RESUMEN
Programmable DNA nucleases such as TALENs and CRISPR/Cas9 are emerging as powerful tools for genome editing. Dual-fluorescent surrogate systems have been demonstrated by several studies to recapitulate DNA nuclease activity and enrich for genetically edited cells. In this study, we created a single-strand annealing-directed, dual-fluorescent surrogate reporter system, referred to as C-Check. We opted for the Golden Gate Cloning strategy to simplify C-Check construction. To demonstrate the utility of the C-Check system, we used the C-Check in combination with TALENs or CRISPR/Cas9 in different scenarios of gene editing experiments. First, we disrupted the endogenous pIAPP gene (3.0 % efficiency) by C-Check-validated TALENs in primary porcine fibroblasts (PPFs). Next, we achieved gene-editing efficiencies of 9.0-20.3 and 4.9 % when performing single- and double-gene targeting (MAPT and SORL1), respectively, in PPFs using C-Check-validated CRISPR/Cas9 vectors. Third, fluorescent tagging of endogenous genes (MYH6 and COL2A1, up to 10.0 % frequency) was achieved in human fibroblasts with C-Check-validated CRISPR/Cas9 vectors. We further demonstrated that the C-Check system could be applied to enrich for IGF1R null HEK293T cells and CBX5 null MCF-7 cells with frequencies of nearly 100.0 and 86.9 %, respectively. Most importantly, we further showed that the C-Check system is compatible with multiplexing and for studying CRISPR/Cas9 sgRNA specificity. The C-Check system may serve as an alternative dual-fluorescent surrogate tool for measuring DNA nuclease activity and enrichment of gene-edited cells, and may thereby aid in streamlining programmable DNA nuclease-mediated genome editing and biological research.
Asunto(s)
Sistemas CRISPR-Cas , Edición Génica/métodos , Animales , Células Cultivadas , Homólogo de la Proteína Chromobox 5 , Proteínas Cromosómicas no Histona/genética , Repeticiones Palindrómicas Cortas Agrupadas y Regularmente Espaciadas , Fibroblastos/citología , Fibroblastos/metabolismo , Fluorescencia , Técnicas de Inactivación de Genes/métodos , Genes Reporteros , Vectores Genéticos/genética , Células HEK293 , Recombinación Homóloga , Humanos , Células MCF-7 , Receptor IGF Tipo 1 , Receptores de Somatomedina/genética , Porcinos , Nucleasas de los Efectores Tipo Activadores de la Transcripción/genética , Nucleasas de los Efectores Tipo Activadores de la Transcripción/metabolismoRESUMEN
During early pancreatic development, Notch signaling represses differentiation of endocrine cells and promotes proliferation of Nkx6-1(+)Ptf1a(+) multipotent progenitor cells (MPCs). Later, antagonistic interactions between Nkx6 transcription factors and Ptf1a function to segregate MPCs into distal Nkx6-1(-)Ptf1a(+) acinar progenitors and proximal Nkx6-1(+)Ptf1a(-) duct and ß-cell progenitors. Distal cells are initially multipotent, but evolve into unipotent, acinar cell progenitors. Conversely, proximal cells are bipotent and give rise to duct cells and late-born endocrine cells, including the insulin producing ß-cells. However, signals that regulate proximodistal (P-D) patterning and thus formation of ß-cell progenitors are unknown. Here we show that Mind bomb 1 (Mib1) is required for correct P-D patterning of the developing pancreas and ß-cell formation. We found that endoderm-specific inactivation of Mib1 caused a loss of Nkx6-1(+)Ptf1a(-) and Hnf1ß(+) cells and a corresponding loss of Neurog3(+) endocrine progenitors and ß-cells. An accompanying increase in Nkx6-1(-)Ptf1a(+) and amylase(+) cells, occupying the proximal domain, suggests that proximal cells adopt a distal fate in the absence of Mib1 activity. Impeding Notch-mediated transcriptional activation by conditional expression of dominant negative Mastermind-like 1 (Maml1) resulted in a similarly distorted P-D patterning and suppressed ß-cell formation, as did conditional inactivation of the Notch target gene Hes1. Our results reveal iterative use of Notch in pancreatic development to ensure correct P-D patterning and adequate ß-cell formation.
Asunto(s)
Embrión de Mamíferos/metabolismo , Células Secretoras de Insulina/metabolismo , Páncreas/metabolismo , Ubiquitina-Proteína Ligasas/metabolismo , Animales , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/genética , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/metabolismo , Western Blotting , Linaje de la Célula , Embrión de Mamíferos/citología , Embrión de Mamíferos/embriología , Femenino , Regulación del Desarrollo de la Expresión Génica , Factor Nuclear 1-beta del Hepatocito/genética , Factor Nuclear 1-beta del Hepatocito/metabolismo , Factor Nuclear 3-beta del Hepatocito/genética , Factor Nuclear 3-beta del Hepatocito/metabolismo , Proteínas de Homeodominio/genética , Proteínas de Homeodominio/metabolismo , Células Secretoras de Insulina/citología , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Ratones Transgénicos , Mutación , Proteínas del Tejido Nervioso/genética , Proteínas del Tejido Nervioso/metabolismo , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Páncreas/citología , Páncreas/embriología , Receptores Notch/genética , Receptores Notch/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Transducción de Señal , Factores de Tiempo , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Ubiquitina-Proteína Ligasas/genéticaRESUMEN
Here we examine how BMP, Wnt, and FGF signaling modulate activin-induced mesendodermal differentiation of mouse ES cells grown under defined conditions in adherent monoculture. We monitor ES cells containing reporter genes for markers of primitive streak (PS) and its progeny and extend previous findings on the ability of increasing concentrations of activin to progressively induce more ES cell progeny to anterior PS and endodermal fates. We find that the number of Sox17- and Gsc-expressing cells increases with increasing activin concentration while the highest number of T-expressing cells is found at the lowest activin concentration. The expression of Gsc and other anterior markers induced by activin is prevented by treatment with BMP4, which induces T expression and subsequent mesodermal development. We show that canonical Wnt signaling is required only during late stages of activin-induced development of Sox17-expressing endodermal cells. Furthermore, Dkk1 treatment is less effective in reducing development of Sox17(+) endodermal cells in adherent culture than in aggregate culture and appears to inhibit nodal-mediated induction of Sox17(+) cells more effectively than activin-mediated induction. Notably, activin induction of Gsc-GFP(+) cells appears refractory to inhibition of canonical Wnt signaling but shows a dependence on early as well as late FGF signaling. Additionally, we find a late dependence on FGF signaling during induction of Sox17(+) cells by activin while BMP4-induced T expression requires FGF signaling in adherent but not aggregate culture. Lastly, we demonstrate that activin-induced definitive endoderm derived from mouse ES cells can incorporate into the developing foregut endoderm in vivo and adopt a mostly anterior foregut character after further culture in vitro.
Asunto(s)
Activinas/farmacología , Células Madre Embrionarias/citología , Endodermo/efectos de los fármacos , Factores de Crecimiento de Fibroblastos/metabolismo , Transducción de Señal , Proteínas Wnt/metabolismo , Animales , Secuencia de Bases , Diferenciación Celular , Células Cultivadas , Embrión de Pollo , Endodermo/citología , Citometría de Flujo , Técnica del Anticuerpo Fluorescente , Ratones , ARN Interferente Pequeño , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
In this study we examined the expression of nestin in islets, the exocrine part, and the big ducts of the adult human pancreas by immunofluorescent double staining. Two different anti-nestin antisera in combination with various pancreatic and endothelial markers were employed. Nestin-immunoreactive cells were found in islets and in the exocrine portion. All nestin-positive cells co-expressed the vascular endothelial markers PECAM-1 (CD31), endoglin (CD105), and CD34 as well as vimentin. Endocrine, acinar, and duct cells did not stain for nestin. We also demonstrated that in the area of big pancreatic ducts, nestin-positive cells represent small capillaries scattered in the connective tissue surrounding the duct epithelium and do not reside between the duct cells. We detected nestin-expressing endothelial cells located adjacent to the duct epithelium where endocrine differentiation occurs. We have shown that nestin is expressed by vascular endothelial cells in human pancreas, and therefore it is unlikely that nestin specifically marks a subpopulation of cells representing endocrine progenitors in the adult pancreas.
Asunto(s)
Proteínas de Filamentos Intermediarios/metabolismo , Proteínas del Tejido Nervioso , Páncreas/metabolismo , Adulto , Humanos , Inmunohistoquímica , Islotes Pancreáticos/metabolismo , Nestina , Conductos Pancreáticos/metabolismoRESUMEN
Wnts are important signaling molecules involved in many normal developmental processes in the human body as well as some forms of cancer. Nineteen Wnt genes are found in the human genome, as well as 10 Wnt receptor genes called Frizzled. Two coreceptors called LRP 5 and 6 are critical for Wnt signal transduction. The interaction of the Wnts with the receptors is regulated by two classes of extracellular Wnt or LRP binding proteins called sFRP and Dickkopf (DKK), which modulate Wnt signaling. We have examined the expression of all Wnt family members both in the exocrine portion and in isolated islets of adult human pancreas. RT-PCR analysis of the 1-day cultured exocrine pellet fraction from the islet isolation procedure showed that Wnt 2, 2b, 3, 4, 5a, 5b, 7a, 7b, 14, and 15 were detectable. All 10 Frizzled (Frz) receptors were expressed but only Frizzled 1, 2, 4, 5, and 6 strongly. RT-PCR performed on purified human islets revealed that Wnt 2b, 3, 4, 5a, 7b, 10a, and 14 and Frz 4, 5, and 6 were the most highly expressed. DKK 1, 3, and 4 as well as sFRP 1, 4, and 5 were expressed in the exocrine fraction. sFRP 2 and 3 were detectable but only at low levels. In situ hybridization for Frz 1-7 showed that expression colocalized with the islets of Langerhans. Together the data suggest that active Wnt signaling occurs in adult pancreas and is probably important for physiological functions.
Asunto(s)
Expresión Génica , Péptidos y Proteínas de Señalización Intercelular/genética , Proteínas de la Membrana/genética , Páncreas/metabolismo , Proteínas/genética , Proteínas Proto-Oncogénicas/genética , Proteínas de Pez Cebra , Adolescente , Adulto , Animales , Femenino , Receptores Frizzled , Glucagón/metabolismo , Humanos , Hibridación in Situ , Insulina/metabolismo , Péptidos y Proteínas de Señalización Intercelular/metabolismo , Islotes Pancreáticos/metabolismo , Masculino , Proteínas de la Membrana/metabolismo , Persona de Mediana Edad , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , Proteínas/metabolismo , Proteínas Proto-Oncogénicas/metabolismo , ARN/genética , ARN/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Proteínas Wnt , Proteína wnt2RESUMEN
The cis-acting elements that regulate Eomes transcription during embryonic development are largely unknown. Here we identify a conserved cis-acting region (EoIV) located ~20kb upstream of the Eomes coding region that faithfully drives reporter expression to sites of Eomes expression during gastrulation. Transgenic EoIV-hsp68-GFP expression was evident in the epiblast of early-streak stage mouse embryos at the site where the primitive streak is initiated. At the mid- and late-streak stages, EoIV-hsp68-GFP expression was found in the streak, node region and definitive endoderm with a particular intensive GFP expression in the node region. At the early head fold stage, GFP was expressed in the node region and the surrounding endoderm. In contrast to earlier reports of Eomes mRNA expression, we confirmed Eomes protein expression in the node of early head fold embryos by immunohistochemistry. In vitro, EoIV-hsp68-GFP expression was activated ES cells differentiating into primitive streak-like progeny in response to Bmp and activin treatment.
Asunto(s)
Mesodermo/metabolismo , Línea Primitiva/metabolismo , Proteínas de Dominio T Box/metabolismo , Activinas/farmacología , Animales , Diferenciación Celular , Células Cultivadas , Secuencia Conservada , Embrión de Mamíferos/citología , Embrión de Mamíferos/metabolismo , Endodermo/citología , Endodermo/metabolismo , Gastrulación , Regulación del Desarrollo de la Expresión Génica , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Inmunohistoquímica , Mesodermo/citología , Mesodermo/embriología , Ratones , Línea Primitiva/citología , Línea Primitiva/embriología , ARN Mensajero/genética , ARN Mensajero/metabolismo , Proteínas de Dominio T Box/genética , Transcripción GenéticaRESUMEN
Extracellular signals in development, physiology, homeostasis and disease often act by regulating transcription. Herein we describe a general method and specific resources for determining where and when such signaling occurs in live animals and for systematically comparing the timing and extent of different signals in different cellular contexts. We used recombinase-mediated cassette exchange (RMCE) to test the effect of successively deleting conserved genomic regions of the ubiquitously active Rosa26 promoter and substituting the deleted regions for regulatory sequences that respond to diverse extracellular signals. We thereby created an allelic series of embryonic stem cells and mice, each containing a signal-responsive sentinel with different fluorescent reporters that respond with sensitivity and specificity to retinoic acids, bone morphogenic proteins, activin A, Wnts or Notch, and that can be adapted to any pathway that acts via DNA elements.
Asunto(s)
Células Madre Embrionarias/metabolismo , Mutación/genética , Regiones Promotoras Genéticas , Transducción de Señal/genética , Transcripción Genética , Activinas/genética , Activinas/metabolismo , Animales , Secuencia de Bases , Proteínas Morfogenéticas Óseas/genética , Proteínas Morfogenéticas Óseas/metabolismo , Embrión de Mamíferos/efectos de los fármacos , Embrión de Mamíferos/metabolismo , Células Madre Embrionarias/efectos de los fármacos , Ingeniería Genética , Sitios Genéticos/genética , Humanos , Ratones , Datos de Secuencia Molecular , Proteínas/genética , ARN no Traducido , Ratas , Receptores Notch/genética , Receptores Notch/metabolismo , Recombinación Genética/genética , Elementos de Respuesta/genética , Eliminación de Secuencia/genética , Transducción de Señal/efectos de los fármacos , Transcripción Genética/efectos de los fármacos , Tretinoina/farmacología , Vía de Señalización Wnt/efectos de los fármacos , Vía de Señalización Wnt/genéticaRESUMEN
Embryonic germ cells (EGC) are cultured pluripotent cells derived from primordial germ cells (PGC). This study explored the possibility of establishing porcine EGC from domestic breeds and Yucatan mini pigs using embryos at Days 17-24 of gestation. In vitro culture of PGC from both pooled and individual embryos resulted in the successful derivation of putative EGC lines from Days 20 to 24 with high efficiency. RT-PCR showed that gene expression among all 31 obtained cell lines was very similar, and only minor changes were detected during in vitro passaging of the cells. Genome-wide RNA-Seq expression profiling showed no expression of the core pluripotency markers OCT4, SOX2, and NANOG, although most other pluripotency genes were expressed at levels comparable to those of mouse embryonic stem cells (ESC). Moreover, germ-specific genes such as BLIMP1 retained their expression. Functional annotation clustering of the gene expression pattern of the putative EGC suggests partial differentiation toward endo/mesodermal lineages. The putative EGC were able to form embryoid bodies in suspension culture and to differentiate into epithelial-like, mesenchymal-like, and neuronal-like cells. However, their injection into immunodeficient mice did not result in teratoma formation. Our results suggest that the PGC-derived cells described in this study are EGC-like, but seem to be multipotent rather than pluripotent cells. Nevertheless, the thorough characterization of these cells in this study, and especially the identification of various genes and pathways involved in pluripotency by RNA-Seq, will serve as a rich resource for further derivation of porcine EGC.
Asunto(s)
Técnicas de Cultivo de Célula/métodos , Diferenciación Celular , Células Madre Embrionarias/citología , Animales , Células Cultivadas , Embrión de Mamíferos , Células Madre Embrionarias/metabolismo , Femenino , Regulación del Desarrollo de la Expresión Génica , Ratones , Embarazo , Porcinos , Porcinos Enanos , Factores de Transcripción/genética , Factores de Transcripción/metabolismoRESUMEN
OBJECTIVES: We aimed to characterize a serum-free culture system resulting in highly enriched duct cells from human exocrine pancreas. In addition, we tested the effect of vascular endothelial growth factor (VEGF) on endothelial cell proliferation and endocrine differentiation of the duct cells. METHODS: The exocrine pellet fraction was cultivated in suspension followed by monolayer culture. Time course analysis of multiple acinar and duct cell markers was performed using reverse transcription-polymerase chain reaction and immunocytochemistry. The effects of VEGF and placental growth factor on the quantities of endothelial, duct, and endocrine cells and fibroblasts were investigated using computerized imaging analysis. RESULTS: Suspension culture of the exocrine material efficiently enriched the cultures for duct cells. Frequent acinar cell death as well as cell selective adherence of acinar cells to the culture dish was the underlying cause of the enrichment. Confocal microscopy demonstrated the virtual absence of cells coexpressing duct cell- and acinar cell-specific markers. The endothelial immunoreactivity of the suspension culture system could be increased 2-fold by VEGF treatment, yet no effect was observed on endocrine cell numbers. CONCLUSIONS: We have characterized a serum-free in vitro culture system to enrich human duct cells and further show that the contribution of acinoductal transdifferentiation to the enrichment of duct cells is negligible.
Asunto(s)
Proliferación Celular , Transdiferenciación Celular , Medio de Cultivo Libre de Suero/metabolismo , Páncreas Exocrino/citología , Conductos Pancreáticos/citología , Adulto , Apoptosis , Biomarcadores/metabolismo , Adhesión Celular , Células Cultivadas , Técnicas de Cocultivo , Células Endoteliales/metabolismo , Fibroblastos/metabolismo , Humanos , Persona de Mediana Edad , Páncreas Exocrino/metabolismo , Conductos Pancreáticos/metabolismo , Factor de Crecimiento Placentario , Proteínas Gestacionales/metabolismo , Factores de Tiempo , Factor A de Crecimiento Endotelial Vascular/metabolismoRESUMEN
Human postnatal pancreatic duct cells are a potential source of new beta cells. Factors regulating proliferation of human pancreatic duct cells in vitro are unknown. In several other cell types, this process is influenced by ligands of the ErbB receptor family. The expression and functionality of the ErbB family members and their possible role in duct cell proliferation were determined. In cultured adult human pancreatic duct cells the different members of the ErbB family (ErbB1-4) were present at transcript and protein level. Stimulation of the duct cells with epidermal growth factor (EGF) and betacellulin results in Tyr-phosphorylation of ErbB1 and ErbB2, followed by activation of Shc, MEK1/2 and ERK1/2. Duct cells with activated ErbB signaling changed morphology and motility. EGF induced proliferation of a fraction of the duct cells and treatment with PD98059 prevented Ki67 expression in EGF-supplemented cells. When transduced with recombinant adenovirus expressing constitutively activated MEK1, duct cells proliferate and spread even in the absence of EGF. Importantly, the adult human duct cells retain their capacity to recapitulate ngn3-induced embryonic (neuro)endocrine differentiation after proliferation. Therefore, the present data support a possible role for human adult pancreatic duct cells, following expansion and transdifferentiation, as a source of insulin by transplantation to type I diabetes patients.