Direct reprogramming hepatocytes into islet-like cells by efficiently targeting and activating the endogenous genes / 中国组织工程研究

ABSTRACT

BACKGROUND: Islet cell transplantation is one of the most effective methods to treat diabetes. However, the shortage of transplanted cells has limited its clinical application. Direct reprogramming of hepatocytes to islet β cells in vitro is a new idea to solve this problem, but differentiation of hepatocytes to islet β cells is a complicated process. OBJECTIVE: Direct reprogramming hepatocytes into islet-like cells by efficient targeting and activating the endogenous genes of hepatocytes with Casilio system (constructed CRISPR/ Cas9-Pumilio system) through modifying guide RNA sequence combined with the transcriptional activator PUFa-P65-HSF1. METHODS: The endogenous PNM (Pdx1, Ngn3, MafA) genes of hepatocytes were targeted and activated by using the Casilio system, which was transfected to the HEK293T cell line by liposome transfection. The expression of endogenous PNM was detected by real-time fluorescence quantitative PCR and immunofluorescence. The Ins-EGFP cell line with stable expression of enhanced green fluorescent protein was constructed by the lentivirus carrying Ins-promoter-EGFP. The Ins-EGFP-HepG2-Cas9-PUFa-p65-HSF1 cell line (referred to as stable translocated cell lines) with stable expression of dCas9 and PUFa-P65-HSF1 in Ins-EGFP-HepG2 cell line was constructed by the PiggyBac(PB) transposon system. By using liposome, the gRNAs were transfected to stable translocated cell line, and the expression level of endogenous gene PNM was detected by real-time fluorescence quantitative PCR and immunofluorescence. Simultaneously, reprogramming efficiency was observed. RESULTS AND CONCLUSION: (1) The activation of endogenous genes by Casilio system was verified in 293T cell line. (2) The expression of EGFP, dCas9 and PUFa-P65-HSF1 in stable cell line was detected by RT-PCR, western blot assay and immunofluorescence. (3) The real-time fluorescence quantitative PCR results confirmed that this new Casilio system could target and activate the PNM which led to the up-regulation of the endogenous PNM gene expression. The efficiency of direct reprogramming was 10%-15%. (4) Based on CRISPR/dCas9, the new Casilio system can efficiently activate the endogenous PNM gene in hepatocytes, enabling the hepatocyte line to be directly reprogrammed as islet-like cells.