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 metabolism and glycogen storage. Additionally, AAV6-based delivery of the CRISPRa SAM system effectively induced the hepatic reprogramming from fibroblasts in mice with live fibrosis. After 8 weeks of induction, the reprogrammed hepatocytes comprised 0.87% of the total hepatocyte population in the mice, significantly reducing liver fibrosis. Conclusion::CRISPRa-induced hepatocyte reprogramming may be a promising strategy for generating functional hepatocytes and treating liver fibrosis caused by hepatic diseases.

Objective To investigate the effect of the loss of myeloid-derived growth factor(Mydgf)on the transformation of cardiac fibroblasts into myofibroblasts after myocardial infarction(MI).Methods Two adult mouse groups,including a wild-type(WT)group and another group with Mydgf knockout(Mydgf-KO),were examined in the study.The mice in these two groups were tested for their cardiac function by measuring left ventricular ejection fraction(LVEF)and left ventricular fractional shortening(LVFS)(n=10).Quantitative real-time PCR(qRT-PCR)(n=3)was performed to determine the mRNA expression levels of myofibroblast markers,including α-smooth muscle actin(α-SMA),periostin(postn),type Ⅷ collagen(col8al),and connective tissue growth factor(ctgf).Western blot(n=3)was performed to verify the protein expression levels of α-SMA.MI modeling was performed on the WT and the Mydgf-KO mice.Postoperative LVEF and LVFS(n=10)were then measured.The hearts were harvested and Masson staining was performed to determine the infarcted area(n=10).The heart samples of Mydgf-KO and WT mice were collected at d 7 and d 14 after MI,respectively,to verify the expression of myofibroblast markers(n=3).Results Compared with WT mice,LVEF and LVFS in adult Mydgf-KO mice showed no significant changes(all P＞0.05).However,the mRNA levels of α-SMA and postn were upregulated,and α-SMA protein expression was also increased(all P＜0.05).After MI,compared with WT mice,LVEF and LVFS in Mydgf-KO mice decreased,and the infarcted area increased significantly(all P＜0.05).Furthermore,mRNA levels of α-SMA,col8al,postn,and ctgf were increased in Mydgf-KO mice.In addition,the α-SMA protein expression level was upregulated and α-SMA-positive fibroblasts were increased(P＜0.05).Conclusion Mydgf deletion promotes the transformation of cardiac fibroblasts into myofibroblasts and aggravates myocardial fibrosis after MI.