Direct reprogramming of fibroblasts into functional hepatocytes via CRISPRa activation of endogenous Gata4 and Foxa3  

作　　者：Jiacheng Li Ruopu Li Xue Bai Wenlong Zhang Yu Nie Shengshou Hu 

机构地区：[1]State Key Laboratory of Cardiovascular Disease,Fuwai Hospital and Cardiovascular Institute,National Center for Cardiovascular Diseases,Chinese Academy of Medical Sciences and Peking Union Medical College,Beijing 100037,China [2]Department of Obstetrics and Gynecology,Beijing Advanced Innovation Center for Genomics,Third Hospital,School of Life Sciences,Peking University,Beijing 100871,China

出　　处：《Chinese Medical Journal》2024年第11期1351-1359,共9页中华医学杂志（英文版）

基　　金：National Key Research and Development Program of China(No.2019YFA0801500);National High Level Hospital Clinical Research Funding(No.2023-GSP-ZD-2-01);Chinese Academy of Medical Sciences Innovation Fund for Medical Sciences(No.2021-I2M-1-008)

摘　　要：Background:The ability to generate functional hepatocytes without relying on donor liver organs holds significant therapeutic promise in the fields of regenerative medicine and potential liver disease treatments.Clustered regularly interspaced short palindromic repeats(CRISPR)activator(CRISPRa)is a powerful tool that can conveniently and efficiently activate the expression of multiple endogenous genes simultaneously,providing a new strategy for cell fate determination.The main purpose of this study is to explore the feasibility of applying CRISPRa for hepatocyte reprogramming and its application in the treatment of mouse liver fibrosis.Method:The differentiation of mouse embryonic fibroblasts(MEFs)into functional induced hepatocyte-like cells(iHeps)was achieved by utilizing the CRISPRa synergistic activation mediator(SAM)system,which drove the combined expression of three endogenous transcription factors-Gata4,Foxa3,and Hnf1a-or alternatively,the expression of two transcription factors,Gata4 and Foxa3.In vivo,we injected adeno-associated virus serotype 6(AAV6)carrying the CRISPRa SAM system into liver fibrotic Col1a1-Cre^(ER);Cas9^(fl/fl)mice,effectively activating the expression of endogenous Gata4 and Foxa3 in fibroblasts.The endogenous transcriptional activation of genes was confirmed using real-time quantitative polymerase chain reaction(RT-qPCR)and RNA-seq,and the morphology and characteristics of the induced hepatocytes were observed through microscopy.The level of hepatocyte reprogramming in vivo is detected by immunofluorescence staining,while the improvement of liver fibrosis is evaluated through Sirius red staining,alpha-smooth muscle actin(α-SMA)immunofluorescence staining,and blood alanine aminotransferase(ALT)examination.Results:Activation of only two factors,Gata4 and Foxa3,via CRISPRa was sufficient to successfully induce the transformation of MEFs into iHeps.These iHeps could be expanded in vitro and displayed functional characteristics similar to those of mature hepatocytes,such as drug metaboli

关 键 词：Liver fibrosis CRISPRa Cell reprogramming Transcriptional activation HEPATOCYTES 

分 类 号：R575[医药卫生—消化系统]