Abstract: PO0630
Mechanistic Elucidation of Nephron Progenitor Cell Expansion Using a Small Molecule, TCS21311, That Replaces BMP7 and Promotes Cell Proliferation
Session Information
- Development, Stem Cells, and Regenerative Medicine
November 04, 2021 | Location: On-Demand, Virtual Only
Abstract Time: 10:00 AM - 12:00 PM
Category: Development, Stem Cells, and Regenerative Medicine
- 500 Development, Stem Cells, and Regenerative Medicine
Authors
- Tsujimoto, Hiraku, Kyoto Daigaku iPS Saibo Kenkyujo, Kyoto, Japan
- Araoka, Toshikazu, Kyoto Daigaku iPS Saibo Kenkyujo, Kyoto, Japan
- Fuji, Hideyoshi, Astellas Seiyaku Kabushiki Kaisha, Chuo-ku, Tokyo, Japan
- Yamagishi, Yukiko, Astellas Seiyaku Kabushiki Kaisha, Chuo-ku, Tokyo, Japan
- Osafune, Kenji, Kyoto Daigaku iPS Saibo Kenkyujo, Kyoto, Japan
Background
Nephron progenitor cells (NPCs) give rise to all epithelial components of the nephron, which is the smallest functional unit of the kidney. The development of a stable supply of NPCs is expected to contribute to kidney regeneration research. Although most reports on the development of NPC expansion culture use BMP7, the detailed mechanisms of action of BMP7 are unknown. To elucidate the roles of BMP7 and improve the NPC expansion culture method, we sought small molecules that can replace BMP7 in the culture system.
Methods
We isolated NPCs from Six2-GFP reporter mice and screened 4,395 chemical compounds using a previously reported expansion culture system. The activity of analogous chemicals from the hits was examined. We predicted the molecular targets of the hit compounds by chemoinformatics analyses of molecular structures. Known downstream signaling pathways were examined by immunoblotting, and differentially expressed genes (DEGs) were analyzed by removing BMP7 from the NPC expansion culture. Furthermore, we improved the expansion culture method using mouse embryonic and human induced pluripotent stem cell (iPSC)-derived NPCs by adding the hit compounds to the expansion culture condition including BMP7.
Results
The chemical screening identified a JAK3 inhibitor, CP690550, in the mouse NPC expansion culture. Although several JAK3 inhibitors as well as some JAK2/3, JAK1/2 and JAK2 inhibitors showed similar activity, one JAK3 inhibitor, TCS21311, showed especially potent effects. A structural analysis of TCS21311 confirmed that JAK3 is its primary target. A pathway analysis of the DEGs by the BMP7 removal indicated STAT3 pathway activation. The phosphorylation of Smad1/5 was increased by TCS21311 even in the absence of BMP7, suggesting a mechanism by which TCS21311 replaces BMP7 via JAK3-STAT3. Furthermore, the addition of TCS21311 to the expansion culture containing BMP7 resulted in more efficient proliferation of mouse embryonic and human iPSC-derived NPCs.
Conclusion
These results will contribute to understanding the roles of BMP7 in NPC proliferation and to the stable supply of NPCs.
Funding
- Commercial Support –