U.S. Department of Health and Human ServicesHHS National Institutes of HealthNIH National Center for Advancing Translational SciencesNCATS

Stress-free cell aggregation by using the CEPT cocktail enhances embryoid body and organoid fitness

Posted on December 11th, 2023 by Hannah Baskir

Seungmi Ryu 1Claire Weber 1Pei-Hsuan Chu 1Ben Ernest 2Vukasin M Jovanovic 1Tao Deng 1Jaroslav Slamecka 1Hyenjong Hong 1Yogita Jethmalani 1Hannah M Baskir 1Jason Inman 1John Braisted 1Marissa B Hirst 2Anton Simeonov 1Ty C Voss 1Carlos A Tristan 1Ilyas Singeç 1

1National Center for Advancing Translational Sciences (NCATS), Stem Cell Translation Laboratory (SCTL), National Institutes of Health (NIH), Rockville, MD 20850, United States of America.

2Rancho Biosciences, 16955 Via Del Campo, #200, San Diego, CA 92127, United States of America.

Abstract

Embryoid bodies (EBs) and self-organizing organoids derived from human pluripotent stem cells (hPSCs) recapitulate tissue development in a dish and hold great promise for disease modeling and drug development. However, current protocols are hampered by cellular stress and apoptosis during cell aggregation, resulting in variability and impaired cell differentiation. Here, we demonstrate that EBs and various organoid models (e.g., brain, gut, kidney) can be optimized by using the small molecule cocktail named CEPT (chroman 1, emricasan, polyamines, trans-ISRIB), a polypharmacological approach that ensures cytoprotection and cell survival. Application of CEPT for just 24 h during cell aggregation has long-lasting consequences affecting morphogenesis, gene expression, cellular differentiation, and organoid function. Various qualification methods confirmed that CEPT treatment enhanced experimental reproducibility and consistently improved EB and organoid fitness as compared to the widely used ROCK inhibitor Y-27632. Collectively, we discovered that stress-free cell aggregation and superior cell survival in the presence of CEPT are critical quality control determinants that establish a robust foundation for bioengineering complex tissue and organ models.