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基于组织特异性启动子的报告系统，用于监测 iPS 细胞向心肌细胞的分化。

Tissue-specific promoter-based reporter system for monitoring cell differentiation from iPSCs to cardiomyocytes.

机构信息

Institute of Human Genetics, Polish Academy of Sciences, Poznan, Poland.

Department of Genetics and Animal Breeding, Poznan University of Life Sciences, Poznan, Poland.

出版信息

Sci Rep. 2020 Feb 5;10(1):1895. doi: 10.1038/s41598-020-58050-2.

DOI:10.1038/s41598-020-58050-2
PMID:32024875
原文链接:https://pmc.ncbi.nlm.nih.gov/articles/PMC7002699/
Abstract

The possibility of using stem cell-derived cardiomyocytes opens a new platform for modeling cardiac cell differentiation and disease or the development of new drugs. Progress in this field can be accelerated by high-throughput screening (HTS) technology combined with promoter reporter system. The goal of the study was to create and evaluate a responsive promoter reporter system that allows monitoring of iPSC differentiation towards cardiomyocytes. The lentiviral promoter reporter system was based on troponin 2 (TNNT2) and alpha cardiac actin (ACTC) with firefly luciferase and mCherry, respectively. The system was evaluated in two in vitro models. First, system followed the differentiation of TNNT2-luc-T2A-Puro-mCMV-GFP and hACTC-mcherry-WPRE-EF1-Neo from transduced iPSC line towards cardiomyocytes and revealed the significant decrease in both inserts copy number during the prolonged in vitro cell culture (confirmed by I-FISH, ddPCR, qPCR). Second, differentiated and contracting control cardiomyocytes (obtained from control non-reporter transduced iPSCs) were subsequently transduced with TNNT2-luc-T2A-Puro-CMV-GFP and hACTC-mcherry-WPRE-EF1-Neo lentiviruses to observe the functionality of obtained cardiomyocytes. Our results indicated that the reporter modified cell lines can be used for HTS applications, but it is essential to monitor the stability of the reporter sequence during extended cell in vitro culture.

摘要

使用干细胞衍生的心肌细胞为心脏细胞分化和疾病建模或开发新药开辟了新的平台。高通量筛选 (HTS) 技术与启动子报告系统相结合，可以加速该领域的进展。本研究的目的是创建和评估一种响应性启动子报告系统，以监测 iPSC 向心肌细胞的分化。该基于慢病毒的启动子报告系统基于肌钙蛋白 2 (TNNT2) 和α心脏肌动蛋白 (ACTC)，分别带有萤火虫荧光素酶和 mCherry。该系统在两个体外模型中进行了评估。首先，该系统跟踪了转导的 iPSC 系向心肌细胞分化的 TNNT2-luc-T2A-Puro-mCMV-GFP 和 hACTC-mcherry-WPRE-EF1-Neo 的分化，并揭示了在延长的体外细胞培养过程中两个插入物拷贝数的显著减少（通过 I-FISH、ddPCR、qPCR 证实）。其次，分化和收缩的对照心肌细胞（从对照非报告转导的 iPSCs 获得）随后用 TNNT2-luc-T2A-Puro-CMV-GFP 和 hACTC-mcherry-WPRE-EF1-Neo 慢病毒转导，以观察获得的心肌细胞的功能。我们的结果表明，报告基因修饰的细胞系可用于 HTS 应用，但在体外延长细胞培养过程中监测报告序列的稳定性至关重要。

https://cdn.ncbi.nlm.nih.gov/pmc/blobs/fefe/7002699/63cf7f5a321c/41598_2020_58050_Fig6_HTML.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/fefe/7002699/25db8031c4d9/41598_2020_58050_Fig1_HTML.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/fefe/7002699/4f9579819270/41598_2020_58050_Fig2_HTML.jpg
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https://cdn.ncbi.nlm.nih.gov/pmc/blobs/fefe/7002699/54c9b8f508ca/41598_2020_58050_Fig5_HTML.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/fefe/7002699/63cf7f5a321c/41598_2020_58050_Fig6_HTML.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/fefe/7002699/25db8031c4d9/41598_2020_58050_Fig1_HTML.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/fefe/7002699/4f9579819270/41598_2020_58050_Fig2_HTML.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/fefe/7002699/dbd5640216cb/41598_2020_58050_Fig3_HTML.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/fefe/7002699/07a667994071/41598_2020_58050_Fig4_HTML.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/fefe/7002699/54c9b8f508ca/41598_2020_58050_Fig5_HTML.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/fefe/7002699/63cf7f5a321c/41598_2020_58050_Fig6_HTML.jpg

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