Project description:This SuperSeries is composed of the following subset Series: GSE24979: MicroRNA-145 Regulates Human Corneal Epithelial Differentiation [Agilent-016436 array data] GSE24980: MicroRNA-145 Regulates Human Corneal Epithelial Differentiation [Agilent-014850 array data] Refer to individual Series
Project description:To investigate the gene expression in human corneal epithelial overexpressing hsa-miR-145 by transfection , we have employed Whole Human Genome Oligo Microarray (Agilent) as a screening platform to identify gene regulation. We discovered a differential gene expression in HCE cells transfected with hsa-mIR-145 against cells with scrambled sequences. Among them, genes related with corneal development, integrity, differentiation and inflammatory responses were found and this was validated by real-time PCR.
Project description:To investigate the microRNA expression in human limbal-peripheral corneal (LPC) epithelia containing corneal epithelial progenitor cells (CEPCs) and early transit amplifying cells, we have employed Human microRNA Microarray V2 (Agilent) as a screening platform to identify specific microRNAs. We discovered a differential expression of 18 microRNAs against central corneal (CC) epithelia, which contains late transit amplifying cells and terminally differentiated cells. Among them, cluster miR-143/145 was expressed strongly in LPC but at low levels in CC epithelia and this was validated by real-time PCR and locked nucleic acid-based in situ hybridization.
Project description:To investigate the microRNA expression in human limbal-peripheral corneal (LPC) epithelia containing corneal epithelial progenitor cells (CEPCs) and early transit amplifying cells, we have employed Human microRNA Microarray V2 (Agilent) as a screening platform to identify specific microRNAs. We discovered a differential expression of 18 microRNAs against central corneal (CC) epithelia, which contains late transit amplifying cells and terminally differentiated cells. Among them, cluster miR-143/145 was expressed strongly in LPC but at low levels in CC epithelia and this was validated by real-time PCR and locked nucleic acid-based in situ hybridization. LPC and CC epithelia, separated by 1-mm in width, were dissected from human cornea for small RNA extraction. Total RNA was extracted by Trizol/chloroform and purified with RNeasy mini spin column. RNA samples with 28S/18S ratios in the range of 1.4 to 1.8 were used for microRNA profiling using an Agilent Human microRNA Microarray V2 platform.
Project description:To investigate the gene expression in human corneal epithelial overexpressing hsa-miR-145 by transfection , we have employed Whole Human Genome Oligo Microarray (Agilent) as a screening platform to identify gene regulation. We discovered a differential gene expression in HCE cells transfected with hsa-mIR-145 against cells with scrambled sequences. Among them, genes related with corneal development, integrity, differentiation and inflammatory responses were found and this was validated by real-time PCR. HCE cells transfected with hsa-miR-145 or scrambled sequences were collected at 24 hours after transfection. Total RNA was extracted by Trizol/chloroform and purified with RNeasy mini spin column. RNA samples with 28S/18S ratios in the range of 1.4 to 1.8 were used for expression RNA profiling using Whole Human Genome Oligo Microarray (Agilent).
Project description:Transplantation of ex vivo expanded limbal stem cells (LSC) is the main treatment for limbal stem cell deficiency though the clinical problem of donor tissues shortage. Recently, as the development of tissue engineering, embryonic stem cells (ESC) derived corneal epithelial-like cells (ESC-CEC) has become a new direction to this issue.Our group successfully induced ESC into corneal epithelial-like cells, and in the present study we explored various aspects of physiological properties of ESC-CEC. The experiment included three samples: hES, the human embryonic stem cell line H1, RA_SB, the corneal epithelial-like cells derived from hES by differentiation with RA and SB, epithelial_cell, the primary human limbal stem cells from cadaver eyes. hES, the human embryonic stem cell line H1, RA_SB, the corneal epithelial-like cells derived from hES by differentiation with RA and SB, epithelial_cell, the primary human limbal stem cells from cadaver eyes.
Project description:Corneal epithelial cells derived from hPSCs provides an important cells source for the construction of the in vitro preclinical models aimed for ophthalmic drugs tests. However, the recent differentiation protocols lack the optimal culturing conditions that hinder the robustness and the quality of cells as well as the scale-up application of cells production. Here we introduce a simplified, yet efficient small molecules-based corneal induction method (SSM-CI) for the generation of corneal epithelial cells from hPSCs. SSM-CI provides the advantage of minimization the cells culturing time and steps using only two defined xenobiotic-free and serum-free culturing mediums in combination with the TGFß pathway, Wnt/ß-catenin pathway signaling chemical inhibitors, and human bFGF growth factor for a period of 25 days. Compared to both conventional human corneal epithelial cell line (HCE-T) as well as the human primary corneal epithelial cells (hPCEpC), human embryonic stem cells derived corneal epithelial cells generated by SSM-CI has highly expressed major differentiation as well as maturation markers such as PAX6 and CK12. RNA-seq analysis indicated the genuine diversion of hPSCs into the corneal epithelium lineage where corneal progenitor and adult corneal epithelial phenotypes were significantly upregulated. Furthermore, despite the inhibition of TGF-β and Wnt/β-catenin at the early stage of differentiation, an upregulation of the TGF-β and Wnt/β-catenin pathways related transcripts we noticed in the late stage which indicated the necessity of these pathways in the generation of mature corneal epithelial cells. Moreover, there was a shift in gene signatures associated with the metabolic characteristics of mature corneal epithelial cells where a decrease of glycolysis related transcripts and an increase in fatty acid oxidation related one was noticed. That was also corresponded by the overexpression of metabolic enzymes and transporters related transcripts that were mainly responsible for the metabolism of fatty acids. Thus SSM-CI provide a comprehensive method for the generation of functional corneal epithelial cells that has the potential for the employment in future preclinical models.
Project description:Exposure to tear fluid can enhance corneal epithelial cells' ability to resist bacterial virulence mechanisms by upregulating epithelial-derived innate defense genes. The aim of this study was to further elucidate the mechanisms by which tear fluid modulates epithelial cell susceptibility to P. aeruginosa internalization and the relationship to known to be upregulated genes with tear fluid exposure. The hypothesis tested was that tear fluid effects on epithelial cells involve the induction of microRNA expression to modify innate defense gene responses to bacterial challenge. Human corneal epithelial cells were incubated in either 40 ul of fresh human tear fluid or high calcium KGM without antibiotics for 16 hours before extraction or before incubation with P. aeruginosa antigens for 3 h then extraction.