CEPT Cocktail

Pluripotent stem cells (PSCs) are highly sensitive to cellular stress in vitro, which presents challenges for their study and use. Small molecule ROCK (Rho-Kinase) inhibitors used as single agents, such as Y-27632 (Cat. No. 1254) and Thiazovivin (Cat. No. 3845), are known to improve viability of stem cells undergoing passaging or cryopreservation.

Chen et al. (2021), from National Center for Advancing Translational Sciences (NCATS) devised a combination of small molecules that enhances the viability of human pluripotent and differentiated cells, comprising the ROCK inhibitor Chroman 1, Emricasan a pan-caspase inhibitor, Polyamines, and the integrated stress response inhibitor Trans-ISRIB (CEPT). This small molecule cocktail simultaneously blocks several stress mechanisms that would otherwise compromise cell structure and function.

Figure 1: Schematic showing a scalable single-cell cloning workflow for hPSCs, combining the CEPT Cocktail and Namocell microfluidic cell dispensing technology, which ensures that single-cell cloning is safe, efficient and practical. The schematic highlights the steps required for streamlined, high-throughput single-cell cloning, cell characterization, and potential downstream applications. Adapted from Tristan et al. (2022) Nat. Protoc.

Chroman 1 was discovered by the team at NCATS to be a potent and selective ROCK inhibitor, that significantly improves cell survival alone or in combination with other small molecules. Emricasan synergizes with Chroman 1 to reduce apoptosis of PSCs, while trans-ISRIB supports protein synthesis and cell attachment during cell passaging. Polyamines are essential polycations that support cell growth, stabilized cellular structures and macromolecules, as well as mediate functions such as transcription, translation, cell cycle and stress response. When combined with Chroman 1, Emricasan and trans-ISRIB in cultures of hPSCs, polyamines have a synergistic effect leading to improved cell attachment and protein translation as well as further improvements in cell survival.

CEPT effectively improves cell survival during routine long-term passaging, single-cell cloning, gene editing, and differentiation of PSCs (Figure 1), as well as embryoid body and organoid formation. In addition, the CEPT cocktail improves survival of differentiated cells following cryopreservation, with cardiomyocyte survival increasing by 36% and motor neuron survival increasing by 63%, compared to DMSO (Cat. No. 3176) controls. It can be used in the large-scale manufacture of hiPSCs under chemically-defined conditions, as well as the standardized large-scale differentiation from hiPSCs of various cell types, including cardiomyocytes, neurons and hepatocytes. The CEPT cocktail therefore has broad potential for improving the manufacture of cells for disease modeling, drug screening, regenerative medicine and cell therapy.

References

1. Chen et al. A versatile polypharmacology platform promotes cytoprotection and viability of human pluripotent and differentiated cells. Nat. Methods 2021, 18 528.
2. Tristan et al. Robotic high-throughput biomanufacturing and functional differentiation of human pluripotent stem cells. Stem Cell Rep. 2021, 16 3076.
3. Tristan et al. Efficient and safe single-cell cloning of human pluripotent stem cells using the CEPT cocktail. Nat. Protoc. 2022, 18 58.