Abstract
Human pluripotent stem cells (hPSCs) are inherently sensitive cells. Single-cell dissociation and the establishment of clonal cell lines have been long-standing challenges. This inefficiency of cell cloning represents a major obstacle for the standardization and streamlining of gene editing in induced pluripotent stem cells for basic and translational research. Here we describe a chemically defined protocol for robust single-cell cloning using microfluidics-based cell sorting in combination with the CEPT small-molecule cocktail. This advanced strategy promotes the viability and cell fitness of self-renewing stem cells. The use of low-pressure microfluidic cell dispensing ensures gentle and rapid dispensing of single cells into 96- and 384-well plates, while the fast-acting CEPT cocktail minimizes cellular stress and maintains cell structure and function immediately after cell dissociation. The protocol also facilitates clone picking and produces genetically stable clonal cell lines from hPSCs in a safe and cost-efficient fashion. Depending on the proliferation rate of the clone derived from a single cell, this protocol can be completed in 7–14 d and requires experience with aseptic cell culture techniques. Altogether, the relative ease, scalability and robustness of this workflow should boost gene editing in hPSCs and leverage a wide range of applications, including cell line development (e.g., reporter and isogenic cell lines), disease modeling and applications in regenerative medicine.
This is a preview of subscription content, access via your institution
Access options
Access Nature and 54 other Nature Portfolio journals
Get Nature+, our best-value online-access subscription
$29.99 / 30 days
cancel any time
Subscribe to this journal
Receive 12 print issues and online access
$259.00 per year
only $21.58 per issue
Buy this article
- Purchase on SpringerLink
- Instant access to full article PDF
Prices may be subject to local taxes which are calculated during checkout
Similar content being viewed by others
Data availability
The main data discussed in this protocol were generated as part of the studies published in the supporting primary research papers in refs. 31,32. Whole-exome files have been deposited to the Sequence Read Archive under BioProject PRJNA552890.
References
Thomson, J. A. et al. Embryonic stem cell lines derived from human blastocysts. Science 282, 1145–1147 (1998).
Takahashi, K. et al. Induction of pluripotent stem cells from adult human fibroblasts by defined factors. Cell 131, 861–872 (2007).
Shi, Y., Inoue, H., Wu, J. C. & Yamanaka, S. Induced pluripotent stem cell technology: a decade of progress. Nat. Rev. Drug Discov. 16, 115–130 (2017).
Sharma, A., Sances, S., Workman, M. J. & Svendsen, C. N. Multi-lineage human iPSC-derived platforms for disease modeling and drug discovery. Cell Stem Cell 26, 309–329 (2020).
Evans, M. J. & Kaufman, M. H. Establishment in culture of pluripotential cells from mouse embryos. Nature 292, 154–156 (1981).
Pal, R. A small-molecule cocktail that beats cellular stress. Nat. Methods 18, 457–458 (2021).
Xu, R. H. et al. Basic FGF and suppression of BMP signaling sustain undifferentiated proliferation of human ES cells. Nat. Methods 2, 185–190 (2005).
Ludwig, T. E. et al. Feeder-independent culture of human embryonic stem cells. Nat. Methods 3, 637–646 (2006).
Ludwig, T. E. et al. Derivation of human embryonic stem cells in defined conditions. Nat. Biotechnol. 24, 185–187 (2006).
Chen, G. et al. Chemically defined conditions for human iPSC derivation and culture. Nat. Methods 8, 424–429 (2011).
Rodin, S. et al. Clonal culturing of human embryonic stem cells on laminin-521/E-cadherin matrix in defined and xeno-free environment. Nat. Commun. 5, 1–13 (2014).
Levenstein, M. E. et al. Basic fibroblast growth factor support of human embryonic stem cell self-renewal. Stem Cells 24, 568–574 (2006).
Garitaonandia, I. et al. Increased risk of genetic and epigenetic instability in human embryonic stem cells associated with specific culture conditions. PLoS ONE 10, 1–25 (2015).
Barbaric, I. et al. Time-lapse analysis of human embryonic stem cells reveals multiple bottlenecks restricting colony formation and their relief upon culture adaptation. Stem Cell Rep. 3, 142–155 (2014).
Watanabe, K. et al. A ROCK inhibitor permits survival of dissociated human embryonic stem cells. Nat. Biotechnol. 25, 681–686 (2007).
Ohgushi, M. et al. Molecular pathway and cell state responsible for dissociation-induced apoptosis in human pluripotent stem cells. Cell Stem Cell 7, 225–239 (2010).
Chen, G., Hou, Z., Gulbranson, D. R. & Thomson, J. A. Actin–myosin contractility is responsible for the reduced viability of dissociated human embryonic stem cells. Cell Stem Cell 7, 240–248 (2010).
Barbaric, I. et al. Pinacidil enhances survival of cryopreserved human embryonic stem cells. Cryobiology 63, 298–305 (2011).
Xu, Y. et al. Revealing a core signaling regulatory mechanism for pluripotent stem cell survival and self-renewal by small molecules. Proc. Natl Acad. Sci. USA 107, 8129–8134 (2010).
Chen, Y. H. & Pruett-Miller, S. M. Improving single-cell cloning workflow for gene editing in human pluripotent stem cells. Stem Cell Res. 31, 186–192 (2018).
Singec, I. et al. Defining the actual sensitivity and specificity of the neurosphere assay in stem cell biology. Nat. Methods 3, 801–806 (2006).
Paull, D. et al. Automated, high-throughput derivation, characterization and differentiation of induced pluripotent stem cells. Nat. Methods 12, 885–892 (2015).
Howden, S. E., Thomson, J. A. & Little, M. H. Simultaneous reprogramming and gene editing of human fibroblasts. Nat. Protoc. 13, 875–898 (2018).
Doudna, J. A. The promise and challenge of therapeutic genome editing. Nature 578, 229–236 (2020).
Yumlu, S. et al. Gene editing and clonal isolation of human induced pluripotent stem cells using CRISPR/Cas9. Methods 121–122, 29–44 (2017).
Merkle, F. T. et al. Human pluripotent stem cells recurrently acquire and expand dominant negative P53 mutations. Nature 545, 229–233 (2017).
Roberts, B. et al. Systematic gene tagging using CRISPR/Cas9 in human stem cells to illuminate cell organization. Mol. Biol. Cell 28, 2854–2874 (2017).
Andrews, P. W. et al. Assessing the safety of human pluripotent stem cells and their derivatives for clinical applications. in. Stem Cell Rep. 9, 1–4 (2017).
Ihry, R. J. et al. p53 inhibits CRISPR–Cas9 engineering in human pluripotent stem cells. Nat. Med. 24, 939–946 (2018).
Li, X. L. et al. Highly efficient genome editing via CRISPR–Cas9 in human pluripotent stem cells is achieved by transient BCL-XL overexpression. Nucleic Acids Res. 46, 10195–10215 (2018).
Chen, Y. et al. A versatile polypharmacology platform promotes cytoprotection and viability of human pluripotent and differentiated cells. Nat. Methods 18, 528–541 (2021).
Tristan, C. A. et al. Robotic high-throughput biomanufacturing and functional differentiation of human pluripotent stem cells. Stem Cell Rep. 16, 3076–3092 (2021).
Ortmann, D. & Vallier, L. Variability of human pluripotent stem cell lines. Curr. Opin. Genet. Dev. 46, 179–185 (2017).
Acknowledgements
We are grateful for the support from the Regenerative Medicine Program (RMP) of the NIH Common Fund, NIH HEAL Initiative and in part by the intramural research program of the National Center for Advancing Translational Sciences (NCATS), NIH. The funders had no role in study design, data collection and data analysis, decision to publish, or preparation of the manuscript. We are also thankful to the NIH Medical Arts Design Section for preparing Fig. 4.
Author information
Authors and Affiliations
Contributions
C.A.T. and I.S. conceived the project and designed the protocol. C.A.T. performed the experiments with help from H.H., Y.J., Y.C., C.W., P-H.C., S.R. and V.M.J. I.H. and T.C.V. performed the 3D rendering data analysis. Data analysis and discussions were done by all authors. C.A.T. and I.S. wrote the manuscript.
Corresponding authors
Ethics declarations
Competing interests
I.S., Y.C. and A.S. are co-inventors on a US Department of Health and Human Services patent application covering the CEPT cocktail and its use.
Peer review
Peer review information
Nature Protocols thanks Andreas Kurtz, Jeanne Loring, Ai Zhang and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.
Additional information
Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Related links
Key references using this protocol
Chen, Y. et al. Nat. Methods 18, 528–541 (2021): https://doi.org/10.1038/s41592-021-01126-2
Tristan, C. A. et al. Stem Cell Rep. 16, 3076–3092 (2021): https://doi.org/10.1016/j.stemcr.2021.11.004
Supplementary information
Source data
Source Data Fig. 2
Cell roundness and impedance data.
Source Data Fig. 3
% Cloning efficiency, growth rate confluency and cell survival data.
Source Data Fig. 6
Unprocessed western blots.
Rights and permissions
About this article
Cite this article
Tristan, C.A., Hong, H., Jethmalani, Y. et al. Efficient and safe single-cell cloning of human pluripotent stem cells using the CEPT cocktail. Nat Protoc 18, 58–80 (2023). https://doi.org/10.1038/s41596-022-00753-z
Received:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1038/s41596-022-00753-z
Comments
By submitting a comment you agree to abide by our Terms and Community Guidelines. If you find something abusive or that does not comply with our terms or guidelines please flag it as inappropriate.
