RESUMEN
Recently, several studies using cultures of human embryos together with single-cell RNA-seq analyses have revealed differences between humans and mice, necessitating the study of human embryos1-8. Despite the importance of human embryology, ethical and legal restrictions have limited post-implantation-stage studies. Thus, recent efforts have focused on developing in vitro self-organizing models using human stem cells9-17. Here, we report genetic and non-genetic approaches to generate authentic hypoblast cells (naive hPSC-derived hypoblast-like cells (nHyCs))-known to give rise to one of the two extraembryonic tissues essential for embryonic development-from naive human pluripotent stem cells (hPSCs). Our nHyCs spontaneously assemble with naive hPSCs to form a three-dimensional bilaminar structure (bilaminoids) with a pro-amniotic-like cavity. In the presence of additional naive hPSC-derived analogues of the second extraembryonic tissue, the trophectoderm, the efficiency of bilaminoid formation increases from 20% to 40%, and the epiblast within the bilaminoids continues to develop in response to trophectoderm-secreted IL-6. Furthermore, we show that bilaminoids robustly recapitulate the patterning of the anterior-posterior axis and the formation of cells reflecting the pregastrula stage, the emergence of which can be shaped by genetically manipulating the DKK1/OTX2 hypoblast-like domain. We have therefore successfully modelled and identified the mechanisms by which the two extraembryonic tissues efficiently guide the stage-specific growth and progression of the epiblast as it establishes the post-implantation landmarks of human embryogenesis.
Asunto(s)
Desarrollo Embrionario , Estratos Germinativos , Células Madre Pluripotentes , Humanos , Diferenciación Celular , Implantación del Embrión , Embrión de Mamíferos/citología , Embrión de Mamíferos/embriología , Embrión de Mamíferos/metabolismo , Desarrollo Embrionario/genética , Desarrollo Embrionario/fisiología , Estratos Germinativos/citología , Estratos Germinativos/embriología , Estratos Germinativos/metabolismo , Células Madre Pluripotentes/citología , Interleucina-6/metabolismo , Gástrula/citología , Gástrula/embriología , Amnios/citología , Amnios/embriología , Amnios/metabolismo , Ectodermo/citología , Ectodermo/embriología , Ectodermo/metabolismo , Péptidos y Proteínas de Señalización Intercelular/genética , Péptidos y Proteínas de Señalización Intercelular/metabolismo , Factores de Transcripción Otx/genética , Factores de Transcripción Otx/metabolismoRESUMEN
Cancer is believed to arise primarily through accumulation of genetic mutations. Although induced pluripotent stem cell (iPSC) generation does not require changes in genomic sequence, iPSCs acquire unlimited growth potential, a characteristic shared with cancer cells. Here, we describe a murine system in which reprogramming factor expression in vivo can be controlled temporally with doxycycline (Dox). Notably, transient expression of reprogramming factors in vivo results in tumor development in various tissues consisting of undifferentiated dysplastic cells exhibiting global changes in DNA methylation patterns. The Dox-withdrawn tumors arising in the kidney share a number of characteristics with Wilms tumor, a common pediatric kidney cancer. We also demonstrate that iPSCs derived from Dox-withdrawn kidney tumor cells give rise to nonneoplastic kidney cells in mice, proving that they have not undergone irreversible genetic transformation. These findings suggest that epigenetic regulation associated with iPSC derivation may drive development of particular types of cancer.
Asunto(s)
Reprogramación Celular , Epigénesis Genética , Regulación Neoplásica de la Expresión Génica , Neoplasias Renales/patología , Animales , Metilación de ADN , Doxiciclina/farmacología , Células Madre Pluripotentes Inducidas/citología , Células Madre Pluripotentes Inducidas/metabolismo , Neoplasias Renales/inducido químicamente , Ratones , Ratones Transgénicos , Factores de Transcripción/metabolismoRESUMEN
Inhibitors of Mek1/2 and Gsk3ß, known as 2i, enhance the derivation of embryonic stem (ES) cells and promote ground-state pluripotency in rodents. Here we show that the derivation of female mouse ES cells in the presence of 2i and leukaemia inhibitory factor (2i/L ES cells) results in a widespread loss of DNA methylation, including a massive erasure of genomic imprints. Despite this global loss of DNA methylation, early-passage 2i/L ES cells efficiently differentiate into somatic cells, and this process requires genome-wide de novo DNA methylation. However, the majority of imprinting control regions (ICRs) remain unmethylated in 2i/L-ES-cell-derived differentiated cells. Consistently, 2i/L ES cells exhibit impaired autonomous embryonic and placental development by tetraploid embryo complementation or nuclear transplantation. We identified the derivation conditions of female ES cells that display 2i/L-ES-cell-like transcriptional signatures while preserving gamete-derived DNA methylation and autonomous developmental potential. Upon prolonged culture, however, female ES cells exhibited ICR demethylation regardless of culture conditions. Our results provide insights into the derivation of female ES cells reminiscent of the inner cell mass of preimplantation embryos.
Asunto(s)
Diferenciación Celular/genética , Metilación de ADN/genética , Células Madre Embrionarias/citología , Animales , Diferenciación Celular/efectos de los fármacos , Metilación de ADN/efectos de los fármacos , Células Madre Embrionarias/efectos de los fármacos , Femenino , Impresión Genómica/efectos de los fármacos , Glucógeno Sintasa Quinasa 3 beta/antagonistas & inhibidores , Factor Inhibidor de Leucemia/farmacología , MAP Quinasa Quinasa 1/antagonistas & inhibidores , MAP Quinasa Quinasa 2/antagonistas & inhibidores , Ratones , Ratones Endogámicos C57BLRESUMEN
Forty years have passed since the first pluripotent stem cells (PSCs), mouse embryonic stem cells (ESCs), were established. Since then, several PSCs have been reported, including human ESCs in 1998, mouse epiblast stem cells (EpiSCs) in 2007, induced PSCs (iPSCs) in 2006 and 2007, and naïve human PSCs in 2014. Naïve PSCs are thought to correspond to pre-implantation epiblast cells, whereas conventional (or primed) human PSCs correspond to post-implantation epiblast cells. Thus, naïve and primed PSCs are classified by their developmental stages and have stage-specific characteristics, despite sharing the common feature of pluripotency. In this review, we discuss the current status of PSCs and their use to model human peri-implantation development.
Asunto(s)
Células Madre Pluripotentes/citología , Diferenciación Celular , Desarrollo Embrionario , HumanosRESUMEN
The spectrum of genetic mutations differs among cancers in different organs, implying a cellular context-dependent effect for genetic aberrations. However, the extent to which the cellular context affects the consequences of oncogenic mutations remains to be fully elucidated. We reprogrammed colon tumor cells in an ApcMin/+ (adenomatous polyposis coli) mouse model, in which the loss of the Apc gene plays a critical role in tumor development and subsequently, established reprogrammed tumor cells (RTCs) that exhibit pluripotent stem cell (PSC)-like signatures of gene expression. We show that the majority of the genes in RTCs that were affected by Apc mutations did not overlap with the genes affected in the intestine. RTCs lacked pluripotency but exhibited an increased expression of Cdx2 and a differentiation propensity that was biased toward the trophectoderm cell lineage. Genetic rescue of the mutated Apc allele conferred pluripotency on RTCs and enabled their differentiation into various cell types in vivo. The redisruption of Apc in RTC-derived differentiated cells resulted in neoplastic growth that was exclusive to the intestine, but the majority of the intestinal lesions remained as pretumoral microadenomas. These results highlight the significant influence of cellular context on gene regulation, cellular plasticity, and cellular behavior in response to the loss of the Apc function. Our results also imply that the transition from microadenomas to macroscopic tumors is reprogrammable, which underscores the importance of epigenetic regulation on tumor promotion.
Asunto(s)
Poliposis Adenomatosa del Colon/genética , Regulación de la Expresión Génica/genética , Genes APC/fisiología , Mutación/genética , Alelos , Animales , Linaje de la Célula/genética , Plasticidad de la Célula/genética , Neoplasias del Colon/genética , Neoplasias del Colon/metabolismo , Epigénesis Genética/genética , Expresión Génica/genética , Regulación Neoplásica de la Expresión Génica/genética , Mucosa Intestinal/metabolismo , Ratones , Células Madre Pluripotentes/metabolismoRESUMEN
BACKGROUND: Blau syndrome, or early-onset sarcoidosis, is a juvenile-onset systemic granulomatosis associated with a mutation in nucleotide-binding oligomerization domain 2 (NOD2). The underlying mechanisms of Blau syndrome leading to autoinflammation are still unclear, and there is currently no effective specific treatment for Blau syndrome. OBJECTIVES: To elucidate the mechanisms of autoinflammation in patients with Blau syndrome, we sought to clarify the relation between disease-associated mutant NOD2 and the inflammatory response in human samples. METHODS: Blau syndrome-specific induced pluripotent stem cell (iPSC) lines were established. The disease-associated NOD2 mutation of iPSCs was corrected by using a CRISPR-Cas9 system to precisely evaluate the in vitro phenotype of iPSC-derived cells. We also introduced the same NOD2 mutation into a control iPSC line. These isogenic iPSCs were then differentiated into monocytic cell lineages, and the statuses of nuclear factor κB pathway and proinflammatory cytokine secretion were investigated. RESULTS: IFN-γ acted as a priming signal through upregulation of NOD2. In iPSC-derived macrophages with mutant NOD2, IFN-γ treatment induced ligand-independent nuclear factor κB activation and proinflammatory cytokine production. RNA sequencing analysis revealed distinct transcriptional profiles of mutant macrophages both before and after IFN-γ treatment. Patient-derived macrophages demonstrated a similar IFN-γ-dependent inflammatory response. CONCLUSIONS: Our data support the significance of ligand-independent autoinflammation in the pathophysiology of Blau syndrome. Our comprehensive isogenic disease-specific iPSC panel provides a useful platform for probing therapeutic and diagnostic clues for the treatment of patients with Blau syndrome.
Asunto(s)
Artritis/etiología , Artritis/metabolismo , Interferón gamma/metabolismo , Macrófagos/metabolismo , Células Madre Pluripotentes/metabolismo , Sinovitis/etiología , Sinovitis/metabolismo , Uveítis/etiología , Uveítis/metabolismo , Linaje de la Célula/genética , Citocinas/metabolismo , Análisis Mutacional de ADN , Exones , Marcación de Gen , Sitios Genéticos , Humanos , Células Madre Pluripotentes Inducidas/citología , Células Madre Pluripotentes Inducidas/metabolismo , Mediadores de Inflamación/metabolismo , Interferón gamma/genética , Ligandos , Macrófagos/inmunología , Masculino , Mutación , FN-kappa B/metabolismo , Proteína Adaptadora de Señalización NOD2/genética , Fenotipo , Células Madre Pluripotentes/citología , SarcoidosisRESUMEN
The forced reduction of global DNA methylation suppresses tumor development in several cancer models in vivo. Nevertheless, the mechanisms underlying these suppressive effects remain unclear. In this report, we describe our findings showing that a genome-wide reduction in the DNA methylation levels induces cellular differentiation in association with decreased cell proliferation in Apc (Min/+) mouse colon tumor cells in vivo. Colon tumor-specific DNA methylation at Cdx1 is reduced in the DNA-hypomethylated tumors accompanied by Cdx1 derepression and an increased expression of intestinal differentiation-related genes. Furthermore, a histological analysis revealed that Cdx1 derepression in the DNA-hypomethylated tumors is correlated with the differentiation of colon tumor cells. Similarly, the treatment of human colon cancer cell lines with a hypomethylating agent induces differentiation-related genes, including CDX1. We herein propose that DNA demethylation exerts a tumor suppressive effect in the colon by inducing tumor cell differentiation.
Asunto(s)
Diferenciación Celular/genética , Neoplasias del Colon/genética , Neoplasias del Colon/patología , Metilación de ADN , Proteína de la Poliposis Adenomatosa del Colon/genética , Animales , Factor de Transcripción CDX2 , Línea Celular Tumoral , ADN (Citosina-5-)-Metiltransferasa 1 , ADN (Citosina-5-)-Metiltransferasas/genética , ADN (Citosina-5-)-Metiltransferasas/metabolismo , Regulación Neoplásica de la Expresión Génica , Proteínas de Homeodominio/genética , Proteínas de Homeodominio/metabolismo , Humanos , Ratones Endogámicos C57BL , Ratones Mutantes , Complejo Represivo Polycomb 2/genética , Complejo Represivo Polycomb 2/metabolismo , Regiones Promotoras Genéticas , Análisis de Matrices Tisulares , Factores de Transcripción/genética , Factores de Transcripción/metabolismoRESUMEN
Cancer arises through the accumulation of both genetic and epigenetic alterations. Although the causal role of genetic mutations on cancer development has been established in vivo, similar evidence for epigenetic alterations is limited. Moreover, mutual interactions between genetic mutations and epigenetic alterations remain unclear. Cellular reprogramming technology can be used to actively modify the epigenome without affecting the underlying genomic sequences. Here we introduce recent studies that have utilized this property for cancer research. We propose that just as it has potential for regenerative medicine and disease modeling, cell reprogramming could also be a powerful tool for dissecting the role of the cancer epigenome in the development and maintenance of cancer cells.
Asunto(s)
Carcinogénesis/genética , Reprogramación Celular/genética , Epigénesis Genética , Epigenómica/métodos , Células Madre Pluripotentes Inducidas/metabolismo , Neoplasias/genética , Desdiferenciación Celular , Humanos , Oncogenes/genéticaRESUMEN
Cancer is generally developed through accumulation of multiple genetic mutations. Epigenetic abnormalities of DNA methylation and histone modification patterns were also found in most cancer cells. Although induced pluripotent stem cells (iPSCs) can be generated through epigenetic reorganization without affecting the underlying genomic sequencing, they have some shared characteristics with cancer cells, which include unlimited growth potential. Taking advantages of such properties of iPSC derivation, the reprogramming technology is applicable not only for regenerative medicine but also for cancer research. Here, we introduce the potential application of iPSC technology for better understandings of cancer biology. Especially, we would like to propose the role of cellular identity in cancer development.
Asunto(s)
Reprogramación Celular , Células Madre Pluripotentes Inducidas/citología , Células Madre Pluripotentes Inducidas/metabolismo , Neoplasias/genética , Animales , Epigénesis Genética , Epigenómica , Humanos , Neoplasias/patología , Transcripción GenéticaRESUMEN
Somatic cells can be reprogrammed into induced pluripotent stem cells (iPSCs) by the transient expression of reprogramming factors. During the reprogramming process, somatic cells acquire the ability to undergo unlimited proliferation, which is also an important characteristic of cancer cells, while their underlying DNA sequence remains unchanged. Based on the characteristics shared between pluripotent stem cells and cancer cells, the potential involvement of the factors leading to reprogramming toward pluripotency in cancer development has been discussed. Recent in vivo reprogramming studies provided some clues to understanding the role of reprogramming-related epigenetic regulation in cancer development. It was shown that premature termination of the in vivo reprogramming result in the development of tumors that resemble pediatric cancers. Given that epigenetic modifications play a central role during reprogramming, failed reprogramming-associated cancer development may have provided a proof of concept for epigenetics-driven cancer development in vivo.
Asunto(s)
Reprogramación Celular , Epigénesis Genética , Neoplasias/genética , Células Madre Pluripotentes Inducidas/metabolismoRESUMEN
Recent studies imply that cancer cells possess the ability to reversibly change their properties between a drug sensitive state and a drug resistant state accompanied by epigenetic changes. This evidence indicates that better understanding of cancer epigenetics is important for efficient cancer therapies. Nevertheless, it had been difficult to deeply examine the epigenetic mechanisms because of lack of the tools to actively modify coordinated epigenetic events. In this stagnant situation, the reprogramming technology established by Yamanaka and coworkers have shed a new light. The novel reprogramming technology has made it possible for researchers to artificially introduce epigenetic remodeling into somatic cells. Accordingly, we might be able to use this technology as a tool to introduce the coordinated epigenetic reorganization. In this review, we introduce the idea of cell state interconversion in cancer cells that is attributable to altered epigenetic regulations. We then depict the epigenetic modifications observed during the process of somatic cell reprogramming and give some examples of the difficulty in cancer cell reprogramming. Finally, we discuss how we can translate this reprogramming refractoriness of cancer cells into uncovering unique epigenetic regulations in cancer cells, which might be applicable eventually to the development of novel cancer therapeutics against drug resistant cancer cells.
Asunto(s)
Diferenciación Celular/fisiología , Reprogramación Celular/fisiología , Resistencia a Antineoplásicos , Epigénesis Genética , Células Madre Pluripotentes Inducidas/citología , Neoplasias/genética , Neoplasias/terapia , Animales , Reprogramación Celular/genética , HumanosRESUMEN
DNA demethylation of astrocyte-specific gene promoters and STAT3 activation in neural precursor cells (NPCs) are essential for astrogliogenesis in the developing brain. To date, it remains unclear whether DNA methylation is the sole epigenetic determinant responsible for suppressing astrocyte-specific genes. Here, we used mouse embryonic stem cells (TKO ESCs) that lacked all 3 DNA methyltransferase genes, Dnmt1, Dnmt3a, and Dnmt3b, and thereby exhibit complete demethylation of the astrocyte-specific glial fibrillary acidic protein (Gfap) gene promoter. We found that although the Gfap promoter was demethylated, STAT3 failed to bind to its cognate element to induce Gfap transcription, whereas it induced transcription of a different target gene, Socs3. Moreover, although the Gfap promoter region containing the STAT3-binding site (GSBS) is enriched with transcription-repressive histone modifications, such as methylation of H3 at lysine 9 (H3K9me3) and H3K27me3, the reduction of these modifications in TKO ESCs was not sufficient for binding of STAT3 at GSBS. Furthermore, GSBS was digested by micrococcal nuclease in late-gestational NPCs that express GFAP upon LIF stimulation, but not in cells that show no expression of GFAP even in the presence of LIF, indicating that STAT3 can access GSBS in the former cells. We further showed that expression of NF-1A, which is known to potentiate differentiation of mid-gestational NPCs into astrocytes, increased its accessibility. Taken together, our results suggest that chromatin accessibility of GSBS plays a critical role in the regulation of Gfap expression.
Asunto(s)
Astrocitos , Diferenciación Celular/genética , Metilación de ADN/genética , Proteínas del Tejido Nervioso , Factor de Transcripción STAT3 , Animales , Astrocitos/citología , Astrocitos/metabolismo , Sitios de Unión , Cromatina/genética , Cromatina/metabolismo , Proteínas de Unión al ADN/genética , Células Madre Embrionarias/citología , Células Madre Embrionarias/metabolismo , Epigénesis Genética , Proteína Ácida Fibrilar de la Glía , Histona Demetilasas con Dominio de Jumonji/metabolismo , Ratones , Proteínas del Tejido Nervioso/genética , Proteínas del Tejido Nervioso/metabolismo , Neurogénesis , Neuronas/citología , Neuronas/metabolismo , Regiones Promotoras Genéticas , Factor de Transcripción STAT3/genética , Factor de Transcripción STAT3/metabolismo , Transcripción GenéticaRESUMEN
Cancer develops through the accumulation of genetic and epigenetic abnormalities. The role of genetic alterations in cancer development has been demonstrated by reverse genetic approaches. However, evidence indicating the functional significance of epigenetic abnormalities remains limited due to the lack of means to actively modify coordinated epigenetic regulations in the genome. Application of the reprogramming technology may help researchers to overcome this limitation and shed new light on cancer research. Reprogramming is accompanied by dynamic changes of epigenetic modifications and is therefore considered to be a useful tool to induce global epigenetic changes in cancer genomes. We herein discuss the similarities between reprogramming processes and carcinogenesis and propose the potential use of reprogramming technology to help understanding of the significance of epigenetic regulations in cancer cells. We, also discuss the application of induced pluripotent stem cell technology to cancer modeling based on the similar characteristics between pluripotent stem cells and cancer cells.
Asunto(s)
Reprogramación Celular/genética , Epigénesis Genética , Neoplasias/genética , Neoplasias/patología , Animales , Metilación de ADN , Proteína Potenciadora del Homólogo Zeste 2 , Genes ras , Impresión Genómica , Humanos , Células Madre Pluripotentes Inducidas/fisiología , Células Madre Pluripotentes Inducidas/trasplante , Neoplasias/etiología , Neoplasias/terapia , Complejo Represivo Polycomb 2/fisiologíaRESUMEN
Because of their ability to self-renew, to differentiate into multiple lineages, and to migrate toward a damaged site, neural stem cells (NSCs), which can be derived from various sources such as fetal tissues and embryonic stem cells, are currently considered to be promising components of cell replacement strategies aimed at treating injuries of the central nervous system, including the spinal cord. Despite their efficiency in promoting functional recovery, these NSCs are not homogeneous and possess variable characteristics depending on their derivation protocols. The advent of induced pluripotent stem (iPS) cells has provided new prospects for regenerative medicine. We used a recently developed robust and stable protocol for the generation of long-term, self-renewing, neuroepithelial-like stem cells from human iPS cells (hiPS-lt-NES cells), which can provide a homogeneous and well-defined population of NSCs for standardized analysis. Here, we show that transplanted hiPS-lt-NES cells differentiate into neural lineages in the mouse model of spinal cord injury (SCI) and promote functional recovery of hind limb motor function. Furthermore, using two different neuronal tracers and ablation of the transplanted cells, we revealed that transplanted hiPS-lt-NES cell-derived neurons, together with the surviving endogenous neurons, contributed to restored motor function. Both types of neurons reconstructed the corticospinal tract by forming synaptic connections and integrating neuronal circuits. Our findings indicate that hiPS-lt-NES transplantation represents a promising avenue for effective cell-based treatment of SCI.
Asunto(s)
Células Madre Pluripotentes Inducidas/trasplante , Células-Madre Neurales/trasplante , Traumatismos de la Médula Espinal/cirugía , Animales , Diferenciación Celular/fisiología , Células Cultivadas , Modelos Animales de Enfermedad , Femenino , Humanos , Inmunohistoquímica , Células Madre Pluripotentes Inducidas/citología , Ratones , Ratones Endogámicos NOD , Ratones SCID , Células-Madre Neurales/metabolismo , Traumatismos de la Médula Espinal/patología , Trasplante de Células Madre/métodosRESUMEN
Reconstitution of the germ cell lineage using pluripotent stem cells provides a unique platform to deepen our understanding of the mechanisms underlying germ cell development and to produce functional gametes for reproduction. This study aimed to establish a culture system that induces a robust number of primordial germ cell-like cells (PGCLCs) from common marmoset (Callithrix jacchus) embryonic stem cells. The robust induction was achieved by not only activation of the conserved PGC-inducing signals, WNT and BMP4, but also temporal inhibitions of WNT and retinoic acid signals, which prevent mesodermal and neural differentiation, respectively, during PGCLC differentiation. Many of the gene expression and differentiation properties of common marmoset PGCLCs were similar to those of human PGCLCs, making this culture system a reliable and useful primate model. Finally, we identified PDPN and KIT as surface marker proteins by which PGCLCs can be isolated from embryonic stem cells without genetic manipulation. This study will expand the opportunities for research on germ cell development and production of functional gametes to the common marmoset.
Asunto(s)
Callithrix , Células Madre Pluripotentes , Animales , Diferenciación Celular , Células Madre Embrionarias , Células Germinativas/metabolismo , Tretinoina/farmacología , Tretinoina/metabolismoRESUMEN
Primate germ cell development remains largely unexplored due to limitations in sample collection and the long duration of development. In mice, primordial germ cell-like cells (PGCLCs) derived from pluripotent stem cells (PSCs) can develop into functional gametes by in vitro culture or in vivo transplantation. Such PGCLC-mediated induction of mature gametes in primates is highly useful for understanding human germ cell development. Since marmosets generate functional sperm earlier than other species, recapitulating the whole male germ cell development process is technically more feasible. Here, we induced the differentiation of iPSCs into gonocyte-like cells via PGCLCs in marmosets. First, we developed an mRNA transfection-based method to efficiently generate PGCLCs. Subsequently, to promote PGCLC differentiation, xenoreconstituted testes (xrtestes) were generated in the mouse kidney capsule. PGCLCs show progressive DNA demethylation and stepwise expression of developmental marker genes. This study provides an efficient platform for the study of marmoset germ cell development.
Asunto(s)
Callithrix , Semen , Humanos , Masculino , Animales , Ratones , Células Germinativas , Diferenciación Celular/genética , ARN Mensajero/genéticaRESUMEN
Trophoblasts are extraembryonic cells that are essential for maintaining pregnancy. Human trophoblasts arise from the morula as trophectoderm (TE), which, after implantation, differentiates into cytotrophoblasts (CTs), syncytiotrophoblasts (STs), and extravillous trophoblasts (EVTs), composing the placenta. Here we show that naïve, but not primed, human pluripotent stem cells (PSCs) recapitulate trophoblast development. Naive PSC-derived TE and CTs (nCTs) recreated human and monkey TE-to-CT transition. nCTs self-renewed as CT stem cells and had the characteristics of proliferating villous CTs and CTs in the cell column of the first trimester. Notably, although primed PSCs differentiated into trophoblast-like cells (BMP4, A83-01, and PD173074 [BAP]-treated primed PSCs [pBAPs]), pBAPs were distinct from nCTs and human placenta-derived CT stem cells, exhibiting properties consistent with the amnion. Our findings establish an authentic paradigm for human trophoblast development, demonstrating the invaluable properties of naive human PSCs. Our system provides a platform to study the molecular mechanisms underlying trophoblast development and related diseases.
Asunto(s)
Células Madre Pluripotentes , Trofoblastos , Diferenciación Celular , Femenino , Humanos , Placenta , EmbarazoRESUMEN
To elucidate the function of the U69 protein kinase of human herpesvirus 6 (HHV-6) in vivo, we first analyzed its subcellular localization in HHV-6-infected Molt 3 cells by using polyclonal antibodies against the U69 protein. Immunofluorescence studies showed that the U69 signal localized to the nucleus in a mesh-like pattern in both HHV-6-infected and HHV6-transfected cells. A computer program predicted two overlapping classic nuclear localization signals (NLSs) in the N-terminal region of the protein; this NLS motif is highly conserved in the N-terminal region of most of the herpesvirus protein kinases examined to date. An N-terminal deletion mutant form of the protein failed to enter the nucleus, whereas a fusion protein of green fluorescent protein (GFP) and/or glutathione S-transferase (GST) and the U69 N-terminal region was transported into the nucleus, demonstrating that the predicted N-terminal NLSs of the protein actually function as NLSs. The nuclear transport of the GST-GFP fusion protein containing the N-terminal NLS of U69 was inhibited by wheat germ agglutinin and by the Q69L Ran-GTP mutant, indicating that the U69 protein is transported into the nucleus from the cytoplasm via classic nuclear transport machinery. A cell-free import assay showed that the nuclear transport of the U69 protein was mediated by importin alpha/beta in conjunction with the small GTPase Ran. When the import assay was performed with a low concentration of each importin-alpha subtype, NPI2/importin-alpha7 elicited more efficient transport activity than did Rch1/importin-alpha1 or Qip1/importin-alpha3. These results suggest a relationship between the localization of NPI2/importin-alpha7 and the cell tropism of HHV-6.
Asunto(s)
Herpesvirus Humano 6/fisiología , Señales de Localización Nuclear , Fosfotransferasas (Aceptor de Grupo Alcohol)/análisis , Fosfotransferasas (Aceptor de Grupo Alcohol)/fisiología , Transporte Activo de Núcleo Celular/fisiología , Línea Celular , Núcleo Celular/química , Células Cultivadas , Glutatión Transferasa/análisis , Glutatión Transferasa/genética , Proteínas Fluorescentes Verdes/análisis , Proteínas Fluorescentes Verdes/genética , Herpesvirus Humano 6/genética , Humanos , Microscopía Fluorescente , Fosfotransferasas (Aceptor de Grupo Alcohol)/genética , Proteínas Recombinantes de Fusión/análisis , Proteínas Recombinantes de Fusión/genética , Eliminación de SecuenciaRESUMEN
Clear cell sarcoma (CCS) is a rare soft tissue sarcoma caused by the EWS/ATF1 fusion gene. Here, we established induced pluripotent stem cells (iPSCs) from EWS/ATF1-controllable murine CCS cells harboring sarcoma-associated genetic abnormalities. Sarcoma-iPSC mice develop secondary sarcomas immediately after EWS/ATF1 induction, but only in soft tissue. EWS/ATF1 expression induces oncogene-induced senescence in most cell types in sarcoma-iPSC mice but prevents it in sarcoma cells. We identify Tppp3-expressing cells in peripheral nerves as a cell-of-origin for these sarcomas. We show cell type-specific recruitment of EWS/ATF1 to enhancer regions in CCS cells. Finally, epigenetic silencing at these enhancers induces senescence and inhibits CCS cell growth through altered EWS/ATF1 binding. Together, we propose that distinct responses to premature senescence are the basis for the cell type-specificity of cancer development.