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High-throughput fingerprinting of human pluripotent stem cell fate responses and lineage bias

Nature Methods volume 10, pages 1225–1231 (2013)Cite this article

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Abstract

Populations of cells create local environments that lead to emergent heterogeneity. This is particularly evident with human pluripotent stem cells (hPSCs): microenvironmental heterogeneity limits hPSC cell fate control. We developed a high-throughput platform to screen hPSCs in configurable microenvironments in which we optimized colony size, cell density and other parameters to achieve rapid and robust cell fate responses to exogenous cues. We used this platform to perform single-cell protein expression profiling, revealing that Oct4 and Sox2 costaining discriminates pluripotent, neuroectoderm, primitive streak and extraembryonic cell fates. We applied this Oct4-Sox2 code to analyze dose responses of 27 developmental factors to obtain lineage-specific concentration optima and to quantify cell line–specific endogenous signaling pathway activation and differentiation bias. We demonstrated that short-term responses predict definitive endoderm induction efficiency and can be used to rescue differentiation of cell lines reticent to cardiac induction. This platform will facilitate high-throughput hPSC-based screening and quantification of lineage-induction bias.

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Figure 1: The 96μCP platform.
Figure 2: Single-cell protein profiling reveals Oct4 and Sox2 mark early cell fates.
Figure 3: Characterization of factors modulating cell fate choices.
Figure 4: Quantitative assessment of cell line–specific endogenous signaling and differentiation.

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Acknowledgements

This work is funded by the Canadian Institutes of Health Research (CIHR) (P.W.Z.). E.J.P.N. is supported by a CIHR Frederick Banting and Charles Best Canada Graduate Scholarships Doctoral Award. P.B.L. is supported by a Heart & Stroke Richard Lewar Centre of Excellence Studentship award. S.S. is supported by a Vanier Canada Graduate Scholarship. P.W.Z. is supported as the Canada Research Chair in Stem Cell Bioengineering. We thank S. Foster for illustrating Supplementary Figure 1. Ligands were provided in kind by the International Stem Cell Initiative. We thank G. Keller (McEwen Centre for Regenerative Medicine/University Health Network), M. Radisic (University of Toronto), A. Nagy (Samuel Lunenfeld Research Institute), D. Melton (Harvard University), A. Elefanty (Monash University) and J. Ellis (The Hospital for Sick Children) for providing cell lines. We thank P. Andrews and M. Pera for helpful discussion.

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Authors and Affiliations

  1. Institute of Biomaterials and Biomedical Engineering, University of Toronto, Toronto, Ontario, Canada

    Emanuel J P Nazareth, Joel E E Ostblom, Petra B Lücker, Shreya Shukla, Manuel M Alvarez, Ting Yin & Peter W Zandstra

  2. Department of Medical Biochemistry and Biophysics, Karolinska Institutet, Stockholm, Sweden

    Joel E E Ostblom

  3. Department of Chemical Engineering and Applied Chemistry, University of Toronto, Toronto, Ontario, Canada

    Petra B Lücker & Peter W Zandstra

  4. Bioprocessing Technology Institute, A*STAR (Agency for Science, Technology and Research), Singapore, Singapore

    Steve K W Oh

  5. McEwen Centre for Regenerative Medicine, University Health Network, Toronto, Ontario, Canada

    Peter W Zandstra

  6. Heart and Stroke Richard Lewar Centre of Excellence, Toronto, Ontario, Canada

    Peter W Zandstra

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Contributions

E.J.P.N. designed, performed and analyzed most experiments. J.E.E.O. assisted with immunocytochemistry and software development. P.B.L. performed cardiac induction experiments. S.S. created hiPSC lines. T.Y. performed endoderm induction experiments. M.M.A. and T.Y. provided cell culture support. S.K.W.O. provided editorial input on the manuscript. E.J.P.N. and P.W.Z. designed the project and wrote the manuscript.

Corresponding author

Correspondence to Peter W Zandstra.

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The authors declare no competing financial interests.

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Supplementary Figures 1–10, Supplementary Table 1 and Supplementary Results (PDF 1736 kb)

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Nazareth, E., Ostblom, J., Lücker, P. et al. High-throughput fingerprinting of human pluripotent stem cell fate responses and lineage bias. Nat Methods 10, 1225–1231 (2013). https://doi.org/10.1038/nmeth.2684

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