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Human keratinocytes have two interconvertible modes of proliferation

Nature Cell Biology volume 18, pages 145–156 (2016)Cite this article

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Abstract

Single stem cells, including those in human epidermis, have a remarkable ability to reconstitute tissues in vitro, but the cellular mechanisms that enable this are ill-defined. Here we used live imaging to track the outcome of thousands of divisions in clonal cultures of primary human epidermal keratinocytes. Two modes of proliferation were seen. In ‘balanced’ mode, similar proportions of proliferating and differentiating cells were generated, achieving the ‘population asymmetry’ that sustains epidermal homeostasis in vivo. In ‘expanding’ mode, an excess of cycling cells was produced, generating large expanding colonies. Cells in expanding mode switched their behaviour to balanced mode once local confluence was attained. However, when a confluent area was wounded in a scratch assay, cells near the scratch switched back to expanding mode until the defect was closed. We conclude that the ability of a single epidermal stem cell to reconstitute an epithelium is explained by two interconvertible modes of proliferation regulated by confluence.

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Figure 1: Live imaging of cultured keratinocytes.
Figure 2: Lineage trees of NFSKs cultured at clonal density.
Figure 3: Division outcomes of sister cells.
Figure 4: Transcriptional analysis of colonies.
Figure 5: Expanding colonies switch towards balance.
Figure 6: Cell density and ROCK2 kinase activity regulate the switch from expanding to balanced mode of proliferation.
Figure 7: Effects of loss of confluence on mode of proliferation in large colonies.
Figure 8: Two interconvertible modes of proliferation underpin epithelial reconstitution in vitro.

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Acknowledgements

The initial association of holoclone and paraclone type behaviour in clonal cultures of NFSKs with stem and balanced progenitor dynamics was recognized by B.D.S. working in collaboration with P.H.J., V.N.-N., D. Doupé and A. Klein, based on the quantitative analysis of published and unpublished colony size distributions6. We thank G. Akdeniz and D. Doupé for experimental work that led up to the project that was analysed by A. Klein and G. Zhang, P. Lombard at the Wellcome Trust-Medical Research Council Cambridge Stem Cell Institute for Bioinformatics analysis and E. Choolun for technical assistance. We acknowledge the support of the Wellcome Trust, Cambridge Cancer Centre, Medical Research Council, the NC3Rs (National Centre for the Replacement, Refinement and Reduction of Animals in Research) and Cancer Research UK (Programme grant C609/A17257).

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  1. Amit Roshan

    Present address: Present address: Norfolk & Norwich University Hospital, Colney Lane, Norwich NR4 7UY, UK.,

Authors and Affiliations

  1. MRC Cancer Unit, University of Cambridge, Hutchison/MRC Research Centre, Box 197, Cambridge Biomedical Campus, Cambridge CB2 0XZ, UK

    Amit Roshan, Varvara Nikolaidou-Neokosmidou & Philip H. Jones

  2. Wellcome Trust Sanger Institute, Hinxton, Cambridge CB10 1SA, UK

    Kasumi Murai, Joanna Fowler & Philip H. Jones

  3. Cavendish Laboratory, TCM, University of Cambridge, JJ Thomson Avenue, Cambridge CB3 0HE, UK

    Benjamin D. Simons

  4. Wellcome Trust/Cancer Research UK Gurdon Institute, The Henry Wellcome Building of Cancer and Developmental Biology, University of Cambridge, Tennis Court Road, Cambridge CB2 1QN, UK

    Benjamin D. Simons

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Contributions

A.R., K.M., J.F., V.N.-N. and P.H.J. designed and performed experiments and analysed and interpreted data. B.D.S. proposed using growth rate from 144 to 168 h to classify division mode in dye-labelled cells. A.R., K.M., J.F. and P.H.J. wrote the manuscript.

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Integrated supplementary information

Supplementary Figure 1 Lineage trees of primary adult human keratinocytes in vitro.

Keratinocytes cultured directly from adult epidermis in standard media. Scale indicates time since plating in hours. Magenta indicates cells that did not divide within 48 h, green cells observed to divide, grey cells those which could not be tracked for 48 h. a, balanced, b expanding trees. Horizontal brackets in b, marked by ∗, indicate representative cells tracked within a single expanding colony.

Supplementary Figure 2 Live imaging of primary adult human keratinocytes and validation of two-mode model.

a–c, Live imaging of keratinocytes cultured directly from adult epidermis. a: Cell cycle time distribution b, c: Division outcomes (% with 95% confidence interval) in balanced, b (547 divisions in 50 colonies), and expanding, c lineages (609 divisions, 6 colonies). See Supplementary Fig. 1a, b for lineage trees. d, e Predictions from a large simulation of keratinocyte growth. Simulated and observed colony size distributions. d: Box plots showing day 7 colony size distributions from a computer simulation assuming two modes of growth with division outcomes shown in Fig. 1 (Simulated, n = 30000 colonies) compared with observed sizes from cultured NFSK (Observed, n = 1,631 colonies pooled from 3 independent experiments). Box boundaries indicate the 25th and 75th percentiles. Line across box is the median. Whiskers indicate 1st and 99th percentiles. There is no statistically significant difference between the distributions (Kolmogorov–Smirnov test P = 0.15). e: Proportions of simulated colonies containing one or more cycling Roshan Keratinocyte Proliferation 2 cells after 7 days, red line indicates balanced colonies, red shading balanced colonies with one or more proliferating cells and green shading expanding colonies. Arrow indicates overlap of colonies containing 50–150 cells, which may be from cells proliferating in either mode.

Supplementary Figure 3 Tracking colony expansion with PKH26 labelled cells.

a: Protocol. NFSK were labelled with PKH26 and plated at clonal density. Every 24 h the coordinates of each colony and the number of cells per colony were determined by fluorescence microscopy. At 168 h dishes were fixed and stained. b,c Images of typical colonies, showing phase contrast images overlaid with PKH26 fluorescence (orange) for time points up to 168 h with the number of cells/colony and the fixed 168 h colonies stained for Dapi, blue, pan cytokeratin (green) and EdU (red). Scale bars: 100 μm. Images are representative examples of 333 colonies in 3 independent experiments.

Supplementary Figure 4 Analysis of PKH26 tracking data.

a: Live imaging data (Fig. 2b) shows that in larger balanced mode colonies the accumulation of differentiating cells (red) resulted in a proportionate increase in cell number between 144 and 168 h 150 cells are expanding. d: Cells per clone in PKH26 labelled colonies. Each line represents a single colony growth with, inset showing enlarged view of colonies between 50 and 150 cells. Full data set is given in Supplementary Table 3. e: Mean cells per colony versus time in balanced mode colonies (n = 304 colonies). The increase is linear (r2 = 0.98), dotted lines indicate 95% confidence intervals. f: Mean cells per colony versus time in expanding mode colonies (n = 29 colonies) showing an exponential increase, dotted lines indicate 95% confidence intervals.

Supplementary Figure 5 Effect of increased cell density on NFSK proliferation.

a: Immunoblot analysis of EGF signalling in NFSK. Lysates were collected at the times indicated after plating keratinocytes in media containing 0, 10 or 20 ng ml−1 added EGF. Arrowheads indicate EGFR degradation product. Position of size markers (kDa) is as indicated. Blots shown are representative of 3 independent experiments. b: Cell cycle time distribution of NFSK cultured in media without supplemented EGF (EGF0). c, d Lineage trees NFSK cultured in EGF0. Scale indicates time since plating in hours. Magenta indicates cells that did not divide within 48 h, green cells observed to divide, grey cells those that could not be tracked for 48 h. c, balanced, d expanding Roshan Keratinocyte Proliferation 4 trees. Horizontal brackets marked by ∗ in b indicate representative cells tracked within a single expanding colony. In d large green circles, arrowed, indicate cells lying in outer third (by area) of expanding colony, other green cells lie within inner two thirds of colonies at 96 h.

Supplementary Figure 6 Effect of Y27632 on NFSK proliferation.

a,b: Lineage trees of NFSK cultured in standard media in the presence of 10 μM Y27632. Scale indicates time since plating in hours. Magenta indicates cells that did not divide within 48 h, green cells observed to divide, grey cells those that could not be tracked for 48 h. a, balanced, b expanding trees. Horizontal brackets in b, marked by ∗, indicate representative cells tracked within a single expanding colony.

Supplementary Figure 7 Scans of Western blots.

Scans of four blots (1 to 4) presented in cropped form in Supplementary Fig. 5a.

Supplementary information

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Time lapse imaging of typical NFSK expanding type colony.

Clock indicates time since plating, yellow dashed line NFSK cells, other cells are mouse 3T3 J2 fibroblast feeder cells, scale bar 100 μm. (MP4 10471 kb)

Time lapse imaging of typical NFSK balanced type colony.

Clock indicates time since plating, yellow dashed line NFSK cells, other cells are mouse 3T3 J2 fibroblast feeder cells, scale bar 100 μm. (MP4 10627 kb)

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Roshan, A., Murai, K., Fowler, J. et al. Human keratinocytes have two interconvertible modes of proliferation. Nat Cell Biol 18, 145–156 (2016). https://doi.org/10.1038/ncb3282

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