Yi Liu, Rachna Balaji, Marcelo A Szymanski de Toledo, Sabrina Ernst, Petra Hautvast, Aylin B Kesdoğan, Jannis Körner, Martin Zenke, Anika Neureiter, Angelika Lampert
{"title":"通过高分辨率成像确定,疼痛靶点 NaV1.7 在人类 iPS 细胞分化为感觉神经元的过程中表达较晚。","authors":"Yi Liu, Rachna Balaji, Marcelo A Szymanski de Toledo, Sabrina Ernst, Petra Hautvast, Aylin B Kesdoğan, Jannis Körner, Martin Zenke, Anika Neureiter, Angelika Lampert","doi":"10.1007/s00424-024-02945-w","DOIUrl":null,"url":null,"abstract":"<p><p>Human-induced pluripotent stem cells (iPS cells) are efficiently differentiated into sensory neurons. These cells express the voltage-gated sodium channel Na<sub>V</sub>1.7, which is a validated pain target. Na<sub>V</sub>1.7 deficiency leads to pain insensitivity, whereas Na<sub>V</sub>1.7 gain-of-function mutants are associated with chronic pain. During differentiation, the sensory neurons start spontaneous action potential firing around day 22, with increasing firing rate until day 40. Here, we used CRISPR/Cas9 genome editing to generate a HA-tag Na<sub>V</sub>1.7 to follow its expression during differentiation. We used two protocols to generate sensory neurons: the classical small molecule approach and a directed differentiation methodology and assessed surface Na<sub>V</sub>1.7 expression by Airyscan high-resolution microscopy. Our results show that maturation of at least 49 days is necessary to observe robust Na<sub>V</sub>1.7 surface expression in both protocols. Electric activity of the sensory neurons precedes Na<sub>V</sub>1.7 surface expression. A clinically effective Na<sub>V</sub>1.7 blocker is still missing, and we expect this iPS cell model system to be useful for drug discovery and disease modeling.</p>","PeriodicalId":19954,"journal":{"name":"Pflugers Archiv : European journal of physiology","volume":null,"pages":null},"PeriodicalIF":2.9000,"publicationDate":"2024-06-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11139713/pdf/","citationCount":"0","resultStr":"{\"title\":\"The pain target Na<sub>V</sub>1.7 is expressed late during human iPS cell differentiation into sensory neurons as determined in high-resolution imaging.\",\"authors\":\"Yi Liu, Rachna Balaji, Marcelo A Szymanski de Toledo, Sabrina Ernst, Petra Hautvast, Aylin B Kesdoğan, Jannis Körner, Martin Zenke, Anika Neureiter, Angelika Lampert\",\"doi\":\"10.1007/s00424-024-02945-w\",\"DOIUrl\":null,\"url\":null,\"abstract\":\"<p><p>Human-induced pluripotent stem cells (iPS cells) are efficiently differentiated into sensory neurons. These cells express the voltage-gated sodium channel Na<sub>V</sub>1.7, which is a validated pain target. Na<sub>V</sub>1.7 deficiency leads to pain insensitivity, whereas Na<sub>V</sub>1.7 gain-of-function mutants are associated with chronic pain. During differentiation, the sensory neurons start spontaneous action potential firing around day 22, with increasing firing rate until day 40. Here, we used CRISPR/Cas9 genome editing to generate a HA-tag Na<sub>V</sub>1.7 to follow its expression during differentiation. We used two protocols to generate sensory neurons: the classical small molecule approach and a directed differentiation methodology and assessed surface Na<sub>V</sub>1.7 expression by Airyscan high-resolution microscopy. Our results show that maturation of at least 49 days is necessary to observe robust Na<sub>V</sub>1.7 surface expression in both protocols. Electric activity of the sensory neurons precedes Na<sub>V</sub>1.7 surface expression. A clinically effective Na<sub>V</sub>1.7 blocker is still missing, and we expect this iPS cell model system to be useful for drug discovery and disease modeling.</p>\",\"PeriodicalId\":19954,\"journal\":{\"name\":\"Pflugers Archiv : European journal of physiology\",\"volume\":null,\"pages\":null},\"PeriodicalIF\":2.9000,\"publicationDate\":\"2024-06-01\",\"publicationTypes\":\"Journal Article\",\"fieldsOfStudy\":null,\"isOpenAccess\":false,\"openAccessPdf\":\"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11139713/pdf/\",\"citationCount\":\"0\",\"resultStr\":null,\"platform\":\"Semanticscholar\",\"paperid\":null,\"PeriodicalName\":\"Pflugers Archiv : European journal of physiology\",\"FirstCategoryId\":\"3\",\"ListUrlMain\":\"https://doi.org/10.1007/s00424-024-02945-w\",\"RegionNum\":4,\"RegionCategory\":\"医学\",\"ArticlePicture\":[],\"TitleCN\":null,\"AbstractTextCN\":null,\"PMCID\":null,\"EPubDate\":\"2024/3/27 0:00:00\",\"PubModel\":\"Epub\",\"JCR\":\"Q2\",\"JCRName\":\"PHYSIOLOGY\",\"Score\":null,\"Total\":0}","platform":"Semanticscholar","paperid":null,"PeriodicalName":"Pflugers Archiv : European journal of physiology","FirstCategoryId":"3","ListUrlMain":"https://doi.org/10.1007/s00424-024-02945-w","RegionNum":4,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":null,"EPubDate":"2024/3/27 0:00:00","PubModel":"Epub","JCR":"Q2","JCRName":"PHYSIOLOGY","Score":null,"Total":0}
The pain target NaV1.7 is expressed late during human iPS cell differentiation into sensory neurons as determined in high-resolution imaging.
Human-induced pluripotent stem cells (iPS cells) are efficiently differentiated into sensory neurons. These cells express the voltage-gated sodium channel NaV1.7, which is a validated pain target. NaV1.7 deficiency leads to pain insensitivity, whereas NaV1.7 gain-of-function mutants are associated with chronic pain. During differentiation, the sensory neurons start spontaneous action potential firing around day 22, with increasing firing rate until day 40. Here, we used CRISPR/Cas9 genome editing to generate a HA-tag NaV1.7 to follow its expression during differentiation. We used two protocols to generate sensory neurons: the classical small molecule approach and a directed differentiation methodology and assessed surface NaV1.7 expression by Airyscan high-resolution microscopy. Our results show that maturation of at least 49 days is necessary to observe robust NaV1.7 surface expression in both protocols. Electric activity of the sensory neurons precedes NaV1.7 surface expression. A clinically effective NaV1.7 blocker is still missing, and we expect this iPS cell model system to be useful for drug discovery and disease modeling.
期刊介绍:
Pflügers Archiv European Journal of Physiology publishes those results of original research that are seen as advancing the physiological sciences, especially those providing mechanistic insights into physiological functions at the molecular and cellular level, and clearly conveying a physiological message. Submissions are encouraged that deal with the evaluation of molecular and cellular mechanisms of disease, ideally resulting in translational research. Purely descriptive papers covering applied physiology or clinical papers will be excluded. Papers on methodological topics will be considered if they contribute to the development of novel tools for further investigation of (patho)physiological mechanisms.