Canonical Wnt signaling activation by chimeric antigen receptors for efficient cardiac differentiation from mouse embryonic stem cells.

IF 5 3区医学 Q2 IMMUNOLOGY

Inflammation and Regeneration Pub Date : 2023-02-10 DOI:10.1186/s41232-023-00258-6

Takahiro Sogo, Shu Nakao, Tasuku Tsukamoto, Tomoe Ueyama, Yukihiro Harada, Dai Ihara, Tomoaki Ishida, Masato Nakahara, Koji Hasegawa, Yuka Akagi, Yasuyuki S Kida, Osamu Nakagawa, Teruyuki Nagamune, Masahiro Kawahara, Teruhisa Kawamura

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引用次数: 1

Abstract

Background: Canonical Wnt signaling is involved in a variety of biological processes including stem cell renewal and differentiation, embryonic development, and tissue regeneration. Previous studies reported the stage-specific roles of the Wnt signaling in heart development. Canonical Wnt signal activation by recombinant Wnt3a in the early phase of differentiation enhances the efficiency of myocardial cell production from pluripotent stem cells. However, the hydrophobicity of Wnt proteins results in high cost to produce the recombinant proteins and presents an obstacle to their preparation and application for therapeutics, cell therapy, or molecular analysis of Wnt signaling.

Methods: To solve this problem, we generated an inexpensive molecule-responsive differentiation-inducing chimeric antigen receptor (designated as diCAR) that can activate Wnt3a signaling. The extracellular domains of low-density-lipoprotein receptor-related protein 6 (LRP6) and frizzeled-8 (FZD8) were replaced with single-chain Fv of anti-fluorescein (FL) antibody, which can respond to FL-conjugated bovine serum albumin (BSA-FL) as a cognate ligand. We then analyzed the effect of this diCAR on Wnt signal activation and cardiomyocyte differentiation of mouse embryonic stem cells in response to BSA-FL treatment.

Results: Embryonic stem cell lines stably expressing this paired diCAR, named Wnt3a-diCAR, showed TCF/β-catenin-dependent transactivation by BSA-FL in a dose-dependent manner. Treatment with either Wnt3a recombinant protein or BSA-FL in the early phase of differentiation revealed similar changes of global gene expressions and resulted in efficient myocardial cell differentiation. Furthermore, BSA-FL-mediated signal activation was not affected by a Wnt3a antagonist, Dkk1, suggesting that the signal transduction via Wnt3a-diCAR is independent of endogenous LRP6 or FZD8.

Conclusion: We anticipate that Wnt3a-diCAR enables target-specific signal activation, and could be an economical and powerful tool for stem cell-based regeneration therapy.

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嵌合抗原受体激活典型Wnt信号以促进小鼠胚胎干细胞的有效心脏分化。

背景:典型Wnt信号参与多种生物过程，包括干细胞更新和分化、胚胎发育和组织再生。先前的研究报道了Wnt信号在心脏发育中的阶段性作用。在分化早期通过重组Wnt3a激活典型Wnt信号可提高多能干细胞产生心肌细胞的效率。然而，Wnt蛋白的疏水性导致重组蛋白的生产成本高，阻碍了其制备和应用于治疗学、细胞治疗或Wnt信号的分子分析。方法:为了解决这一问题，我们制作了一种廉价的分子反应性诱导分化嵌合抗原受体(指定为diCAR)，可以激活Wnt3a信号传导。将低密度脂蛋白受体相关蛋白6 (LRP6)和frizzed -8 (FZD8)的胞外结构域替换为抗荧光素(FL)抗体的单链Fv，该抗体可作为同源配体响应FL偶联的牛血清白蛋白(BSA-FL)。然后，我们分析了该diCAR对BSA-FL处理后小鼠胚胎干细胞Wnt信号激活和心肌细胞分化的影响。结果:稳定表达该配对diCAR(命名为Wnt3a-diCAR)的胚胎干细胞系显示出BSA-FL对TCF/β-catenin依赖的剂量依赖性转激活。在分化早期用Wnt3a重组蛋白或BSA-FL处理心肌细胞，均显示出相似的基因表达变化，并导致有效的心肌细胞分化。此外，bsa - fl介导的信号激活不受Wnt3a拮抗剂Dkk1的影响，这表明通过Wnt3a- dicar的信号转导不依赖于内源性LRP6或FZD8。结论:我们预计Wnt3a-diCAR能够实现靶向性信号激活，并可能成为干细胞再生治疗的一种经济而强大的工具。

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来源期刊

Inflammation and Regeneration Multiple-

CiteScore

11.10

自引率

1.20%

发文量

审稿时长

11 weeks

期刊介绍： Inflammation and Regeneration is the official journal of the Japanese Society of Inflammation and Regeneration (JSIR). This journal provides an open access forum which covers a wide range of scientific topics in the basic and clinical researches on inflammation and regenerative medicine. It also covers investigations of infectious diseases, including COVID-19 and other emerging infectious diseases, which involve the inflammatory responses. Inflammation and Regeneration publishes papers in the following categories: research article, note, rapid communication, case report, review and clinical drug evaluation.