1 Laboratory of Pathology, Key Laboratory of Transplant Engineering and Immunology, NHFPC, West China Hospital, Sichuan University, Chengdu, China.
2 Laboratory of Pathology, West China School of Medicine, Sichuan University, Chengdu, China.
DNA Cell Biol. 2019 Apr;38(4):314-321. doi: 10.1089/dna.2018.4493. Epub 2019 Feb 14.
The shortage of human hepatocytes continues to be a significant limitation for the widespread application of hepatocyte transplantation and bioartificial liver (BAL) support therapy. Recombinant activation gene 2 (Rag2) and fumarylacetoacetate hydrolase (Fah)-deficient mice could be highly repopulated with human hepatocytes. However, Fah/Rag2-deficient mice can only produce up to 1 × 10 human hepatocytes per mouse. We hypothesized that 2-10 × 10 human hepatocytes can be produced per Fah/Rag2-deficient pig, which is an adequate supply for hepatocyte transplantation and BAL therapy. In a novel approach, we used stably transfected Cas9 cells and single-guide RNA adenoviruses containing fluorescent reporters to enrich porcine cells with Fah/Rag2 dual gene mutations. This resulted in the construction of Fah/Rag2 double knockout porcine iliac artery endothelial cells, which were subsequently used for generating Fah/Rag2-deficient pigs.
人源肝细胞的短缺仍然是肝细胞移植和生物人工肝（BAL）支持治疗广泛应用的重大限制。重组激活基因 2（Rag2）和延胡索酰乙酰乙酸水解酶（Fah）缺陷型小鼠可以被高度重构成人肝细胞。然而，Fah/Rag2 缺陷型小鼠每只小鼠最多只能产生 1×10 个人源肝细胞。我们假设 Fah/Rag2 缺陷型猪每只猪可以产生 2-10×10 个人源肝细胞，这足以满足肝细胞移植和 BAL 治疗的需要。在一种新方法中，我们使用稳定转染的 Cas9 细胞和含有荧光报告基因的单导向 RNA 腺病毒，富集 Fah/Rag2 双基因突变的猪细胞。这导致 Fah/Rag2 双基因敲除猪髂动脉内皮细胞的构建，随后用于生成 Fah/Rag2 缺陷型猪。
