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A method for the generation of human stem cell-derived alpha cells

ABSTRACT: A method for the generation of human stem cell-derived alpha cells

PROVIDER: PRJNA577625 | ENA |

REPOSITORIES: ENA

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			Action	DRS
	SRR10278791.fastq.gz	Fastqsanger.gz
	SRR10278792.fastq.gz	Fastqsanger.gz

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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.

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A method for the generation of human stem cell-derived alpha cells

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