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. 2022 Aug;49(8):7887-7898.
doi: 10.1007/s11033-022-07621-9. Epub 2022 May 30.

Development of an efficient single-cell cloning and expansion strategy for genome edited induced pluripotent stem cells

Nupur Bhargava #  1   2 , Priya Thakur #  1   2 , Thulasi Priyadharshini Muruganandam  1   2 , Shashank Jaitly  1   2 , Pragya Gupta  1   2 , Neelam Lohani  1   2 , Sangam Giri Goswami  1   2 , Vinodh Saravanakumar  1 , Saurabh Kumar Bhattacharya  3 , Suman Jain  4 , Sivaprakash Ramalingam  5   6
Affiliations

Development of an efficient single-cell cloning and expansion strategy for genome edited induced pluripotent stem cells

Nupur Bhargava et al. Mol Biol Rep. 2022 Aug.

Abstract

Background: Disease-specific human induced pluripotent stem cells (hiPSCs) can be generated directly from individuals with known disease characteristics or alternatively be modified using genome editing approaches to introduce disease causing genetic mutations to study the biological response of those mutations. The genome editing procedure in hiPSCs is still inefficient, particularly when it comes to homology directed repair (HDR) of genetic mutations or targeted transgene insertion in the genome and single cell cloning of edited cells. In addition, genome editing processes also involve additional cellular stresses such as poor cell viability and genetic stability of hiPSCs. Therefore, efficient workflows are desired to increase genome editing application to hiPSC disease models and therapeutic applications.

Methods and results: To this end, we demonstrate an efficient workflow for feeder-free single cell clone generation and expansion in both CRISPR-mediated knock-out (KO) and knock-in (KI) hiPSC lines. Using StemFlex medium and CloneR supplement in conjunction with Matrigel cell culture matrix, we show that cell viability and expansion during single-cell cloning in edited and unedited cells is significantly enhanced. Keeping all factors into account, we have successfully achieved hiPSC single-cell survival and cloning in both edited and unedited cells with rates as maximum as 70% in less than 2 weeks.

Conclusion: This simplified and efficient workflow will allow for a new level of sophistication in generating hiPSC-based disease models to promote rapid advancement in basic research and also the development of novel cellular therapeutics.

Keywords: Disease modeling; Drug discovery; Genome-editing; Induced pluripotent stem cells; Regenerative medicine; Single-cell cloning.

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References

    1. Rowe R, Daley G (2019) Induced pluripotent stem cells in disease modelling and drug discovery. Nat Rev Genet 20(7):377–388. https://doi.org/10.1038/s41576-019-0100-z - DOI - PubMed - PMC
    1. Liu G, David B, Trawczynski M, Fessler R (2019) Advances in pluripotent stem cells: history, mechanisms, technologies, and applications. Stem Cell Rev Rep 16(1):3–32. https://doi.org/10.1007/s12015-019-09935-x - DOI - PMC
    1. Govindan G, Ramalingam S (2016) Programmable site-specific nucleases for targeted genome engineering in higher eukaryotes. J Cell Physiol 231(11):2380–2392. https://doi.org/10.1002/jcp.25367 - DOI - PubMed
    1. Kawatani K, Nambara T, Nawa N, Yoshimatsu H, Kusakabe H, Hirata K et al (2021) A human isogenic iPSC-derived cell line panel identifies major regulators of aberrant astrocyte proliferation in Down syndrome. Commun Biol. https://doi.org/10.1038/s42003-021-02242-7 - DOI - PubMed - PMC
    1. Dakhore S, Nayer B, Hasegawa K (2018) Human pluripotent stem cell culture: current status, challenges, and advancement. Stem Cells International 2018:1–17. https://doi.org/10.1155/2018/7396905 - DOI

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