RESUMEN
Endothelial cells (ECs) lining arteries and veins have distinct molecular/functional signatures. The underlying regulatory mechanisms are incompletely understood. Here, we established a specific fingerprint of freshly isolated arterial and venous ECs from human umbilical cord comprising 64 arterial and 12 venous genes, representing distinct functions/pathways. Among the arterial genes were 8 transcription factors (TFs), including Notch target HEY2, the current "gold standard" determinant for arterial EC (aEC) specification. Culture abrogated differential gene expression in part due to gradual loss of canonical Notch activity and HEY2 expression. Notably, restoring HEY2 expression or Delta-like4-induced Notch signaling in cultured ECs only partially reinstated the aEC gene signature, whereas combined overexpression of the 8 TFs restored this fingerprint more robustly. Whereas some TFs stimulated few genes, others boosted a large proportion of arterial genes. Although there was some overlap and cross-regulation, the TFs largely complemented each other in regulating the aEC gene profile. Finally, overexpression of the 8 TFs in human umbilical vein ECs conveyed an arterial-like behavior upon their implantation in a Matrigel plug in vivo. Thus, our study shows that Notch signaling determines only part of the aEC signature and identifies additional novel and complementary transcriptional players in the complex regulation of human arteriovenous EC identity.
Asunto(s)
Arterias/citología , Células Endoteliales/metabolismo , Factores de Transcripción/genética , Transcriptoma , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/genética , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/metabolismo , Western Blotting , Línea Celular , Células Cultivadas , Análisis por Conglomerados , Redes Reguladoras de Genes , Células Endoteliales de la Vena Umbilical Humana/metabolismo , Humanos , Modelos Genéticos , Análisis de Secuencia por Matrices de Oligonucleótidos , Interferencia de ARN , Receptores Notch/genética , Receptores Notch/metabolismo , Proteínas Represoras/genética , Proteínas Represoras/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Transducción de Señal/genética , Factores de Transcripción/metabolismoRESUMEN
We generated ATCi-MF1 induced pluripotent stem (iPS) cell line from Macaca fascicularis adult skin fibroblasts using non-integrative Sendai viruses carrying OCT3/4, KLF4, SOX2 and c-MYC. Once established, ATCi-MF1 cells present a normal karyotype, are Sendai virus-free and express pluripotency associated markers. Microsatellite markers analysis confirmed the origin of the iPS cells from the parental fibroblasts. Pluripotency was tested with the in vivo teratoma formation assay. ATCi-MF1 cell line may be a useful primate iPS cell model to test different experimental conditions where the use of human cells can imply ethical issues, as microinjection of pluripotent stem cells in pre-implantational embryos.
Asunto(s)
Fibroblastos/metabolismo , Células Madre Pluripotentes Inducidas/metabolismo , Virus Sendai , Piel/metabolismo , Factores de Transcripción , Transducción Genética , Animales , Línea Celular , Fibroblastos/citología , Células Madre Pluripotentes Inducidas/citología , Factor 4 Similar a Kruppel , Macaca fascicularis , Piel/citología , Factores de Transcripción/biosíntesis , Factores de Transcripción/genéticaRESUMEN
We generated two rat embryonic stem cell (ESC) lines: ATCe-SD7.8 from Sprague-Dawley strain and ATCe-WK1 from Wistar Kyoto strain. Cells were marked with enhanced green fluorescent protein (eGFP) by transduction with a lentiviral vector. Cells present a normal karyotype and express pluripotency-associated markers. Pluripotency was tested in vivo with the teratoma formation assay. Cells maintain eGFP expression upon differentiation to the three-germ layers. These cells can be a useful tool for cell therapy studies and chimera generation as they can be easily tracked by eGFP expression.
Asunto(s)
Separación Celular , Células Madre Embrionarias/metabolismo , Proteínas Fluorescentes Verdes/metabolismo , Animales , Células Madre Embrionarias/citología , Proteínas Fluorescentes Verdes/genética , Ratas , Ratas Endogámicas WKY , Ratas Sprague-Dawley , Ratas TransgénicasRESUMEN
We generated a rat iPSC line called ATCi-rSD95 from transgenic Sprague-Dawley GFP fetal fibroblasts. Established ATCi-rSD95 cells present a normal karyotype, silencing of the transgenes and express pluripotency-associated markers. Additionally, ATCi-rSD95 cells are able to form teratoma with differentiated cells derived from the three germ-layers that maintain the GFP expression.
Asunto(s)
Proteínas Fluorescentes Verdes/biosíntesis , Células Madre Pluripotentes Inducidas/metabolismo , Animales , Línea Celular , Proteínas Fluorescentes Verdes/genética , Células Madre Pluripotentes Inducidas/citología , Ratas , Ratas TransgénicasRESUMEN
Although transplantation of adipose-derived stem cells (ADSC) in chronic myocardial infarction (MI) models is associated with functional improvement, its therapeutic value is limited due to poor long-term cell engraftment and survival. Thus, the objective of this study was to examine whether transplantation of collagen patches seeded with ADSC could enhance cell engraftment and improve cardiac function in models of chronic MI. With that purpose, chronically infarcted Sprague-Dawley rats (n = 58) were divided into four groups and transplanted with media, collagen scaffold (CS), rat ADSC, or CS seeded with rat ADSC (CS-rADSC). Cell engraftment, histological changes, and cardiac function were assessed 4 months after transplantation. In addition, Göttingen minipigs (n = 18) were subjected to MI and then transplanted 2 months later with CS or CS seeded with autologous minipig ADSC (CS-pADSC). Functional and histological assessments were performed 3 months post-transplantation. Transplantation of CS-rADSC was associated with increased cell engraftment, significant improvement in cardiac function, myocardial remodeling, and revascularization. Moreover, transplantation of CS-pADSC in the pre-clinical swine model improved cardiac function and was associated with decreased fibrosis and increased vasculogenesis. In summary, transplantation of CS-ADSC resulted in enhanced cell engraftment and was associated with a significant improvement in cardiac function and myocardial remodeling.