Efficient single-cell cloning of hPSCs using the CEPT strategy

Single-cell cloning and gene editing patient-specific hPSCs, particularly through CRISPR/Cas9 techniques, has enormous potential to advance the field of regenerative medicine. The main challenges to the hPSC gene editing field are 1) low cloning efficiencies, 2) severe observed cellular stress, 3) risk of inducing genetic mutations, 4) identification of correctly edited cell populations, and 5) establishment of a healthy, self-renewing clonal cell line. 

The CEPT strategy is a published, chemically defined small molecule combination of Chroman 1, Emricasan, and Trans-ISRIB (CET), along and a polyamine solution (CEPT), proven to improve cell viability and genomic stability of hPSCs in single-cell and low-density cultures through potent ROCK pathway inhibition, promotion of cell adhesion, maintenance of cellular structure and membrane integrity, and reduction in DNA fracture. 

Cytoprotection from the CEPT strategy provides superior cell survival under stressful conditions, including single-cell passaging, cloning, and gene editing protocols. This research spotlight highlights a 2022 Nature Protocols publication by Tristan et al., outlining a reproducible and scalable workflow for establishing clonal cell lines and a standardized gene editing procedure. The process combines the unique cellular protection from CEPT with a streamlined and gentle process for single-cell dispensing into clonal wells through a low-pressure microfluidic platform.

The Spotlight

Keywords: hPSC culture, clonal cell line generation, reporter and isogenic cell line establishment, gene editing, hPSC single-cell dissociation, disease modeling, regenerative medicine, cell-based therapies

Cell type(s): hPSCs, hiPSCs, hESCs

Experiment purpose: Efficient hPSC single-cell cloning protocol that provides a low-cost, chemically defined method to generate genetically stable clones, overcomes hPSC’s sensitivity to dissociation, and allows significant improvement in gene editing workflows.

Product highlights: This study highlights a combination of small molecules comprised of Chroman 1, Emricasan, and Trans-IRIB. Captivate Bio has launched a CET Cocktail pack which includes the powdered small molecules found in the CEPT publication. The Captivate Bio CET Cocktail pack enhances stem cells in survival, promotes clonal growth of genetically stable hPSCs, and provides comprehensive cytoprotection.

Reference: Tristan et al. 2022. Efficient and safe single-cell cloning of human pluripotent stem cells using the CEPT cocktail. Nature Protocols. 

Conclusion

This study found that CEPT supplementation provides optimal physiochemical conditions for efficient single-cell cloning of hPSCs and therefore allows standardization and scalability of hPSC gene editing workflows. This protocol ultimately improves the efficiency and utilization of these cells in a wide range of regenerative medicine and disease modeling applications.  

Want to know more about the CET Cocktail Kit or CEPT protocol?
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Image source: Tristan et al. 2022. Nature Methods.