Direct reprogramming hepatocytes into islet-like cells by efficiently targeting and activating the endogenous genes

doi:10.3969/j.issn.2095-4344.2172

Chinese Journal of Tissue Engineering Research ›› 2021, Vol. 25 ›› Issue (7): 1056-1063.doi: 10.3969/j.issn.2095-4344.2172

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Direct reprogramming hepatocytes into islet-like cells by efficiently targeting and activating the endogenous genes

Wang Hanyue1, 2, Li Furong2, Yang Xiaofei1, 2, Hu Chaofeng1

1Department of Pathology and Pathophysiology, School of Medicine, Jinan University, Guangzhou 510632, Guangdong Province, China; 2Translational Medicine Collaborative Innovation Center, Second Clinical Medical College (Shenzhen People’s Hospital) of Jinan University, Shenzhen 518020, Guangdong Province, China

Received:2020-01-19 Revised:2020-01-20 Accepted:2020-03-13 Online:2021-03-08 Published:2020-12-08
Contact: Hu Chaofeng, MD, Professor, Department of Pathology and Pathophysiology, School of Medicine, Jinan University, Guangzhou 510632, Guangdong Province, China Yang Xiaofei, MD, Assistant researcher, Department of Pathology and Pathophysiology, School of Medicine, Jinan University, Guangzhou 510632, Guangdong Province, China; Translational Medicine Collaborative Innovation Center, Second Clinical Medical College (Shenzhen People’s Hospital) of Jinan University, Shenzhen 518020, Guangdong Province, China
About author:Wang Hanyue, Mater candidate, Department of Pathology and Pathophysiology, School of Medicine, Jinan University, Guangzhou 510632, Guangdong Province, China; Translational Medicine Collaborative Innovation Center, Second Clinical Medical College (Shenzhen People’s Hospital) of Jinan University, Shenzhen 518020, Guangdong Province, China
Supported by:
the National Natural Science Foundation for the Youth of China, No. 81800685; the National Natural Science Foundation of China, No. 81670702; the National Natural Science Foundation for the Youth of Guangdong Province, No. 2018A030310039; the Free Exploration Project of Basic Research of Science and Technology Innovation Commission of Shenzhen, No. JCYJ20170307100154602

Abstract

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.

Key words: somatic cell, islet-like cell, liver, cell gene transfer, diabetes, CRISPR/dCas9, factor, protein, pathway

CLC Number:

Wang Hanyue, Li Furong, Yang Xiaofei, Hu Chaofeng. Direct reprogramming hepatocytes into islet-like cells by efficiently targeting and activating the endogenous genes[J]. Chinese Journal of Tissue Engineering Research, 2021, 25(7): 1056-1063.

Figures/Tables 5

2.2 筛选针对Pdx1，Ngn3，MafA启动子区域的最佳gRNA 为了筛选Casilio体系中激活作用最佳的gRNA，将dCas9、PUFa-P65-HSF1表达质粒与分别针对Pdx1，Ngn3及MafA启动子区域的4条gRNA的表达质粒按照1∶1∶1的比例单独或联合转染293T和HepG2细胞。如图6所示，在293T细胞系中Pdx1基因的gRNA3上调26倍 (P < 0.000 1)，gRNA2上调16倍(P < 0.000 1)，在4条gRNA中上调倍数最高；针对Ngn3基因的gRNA3和gRNA2上调倍数最高，分别上调501倍(P=0.000 8)和398倍(P=0.001 0)；MafA基因gRNA2和gRNA4上调倍数最高，分别上调3倍和2倍。在HepG2细胞系中针对Pdx1基因的gRNA4和 gRNA3上调倍数最高，分别上调223倍(P=0.000 4)和82倍(P=0.000 3)；针对Ngn3基因的gRNA3和 gRNA2上调倍数最高，分别上调 234倍(P=0.000 1) 和95倍(P=0.001 1)。针对MafA基因的gRNA2和gRNA4上调倍数最高，分别上调6倍和5倍。针对Pdx1，Ngn3和MafA基因的4条gRNA在293T和HepG2细胞中均有协同效应，即4条gRNA同时转染的激活效率高于单个gRNA转染。 2.3 Ins-EGFP-HepG2-dCas9-PUFa-p65-HSF1稳转细胞系的构建及鉴定为了验证PNM的重编程效率，利用慢病毒构建针对Insulin基因启动子的绿色荧光报告基因(EGFP)细胞系，用以标记胰岛素基因表达阳性的细胞，见图7A，结果显示EGFP报告基因插入稳转细胞系基因组中。由于dCas9及PUFa-p65-HSF1序列较长，质粒的表达效率低，因此，利用PB转座子体系在Ins-EGFP-HepG2细胞系的基础上，构建了稳定表达dCas9和PUFa-P65-HSF1的Ins-EGFP-HepG2-dCas9-PUFa-p65-HSF1稳转细胞系，PB携带的dCas9和Activator载体标签，见图7B。免疫印迹和免疫荧光结果均显示，经过筛选及多西环素诱导后的稳转细胞系中有dCas9和P65-HSF1蛋白的表达，见图7C-F，表明PB转座子成功实现了dCas9，P65-HSF1片段在HepG2肝细胞系基因组的整合，并且稳转细胞系构建过程中未见细胞形态的明显改变。稳定表达dCas9和PUFa-p65-HSF1蛋白的稳转细胞系只需导入gRNA即可实现内源基因激活作用。"

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Direct reprogramming hepatocytes into islet-like cells by efficiently targeting and activating the endogenous genes

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