Blöchinger Anna Karolina, Siehler Johanna, Wißmiller Katharina, Shahryari Alireza, Burtscher Ingo, Lickert Heiko
Institute of Diabetes and Regeneration Research, Helmholtz Zentrum München, 85764 Neuherberg, Germany; Institute of Stem Cell Research, Helmholtz Zentrum München, 85764 Neuherberg, Germany; Technische Universität München, Ismaninger Straße 22, 81675 München, Germany.
Institute of Diabetes and Regeneration Research, Helmholtz Zentrum München, 85764 Neuherberg, Germany; Institute of Stem Cell Research, Helmholtz Zentrum München, 85764 Neuherberg, Germany; German Center for Diabetes Research (DZD), 85764 Neuherberg, Germany.
Stem Cell Res. 2020 May;45:101797. doi: 10.1016/j.scr.2020.101797. Epub 2020 Apr 22.
Differentiating human induced pluripotent stem cells (hiPSCs) into insulin (INS)-producing β-like cells has potential for diabetes research and therapy. Here, we generated a heterozygous fluorescent hiPSC reporter, labeling INS-producing β-like cells. We used CRISPR/Cas9 technology to knock-in a T2A-H2B-Cherry cassette to replace the translational INS stop codon, enabling co-transcription and T2A-peptide mediated co-translational cleavage of INS-T2A and H2B-Cherry. The hiPSC-INS-T2A-H2B-Cherry reporter cells were pluripotent and showed multi-lineage differentiation potential. Cells expressing the β-cell specific hormone INS are identified by nuclear localized H2B-Cherry reporter upon pancreatic endocrine differentiation. Thus, the generated reporter hiPSCs enable live identification of INS hormone-producing β-like cells.
将人诱导多能干细胞（hiPSC）分化为产生胰岛素（INS）的β样细胞在糖尿病研究和治疗方面具有潜力。在此，我们生成了一种杂合荧光hiPSC报告基因，用于标记产生INS的β样细胞。我们使用CRISPR/Cas9技术敲入一个T2A-H2B-樱桃红盒，以取代翻译后的INS终止密码子，从而实现INS-T2A和H2B-樱桃红的共转录以及T2A肽介导的共翻译切割。hiPSC-INS-T2A-H2B-樱桃红报告基因细胞具有多能性，并显示出多谱系分化潜能。在胰腺内分泌分化后，通过核定位的H2B-樱桃红报告基因来鉴定表达β细胞特异性激素INS的细胞。因此，所生成的报告基因hiPSC能够对产生INS激素的β样细胞进行活体鉴定。
