Integration of early-stage cryopreservation and cell cycle modulation into a flexible kidney organoid differentiation system
Yan, X.; Wang, J.; Xu, M.; Hu, C.; Chen, S.; Zhao, Y.; Wang, J.; Rong, R.; Zhu, T.; Zhang, W.
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Kidney organoids derived from human pluripotent stem cells (hPSCs) represent a promising platform for modeling nephrogenesis and renal diseases. However, conventional differentiation protocols are continuous and time-sensitive, limiting their scalability and reproducibility. Here, we developed a method to pause and resume organoid formation through cryopreservation at an early differentiation stage using a chemically defined formulation that maintains high post-thaw viability and differentiation potential. We further found that synchronizing hPSCs in the G1 phase with PD-0332991 enhanced post-thaw organoid formation and transcriptional fidelity, while G2 phase enrichment with Ro-3306 promoted the development of SLC12A3-positive distal convoluted tubules. The post-thaw organoids exhibited well-organized nephron architecture and function comparable to uninterrupted cultured controls. This platform proved effective for modeling BK polyomavirus (BKV) infection, drug-induced nephrotoxicity, and renal fibrosis. Together, our cryopreservation and cell cycle synchronization strategy provides a flexible, practical framework to advance organoid-based research and translation.
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