Efficient and safe single-cell cloning of human pluripotent stem cells using the CEPT cocktail

Carlos A. Tristan, Hyenjong Hong, Yogita Jethmalani, Yu Chen, Claire Weber, Pei-Hsuan Chu, Seungmi Ryu, Vukasin M. Jovanovic, Inae Hur, Ty C. Voss, Anton Simeonov, Ilyas Singeç

Published: 2022-10-18 DOI: 10.1038/s41596-022-00753-z

microfluidics

hPSCs

single-cell cloning

gene editing

CEPT cocktail

AI 解读

Supplementary information

Reporting Summary

Tutorial: guidelines for the computational analysis of single-cell RNA sequencing data

ProBac-seq, a bacterial single-cell RNA sequencing methodology using droplet microfluidics and large oligonucleotide probe sets

Identification of cancer-related mutations in human pluripotent stem cells using RNA-seq analysis

Spiral volumetric optoacoustic tomography for imaging whole-body biodynamics in small animals

Genetic manipulation and immortalized culture of ex vivo primary human germinal center B cells

Tutorial: design and execution of CRISPR in vivo screens

Reproducible strategy for excisional skin-wound-healing studies in mice

CRISPR–Cas9-mediated chromosome engineering inArabidopsis thaliana

Human primordial germ cell-like cells specified from resetting precursors develop in human hindgut organoids

Tracking endocytosis and intracellular distribution of spherical nucleic acids with correlative single-cell imaging

查看全部

Sections

Figures

References

Supplementary information

Ludwig, T. E. et al. Feeder-independent culture of human embryonic stem cells. Nat. Methods 3, 637–646 (2006).
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. 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).

Efficient and safe single-cell cloning of human pluripotent stem cells using the CEPT cocktail

Supplementary information

推荐阅读