Chemically defined and small molecules-based generation of sinoatrial node-like cells
Abstract Background Existing methods for in vitro differentiation of human pluripotent stem cells (hPSCs) into sinoatrial node-like cells (SANLCs) require complex and undefined medium constituents. This might hinder the elucidation of the molecular mechanisms involved in cardiac subtype specificatio...
| Main Authors: | , , , , , , , , , , |
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| Format: | Article |
| Language: | English |
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BMC
2022-04-01
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| Series: | Stem Cell Research & Therapy |
| Subjects: | |
| Online Access: | https://doi.org/10.1186/s13287-022-02834-y |
| _version_ | 1818273909431074816 |
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| author | Xiaojie Hou Shuhong Ma Wei Fan Fang Li Miaomiao Xu Chao Yang Feng Liu Ying Yan Juyi Wan Feng Lan Bin Liao |
| author_facet | Xiaojie Hou Shuhong Ma Wei Fan Fang Li Miaomiao Xu Chao Yang Feng Liu Ying Yan Juyi Wan Feng Lan Bin Liao |
| author_sort | Xiaojie Hou |
| collection | DOAJ |
| description | Abstract Background Existing methods for in vitro differentiation of human pluripotent stem cells (hPSCs) into sinoatrial node-like cells (SANLCs) require complex and undefined medium constituents. This might hinder the elucidation of the molecular mechanisms involved in cardiac subtype specification and prevent translational application. In our study, we aimed to establish a chemically defined differentiation methods to generate SANLCs effectively and stably. Methods We induced human embryonic stem cells (hESCs)/induced PSCs (hiPSCs) to pan-cardiomyocytes by temporal modulation of the WNT/β-catenin (WNT) signaling pathway with GSK3 inhibitor and WNT inhibitor. During cardiac mesoderm stage of the differentiation process, signaling of WNT, retinoid acid (RA), and fibroblast growth factor (FGF) was manipulated by three specific molecules. Moreover, metabolic selection was designed to improve the enrichment of SANLCs. Finally, RT-PCR, immunofluorescence, flow cytometry, and whole cell patch clamp were used to identify the SANLCs. Results WNT, RA, and FGF signaling promote the differentiation of hPSCs into SANLCs in a concentration- and time window-sensitive manner, respectively. Synergetic modulation of WNT, FGF, and RA signaling pathways enhance the pacemaker phenotype and improve the differentiation efficiency of SANLCs (up to 45%). Moreover, the purification based on lactate metabolism and glucose starvation further reached approximately 50% of SANLCs. Finally, the electrophysiological data demonstrate that cells differentiated with the proposed protocol produce a considerable number of SANLCs that display typical electrophysiological characteristics of pacemaker cells in vitro. Conclusion We provide an optimized and chemically defined protocol to generate SANLCs by combined modulation of WNT, RA, and FGF signaling pathways and metabolic selection by lactate enrichment and glucose starvation. This chemically defined method for generating SANLCs might provide a platform for disease modeling, drug discovery, predictive toxicology, and biological pacemaker construction. |
| first_indexed | 2024-12-12T22:05:27Z |
| format | Article |
| id | doaj.art-f66ff35127754e3184dcf2bd1302d2f2 |
| institution | Directory Open Access Journal |
| issn | 1757-6512 |
| language | English |
| last_indexed | 2024-12-12T22:05:27Z |
| publishDate | 2022-04-01 |
| publisher | BMC |
| record_format | Article |
| series | Stem Cell Research & Therapy |
| spelling | doaj.art-f66ff35127754e3184dcf2bd1302d2f22022-12-22T00:10:24ZengBMCStem Cell Research & Therapy1757-65122022-04-0113111710.1186/s13287-022-02834-yChemically defined and small molecules-based generation of sinoatrial node-like cellsXiaojie Hou0Shuhong Ma1Wei Fan2Fang Li3Miaomiao Xu4Chao Yang5Feng Liu6Ying Yan7Juyi Wan8Feng Lan9Bin Liao10Department of Cardiovascular Surgery, Affiliated Hospital of Southwest Medical UniversityShenzhen Key Laboratory of Cardiovascular Disease, Fuwai Hospital Chinese Academy of Medical Sciences, State Key Laboratory of Cardiovascular Disease, Key Laboratory of Pluripotent Stem Cells in Cardiac Repair and Regeneration, Chinese Academy of Medical Sciences and Peking Union Medical CollegeDepartment of Cardiovascular Surgery, Affiliated Hospital of Southwest Medical UniversityKey Laboratory of Medical Electrophysiology, Ministry of Education & Medical Electrophysiological Key Laboratory of Sichuan Province, (Collaborative Innovation Center for Prevention of Cardiovascular Diseases) Institute of Cardiovascular Research, Southwest Medical UniversityShenzhen Key Laboratory of Cardiovascular Disease, Fuwai Hospital Chinese Academy of Medical Sciences, State Key Laboratory of Cardiovascular Disease, Key Laboratory of Pluripotent Stem Cells in Cardiac Repair and Regeneration, Chinese Academy of Medical Sciences and Peking Union Medical CollegeDepartment of Cardiovascular Surgery, Affiliated Hospital of Southwest Medical UniversityDepartment of Cardiovascular Surgery, Affiliated Hospital of Southwest Medical UniversityDepartment of Cardiovascular Surgery, Affiliated Hospital of Southwest Medical UniversityDepartment of Cardiovascular Surgery, Affiliated Hospital of Southwest Medical UniversityShenzhen Key Laboratory of Cardiovascular Disease, Fuwai Hospital Chinese Academy of Medical Sciences, State Key Laboratory of Cardiovascular Disease, Key Laboratory of Pluripotent Stem Cells in Cardiac Repair and Regeneration, Chinese Academy of Medical Sciences and Peking Union Medical CollegeDepartment of Cardiovascular Surgery, Affiliated Hospital of Southwest Medical UniversityAbstract Background Existing methods for in vitro differentiation of human pluripotent stem cells (hPSCs) into sinoatrial node-like cells (SANLCs) require complex and undefined medium constituents. This might hinder the elucidation of the molecular mechanisms involved in cardiac subtype specification and prevent translational application. In our study, we aimed to establish a chemically defined differentiation methods to generate SANLCs effectively and stably. Methods We induced human embryonic stem cells (hESCs)/induced PSCs (hiPSCs) to pan-cardiomyocytes by temporal modulation of the WNT/β-catenin (WNT) signaling pathway with GSK3 inhibitor and WNT inhibitor. During cardiac mesoderm stage of the differentiation process, signaling of WNT, retinoid acid (RA), and fibroblast growth factor (FGF) was manipulated by three specific molecules. Moreover, metabolic selection was designed to improve the enrichment of SANLCs. Finally, RT-PCR, immunofluorescence, flow cytometry, and whole cell patch clamp were used to identify the SANLCs. Results WNT, RA, and FGF signaling promote the differentiation of hPSCs into SANLCs in a concentration- and time window-sensitive manner, respectively. Synergetic modulation of WNT, FGF, and RA signaling pathways enhance the pacemaker phenotype and improve the differentiation efficiency of SANLCs (up to 45%). Moreover, the purification based on lactate metabolism and glucose starvation further reached approximately 50% of SANLCs. Finally, the electrophysiological data demonstrate that cells differentiated with the proposed protocol produce a considerable number of SANLCs that display typical electrophysiological characteristics of pacemaker cells in vitro. Conclusion We provide an optimized and chemically defined protocol to generate SANLCs by combined modulation of WNT, RA, and FGF signaling pathways and metabolic selection by lactate enrichment and glucose starvation. This chemically defined method for generating SANLCs might provide a platform for disease modeling, drug discovery, predictive toxicology, and biological pacemaker construction.https://doi.org/10.1186/s13287-022-02834-yHuman pluripotent stem cellsSinoatrial node-like cellsDifferentiationChemically defined mediumSmall moleculeSignaling pathway |
| spellingShingle | Xiaojie Hou Shuhong Ma Wei Fan Fang Li Miaomiao Xu Chao Yang Feng Liu Ying Yan Juyi Wan Feng Lan Bin Liao Chemically defined and small molecules-based generation of sinoatrial node-like cells Stem Cell Research & Therapy Human pluripotent stem cells Sinoatrial node-like cells Differentiation Chemically defined medium Small molecule Signaling pathway |
| title | Chemically defined and small molecules-based generation of sinoatrial node-like cells |
| title_full | Chemically defined and small molecules-based generation of sinoatrial node-like cells |
| title_fullStr | Chemically defined and small molecules-based generation of sinoatrial node-like cells |
| title_full_unstemmed | Chemically defined and small molecules-based generation of sinoatrial node-like cells |
| title_short | Chemically defined and small molecules-based generation of sinoatrial node-like cells |
| title_sort | chemically defined and small molecules based generation of sinoatrial node like cells |
| topic | Human pluripotent stem cells Sinoatrial node-like cells Differentiation Chemically defined medium Small molecule Signaling pathway |
| url | https://doi.org/10.1186/s13287-022-02834-y |
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