Biomimetic niches reveal the minimal cues to trigger apical lumen formation in single hepatocytes

Abstract

The symmetry breaking of protein distribution and cytoskeleton organization is an essential aspect for the development of apicobasal polarity. In embryonic cells this process is largely cell autonomous, while differentiated epithelial cells collectively polarize during epithelium formation. Here, we demonstrate that the de novo polarization of mature hepatocytes does not require the synchronized development of apical poles on neighbouring cells. De novo polarization at the single-cell level by mere contact with the extracellular matrix and immobilized cadherin defining a polarizing axis. The creation of these single-cell liver hemi-canaliculi allows unprecedented imaging resolution and control and over the lumenogenesis process. We show that the density and localization of cadherins along the initial cell–cell contact act as key triggers of the reorganization from lateral to apical actin cortex. The minimal cues necessary to trigger the polarization of hepatocytes enable them to develop asymmetric lumens with ectopic epithelial cells originating from the kidney, breast or colon.

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Fig. 1: Development of polarity of primary hepatocytes in artificial micro-niches.
Fig. 2: Characterization of protein organization of single-cell hemi-lumen.
Fig. 3: Functional characterization of bile secretion of single-cell hemi-lumen.
Fig. 4: Hepatocytes form lumens with epithelial cells but not with fibroblasts.
Fig. 5: Cadherin distribution orchestrates the establishment of polarity.
Fig. 6: Graphical summary of the proposed mechanism.

Data availability

The data supporting the findings of this study are available within this article and its Supplementary Information, or from the corresponding author on reasonable request.

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Acknowledgements

We acknowledge the help of the MBI editorial team for a critical reading of the manuscript. R.T. and S.S.N. acknowledge support by the NIHR Clinical Research Facility at Guy’s & St. Thomas’ NHS Foundation Trust, UK; NIHR Biomedical Research Centre based at Guy’s and St. Thomas’ NHS Foundation Trust, UK; King’s College London, UK; and the Medical Research Council (MRC) (MR/L006537/1), UK. The views expressed are those of the authors and not necessarily those of the NHS, the NIHR or the Department of Health. H.Y. acknowledges support from MOE-ARC (R-185-000-342-112). N.V.H. acknowledges support from grant OFIRG15nov120 from the National Medical Research Council, Singapore. C.M. acknowledges financial support by the Volkswagen Foundation under the Freigeist Fellowship, agreement no. 94195. V.V. acknowledges funding from NRF investigatorship NRF-NRFI2018-07 and MBI core funding. The 0.5 million MDCK cells expressing histone-GFP were a generous gift from Benoit Ladoux, Institut Jacques Monod, Paris.

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Authors

Contributions

Y.Z. and V.A. performed most of the single-cell experiment and imaging. R.T. and S.S.N. developed and provided the hiPSC-induced hepatocytes. H.Y. and I.C.N. isolated and provided the primary rat hepatocytes. R.M. and Y.Z. performed the quantitative analysis. R.M. and P.T. performed the E-cad titration experiment. C.M. helped and quantified the RICM experiments. F.L.C. provided invaluable support for imaging. N.V.H. and Y.Z. performed the experiments on mouse hepatocytes. Y.Z., R.M. and V.V. conceived the experiments. V.V. supervised the work. Y.Z., R.M., V.A. and V.V. wrote the manuscript.

Corresponding author

Correspondence to Virgile Viasnoff.

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Supplementary information

41563_2020_662_MOESM3_ESM.avi

Live RICM imaging of the hemi-lumen in control conditions. Note the central ring that moves radially indicating a dome-shaped structure of about 200 nm height on top of the coverslip.

41563_2020_662_MOESM4_ESM.avi

Live RICM imaging of the hemi-lumen under 10 uM ketokonazole. The inhibition of bile secretion leads to a large reduction of vertical fluctuation (amplitude and frequency). The fluctuations are no longer radial (lumen pulsating) but rather diffused and lateral, indicating a mere membrane fluctuation.

41563_2020_662_MOESM5_ESM.avi

Live RICM imaging of the hemi-lumen in cells treated with UDCA (40 uM). This treatment stimulates bile salt secretion. Multiple interference rings in the centre of the lumen indicate a much-inflated geometry compared with the control case.

41563_2020_662_MOESM6_ESM.avi

Live RICM imaging of the hemi-lumen on doughnut patterns in control conditions. The central fluctuations are fully random compared with disc patterns. This indicates the absence of coordinated pulsations that are replaced by simple fluctuations of the free membrane.

Supplementary Information

Supplementary Figs. 1–10

Reporting Summary

Supplementary Video 1

Live RICM imaging of the hemi-lumen in control conditions. Note the central ring that moves radially indicating a dome-shaped structure of about 200 nm height on top of the coverslip.

Supplementary Video 2

Live RICM imaging of the hemi-lumen under 10 uM ketokonazole. The inhibition of bile secretion leads to a large reduction of vertical fluctuation (amplitude and frequency). The fluctuations are no longer radial (lumen pulsating) but rather diffused and lateral, indicating a mere membrane fluctuation.

Supplementary Video 3

Live RICM imaging of the hemi-lumen in cells treated with UDCA (40 uM). This treatment stimulates bile salt secretion. Multiple interference rings in the centre of the lumen indicate a much-inflated geometry compared with the control case.

Supplementary Video 4

Live RICM imaging of the hemi-lumen on doughnut patterns in control conditions. The central fluctuations are fully random compared with disc patterns. This indicates the absence of coordinated pulsations that are replaced by simple fluctuations of the free membrane.

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Zhang, Y., De Mets, R., Monzel, C. et al. Biomimetic niches reveal the minimal cues to trigger apical lumen formation in single hepatocytes. Nat. Mater. (2020). https://doi.org/10.1038/s41563-020-0662-3

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