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

A Small Molecule Cocktail Promotes Survival Of Human Pluripotent and Differentiated Cells

Posted on September 3rd, 2019 by claire.malley@nih.gov

Members of SCTL presented a poster at the ISSCR 2019. The poster is entitled: A Small Molecule Cocktail Promotes Survival Of Human Pluripotent and Differentiated Cells.

Authors (underlined, presenting): Yu Chen, Carlos A. Tristan, Lu Chen, Vukasin M. Jovanovic, Claire Malley, Pei-Hsuan Chu, Seungmi Ryu, Tao Deng, Pinar Ormanoglu, Dingyin Tao, Jaroslav Slamecka, Sam Michael, Christopher P. Austin, Anton Simeonov, Ilyas Singeç

Download the full-size poster here.

More information about ISSCR 2019.


Access to human induced pluripotent stem cells (iPSCs) has created novel paradigms for drug discovery and regenerative medicine. However, poor survival of iPSCs during routine passaging, cell differentiation, and cryopreservation poses major challenges to the establishment of standard operating procedures to produce and store iPSCs on a large scale, and to the development of efficient genome editing protocols based on optimized single-cell cloning procedures. The ROCK inhibitor Y-27632 has been widely used to improve cell survival, but significant amounts of cell death remain evident in many iPSC applications. Here, we developed a four-component small- molecule cocktail named “CEPT” that dramatically improves iPSC viability. Testing 15,333 compounds in quantitative high-throughput screening (qHTS), we first identified 113 hits that improved iPSC survival. Advancing 29 hits to combination screening based on their diverse modes of action, we discovered compound C and compound E as a synergistic pair (C+E) that improved iPSC survival during routine passaging by approximately 50% as compared to Y- 27632. Despite the dramatic effect of C+E during routine passaging, only modest improvement was obtained when iPSCs were seeded at a low density (25 cells/cm2) or in a 1 cell/well condition. Therefore, we designed another combination screening assay to search for additional compounds that, when applied together with C+E, can further enhance iPSC survival at ultra-low cell density conditions. Screening 7,599 compounds using the new assay, we found that C+E together with two additional compounds (compounds P and T) dramatically improved iPSC survival at ultra- low cell density, increasing single-cell cloning efficiency up to ~55% as compared to the ~10% with Y-27632. We then extensively tested CEPT and demonstrated that this cocktail was highly efficient in improving cell survival during routine cell passaging, embryoid body formation, genome editing using CRISPR/Cas9, the establishment of new iPSC lines, and cryopreservation/thawing various differentiated cells. Hence, the versatility of CEPT provides a powerful chemical platform for establishing efficient protocols and may become a widely used approach in cryobiology, drug development, and regenerative medicine.