Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Article
  • Published:

A versatile polypharmacology platform promotes cytoprotection and viability of human pluripotent and differentiated cells

Abstract

Human pluripotent stem cells (hPSCs) are capable of extensive self-renewal yet remain highly sensitive to environmental perturbations in vitro, posing challenges to their therapeutic use. There is an urgent need to advance strategies that ensure safe and robust long-term growth and functional differentiation of these cells. Here, we deployed high-throughput screening strategies to identify a small-molecule cocktail that improves viability of hPSCs and their differentiated progeny. The combination of chroman 1, emricasan, polyamines, and trans-ISRIB (CEPT) enhanced cell survival of genetically stable hPSCs by simultaneously blocking several stress mechanisms that otherwise compromise cell structure and function. CEPT provided strong improvements for several key applications in stem-cell research, including routine cell passaging, cryopreservation of pluripotent and differentiated cells, embryoid body (EB) and organoid formation, single-cell cloning, and genome editing. Thus, CEPT represents a unique poly-pharmacological strategy for comprehensive cytoprotection, providing a rationale for efficient and safe utilization of hPSCs.

This is a preview of subscription content, access via your institution

Access options

Rent or buy this article

Prices vary by article type

from$1.95

to$39.95

Prices may be subject to local taxes which are calculated during checkout

Fig. 1: Quantitative HTS identifies chroman 1 as a best-in-class ROCK inhibitor.
Fig. 2: Combinatorial matrix screen identifies compounds with synergistic activities.
Fig. 3: The combination of chroman 1, emricasan, polyamines, and trans-ISRIB promotes clonal growth and expansion of genetically stable hPSCs.
Fig. 4: Optimized EB and organoid formation.
Fig. 5: CEPT enables superior cryopreservation of pluripotent and differentiated cells.
Fig. 6: CEPT protects dissociated hPSCs from multiple cellular stress mechanisms.

Similar content being viewed by others

Data availability

Extended results of small-molecule screening associated with Figs. 1–3, Extended Data Figs. 2 and 3, and Supplementary Figs. 1 and 2 are available at the following link: https://tripod.nih.gov/matrix-client/?project=2621.

Whole-exome and RNA-seq files have been deposited to the Sequence Read Archive under BioProject PRJNA552890. An interactive R Shiny app is available to browse the RNA-seq data at https://ipsceq.ncats.io. Sequencing data analysis code is available at https://github.com/cemalley/Chen_methods. ANNOVAR and annotation databases are available for download at https://annovar.openbioinformatics.org/en/latest/user-guide/download/. Source data is available for Figs. 16 and Extended Data Figs. 3, 6, 810 with this paper.

References

  1. 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).

    Article  CAS  PubMed  Google Scholar 

  2. Chen, G. et al. Chemically defined conditions for human iPSC derivation and culture. Nat. Methods 8, 424–429 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  3. 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).

  4. Chen, Y. & Pruett-Miller, S. M. Improving single-cell cloning work flow for gene editing in human pluripotent stem cells. Stem Cell Res. 31, 186–192 (2018).

  5. Ihry, R. J. et al. P53 inhibits CRISPR–Cas9 engineering in human pluripotent stem cells. Nat. Med. 24, 939–946 (2018).

    Article  CAS  PubMed  Google Scholar 

  6. Watanabe, K. et al. A ROCK inhibitor permits survival of dissociated human embryonic stem cells. Nat. Biotechnol. 25, 681–686 (2007).

    Article  CAS  PubMed  Google Scholar 

  7. 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).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  8. 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).

    Article  CAS  PubMed  Google Scholar 

  9. 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).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  10. Tilson, S. G. et al. ROCK inhibition facilitates in vitro expansion of glioblastoma stem-like cells. PLoS ONE 10, e0132823 (2015).

    Article  PubMed  PubMed Central  Google Scholar 

  11. Zhao, M. et al. Y-27632 preconditioning enhances transplantation of human-induced pluripotent stem cell-derived cardiomyocytes in myocardial infarction mice. Cardiovasc. Res. 115, 343–356 (2019).

    Article  CAS  PubMed  Google Scholar 

  12. Huang, R. et al. The NCGC Pharmaceutical Collection: a comprehensive resource of clinically approved drugs enabling repurposing and chemical genomics. Sci. Transl. Med. 3, 80ps16 (2011).

    Article  PubMed  PubMed Central  Google Scholar 

  13. Mathews Griner, L. A. et al. High-throughput combinatorial screening identifies drugs that cooperate with ibrutinib to kill activated B-cell-like diffuse large B-cell lymphoma cells. Proc. Natl Acad. Sci. USA 111, 2349–2354 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  14. Huang, R. et al. Chemical genomics profiling of environmental chemical modulation of human nuclear receptors. Environ. Health Perspect. 119, 1142–1148 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  15. Chen, Y. T. et al. Asymmetric synthesis of potent chroman-based Rho kinase (ROCK-II) inhibitors. Med. Chem. Commun. 2, 73–75 (2011).

    Article  CAS  Google Scholar 

  16. Anastassiadis, T., Deacon, S. W., Devarajan, K., Ma, H. & Peterson, J. R. Comprehensive assay of kinase catalytic activity reveals features of kinase inhibitor selectivity. Nat. Biotechnol. 29, 1039–1045 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  17. Vallabhaneni, H. et al. High basal levels of γH2AX in human induced pluripotent stem cells are linked to replication-associated DNA damage and repair. Stem Cells 36, 1501–1513 (2018).

  18. Närvä, E. et al. A strong contractile actin fence and large adhesions direct human pluripotent colony morphology and adhesion. Stem Cell Rep. 9, 67–76 (2017).

  19. 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).

    Article  Google Scholar 

  20. Inglese, J. et al. Quantitative high-throughput screening: a titration-based approach that efficiently identifies biological activities in large chemical libraries. Proc. Natl Acad. Sci. USA 103, 11473–11478 (2006).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  21. Chang, M.-Y. et al. Doxycycline enhances survival and self-renewal of human pluripotent stem cells. Stem Cell Rep. 3, 353–364 (2014).

  22. Sidrauski, C., McGeachy, A. M., Ingolia, N. T. & Walter, P. The small molecule ISRIB reverses the effects of eIF2α phosphorylation on translation and stress granule assembly. eLife 4, e05033 (2015).

  23. Miller-Fleming, L., Olin-Sandoval, V., Campbell, K. & Ralser, M. Remaining mysteries of molecular biology: the role of polyamines in the cell. J. Mol. Biol. 427, 3389–3406 (2015).

    Article  CAS  PubMed  Google Scholar 

  24. Zhao, T., Goh, K. J., Ng, H. H. & Vardy, L. A. A role for polyamine regulators in ESC self-renewal. Cell Cycle 11, 4517–4523 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  25. Valamehr, B. et al. A novel platform to enable the high-throughput derivation and characterization of feeder-free human iPSCs. Sci. Rep. 2, 213 (2012).

  26. Chang, M. T. et al. Identifying recurrent mutations in cancer reveals widespread lineage diversity and mutational specificity. Nat. Biotechnol. 34, 155–163 (2016).

    Article  CAS  PubMed  Google Scholar 

  27. Tate, J. G. et al. COSMIC: The Catalogue Of Somatic Mutations In Cancer. Nucleic Acids Res. 47, 941–947 (2018).

    Article  Google Scholar 

  28. O’Leary, N. A. et al. Reference sequence (RefSeq) database at NCBI: current status, taxonomic expansion, and functional annotation. Nucleic Acids Res. 44, D733–D745 (2016).

    Article  PubMed  Google Scholar 

  29. Sherry, S. T. dbSNP: the NCBI database of genetic variation. Nucleic Acids Res. 29, 308–311 (2001).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  30. Auton, A. et al. A global reference for human genetic variation. Nature 526, 68–74 (2015).

    Article  PubMed  Google Scholar 

  31. Lek, M. et al. Analysis of protein-coding genetic variation in 60,706 humans. Nature 536, 285–291 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  32. Adzhubei, I. A. et al. A method and server for predicting damaging missense mutations. Nat. Methods 7, 248–249 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  33. Landrum, M. J. et al. ClinVar: public archive of interpretations of clinically relevant variants. Nucleic Acids Res. 44, D862–D868 (2016).

    Article  CAS  PubMed  Google Scholar 

  34. Merkle, F. T. et al. Human pluripotent stem cells recurrently acquire and expand dominant negative P53 mutations. Nature 545, 229–233 (2017).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  35. 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).

  36. 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).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  37. Tsankov, A. M. et al. A qPCR ScoreCard quantifies the differentiation potential of human pluripotent stem cells. Nat. Biotechnol. 33, 1182–1192 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  38. Lancaster, M. A. et al. Cerebral organoids model human brain development and microcephaly. Nature 501, 373–379 (2013).

    Article  CAS  PubMed  Google Scholar 

  39. Lachmann, A. et al. Massive mining of publicly available RNA-seq data from human and mouse. Nat. Commun. 9, 1366 (2018).

    Article  PubMed  PubMed Central  Google Scholar 

  40. Wagh, V. et al. Effects of cryopreservation on the transcriptome of human embryonic stem cells after thawing and culturing. Stem Cell Rev. Rep. 7, 506–517 (2011).

  41. Wong, K. G. et al. CryoPause: a new method to immediately initiate experiments after cryopreservation of pluripotent stem cells. Stem Cell Rep. 9, 355–365 (2017).

  42. Schlaeger, T. M. et al. A comparison of non-integrating reprogramming methods. Nat. Biotechnol. 33, 58–63 (2014).

    Article  PubMed  PubMed Central  Google Scholar 

  43. Paull, D. et al. Automated, high-throughput derivation, characterization and differentiation of induced pluripotent stem cells. Nat. Methods 12, 885–892 (2015).

    Article  CAS  PubMed  Google Scholar 

  44. Takaki, T. et al. Actomyosin drives cancer cell nuclear dysmorphia and threatens genome stability. Nat. Commun. 8, 16013 (2017).

    Article  PubMed  PubMed Central  Google Scholar 

  45. Zannini, L., Delia, D. & Buscemi, G. CHK2 kinase in the DNA damage response and beyond. J. Mol. Cell. Biol. 6, 442–457 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  46. Wortel, I. M. N., van der Meer, L. T., Kilberg, M. S. & van Leeuwen, F. N. Surviving stress: modulation of ATF4-mediated stress responses in normal and malignant cells. Trends Endocrinol. Metab. 28, 794–806 (2017).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  47. Schmidt, E. K., Clavarino, G., Ceppi, M. & Pierre, P. SUnSET, a nonradioactive method to monitor protein synthesis. Nat. Methods 6, 275–277 (2009).

    Article  CAS  PubMed  Google Scholar 

  48. Bigarella, C. L., Liang, R. & Ghaffari, S. Stem cells and the impact of ROS signaling. Development 141, 4206–4218 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  49. Collins, A. R. The comet assay for DNA damage and repair: principles, applications, and limitations. Mol. Biotechnol. 26, 249–261 (2004).

    Article  CAS  PubMed  Google Scholar 

  50. Raj, D. et al. Switchable CAR-T cells mediate remission in metastatic pancreatic ductal adenocarcinoma. Gut 68, 1052–1064 (2018).

  51. Themeli, M. et al. Generation of tumor-targeted human T lymphocytes from induced pluripotent stem cells for cancer therapy. Nat. Biotechnol. 31, 928–933 (2013).

  52. Enver, T. et al. Cellular differentiation hierarchies in normal and culture-adapted human embryonic stem cells. Hum. Mol. Genet. 14, 3129–3140 (2005).

    Article  CAS  PubMed  Google Scholar 

  53. Baker, D. E. C. et al. Adaptation to culture of human embryonic stem cells and oncogenesis in vivo. Nat. Biotechnol. 25, 207–215 (2007).

    Article  CAS  PubMed  Google Scholar 

  54. Taapken, S. M. et al. Karyotypic abnormalities in human induced pluripotent stem cells and embryonic stem cells. Nat. Biotechnol. 29, 313–314 (2011).

    Article  CAS  PubMed  Google Scholar 

  55. Andrews, P. W. et al. Assessing the safety of human pluripotent stem cells and their derivatives for clinical applications. Stem Cell Rep. 9, 1–4 (2017).

    Article  Google Scholar 

  56. Rosler, E. S. et al. Long-term culture of human embryonic stem cells in feeder-free conditions. Dev. Dyn. 229, 259–274 (2004).

    Article  CAS  PubMed  Google Scholar 

  57. 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).

    Article  Google Scholar 

  58. Liu, L., Michowski, W., Kolodziejczyk, A. & Sicinski, P. The cell cycle in stem cell proliferation, pluripotency and differentiation. Nat. Cell Biol. 21, 1060–1067 (2019).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

Download references

Acknowledgements

We thank P. Shinn, M. Itkin, Z. Itkin, C. Klumpp-Thomas, J. Braisted, J. Freilino, T. Voss, M. Iannotti, C. Pepper Bonney, Y. Gedik, D. Ngan, A. Rossoshek, and A. Knebel for their support throughout this work. We are grateful to A. Hoofring and E. Tyler from the NIH Medical Arts Design Section and D. C. Gershlick (University of Cambridge) for their art designs. We would like to thank D. Panchision for critical reading of the manuscript. We also gratefully acknowledge funding from the Regenerative Medicine Program (RMP) of the NIH Common Fund 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 analysis; decision to publish; or preparation of the manuscript.

Author information

Authors and Affiliations

Authors

Contributions

Y.C. and I.S. conceived the project. Experiments and screening: Y.C., C.A.T., V.M.J., P.O., P.-H.C., S.R., T.D., D.T., Y.F., J.S., H.H., C.A.L. and S.M. Data analysis and discussions: Y.C., C.A.T., L.C., V.M.J., C.M., C.P.A., A.S., I.S. Manuscript writing: Y.C., C.A.T., A.S., I.S.

Corresponding author

Correspondence to Ilyas Singeç.

Ethics declarations

Competing interests

Y.C., A.S., and I.S. are coinventors on a US Department of Health and Human Services patent application covering CEPT and its use.

Additional information

Peer review information Nature Methods thanks the anonymous reviewers for their contribution to the peer review of this work. Madhura Mukhopadhyay was the primary editor on this article and managed its editorial process and peer review in collaboration with the rest of the editorial team.

Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Extended data

Extended Data Fig. 1 Target specificity and cell passaging with Chroman 1.

a, HotSpot kinase profiling was used to individually inhibit a panel of 369 human wild-type kinases using 50 nM Chroman 1 or 10 µM Y-27632 (concentrations were chosen based on dose-response curves and maximum cell survival in the primary screen; different colors in the tree indicate different kinase families). b, Table summarizing the inhibitory activity of 50 nM Chroman 1 and 10 μM Y-27632 as determined by HotSpot kinase profiling shown in panel (a). The table shows values for ROCK1 and ROCK2 as the primary targets. All off-target kinases that Y-27632 inhibits more strongly than Chroman 1 are shown and include PKCeta (also known as PKCη or PRKCH), PKCepsilon (also known as PKCε or PRKCE), PKCdelta (also known as PKCδ or PRKCD), PKN1, PKN2 and PRKX. Two negligible off-target kinases that Chroman 1 inhibits relatively better than Y-27632 are LATS2 (15.32%) and DMPK2 (16.44%). c, hESCs (WA09) maintained pluripotency and multilineage differentiation potential after serial passaging with Chroman 1 for 40 passages. Scale bar, 2.5 µm.

Extended Data Fig. 2 Synergy score examples and long-term culture in C + E.

a-d, Checkerboard examples from the matrix screening data. The 10 × 10 viability matrices (a,c) and the delta HSA value matrices (b,d) for the combination of Chroman 1 + Emricasan (a,b), and Chroman 1 + Blebbistatin (c,d). Yellow boxes highlight the maximum survival and synergy level achieved by the combinations. e,f, hESCs (WA09) were serially passaged for a total of 40 passages with C + E applied for the initial 24 h during every passage. Cells expressed pluripotent markers NANOG and OCT4, and differentiated into ectoderm (PAX6), mesoderm (Brachyury) and endoderm (SOX17) lineages both by directed differentiation in adherent cultures (e) and spontaneous differentiation in EB cultures followed by Scorecard analysis (f). Scale bar, 2.5 µm.

Extended Data Fig. 3 Assay development for ultra-low cell density HTS.

a, hESCs (WA09) were single-cell dissociated with Accutase and dispensed into 1536-well plates at the indicated cell densities. Two plates were prepared for each seeding density. CTG reading was carried out with one plate immediately following cell dispensing (read 1) and with the second plate 24 h later (read 2). CTG-fold change was calculated as read 2 divided by read 1 to indicate cell recovery and growth within 24 h. b, Assay development for hESCs (WA09) in 1536-well format to model the stress that is associated with ultra-low cell density on cell survival. Fewer than 50 cells per well was determined as ultra-low cell density (n = 128 wells for the density of 0 and 5; n = 256 wells for the density of 50, 100, 500, and 2000). c, Summary of qHTS performed at ultra-low cell density (10 cells/well) that identified 316 hits showing synergy with C + E (see Supplementary Table 2 for details). All compounds were used in combination with C + E, tested in triplicates, and ranked based on their median CTG readings. Data were normalized to the average CTG reading obtained with C + E. d, Example dose-response curves of Trans-ISRIB, Isotretinoin, and Y-27632. Data represent median ± s.d. (n = 6 wells for each concentrations of all groups).

Source data

Extended Data Fig. 4 Characterization of hPSC lines after long-term culture with CEPT.

a, Phase-contrast images of hESCs (WA09) at 24 h and 72 h post-passage comparing DMSO (control), Y-27632, and CEPT. Twice as many cells were plated in the DMSO group to compensate for poor cell survival after single-cell dissociation with Accutase and plating in E8 Medium without a ROCK inhibitor. Scale bar, upper row, 200 µm; lower row, 50 µm. b, Comprehensive analysis of eight cell lines passaged by using CEPT (24 h exposure at each passage). High-content imaging and quantification confirm that the vast majority of cells expressed pluripotency-associated markers. Staining and quantification was performed 3 days after plating using the method B algorithm of the Columbus image analysis system (Perkin-Elmer). Data are mean ± s.d. (n = 4 wells for each group). Karyotyping was carried out at the indicated passage numbers. c, Representative images showing that hESCs grow in colonies and express typical pluripotency-associated markers (Alkaline Phosphatase, NANOG, OCT4, SOX2) after long-term serial passaging with CEPT. Scale bar, 200 µm. d, Representative example demonstrating that hESCs (WA09) passaged by CEPT can be differentiated into neurons and other lineages (data not shown). Scale bar, 200 µm.

Extended Data Fig. 5 Whole-exome sequencing (WES) of hESCs and iPSCs after serial passaging using CEPT.

a,b, To demonstrate that CEPT treatment was safe, hESCs (WA09) and iPSCs (LiPSC-GR1.1) were cultured for 20 passages and exposed to CEPT for 24 h during every passage. Western blots and immunocytochemistry show that both cell lines maintained OCT4 expression and differentiated into ectoderm, mesoderm and endoderm lineages by directed differentiation in adherent monolayer cultures. Scale bars, 100 µm. c-f, WES variant annotations, genotypes, and CNV analysis show genetic stability of cell lines. COSMIC variants by passage number for only exonic, nonsynonymous SNPs in cancer hotspots, split by cell line. There was no change in rarity or genotype over passaging (c). Genotype frequencies by passage number for all SNPs (upper panel) and SNPs in cancer hotspots (lower panel), which were proportionally constant (d). Functional SNP annotations by passage number (left) and exonic functional SNP annotations (right), a subset of the former, with unchanging variant counts per annotation category (e). Indel correlogram showing no significant correlation between key variables, passage number, and genotype. The CLINSIG database was used to analyze indels (f).

Extended Data Fig. 6 Improved single-cell cloning by CEPT and karyotype analysis of clonal lines.

a, Single-cell cloning experiment in 96-well plates. Single cells were deposited using BD FACSAria Fusion and treated with Y-27632 or CEPT. First medium change was on day 3 in order not to disturb single cells. Whole-well images were captured using a 2x objective after calcein green AM staining to quantify clone numbers on day 9. Arrows show single clones in each well. Scale bar, 3.2 mm. b, Quantification of single-cell cloning experiment showing higher colony formation rate after CEPT treatment. Data represent mean ± s.d. (n = 3 plates for each group), ***p = 0.0006, unpaired two-tailed Student’s t-test. c, Single-cell cloning efficiency in mTeSR1 medium with microfluidic cell-dispenser Hana shows that CEPT is superior to Y-27632 and comparable to CloneR (WA09 cell line) confirming the broad applicability of CEPT with different cell-culture media. Data represent mean ± s.d. (n = 6 plates for each group). ***p = 0.001, one-way ANOVA (see also Fig. 3g–k using StemFlex medium). d, Time-lapse images recorded by Cytena to document stringent single-cell deposition. A single cell was identified by the system in the outgoing droplet (the volume enclosed by the two concentric circles) and dispensed into well A11 in a 96-well plate. e,f, CEPT improves single-cell cloning when Cytena single-cell dispenser is used. Single cells were dispensed using Cytena into 96-well plates coated with laminin 521 or vitronectin containing StemFlex media following Accutase dissociation. Single cell clones were stained with calcein green AM 9 days after dispensing and images of the plates were scanned with a 2x objective using IN Cell Analyzer high content analysis (HCA) System. Data are mean ± s.d. (e: n = 3 plates for all groups. WA01, **p = 0.0054; WA09, ***p = 0.0005; GM25256, **p = 0.0061; LiPSC-GR1.1, **p = 0.01; f: n = 3 plates for all groups. WA01, **p = 0.0096; WA09, **p = 0.0011; GM25256, ***p = 0.0006; LiPSC-GR1.1, **p = 0.0025; unpaired two-tailed Student’s t-test). g, Time-lapse microscopic images documenting multiple clusters of pluripotent cells (WA09) arose from a single cell due to cell migration on LN521. Scale bar, 200 µm. h, Single-cell cloning and establishment of eight clonal cell lines from hESCs (WA09) and iPSCs (LiPSC-GR1.1) by using CEPT treatment. All clonal cell lines maintained normal karyotypes as analyzed after passage 4. i-l, CEPT supports cell survival after electroporation. The recovery of one hESC (WA09) and two iPSC (LiPSC GR1.1 and NCRM5) lines from electroporation was quantified using the CTG assay 24 h post-electroporation. Representative images of recovered cells (WA09) are shown in l. Data represent mean ± s.d. (n = 9 wells for each group). Scale bar, 200 µm.

Source data

Extended Data Fig. 7 Efficient establishment of clonal lines of gene-edited iPSCs.

a, Improved workflow to generate gene-edited clonal cell lines from iPSCs. b, Detection of GFP+ cells 3 days after electroporation. Scale bar, 5 µm. c, Schematic of potential gene editing results with or without the integration of the plasmid backbone (AMP), both of which may lead to the expression and correct localization of GFP. d, Randomly picked GFP+ clones (n = 8) were analyzed for genomic copy numbers of GFP and the plasmid backbone (AMP) using ddPCR. One clone showed bi-allelic correct editing (blue circle), five clones showed monoallelic correct editing (green circles), and two clones were edited with plasmid integration (red circles). e, Microscopic images of one of the mono-allelic correctly edited clones (clone #5) and the bi-allelic clone (clone #8). Note the stronger GFP signal intensity in the bi-allelic clone. Scale bar, 10 µm; inset, 2 µm. f, Junction PCR to detect the GFP insertion into the N-terminus of LMNB1 in clone #5 and #8. GM25256 represents the parental iPSC line. g, Sanger sequencing confirming edited and unedited alleles. h, Western blot analysis of LMNB1 expression in cell lines with mono- and bi-allelic modification and the parental iPSC line (GM25256). i, Gene-edited clonal cell lines (clones #5 and #8) maintained normal karyotypes after expansion. j,k, Differentiation of LMNB1-edited cells (clone #8) into ectoderm (PAX6), mesoderm (Brachyury), and endoderm (SOX17), and neurons (TUJ1).

Extended Data Fig. 8 CEPT improves EB differentiation and formation of kidney organoids.

a, Graphic summary of spontaneous multi-lineage differentiation of single EBs cultured individually in 96-well ULA plates in chemically-defined E6 Medium. The expression of PAX6, SOX17 and Brachyury (TBXT) was measured using RT-PCR (see also Fig. 4f). b, Overview of kidney organoids (one well of 6-well plate) using phase-contrast microscopy and immunocytochemical staining of nephron segment with LTL (Tetragonolobus Lectin), PODXL (Podocalyxin), and ECAD (E-Cadherin). Scale bars, 500 µm. c, Quantification showing that CEPT treatment generated more kidney organoids as compared to Y-27632 and this effect is more pronounced when fewer cells are plated. Data are mean ± s.d. (n = 4 wells for each group), **p = 0.0049, two-way ANOVA.

Source data

Extended Data Fig. 9 CEPT improves thawing of cryopreserved cells and facilitates colony picking during iPSC line establishment.

a, Experiments in upper panel show that long-term cryopreservation and thawing of undifferentiated and differentiated cells (hESCs, iPSCs, astrocytes) in the presence of CEPT is superior to DMSO, Y-27632, and CloneR. Cell survival was quantified using the CTG assay. In independent experiments (lower panel), long-term cryopreserved cells were placed on dry-ice for 72 h to simulate shipment and then thawed and analyzed at 24 h post-plating. Data are mean ± s.d. (n = 3 wells for each group). Upper row: WA09, ***p = 0.0002 for Y-27632 vs. CEPT, ***p = 0.0009 for CloneR vs. CEPT; LiPSC-GR1.1, ***p = 0.0009 for Y-27632 vs. CEPT, **p = 0.0015 for CloneR vs. CEPT; Astrocytes, ****p = 0.0006 for Y-27632 vs. CEPT, ****p = 0.0002 for CloneR vs. CEPT; Lower row: WA09, ****p = 0.0003 for Y-27632 vs. CEPT, ****p = 0.0004 for CloneR vs. CEPT; LiPSC-GR1.1, **p = 0.0023 for Y-27632 vs. CEPT, *p = 0.0126 for CloneR vs. CEPT; Astrocytes, ****p < 0.0001 for both comparisons. One-way ANOVA with Tukey post-hoc test. b,c, Frozen vials of iPSC-derived motor neurons (FUJIFILM CDI) were thawed and treated with Y-27632 and CEPT for 24 h. Electrophysiological characterization of motor neurons was recorded using multi-electrode array technology. Note the higher spontaneous activity of neuronal cultures thawed with CEPT (recordings performed at 7 days after thawing and plating cells). Data are mean ± s.d. (n = 3 wells for each group). p = 0.5023, one-way ANOVA. d,e, Human skin fibroblasts were reprogrammed using the Yamanaka factors and emerging individual iPSC colonies were manually picked and transferred to new plates. At day 8, cell confluency was measured demonstrating that CEPT yields more cellular material for cell line establishment. Data represent mean ± s.d. (n = 10 wells for each group), **p = 0.0044, *p = 0.0292, one-way ANOVA.

Source data

Extended Data Fig. 10 CEPT confers cytoprotection during passaging of hPSCs and normal cellular stress response in the presence of CEPT.

a, Western blot analysis of iPSCs (LiPSC-GR1.1) showing cellular stress at 3 h post-passage in the presence of DMSO and Y-27632. b, Western blot analysis of cell membrane-associated proteins. Human iPSCs (LiPSC-GR1.1) were dissociated and exposed to Y-27632 or CEPT for 24 h. GAPDH was used as a loading control. c, Puromycin pulse–chase experiment demonstrating that CEPT-treated iPSCs (LiPSC-GR1.1) show higher protein synthesis capacity than cultures passaged with Y-27632. All experiments were performed at 3 h post-passage and samples were collected after 50 min of puromycin exposure. d, Measurement of glutathione levels in two hESC lines and three iPSC lines. Glutathione levels were consistently higher in CEPT treated cultures compared to DMSO and Y-27632. Data represent mean ± s.d. (n = 4 wells for each group). WA01, ****p < 0.0001 for both comparison; WA07, **p = 0.0071 and ***p = 0.0002; GM23476, ****p < 0.0001 and ***p = 0.0001; GM25256, ****p < 0.0001 and ns = 0.0952; GM26107, ****p < 0.0001 and **p = 0.0075; one-way ANOVA. e, Dose-response experiment showing that increasing concentrations of etoposide correlate with enhanced signal for γH2AX in hESCs (WA09) as measured by Western blotting. f, hESCs (WA09) were treated with 340 µM etoposide for 3 h. Note that CEPT treatment reduces γH2AX levels with and without etoposide treatment. g, hESCs (WA09) treated with CEPT show expected physiological stress-response to etoposide-induced DNA damage and strongly induce γH2AX, p21, p53, and phosphorylation of BRCA1, CHK2, and p53. Cells were treated with 340 µM etoposide for 3 h.

Source data

Supplementary information

Supplementary Information

Supplementary Figures 1–4 and Table 3

Reporting Summary

Supplementary Tables 1 and 2

Table 1. List of 113 hits identified in the primary single-agent screen. Table 2. List of 316 hits identified in ultralow cell density combination screens.

Supplementary Movie 1

Cell attachment and survival strongly depend on the presence of chroman 1

Supplementary Movie 2

CEPT enables single-cell cloning and monitoring of clone formation

Supplementary Data 1

Source data for supplementary figures

Source data

Source Data Fig. 1

Statistical Source Data

Source Data Fig. 2

Statistical Source Data

Source Data Fig. 3

Statistical Source Data

Source Data Fig. 4

Statistical Source Data

Source Data Fig. 5

Statistical Source Data

Source Data Fig. 6

Statistical Source Data

Source Data Extended Data Fig. 3

Statistical Source Data

Source Data Extended Data Fig. 6

Statistical Source Data

Source Data Extended Data Fig. 8

Statistical Source Data

Source Data Extended Data Fig. 9

Statistical Source Data

Source Data Extended Data Fig. 10

Statistical Source Data

Source Data Figs. 2, 3, and 6 and Extended Data Fig. 5

Unprocessed Western Blots and/or gels

Source Data Extended Data Figs. 7 and 10

Unprocessed Western Blots and/or gels

Rights and permissions

Springer Nature or its licensor (e.g. a society or other partner) holds exclusive rights to this article under a publishing agreement with the author(s) or other rightsholder(s); author self-archiving of the accepted manuscript version of this article is solely governed by the terms of such publishing agreement and applicable law.

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Chen, Y., Tristan, C.A., Chen, L. et al. A versatile polypharmacology platform promotes cytoprotection and viability of human pluripotent and differentiated cells. Nat Methods 18, 528–541 (2021). https://doi.org/10.1038/s41592-021-01126-2

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/s41592-021-01126-2

This article is cited by

Search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing