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Artificial niche microarrays for probing single stem cell fate in high throughput

Nature Methods volume 8, pages 949–955 (2011)Cite this article

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Abstract

To understand the regulatory role of niches in maintaining stem-cell fate, multifactorial in vitro models are required. These systems should enable analysis of biochemical and biophysical niche effectors in a combinatorial fashion and in the context of a physiologically relevant cell-culture substrate. We report a microengineered platform comprised of soft hydrogel microwell arrays with modular stiffness (shear moduli of 1–50 kPa) in which individual microwells can be functionalized with combinations of proteins spotted by robotic technology. To validate the platform, we tested the effect of cell-cell interactions on adipogenic differentiation of adherent human mesenchymal stem cells (MSCs) and the effect of substrate stiffness on osteogenic MSC differentiation. We also identified artificial niches supporting extensive self-renewal of nonadherent mouse neural stem cells (NSCs). Using this method, it is possible to probe the effect of key microenvironmental perturbations on the fate of any stem cell type in single cells and in high throughput.

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Figure 1: Production of artificial niche microarrays.
Figure 2: Modulation of MSC seeding concentration and its effect on cell fate.
Figure 3: Biochemical recapitulation of the cell density effect on MSC adipogenic differentiation.
Figure 4: Effect of matrix stiffness on MSC osteogenic differentiation.
Figure 5: Screening for the effect of niche proteins on NSC fate.

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Acknowledgements

We thank B. Wehrle-Haller, M. Knobloch, A. Grapin-Botton, M. Ehrbar and A. Ranga for helpful discussions and critical reading of the manuscript, C. Huei Tan for help with the development of hydrogel printing strategies, A. Griffa for support with image analysis, K. Johnsson for the gift of the Qarray mini robotic spotter, V. Taylor for providing Hes5-GFP neural stem cells (University of Sheffield), M. Emmert and S. Hoerstrupp (Zurich University Hospital) for providing mesenchymal stem cells, and M. Martino and J. Hubbell (Ecole Polytechnique Fédérale de Lausanne) for providing recombinant fibronectin fragment 9–10. The work was funded by a European Young Investigator award (PE002-117115/1) to M.P.L., the Swiss National Science Foundation grants CR32I3_125426 and CR23I2_125290, and the Juvenile Diabetes Research Funding grant JDRF41-2009-775.

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Authors and Affiliations

  1. Laboratory of Stem Cell Bioengineering and Institute of Bioengineering, Ecole Polytechnique Fédérale de Lausanne, Lausanne, Switzerland

    Samy Gobaa, Sylke Hoehnel, Marta Roccio, Andrea Negro, Stefan Kobel & Matthias P Lutolf

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  1. Samy Gobaa
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  2. Sylke Hoehnel
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  3. Marta Roccio
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  4. Andrea Negro
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  5. Stefan Kobel
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  6. Matthias P Lutolf
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Contributions

S.G. and M.P.L. conceived the study, interpreted results and wrote the manuscript; S.G. conducted most experimental work (mask and press design, microwell array production, image acquisition, cell counts) and performed statistical analyses; S.H. contributed to stem cell culture, fate analyses and microfabrication; M.R. contributed to stem cell culture and fate analyses; A.N. contributed to technology development (design of press) and wrote image-analysis scripts; S.K. contributed to technology development (mask design and stamp production) and image acquisition scripts.

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Correspondence to Matthias P Lutolf.

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The authors declare no competing financial interests.

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Supplementary Figures 1–11, Supplementary Tables 1–2 (PDF 6841 kb)

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Gobaa, S., Hoehnel, S., Roccio, M. et al. Artificial niche microarrays for probing single stem cell fate in high throughput. Nat Methods 8, 949–955 (2011). https://doi.org/10.1038/nmeth.1732

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