RESUMEN
Reprogramming of somatic cells to induced pluripotent stem cells (iPSC) provides an important cell source to derive patient-specific cells for potential therapeutic applications. However, it is not yet clear whether reprogramming through pluripotency allows the production of differentiated cells with improved functional properties that may be beneficial in regenerative therapies. To address this, we compared the production and assembly of extracellular matrix (ECM) by iPSC-derived fibroblasts to that of the parental, dermal fibroblasts (BJ), from which these iPSC were initially reprogrammed, and to fibroblasts differentiated from human embryonic stem cells (hESC). iPSC- and hESC-derived fibroblasts demonstrated stable expression of surface markers characteristic of stromal fibroblasts during prolonged culture and showed an elevated growth potential when compared to the parental BJ fibroblasts. We found that in the presence of L: -ascorbic acid-2-phosphate, iPSC- and hESC-derived fibroblasts increased their expression of collagen genes, secretion of soluble collagen, and extracellular deposition of type I collagen to a significantly greater degree than that seen in the parental BJ fibroblasts. Under culture conditions that enabled the self-assembly of a 3D stromal tissue, iPSC- and hESC-derived fibroblasts generated a well organized, ECM that was enriched in type III collagen. By characterizing the functional properties of iPSC-derived fibroblasts compared to their parental fibroblasts, we demonstrate that these cells represent a promising, alternative source of fibroblasts to advance future regenerative therapies.
Asunto(s)
Diferenciación Celular , Células Madre Embrionarias/metabolismo , Proteínas de la Matriz Extracelular/metabolismo , Fibroblastos/metabolismo , Células Madre Pluripotentes Inducidas/metabolismo , Ácido Ascórbico/análogos & derivados , Ácido Ascórbico/farmacología , Biomarcadores/metabolismo , Técnicas de Cultivo de Célula , Células Cultivadas , Colágeno/biosíntesis , Células Madre Embrionarias/citología , Matriz Extracelular/metabolismo , Fibroblastos/citología , Humanos , Células Madre Pluripotentes Inducidas/citologíaRESUMEN
A common integration site, cloned from MoMuLV-induced rat T cell lymphomas, was mapped immediately upstream of Not dead yet-1 (Ndy1)/KDM2B, a gene expressed primarily in testis, spleen, and thymus, that is also known as FBXL10 or JHDM1B. Ndy1 encodes a nuclear, chromatin-associated protein that harbors Jumonji C (JmjC), CXXC, PHD, proline-rich, F-box, and leucine-rich repeat domains. Ndy1 and its homolog Ndy2/KDM2A (FBXL11 or JHDM1A), which is also a target of provirus integration in retrovirus-induced lymphomas, encode proteins that were recently shown to possess Jumonji C-dependent histone H3 K36 dimethyl-demethylase or histone H3 K4 trimethyl-demethylase activities. Here, we show that mouse embryo fibroblasts engineered to express Ndy1 or Ndy2 undergo immortalization in the absence of replicative senescence via a JmjC domain-dependent process that targets the Rb and p53 pathways. Knockdown of endogenous Ndy1 or expression of JmjC domain mutants of Ndy1 promote senescence, suggesting that Ndy1 is a physiological inhibitor of senescence in dividing cells and that inhibition of senescence depends on histone H3 demethylation.
Asunto(s)
Transformación Celular Neoplásica , Embrión de Mamíferos/química , Proteínas Oncogénicas/fisiología , Secuencia de Aminoácidos , Animales , Animales Recién Nacidos , Northern Blotting , Western Blotting , Transformación Celular Viral , Fibroblastos/citología , Linfoma de Células T/patología , Linfoma de Células T/virología , Ratones , Datos de Secuencia Molecular , Virus de la Leucemia Murina de Moloney/fisiología , Ratas , Ratas Endogámicas F344 , Proteína de Retinoblastoma/metabolismo , Proteína p53 Supresora de Tumor/metabolismoRESUMEN
Pre-TCR signals regulate the transition of the double-negative (DN) 3 thymocytes to the DN4, and subsequently to the double-positive (DP) stage. In this study, we show that pre-TCR signals activate Akt and that pharmacological inhibition of the PI3K/Akt pathway, or combined ablation of Akt1 and Akt2, and to a lesser extent Akt1 and Akt3, interfere with the differentiation of DN3 and the accumulation of DP thymocytes. Combined ablation of Akt1 and Akt2 inhibits the proliferation of DN4 cells, while combined ablation of all Akt isoforms also inhibits the survival of all the DN thymocytes. Finally, the combined ablation of Akt1 and Akt2 inhibits the survival of DP thymocytes. Constitutively active Lck-Akt1 transgenes had the opposite effects. We conclude that, following their activation by pre-TCR signals, Akt1, Akt2, and, to a lesser extent, Akt3 promote the transition of DN thymocytes to the DP stage, in part by enhancing the proliferation and survival of cells undergoing beta-selection. Akt1 and Akt2 also contribute to the differentiation process by promoting the survival of the DP thymocytes.
Asunto(s)
Proteínas Proto-Oncogénicas c-akt/fisiología , Receptores de Antígenos de Linfocitos T/inmunología , Linfocitos T/inmunología , Timo/inmunología , Animales , Proliferación Celular , Supervivencia Celular , Expresión Génica , Activación de Linfocitos/genética , Ratones , Ratones Noqueados , Fosfatidilinositol 3-Quinasas/metabolismo , Inhibidores de las Quinasa Fosfoinosítidos-3 , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Isoformas de Proteínas/fisiología , Proteínas Proto-Oncogénicas c-akt/genética , Proteínas Proto-Oncogénicas c-akt/metabolismo , Timo/citología , TransgenesRESUMEN
T cell differentiation in the thymus is dependent upon signaling through the TCR and is characterized by the resulting changes in expression patterns of CD4 and CD8 surface coreceptor molecules. Although recent studies have characterized the effects of proximal TCR signaling on T cell differentiation, the downstream integration of these signals remains largely unknown. The growth factor independence-1 (GFI1) and GFI1B transcriptional repressors may regulate cytokine signaling pathways to affect lymphocyte growth and survival. In this study, we show that Gfi1 expression is induced upon induction of the T cell program. Gfi1B expression is low and dynamic during T cell development, but is terminated in mature thymocytes. Transgenic expression of GFI1 and GFI1B in T cells allowed us to determine the functional consequences of constitutive expression. GFI1 potentiates response to TCR stimulation and IL-2, whereas GFI1B-transgenic T cells are defective in T cell activation. Moreover, GFI1B-transgenic thymocytes display reduced expression of the late-activation marker IL-7R alpha, and a decrease in CD4(-)8(+) single-positive T cells that can be mitigated by transgenic expression of BCL2 or GFI1. These data show that GFI1 and GFI1B are functionally unique, and implicate a role for GFI1 in the integration of activation and survival signals.