The maturation of iPS cell-derived brain microvascular endothelial cells by inducible-SOX18 expression
Abstract Background Brain microvascular endothelial cells (BMECs) play a major role in the blood–brain barrier (BBB), and are critical for establishing an in vitro BBB model. Currently, iPSC-derived BMECs (iBMECs) have been used to construct in vitro BBB models with physiological barrier functions,...
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| Format: | Article |
| Language: | English |
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BMC
2023-02-01
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| Series: | Fluids and Barriers of the CNS |
| Subjects: | |
| Online Access: | https://doi.org/10.1186/s12987-023-00408-5 |
| _version_ | 1811171551513411584 |
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| author | Hongyan Zhang Tomoko Yamaguchi Kenji Kawabata |
| author_facet | Hongyan Zhang Tomoko Yamaguchi Kenji Kawabata |
| author_sort | Hongyan Zhang |
| collection | DOAJ |
| description | Abstract Background Brain microvascular endothelial cells (BMECs) play a major role in the blood–brain barrier (BBB), and are critical for establishing an in vitro BBB model. Currently, iPSC-derived BMECs (iBMECs) have been used to construct in vitro BBB models with physiological barrier functions, such as high trans-endothelial electrical resistance (TEER) and expression of transporter proteins. However, the relatively low p-glycoprotein (P-gp) level and a decrease in the efflux ratio of its substrates in iBMECs suggest their immature nature. Therefore, more mature iBMECs by optimizing the differentiation induction protocol is beneficial for establishing a more reliable in vitro BBB model for studying central nervous system (CNS) drug transport. Methods To identify human brain endothelial cell fate-inducing factors, HUVEC was transfected with Zic3A-, Zic3B-, and Sox18-expressing lentivirus vector. Since SOX18 was found to induce BMEC properties, we used a Dox-inducible Tet-on system to express SOX18 during iBMEC differentiation and explored the impact of SOX18 expression on iBMEC maturation. Results Sox18-mediated iBMECs achieved a higher TEER value than normal iBMECs (> 3000 Ω cm2). From day 6 to day 10 (d6–10 group), the iBMECs with SOX18 expression expressed a series of tight junction markers and showed upregulation of Mfsd2a, a specific marker of the BBB. The d6–10 group also expressed SLC2A1/Glut1 at levels as high as normal iBMECs, and upregulated ABCB1/P-gp and ABCC1/MRP1 expression. Moreover, Sox18-mediated iBMECs showed higher viability than normal iBMECs after puromycin treatment, indicating that SOX18 expression could upregulate P-gp activity in iBMECs. Conclusions Inducible SOX18 expression in iBMECs gained BBB phenotypes, including high TEER values and upregulation of tight junction-related genes, endothelial cell (EC) markers, BBB transporters, and higher cell viability after treatment with puromycin. Collectively, we provide a differentiation method for the maturation of human iPS cell-derived BMECs with SOX18 expression, describing its contribution to form an in vitro BBB model for CNS drug transport studies. |
| first_indexed | 2024-04-10T17:15:54Z |
| format | Article |
| id | doaj.art-2770f4cc566949d2b548b199bddc7cf7 |
| institution | Directory Open Access Journal |
| issn | 2045-8118 |
| language | English |
| last_indexed | 2024-04-10T17:15:54Z |
| publishDate | 2023-02-01 |
| publisher | BMC |
| record_format | Article |
| series | Fluids and Barriers of the CNS |
| spelling | doaj.art-2770f4cc566949d2b548b199bddc7cf72023-02-05T12:21:17ZengBMCFluids and Barriers of the CNS2045-81182023-02-0120111010.1186/s12987-023-00408-5The maturation of iPS cell-derived brain microvascular endothelial cells by inducible-SOX18 expressionHongyan Zhang0Tomoko Yamaguchi1Kenji Kawabata2Laboratory of Biomedical Innovation, Graduate School of Pharmaceutical Sciences, Osaka UniversityLaboratory of Cell Model for Drug Discovery, National Institutes of Biomedical Innovation, Health, and NutritionLaboratory of Biomedical Innovation, Graduate School of Pharmaceutical Sciences, Osaka UniversityAbstract Background Brain microvascular endothelial cells (BMECs) play a major role in the blood–brain barrier (BBB), and are critical for establishing an in vitro BBB model. Currently, iPSC-derived BMECs (iBMECs) have been used to construct in vitro BBB models with physiological barrier functions, such as high trans-endothelial electrical resistance (TEER) and expression of transporter proteins. However, the relatively low p-glycoprotein (P-gp) level and a decrease in the efflux ratio of its substrates in iBMECs suggest their immature nature. Therefore, more mature iBMECs by optimizing the differentiation induction protocol is beneficial for establishing a more reliable in vitro BBB model for studying central nervous system (CNS) drug transport. Methods To identify human brain endothelial cell fate-inducing factors, HUVEC was transfected with Zic3A-, Zic3B-, and Sox18-expressing lentivirus vector. Since SOX18 was found to induce BMEC properties, we used a Dox-inducible Tet-on system to express SOX18 during iBMEC differentiation and explored the impact of SOX18 expression on iBMEC maturation. Results Sox18-mediated iBMECs achieved a higher TEER value than normal iBMECs (> 3000 Ω cm2). From day 6 to day 10 (d6–10 group), the iBMECs with SOX18 expression expressed a series of tight junction markers and showed upregulation of Mfsd2a, a specific marker of the BBB. The d6–10 group also expressed SLC2A1/Glut1 at levels as high as normal iBMECs, and upregulated ABCB1/P-gp and ABCC1/MRP1 expression. Moreover, Sox18-mediated iBMECs showed higher viability than normal iBMECs after puromycin treatment, indicating that SOX18 expression could upregulate P-gp activity in iBMECs. Conclusions Inducible SOX18 expression in iBMECs gained BBB phenotypes, including high TEER values and upregulation of tight junction-related genes, endothelial cell (EC) markers, BBB transporters, and higher cell viability after treatment with puromycin. Collectively, we provide a differentiation method for the maturation of human iPS cell-derived BMECs with SOX18 expression, describing its contribution to form an in vitro BBB model for CNS drug transport studies.https://doi.org/10.1186/s12987-023-00408-5Blood-brain-barrierBrain microvascular endothelial cellsSox18Transcription factorsMaturationDifferentiation |
| spellingShingle | Hongyan Zhang Tomoko Yamaguchi Kenji Kawabata The maturation of iPS cell-derived brain microvascular endothelial cells by inducible-SOX18 expression Fluids and Barriers of the CNS Blood-brain-barrier Brain microvascular endothelial cells Sox18 Transcription factors Maturation Differentiation |
| title | The maturation of iPS cell-derived brain microvascular endothelial cells by inducible-SOX18 expression |
| title_full | The maturation of iPS cell-derived brain microvascular endothelial cells by inducible-SOX18 expression |
| title_fullStr | The maturation of iPS cell-derived brain microvascular endothelial cells by inducible-SOX18 expression |
| title_full_unstemmed | The maturation of iPS cell-derived brain microvascular endothelial cells by inducible-SOX18 expression |
| title_short | The maturation of iPS cell-derived brain microvascular endothelial cells by inducible-SOX18 expression |
| title_sort | maturation of ips cell derived brain microvascular endothelial cells by inducible sox18 expression |
| topic | Blood-brain-barrier Brain microvascular endothelial cells Sox18 Transcription factors Maturation Differentiation |
| url | https://doi.org/10.1186/s12987-023-00408-5 |
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