技术资料

Induced pluripotent stem cell (iPSC)-derived retinal pigment epithelium (RPE) has widely been appreciated as a promising tool to model human ocular disease emanating from primary RPE pathology. Here,we describe the successful reprogramming of adult human dermal fibroblasts to iPSCs and their differentiation to pure expandable RPE cells with structural and functional features characteristic for native RPE. Fibroblast cultures were established from skin biopsy material and subsequently reprogrammed following polycistronic lentiviral transduction with OCT4,SOX2,KLF4 and L-Myc. Fibroblast-derived iPSCs showed typical morphology,chromosomal integrity and a distinctive stem cell marker profile. Subsequent differentiation resulted in expandable pigmented hexagonal RPE cells. The cells revealed stable RNA expression of mature RPE markers RPE65,RLBP and BEST1. Immunolabelling verified localisation of BEST1 at the basolateral plasma membrane,and scanning electron microscopy showed typical microvilli at the apical side of iPSC-derived RPE cells. Transepithelial resistance was maintained at high levels during cell culture indicating functional formation of tight junctions. Secretion capacity was demonstrated for VEGF-A. Feeding of porcine photoreceptor outer segments revealed the proper ability of these cells for phagocytosis. IPSC-derived RPE cells largely maintained these properties after cryopreservation. Together,our study underlines that adult dermal fibroblasts can serve as a valuable resource for iPSC-derived RPE with characteristics highly reminiscent of true RPE cells. This will allow its broad application to establish cellular models for RPE-related human diseases. View Publication

BACKGROUND Induced pluripotent stem cells (iPSCs) hold tremendous potential,both as a biological tool to uncover the pathophysiology of disease by creating relevant human cell models and as a source of cells for cell-based therapeutic applications. Studying the reprogramming process will also provide significant insight into tissue development. OBJECTIVE We sought to characterize the derivation of iPSC lines from nasal epithelial cells (NECs) isolated from nasal mucosa samples of children,a highly relevant and easily accessible tissue for pediatric populations. METHODS We performed detailed comparative analysis on the transcriptomes and methylomes of NECs,iPSCs derived from NECs (NEC-iPSCs),and embryonic stem cells (ESCs). RESULTS NEC-iPSCs express pluripotent cell markers,can differentiate into all 3 germ layers in vivo and in vitro,and have a transcriptome and methylome remarkably similar to those of ESCs. However,residual DNA methylation marks exist,which are differentially methylated between NEC-iPSCs and ESCs. A subset of these methylation markers related to epithelium development and asthma and specific to NEC-iPSCs persisted after several passages in vitro,suggesting the retention of an epigenetic memory of their tissue of origin. Our analysis also identified novel candidate genes with dynamic gene expression and DNA methylation changes during reprogramming,which are indicative of possible roles in airway epithelium development. CONCLUSION NECs are an excellent tissue source to generate iPSCs in pediatric asthmatic patients,and detailed characterization of the resulting iPSC lines would help us better understand the reprogramming process and retention of epigenetic memory. View Publication

Not provided. View Publication

Type I and type III interferons (IFNs) are crucial components of the first-line antiviral host response. While specific receptors for both IFN types exist,intracellular signaling shares the same Jak-STAT pathway. Due to its receptor expression,IFN-λ responsiveness is restricted mainly to epithelial cells. Here,we display IFN-stimulated gene induction at the single cell level to comparatively analyze the activities of both IFN types in intestinal epithelial cells and mini-gut organoids. Initially,we noticed that the response to both types of IFNs at low concentrations is based on a single cell decision-making determining the total cell intrinsic antiviral activity. We identified histone deacetylase (HDAC) activity as a crucial restriction factor controlling the cell frequency of IFN-stimulated gene (ISG) induction upon IFN-λ but not IFN-β stimulation. Consistently,HDAC blockade confers antiviral activity to an elsewise non-responding subpopulation. Second,in contrast to the type I IFN system,polarization of intestinal epithelial cells strongly enhances their ability to respond to IFN-λ signaling and raises the kinetics of gene induction. Finally,we show that ISG induction in mini-gut organoids by low amounts of IFN is characterized by a scattered heterogeneous responsiveness of the epithelial cells and HDAC activity fine-tunes exclusively IFN-λ activity. This study provides a comprehensive description of the differential response to type I and type III IFNs and demonstrates that cell polarization in gut epithelial cells specifically increases IFN-λ activity. View Publication