RESUMEN
BACKGROUND & AIMS: The differentiation of stem cells to hepatocyte-like cells (HLC) offers the perspective of unlimited supply of human hepatocytes. However, the degree of differentiation of HLC remains controversial. To obtain an unbiased characterization, we performed a transcriptomic study with HLC derived from human embryonic and induced stem cells (ESC, hiPSC) from three different laboratories. METHODS: Genome-wide gene expression profiles of ESC and HLC were compared to freshly isolated and up to 14days cultivated primary human hepatocytes. Gene networks representing successful and failed hepatocyte differentiation, and the transcription factors involved in their regulation were identified. RESULTS: Gene regulatory network analysis demonstrated that HLC represent a mixed cell type with features of liver, intestine, fibroblast and stem cells. The "unwanted" intestinal features were associated with KLF5 and CDX2 transcriptional networks. Cluster analysis identified highly correlated groups of genes associated with mature liver functions (n=1057) and downregulated proliferation associated genes (n=1562) that approach levels of primary hepatocytes. However, three further clusters containing 447, 101, and 505 genes failed to reach levels of hepatocytes. Key TF of two of these clusters include SOX11, FOXQ1, and YBX3. The third unsuccessful cluster, controlled by HNF1, CAR, FXR, and PXR, strongly overlaps with genes repressed in cultivated hepatocytes compared to freshly isolated hepatocytes, suggesting that current in vitro conditions lack stimuli required to maintain gene expression in hepatocytes, which consequently also explains a corresponding deficiency of HLC. CONCLUSIONS: The present gene regulatory network approach identifies key transcription factors which require modulation to improve HLC differentiation.
Asunto(s)
Células Madre Embrionarias/citología , Hepatocitos/citología , Células Madre Pluripotentes Inducidas/citología , Hígado/metabolismo , ARN/genética , Factores de Transcripción/genética , Transcriptoma , Diferenciación Celular , Células Cultivadas , Células Madre Embrionarias/metabolismo , Redes Reguladoras de Genes , Hepatocitos/metabolismo , Humanos , Células Madre Pluripotentes Inducidas/metabolismo , Hígado/citología , Factores de Transcripción/biosíntesisRESUMEN
Objetivo: Demostrar que el óxido nítrico producido por el hígado juega un papel protector en este órgano durante episodios de sepsis. Diseño: Estudio experimental en animales, prospectivo con grupo control. Material y métodos: Se utilizaron ratas Sprague-Dawley machos con peso entre 200 y 250 g. Para experimento in vivo, los animales recibieron una sola inyección de 28 mg/kg/IV de Corynebacterium parvum; ratas normales se emplearon como controles y donadores de hepatocitos para los estudios in vitro. Los hepatocitos normales y estimulados con C. parvum fueron aislados utilizando una modificación a la técnica de perfusión de colagenasa in situ; fueron colocados en platos de Petri con una capa de gelatina de 100 mm en 6 ml de medio de cultivo que contenía: L-arginina, insulina, L-glutaminam, penicilina, streptomicina y 10 por ciento de suero bajo en endotoxina. Después e 24 h en cultivo los hepatocitos recibieron medio frasco adicionado de citoquinas (INF+TNF+IL-1) y endotoxina (LPS para estimular la producción de óxido nítrico in vitro. Los sobrenadantes fueron colectados, almacenados y sujetos a medición de NO2 + NO3 empleando un método automático colorimétrico