1,25-Dihydroxyvitamin D Enhances the Regenerative Function of Lgr5+ Intestinal Stem Cells In Vitro and In Vivo
Abstract
:1. Introduction
2. Materials and Methods
2.1. Animal Subjects
2.2. Crypt Single-Cell Isolation
2.3. High-Purity Lgr5+ Single ISC Culture
2.4. Organoid Culture
2.5. Generation of VDR Knockdown ISC Lines
2.6. Plasmid Constructs
2.7. Preparation of Lentiviruses
2.8. Generation of Inflammation- and Gut-Homing Macrophages (CD11b+Gr1+) (MAC Cells) from Bone Marrow and Lentivirus Transduction
2.9. Experiments for Tracking Lgr5+ ISCs In Vivo
2.10. Experiments Using DC-CYP and DC-Ctr Cells
2.11. Induction and Treatment of Experimental Colitis
2.12. Histological Analysis
2.13. Immunocytochemistry Analysis
2.14. Flow Cytometry Analysis
2.15. Quantitative Real-Time RT-qPCR
2.16. Western Blot Analysis
2.17. Statistical Analysis
3. Results
3.1. High-Purity Lgr5+ ISC Lines Express Stemness Markers and Molecules Necessary for 1,25(OH)2D Signaling
3.2. High-Purity Lgr5+ ISC Lines Initiate Differentiation after Removing Chemical Inhibitors (Valproic Acid and CHIR99021)
3.3. The 1,25(OH)2D at Concentrations Higher than Physiological Levels Strongly Enhances the Gene and Protein Expression of Mature Intestinal Epithelial Markers in the Lgr5+ ISC Lines in the Presence and Absence of Valproic Acid and CHIR99021
3.4. The 1,25(OH)2D at Concentrations Higher than Physiological Levels Vigorously Augments Mature Intestinal Epithelial Markers’ Gene and Protein Expression in Colon Organoid Cultures
3.5. The 1,25(OH)2D’s Functions in Lgr5+ ISCs are Primarily Mediated by VDR
3.6. De Novo Synthesized Locally High 1,25(OH)2D Concentrations Modulate Local Vitamin D Metabolism
3.7. De Novo Synthesized Locally High 1,25(OH)2D Concentrations Enhance Lgr5+ ISC Differentiation In Vivo
4. Discussion
5. Conclusions
Supplementary Materials
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Acknowledgments
Conflicts of Interest
References
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Shaikh, N.A.; Liu, C.; Yin, Y.; Baylink, D.J.; Tang, X. 1,25-Dihydroxyvitamin D Enhances the Regenerative Function of Lgr5+ Intestinal Stem Cells In Vitro and In Vivo. Cells 2024, 13, 1465. https://doi.org/10.3390/cells13171465
Shaikh NA, Liu C, Yin Y, Baylink DJ, Tang X. 1,25-Dihydroxyvitamin D Enhances the Regenerative Function of Lgr5+ Intestinal Stem Cells In Vitro and In Vivo. Cells. 2024; 13(17):1465. https://doi.org/10.3390/cells13171465
Chicago/Turabian StyleShaikh, Nisar Ali, Chenfan Liu, Yue Yin, David J. Baylink, and Xiaolei Tang. 2024. "1,25-Dihydroxyvitamin D Enhances the Regenerative Function of Lgr5+ Intestinal Stem Cells In Vitro and In Vivo" Cells 13, no. 17: 1465. https://doi.org/10.3390/cells13171465