XPRESSO: Rapid genetic engineering of human pluripotent stem cells for durable overexpression using a modular anti-silencing vector.

IF 5.1 2区医学 Q1 CELL & TISSUE ENGINEERING

Stem Cell Reports Pub Date : 2025-08-19 DOI:10.1016/j.stemcr.2025.102603

Yehuda Wexler, Harel Grinstein, Irit Huber, Shany Glatstein, Matteo Ghiringhelli, Oded Edri, Michal Landesberg, Daniel Shiff, Gil Arbel, Idan Rosh, Ashwani Choudhary, Shani Stern, Lior Gepstein

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引用次数: 0

Abstract

Ectopic expression of proteins in human pluripotent stem cells (hPSCs) is highly desirable as a research tool and important for clinical translation. However, genetically engineering hPSCs for long-term overexpression of proteins remains inefficient, labor-intensive, and plagued by epigenetic silencing, necessitating dedication of significant resources, and entailing laborious workflows. To address these limitations, we report the development of XPRESSO (expedited persistent and robust engineering of stem cells with sleeping beauty for overexpression), a modular "anti-silencing" transposon vector, which we have combined with a highly efficient and accessible methodology for the rapid generation of genetically modified hPSC lines in a gene-independent manner. Using this method, we successfully generated dozens of stable hPSC lines with robust and continuous functional expression of optogenetic proteins, Cas9, shRNA, and a calcium indicator in both undifferentiated and differentiated (cardiomyocyte and neuronal) cells.

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XPRESSO：使用模块化抗沉默载体对人类多能干细胞进行持久过表达的快速基因工程。

人类多能干细胞（hPSCs）中蛋白质的异位表达是一种非常理想的研究工具，对临床翻译非常重要。然而，用于长期过表达蛋白质的基因工程hPSCs仍然效率低下，劳动密集型，并且受到表观遗传沉默的困扰，需要投入大量资源，并且需要繁琐的工作流程。为了解决这些限制，我们报告了XPRESSO（快速持久和强大的干细胞工程与睡美人过度表达）的开发，这是一种模块化的“抗沉默”转座子载体，我们结合了一种高效和可访问的方法，以基因独立的方式快速生成转基因hPSC系。利用这种方法，我们成功地生成了数十个稳定的hPSC细胞系，这些细胞系在未分化和分化的（心肌细胞和神经元）细胞中都具有光遗传蛋白、Cas9、shRNA和钙指示剂的稳健和连续的功能表达。

本文章由计算机程序翻译，如有差异，请以英文原文为准。

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来源期刊

Stem Cell Reports CELL & TISSUE ENGINEERING-CELL BIOLOGY

CiteScore

10.50

自引率

1.70%

发文量

200

审稿时长

28 weeks

期刊介绍： Stem Cell Reports publishes high-quality, peer-reviewed research presenting conceptual or practical advances across the breadth of stem cell research and its applications to medicine. Our particular focus on shorter, single-point articles, timely publication, strong editorial decision-making and scientific input by leaders in the field and a "scoop protection" mechanism are reasons to submit your best papers.