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.
Department of Obstetrics and Gynecology, Beijing Advanced Innovation Center for Genomics, Third Hospital, School of Life Sciences, Peking University, Beijing 100871, China.
Chin Med J (Engl). 2024 Jun 5;137(11):1351-1359. doi: 10.1097/CM9.0000000000003088. Epub 2024 May 9.
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.
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-CreER ; Cas9fl/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.
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.
CRISPRa-induced hepatocyte reprogramming may be a promising strategy for generating functional hepatocytes and treating liver fibrosis caused by hepatic diseases.
在再生医学和潜在肝脏疾病治疗领域，不依赖供体肝脏器官就能生成具有功能的肝细胞具有重要的治疗意义。规律成簇间隔短回文重复（CRISPR）激活（CRISPRa）是一种强大的工具，可以方便高效地同时激活多个内源性基因的表达，为细胞命运决定提供了新策略。本研究的主要目的是探索应用 CRISPRa 进行肝细胞重编程及其在治疗小鼠肝纤维化中的应用。
利用 CRISPRa 协同激活介体（SAM）系统将小鼠胚胎成纤维细胞（MEFs）分化为功能性诱导的肝样细胞（iHeps），该系统驱动三个内源性转录因子-Gata4、Foxa3 和 Hnf1a-的联合表达，或者表达两个转录因子 Gata4 和 Foxa3。在体内，我们将携带 CRISPRa SAM 系统的腺相关病毒血清型 6（AAV6）注射到 Col1a1-CreER；Cas9fl/fl 小鼠的肝纤维化中，有效地激活了成纤维细胞中内源性 Gata4 和 Foxa3 的表达。通过实时定量聚合酶链反应（RT-qPCR）和 RNA 测序证实基因的内源性转录激活，并通过显微镜观察诱导的肝细胞的形态和特征。通过免疫荧光染色检测体内诱导的肝细胞的程度，通过天狼猩红染色、α-平滑肌肌动蛋白（α-SMA）免疫荧光染色和血液丙氨酸氨基转移酶（ALT）检查评估肝纤维化的改善。
通过 CRISPRa 仅激活两个因子 Gata4 和 Foxa3 就足以成功地将 MEFs 诱导为 iHeps。这些 iHeps 可以在体外扩增，并表现出与成熟肝细胞相似的功能特征，如药物代谢和糖原储存。此外，基于 AAV6 的 CRISPRa SAM 系统的递送有效地诱导了活纤维化小鼠中从成纤维细胞到肝细胞的重编程。诱导 8 周后，重编程的肝细胞占小鼠总肝细胞群体的 0.87%，显著减少了肝纤维化。
CRISPRa 诱导的肝细胞重编程可能是生成功能性肝细胞和治疗由肝脏疾病引起的肝纤维化的一种有前途的策略。
