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
{"title":"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。","authors":"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","doi":"10.1186/s41232-023-00258-6","DOIUrl":null,"url":null,"abstract":"<p><strong>Background: </strong>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.</p><p><strong>Methods: </strong>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.</p><p><strong>Results: </strong>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.</p><p><strong>Conclusion: </strong>We anticipate that Wnt3a-diCAR enables target-specific signal activation, and could be an economical and powerful tool for stem cell-based regeneration therapy.</p>","PeriodicalId":13588,"journal":{"name":"Inflammation and Regeneration","volume":null,"pages":null},"PeriodicalIF":5.0000,"publicationDate":"2023-02-10","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9912504/pdf/","citationCount":"1","resultStr":"{\"title\":\"Canonical Wnt signaling activation by chimeric antigen receptors for efficient cardiac differentiation from mouse embryonic stem cells.\",\"authors\":\"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\",\"doi\":\"10.1186/s41232-023-00258-6\",\"DOIUrl\":null,\"url\":null,\"abstract\":\"<p><strong>Background: </strong>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.</p><p><strong>Methods: </strong>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.</p><p><strong>Results: </strong>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.</p><p><strong>Conclusion: </strong>We anticipate that Wnt3a-diCAR enables target-specific signal activation, and could be an economical and powerful tool for stem cell-based regeneration therapy.</p>\",\"PeriodicalId\":13588,\"journal\":{\"name\":\"Inflammation and Regeneration\",\"volume\":null,\"pages\":null},\"PeriodicalIF\":5.0000,\"publicationDate\":\"2023-02-10\",\"publicationTypes\":\"Journal Article\",\"fieldsOfStudy\":null,\"isOpenAccess\":false,\"openAccessPdf\":\"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9912504/pdf/\",\"citationCount\":\"1\",\"resultStr\":null,\"platform\":\"Semanticscholar\",\"paperid\":null,\"PeriodicalName\":\"Inflammation and Regeneration\",\"FirstCategoryId\":\"3\",\"ListUrlMain\":\"https://doi.org/10.1186/s41232-023-00258-6\",\"RegionNum\":3,\"RegionCategory\":\"医学\",\"ArticlePicture\":[],\"TitleCN\":null,\"AbstractTextCN\":null,\"PMCID\":null,\"EPubDate\":\"\",\"PubModel\":\"\",\"JCR\":\"Q2\",\"JCRName\":\"IMMUNOLOGY\",\"Score\":null,\"Total\":0}","platform":"Semanticscholar","paperid":null,"PeriodicalName":"Inflammation and Regeneration","FirstCategoryId":"3","ListUrlMain":"https://doi.org/10.1186/s41232-023-00258-6","RegionNum":3,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":null,"EPubDate":"","PubModel":"","JCR":"Q2","JCRName":"IMMUNOLOGY","Score":null,"Total":0}
Canonical Wnt signaling activation by chimeric antigen receptors for efficient cardiac differentiation from mouse embryonic stem cells.
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.
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.
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