EPI-SMCs could serve as important tools for studying human cardiogenesis, and as a platform for vascular disease modelling and drug screening.
Assuntos
Pericárdio/citologia , Pericárdio/metabolismo , Células-Tronco Pluripotentes/citologia , Western Blotting , Diferenciação Celular/genética , Diferenciação Celular/fisiologia , Células Cultivadas , Citometria de Fluxo , Humanos , Imuno-Histoquímica , Músculo Liso Vascular/citologia , Músculo Liso Vascular/metabolismo , Músculo Liso Vascular/fisiologia , Miócitos de Músculo Liso/citologia , Miócitos de Músculo Liso/metabolismo , Miócitos de Músculo Liso/fisiologia , Células-Tronco Pluripotentes/metabolismo , Células-Tronco Pluripotentes/fisiologia , Reação em Cadeia da Polimerase em Tempo RealRESUMO
The transcription factor brachyury (T, BRA) is one of the first markers of gastrulation and lineage specification in vertebrates. Despite its wide use and importance in stem cell and developmental biology, its functional genomic targets in human cells are largely unknown. Here, we use differentiating human embryonic stem cells to study the role of BRA in activin A-induced endoderm and BMP4-induced mesoderm progenitors. We show that BRA has distinct genome-wide binding landscapes in these two cell populations, and that BRA interacts and collaborates with SMAD1 or SMAD2/3 signalling to regulate the expression of its target genes in a cell-specific manner. Importantly, by manipulating the levels of BRA in cells exposed to different signalling environments, we demonstrate that BRA is essential for mesoderm but not for endoderm formation. Together, our data illuminate the function of BRA in the context of human embryonic development and show that the regulatory role of BRA is context dependent. Our study reinforces the importance of analysing the functions of a transcription factor in different cellular and signalling environments.
Assuntos
Células-Tronco Embrionárias/citologia , Proteínas Fetais/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Neurogênese/fisiologia , Proteína Smad1/metabolismo , Proteínas com Domínio T/metabolismo , Animais , Proteína Morfogenética Óssea 4/metabolismo , Linhagem Celular , Células-Tronco Embrionárias/metabolismo , Endoderma/citologia , Gastrulação/fisiologia , Humanos , Mesoderma/citologia , Camundongos , Camundongos Transgênicos , Proteína Smad2/metabolismo , Proteína Smad3/metabolismoRESUMO
Genetic defects in bone morphogenetic protein type II receptor (BMPRII) signalling and inflammation contribute to the pathogenesis of pulmonary arterial hypertension (PAH). The receptor is activated by bone morphogenetic protein (BMP) ligands, which also enhance BMPR2 transcription. A small-molecule BMP upregulator with selectivity on vascular endothelium would be a desirable therapeutic intervention for PAH.We assayed compounds identified in the screening of BMP2 upregulators for their ability to increase the expression of inhibitor of DNA binding 1 (Id1), using a dual reporter driven specifically in human embryonic stem cell-derived endothelial cells. These assays identified a novel piperidine, BMP upregulator 1 (BUR1), that increased endothelial Id1 expression with a half-maximal effective concentration of 0.098â µmol·L-1 Microarray analyses and immunoblotting showed that BUR1 induced BMP2 and prostaglandin-endoperoxide synthase 2 (PTGS2) expression. BUR1 effectively rescued deficient angiogenesis in autologous BMPR2+/R899X endothelial cells generated by CRISPR/Cas9 and patient cells.BUR1 prevented and reversed PAH in monocrotaline rats, and restored BMPRII downstream signalling and modulated the arachidonic acid pathway in the pulmonary arterial endothelium in the Sugen 5416/hypoxia PAH mouse model.In conclusion, using stem cell technology we have provided a novel small-molecule compound which regulates BMP2 and PTGS2 levels that might be useful for the treatment of PAH.
Assuntos
Proteína Morfogenética Óssea 2/metabolismo , Ciclo-Oxigenase 2/metabolismo , Células Endoteliais/efeitos dos fármacos , Hipertensão Pulmonar/tratamento farmacológico , Hipertensão Pulmonar/metabolismo , Piperidinas/farmacologia , Animais , Linhagem Celular , Proliferação de Células , Dinoprostona/sangue , Modelos Animais de Doenças , Endotélio Vascular/efeitos dos fármacos , Endotélio Vascular/metabolismo , Humanos , Leucotrieno B4/sangue , Artéria Pulmonar/efeitos dos fármacos , Ratos , Transdução de Sinais/efeitos dos fármacos , Células-Tronco/citologiaRESUMO
Understanding human pre-implantation development has important implications for assisted reproductive technology (ART) and for human embryonic stem cell (hESC)-based therapies. Owing to limited resources, the cellular and molecular mechanisms governing this early stage of human development are poorly understood. Nonetheless, recent advances in non-invasive imaging techniques and molecular and genomic technologies have helped to increase our understanding of this fascinating stage of human development. Here, we summarize what is currently known about human pre-implantation embryo development and highlight how further studies of human pre-implantation embryos can be used to improve ART and to fully harness the potential of hESCs for therapeutic goals.
Assuntos
Terapia Baseada em Transplante de Células e Tecidos/métodos , Desenvolvimento Embrionário , Técnicas de Reprodução Assistida , Aneuploidia , Animais , Blastocisto/citologia , Blastocisto/fisiologia , Embrião de Mamíferos/anatomia & histologia , Embrião de Mamíferos/fisiologia , Células-Tronco Embrionárias/fisiologia , Redes Reguladoras de Genes , Humanos , Peptídeos e Proteínas de Sinalização Intercelular/metabolismoRESUMO
The inner cell mass of the mouse pre-implantation blastocyst comprises epiblast progenitor and primitive endoderm cells of which cognate embryonic (mESCs) or extra-embryonic (XEN) stem cell lines can be derived. Importantly, each stem cell type retains the defining properties and lineage restriction of their in vivo tissue of origin. Recently, we demonstrated that XEN-like cells arise within mESC cultures. This raises the possibility that mESCs can generate self-renewing XEN cells without the requirement for gene manipulation. We have developed a novel approach to convert mESCs to XEN cells (cXEN) using growth factors. We confirm that the downregulation of the pluripotency transcription factor Nanog and the expression of primitive endoderm-associated genes Gata6, Gata4, Sox17 and Pdgfra are necessary for cXEN cell derivation. This approach highlights an important function for Fgf4 in cXEN cell derivation. Paracrine FGF signalling compensates for the loss of endogenous Fgf4, which is necessary to exit mESC self-renewal, but not for XEN cell maintenance. Our cXEN protocol also reveals that distinct pluripotent stem cells respond uniquely to differentiation promoting signals. cXEN cells can be derived from mESCs cultured with Erk and Gsk3 inhibitors (2i), and LIF, similar to conventional mESCs. However, we find that epiblast stem cells (EpiSCs) derived from the post-implantation embryo are refractory to cXEN cell establishment, consistent with the hypothesis that EpiSCs represent a pluripotent state distinct from mESCs. In all, these findings suggest that the potential of mESCs includes the capacity to give rise to both extra-embryonic and embryonic lineages.
Assuntos
Células-Tronco Embrionárias/citologia , Endoderma/citologia , Endoderma/embriologia , Células-Tronco Pluripotentes/citologia , Ativinas/administração & dosagem , Animais , Proteína Morfogenética Óssea 4/farmacologia , Diferenciação Celular/efeitos dos fármacos , Linhagem da Célula , Células Cultivadas , Células-Tronco Embrionárias/efeitos dos fármacos , Células-Tronco Embrionárias/metabolismo , Endoderma/metabolismo , Fator 4 de Crescimento de Fibroblastos/deficiência , Fator 4 de Crescimento de Fibroblastos/genética , Fator 4 de Crescimento de Fibroblastos/metabolismo , Fator de Transcrição GATA4/genética , Fator de Transcrição GATA6/genética , Regulação da Expressão Gênica no Desenvolvimento , Proteínas HMGB/genética , Camundongos , Camundongos Knockout , Camundongos Transgênicos , Modelos Biológicos , Comunicação Parácrina , Células-Tronco Pluripotentes/efeitos dos fármacos , Células-Tronco Pluripotentes/metabolismo , Receptor alfa de Fator de Crescimento Derivado de Plaquetas/genética , Fatores de Transcrição SOXF/genética , Tretinoína/administração & dosagemRESUMO
Recent progress in deriving human embryonic stem (hES) cells and defining their capacity to differentiate has inspired hope that they could become a source of replacement cells for damaged or diseased tissues. We review the immunological barriers to transplanting hES cells and consider several potential solutions, including stem-cell banking, modification of the immunogenicity of donor cells and induction of tolerance to the graft. We evaluate the probable efficacy of these approaches with a view to facilitating the use of hES cells in clinical practice.
Assuntos
Sistema Imunitário/fisiologia , Transplante de Células-Tronco , Células-Tronco/imunologia , Sistema ABO de Grupos Sanguíneos/imunologia , Animais , Incompatibilidade de Grupos Sanguíneos , Quimera/imunologia , Histocompatibilidade , Teste de Histocompatibilidade , Humanos , Tolerância Imunológica , Imunossupressores/uso terapêuticoRESUMO
The Hairy-related transcription factor family of Notch- and ALK1-downstream transcriptional repressors, called Hrt/Hey/Hesr/Chf/Herp/Gridlock, has complementary and indispensable functions for vascular development. While mouse embryos null for either Hrt1/Hey1 or Hrt2/Hey2 did not show early vascular phenotypes, Hrt1/Hey1; Hrt2/Hey2 double null mice (H1(ko) /H2(ko) ) showed embryonic lethality with severe impairment of vascular morphogenesis. It remained unclear, however, whether Hrt/Hey functions are required in endothelial cells or vascular smooth muscle cells. In this study, we demonstrate that mice with endothelial-specific deletion of Hrt2/Hey2 combined with global Hrt1/Hey1 deletion (H1(ko) /H2(eko) ) show abnormal vascular morphogenesis and embryonic lethality. Their defects were characterized by the failure of vascular network formation in the yolk sac, abnormalities of embryonic vascular structures and impaired smooth muscle cell recruitment, and were virtually identical to the H1(ko) /H2(ko) phenotypes. Among signaling molecules implicated in vascular development, Robo4 expression was significantly increased and activation of Src family kinases was suppressed in endothelial cells of H1(ko) /H2(eko) embryos. The present study indicates an important role of Hrt1/Hey1 and Hrt2/Hey2 in endothelial cells during early vascular development, and further suggests involvement of Robo4 and Src family kinases in the mechanisms of embryonic vascular defects caused by the Hrt/Hey deficiency.
Assuntos
Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Sistema Cardiovascular/embriologia , Regulação da Expressão Gênica no Desenvolvimento/fisiologia , Morfogênese/fisiologia , Proteínas Repressoras/metabolismo , Transdução de Sinais/genética , Animais , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Western Blotting , Endotélio/metabolismo , Deleção de Genes , Regulação da Expressão Gênica no Desenvolvimento/genética , Imuno-Histoquímica , Hibridização In Situ , Camundongos , Morfogênese/genética , Proteínas do Tecido Nervoso/metabolismo , Receptores de Superfície Celular , Receptores Imunológicos/metabolismo , Proteínas Repressoras/genética , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
It is imperative to unveil the full range of differentiated cell types into which human pluripotent stem cells (hPSCs) can develop. The need is twofold: it will delimit the therapeutic utility of these stem cells and is necessary to place their position accurately in the developmental hierarchy of lineage potential. Accumulated evidence suggested that hPSC could develop in vitro into an extraembryonic lineage (trophoblast (TB)) that is typically inaccessible to pluripotent embryonic cells during embryogenesis. However, whether these differentiated cells are truly authentic TB has been challenged. In this debate, we present a case for and a case against TB differentiation from hPSCs. By analogy to other differentiation systems, our debate is broadly applicable, as it articulates higher and more challenging standards for judging whether a given cell type has been genuinely produced from hPSC differentiation.
Assuntos
Diferenciação Celular/fisiologia , Células-Tronco Embrionárias/citologia , Trofoblastos/citologia , Linhagem da Célula , Células Cultivadas , Células-Tronco Embrionárias/fisiologia , Feminino , Humanos , Técnicas In Vitro , Morfogênese/fisiologia , Placenta/citologia , Placenta/fisiologia , Gravidez , Trofoblastos/fisiologiaRESUMO
We examined the allele-specific expression of six imprinted genes and the methylation profiles of three imprinting control regions to assess the epigenetic status of human embryonic stem cells. We identified generally monoallelic gene expression and normal methylation patterns. During prolonged passage, one cell line became biallelic with respect to H19, but without loss of the gametic methylation imprint. These data argue for a substantial degree of epigenetic stability in human embryonic stem cells.
Assuntos
Epigênese Genética , Impressão Genômica , Células-Tronco , Alelos , Linhagem Celular , Células Cultivadas , Metilação de DNA , Embrião de Mamíferos , Expressão Gênica , Genes Reguladores , Humanos , Modelos Biológicos , Dados de Sequência Molecular , RNA Longo não Codificante , RNA não Traduzido/metabolismoRESUMO
Mouse epiblast stem cells (EpiSCs) derived from postimplantation embryos are developmentally and functionally different from embryonic stem cells (ESCs) generated from blastocysts. EpiSCs require Activin A and FGF2 signaling for self-renewal, similar to human ESCs (hESCs), while mouse ESCs require LIF and BMP4. Unlike ESCs, EpiSCs have undergone X-inactivation, similar to the tendency of hESCs. The shared self-renewal and X-inactivation properties of EpiSCs and hESCs suggest that they have an epigenetic state distinct from ESCs. This hypothesis predicts that EpiSCs would have monoallelic expression of most imprinted genes, like that observed in hESCs. Here, we confirm this prediction. By contrast, we find that mouse induced pluripotent stem cells (iPSCs) tend to lose imprinting similar to mouse ESCs. These findings reveal that iPSCs have an epigenetic status associated with their pluripotent state rather than their developmental origin. Our results also reinforce the view that hESCs and EpiSCs are in vitro counterparts, sharing an epigenetic status distinct from ESCs and iPSCs.
Assuntos
Epigênese Genética , Impressão Genômica , Células-Tronco Pluripotentes/metabolismo , Animais , Antígenos de Diferenciação/genética , Antígenos de Diferenciação/metabolismo , Células Cultivadas , Metilação de DNA , Células-Tronco Embrionárias/metabolismo , Perfilação da Expressão Gênica , Regulação da Expressão Gênica no Desenvolvimento , Camadas Germinativas/citologia , Proteínas de Homeodomínio/genética , Proteínas de Homeodomínio/metabolismo , Humanos , Células-Tronco Pluripotentes Induzidas/metabolismo , Camundongos , Proteína Homeobox Nanog , Fator 3 de Transcrição de Octâmero/genética , Fator 3 de Transcrição de Octâmero/metabolismo , Reação em Cadeia da Polimerase em Tempo Real , Fatores de Transcrição SOXB1/genética , Fatores de Transcrição SOXB1/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismoRESUMO
Although the first mouse embryonic stem (ES) cell lines were derived 25 years ago using feeder-layer-based blastocyst cultures, subsequent efforts to extend the approach to other mammals, including both laboratory and domestic species, have been relatively unsuccessful. The most notable exceptions were the derivation of non-human primate ES cell lines followed shortly thereafter by their derivation of human ES cells. Despite the apparent common origin and the similar pluripotency of mouse and human embryonic stem cells, recent studies have revealed that they use different signalling pathways to maintain their pluripotent status. Mouse ES cells depend on leukaemia inhibitory factor and bone morphogenetic protein, whereas their human counterparts rely on activin (INHBA)/nodal (NODAL) and fibroblast growth factor (FGF). Here we show that pluripotent stem cells can be derived from the late epiblast layer of post-implantation mouse and rat embryos using chemically defined, activin-containing culture medium that is sufficient for long-term maintenance of human embryonic stem cells. Our results demonstrate that activin/Nodal signalling has an evolutionarily conserved role in the derivation and the maintenance of pluripotency in these novel stem cells. Epiblast stem cells provide a valuable experimental system for determining whether distinctions between mouse and human embryonic stem cells reflect species differences or diverse temporal origins.
Assuntos
Embrião de Mamíferos/citologia , Células-Tronco Embrionárias/citologia , Células-Tronco Pluripotentes/citologia , Ativinas/metabolismo , Animais , Técnicas de Cultura de Células , Linhagem Celular , Meios de Cultura/química , Implantação do Embrião , Células-Tronco Embrionárias/metabolismo , Feminino , Perfilação da Expressão Gênica , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos CBA , Análise de Sequência com Séries de Oligonucleotídeos , Células-Tronco Pluripotentes/metabolismo , Ratos , Ratos Sprague-Dawley , Ratos Wistar , Transdução de SinaisRESUMO
Cardiogenesis relies on the precise spatiotemporal coordination of multiple progenitor populations. Understanding the specification and differentiation of these distinct progenitor pools during human embryonic development is crucial for advancing our knowledge of congenital cardiac malformations and designing new regenerative therapies. By combining genetic labelling, single-cell transcriptomics, and ex vivo human-mouse embryonic chimeras we uncovered that modulation of retinoic acid signaling instructs human pluripotent stem cells to form heart field-specific progenitors with distinct fate potentials. In addition to the classical first and second heart fields, we observed the appearance of juxta-cardiac field progenitors giving rise to both myocardial and epicardial cells. Applying these findings to stem-cell based disease modelling we identified specific transcriptional dysregulation in first and second heart field progenitors derived from stem cells of patients with hypoplastic left heart syndrome. This highlights the suitability of our in vitro differentiation platform for studying human cardiac development and disease.
Assuntos
Células-Tronco Pluripotentes , Tretinoína , Humanos , Animais , Camundongos , Tretinoína/farmacologia , Coração , Miocárdio , Diferenciação Celular , Miócitos CardíacosRESUMO
Activin/Nodal signaling is necessary to maintain pluripotency of human embryonic stem cells (hESCs) and to induce their differentiation toward endoderm. However, the mechanisms by which Activin/Nodal signaling achieves these opposite functions remain unclear. To unravel these mechanisms, we examined the transcriptional network controlled in hESCs by Smad2 and Smad3, which represent the direct effectors of Activin/Nodal signaling. These analyses reveal that Smad2/3 participate in the control of the core transcriptional network characterizing pluripotency, which includes Oct-4, Nanog, FoxD3, Dppa4, Tert, Myc, and UTF1. In addition, similar experiments performed on endoderm cells confirm that a broad part of the transcriptional network directing differentiation is downstream of Smad2/3. Therefore, Activin/Nodal signaling appears to control divergent transcriptional networks in hESCs and in endoderm. Importantly, we observed an overlap between the transcriptional network downstream of Nanog and Smad2/3 in hESCs; whereas, functional studies showed that both factors cooperate to control the expression of pluripotency genes. Therefore, the effect of Activin/Nodal signaling on pluripotency and differentiation could be dictated by tissue specific Smad2/3 partners such as Nanog, explaining the mechanisms by which signaling pathways can orchestrate divergent cell fate decisions.
Assuntos
Ativinas/metabolismo , Endoderma/citologia , Redes Reguladoras de Genes , Proteína Nodal/metabolismo , Células-Tronco/metabolismo , Sequência de Bases , Diferenciação Celular , Linhagem Celular , Imunoprecipitação da Cromatina , Células-Tronco Embrionárias/metabolismo , Endoderma/metabolismo , Genes Reporter , Proteínas de Homeodomínio/metabolismo , Humanos , Luciferases de Vaga-Lume/biossíntese , Luciferases de Vaga-Lume/genética , Proteína Homeobox Nanog , Regiões Promotoras Genéticas , Análise de Sequência de DNA , Proteína Smad2/metabolismo , Proteína Smad3/metabolismo , Células-Tronco/citologiaRESUMO
UNLABELLED: Generation of hepatocytes from human embryonic stem cells (hESCs) could represent an advantageous source of cells for cell therapy approaches as an alternative to orthotopic liver transplantation. However, the generation of differentiated hepatocytes from hESCs remains a major challenge, especially using a method compatible with clinical applications. We report a novel approach to differentiate hESCs into functional hepatic cells using fully defined culture conditions, which recapitulate essential stages of liver development. hESCs were first differentiated into a homogenous population of endoderm cells using a combination of activin, fibroblast growth factor 2, and bone morphogenetic protein 4 together with phosphoinositide 3-kinase inhibition. The endoderm cells were then induced to differentiate further into hepatic progenitors using fibroblast growth factor 10, retinoic acid, and an inhibitor of activin/nodal receptor. After further maturation, these cells expressed markers of mature hepatocytes, including asialoglycoprotein receptor, tyrosine aminotransferase, alpha1-antitrypsin, Cyp7A1, and hepatic transcription factors such as hepatocyte nuclear factors 4alpha and 6. Furthermore, the cells generated under these conditions exhibited hepatic functions in vitro, including glycogen storage, cytochrome activity, and low-density lipoprotein uptake. After transduction with a green fluorescent protein-expressing lentivector and transplantation into immunodeficient uPA transgenic mice, differentiated cells engrafted into the liver, grew, and expressed human albumin and alpha1-antitrypsin as well as green fluorescent protein for at least 8 weeks. In addition, we showed that hepatic cells could be generated from human-induced pluripotent cells derived from reprogrammed fibroblasts, demonstrating the efficacy of this approach with pluripotent stem cells of diverse origins. CONCLUSION: We have developed a robust and efficient method to differentiate pluripotent stem cells into hepatic cells, which exhibit characteristics of human hepatocytes. Our approach should facilitate the development of clinical grade hepatocytes for transplantation and for research on drug discovery.
Assuntos
Diferenciação Celular , Células-Tronco Embrionárias/citologia , Hepatócitos/citologia , Fígado/embriologia , Ativinas/farmacologia , Animais , Benzamidas/farmacologia , Proteína Morfogenética Óssea 4/farmacologia , Diferenciação Celular/fisiologia , Cromonas/farmacologia , Dioxóis/farmacologia , Fator 2 de Crescimento de Fibroblastos/farmacologia , Regulação da Expressão Gênica no Desenvolvimento/efeitos dos fármacos , Humanos , Camundongos , Morfolinas/farmacologia , Células-Tronco Pluripotentes/citologia , Tretinoína/farmacologiaRESUMO
Mouse embryonic pluripotent stem cells can be obtained from the inner cell mass at the blastocyst stage (embryonic stem cells, ESCs) or from the late epiblast of postimplantation embryos (epiblast stem cells, EpiSCs). During normal development, the transition between these two stages is marked by major epigenetic and transcriptional changes including DNA de novo methylation. These modifications represent an epigenetic mark conserved in ESCs and EpiSCs. Pluripotent ESCs derived from blastocysts generated by nuclear transfer (NT) have been shown to be correctly reprogrammed. However, NT embryos frequently undergo abnormal development. In the present study, we have examined whether pluripotent cells could be derived from the epiblast of postimplantation NT embryos and whether the reprogramming process would affect the epigenetic changes occurring at this stage, which could explain abnormal development of NT embryos. We showed that EpiSCs could be derived with the same efficiency from NT embryos and from their fertilized counterparts. However, gene expression profile analyses showed divergence between fertilized- and nuclear transfer-EpiSCs with a surprising bias in the distribution of the differentially expressed genes, 30% of them being localized on chromosome 11. A majority of these genes were downregulated in NT-EpiSCs and imprinted genes represented a significant fraction of them. Notably, analysis of the epigenetic status of a downregulated imprinted gene in NT-EpiSCs revealed complete methylation of the two alleles. Therefore, EpiSCs derived from NT embryos appear to be incorrectly reprogrammed, indicating that abnormal epigenetic marks are imposed on cells in NT embryos during the transition from early to late epiblast.
Assuntos
Camadas Germinativas/metabolismo , Células-Tronco/metabolismo , Animais , Biomarcadores , Linhagem Celular , Proliferação de Células , Forma Celular , Epigênese Genética , Fertilização in vitro , Perfilação da Expressão Gênica , Camadas Germinativas/citologia , Camundongos , Camundongos Endogâmicos C57BL , Técnicas de Transferência Nuclear , Células-Tronco/citologiaRESUMO
Recently in Cell, Tan et al. (2021) report the successful generation of human-monkey chimeras in vitro, providing an opportunity for new insights into the biology of human stem cells and early human development in an embryonic environment that is evolutionary closer to human than previously studied rodent and domestic species.
Assuntos
Quimera , Células-Tronco Pluripotentes , Animais , Haplorrinos , HumanosRESUMO
The newly revised 2021 ISSCR Guidelines for Stem Cell Research and Clinical Translation includes scientific and ethical guidance for the transfer of human pluripotent stem cells and their direct derivatives into animal models. In this white paper, the ISSCR subcommittee that drafted these guidelines for research involving the use of nonhuman embryos and postnatal animals explains and summarizes their recommendations.