Shanghai Frontiers Science Center of Genome Editing and Cell Therapy, Biomedical Synthetic Biology Research Center, Shanghai Key Laboratory of Regulatory Biology, Institute of Biomedical Sciences and School of Life Sciences, East China Normal University, Dongchuan Road 500, Shanghai, 200241, China.
Department of Animal Sciences, College of Agriculture and Natural Resources, Michigan State University, East Lansing, MI, 48824, USA.
Adv Sci (Weinh). 2023 Feb;10(4):e2202858. doi: 10.1002/advs.202202858. Epub 2022 Dec 11.
Pluripotent stem cells (PSCs) hold great promise for cell-based therapies, disease modeling, and drug discovery. Classic somatic cell reprogramming to generate induced pluripotent stem cells (iPSCs) is often achieved based on overexpression of transcription factors (TFs). However, this process is limited by side effect of overexpressed TFs and unpredicted targeting of TFs. Pinpoint control over endogenous TFs expression can provide the ability to reprogram cell fate and tissue function. Here, a light-inducible cell reprogramming (LIRE) system is developed based on a photoreceptor protein cryptochrome system and clustered regularly interspaced short palindromic repeats/nuclease-deficient CRISPR-associated protein 9 for induced PSCs reprogramming. This system enables remote, non-invasive optogenetical regulation of endogenous Sox2 and Oct4 loci to reprogram mouse embryonic fibroblasts into iPSCs (iPSC ) under light-emitting diode-based illumination. iPSC cells can be efficiently differentiated into different cells by upregulating a corresponding TF. iPSC cells are used for blastocyst injection and optogenetic chimeric mice are successfully generated, which enables non-invasive control of user-defined endogenous genes in vivo, providing a valuable tool for facile and traceless controlled gene expression studies and genetic screens in mice. This LIRE system offers a remote, traceless, and non-invasive approach for cellular reprogramming and modeling of complex human diseases in basic biological research and regenerative medicine applications.
多能干细胞 (PSCs) 在细胞治疗、疾病建模和药物发现方面具有巨大的应用潜力。经典的体细胞重编程以生成诱导多能干细胞 (iPSCs) 通常是通过过表达转录因子 (TFs) 来实现的。然而，该过程受到过表达 TFs 的副作用和 TFs 不可预测的靶向性的限制。对内源性 TFs 表达的精确定制控制可以提供重编程细胞命运和组织功能的能力。本文开发了一种基于光受体蛋白隐花色素系统和无核酸酶缺陷 CRISPR 相关蛋白 9 的光诱导细胞重编程 (LIRE) 系统，用于诱导 PSCs 重编程。该系统可实现远程、非侵入性的光遗传学调控内源性 Sox2 和 Oct4 基因座，从而在基于发光二极管的光照下将小鼠胚胎成纤维细胞重编程为 iPSCs (iPSC)。通过上调相应的 TF，可有效地将 iPSC 细胞分化为不同的细胞。将 iPSC 细胞用于胚胎注射，并成功生成了光遗传嵌合小鼠，这使得可以在体内对用户定义的内源性基因进行非侵入性控制，为在小鼠中进行简便、无痕的可控基因表达研究和遗传筛选提供了有价值的工具。该 LIRE 系统为基础生物学研究和再生医学应用中的细胞重编程和复杂人类疾病建模提供了一种远程、无痕和非侵入性的方法。
