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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References
Rheinwald, J. G. & Green, H. Serial cultivation of strains of human epidermal keratinocytes: the formation of keratinizing colonies from single cells. Cell 6, 331–343 (1975).
Watt, F. M. & Green, H. Stratification and terminal differentiation of cultured epidermal cells. Nature 295, 434–436 (1982).
Sato, T. & Clevers, H. Growing self-organizing mini-guts from a single intestinal stem cell: mechanism and applications. Science 340, 1190–1194 (2013).
Huch, M. et al. Unlimited in vitro expansion of adult bi-potent pancreas progenitors through the Lgr5/R-spondin axis. EMBO J. 32, 2708–2721 (2013).
Huch, M. et al. In vitro expansion of single Lgr5+ liver stem cells induced by Wnt-driven regeneration. Nature 494, 247–250 (2013).
Jones, P. H. & Watt, F. M. Separation of human epidermal stem cells from transit amplifying cells on the basis of differences in integrin function and expression. Cell 73, 713–724 (1993).
Barrandon, Y. & Green, H. Three clonal types of keratinocyte with different capacities for multiplication. Proc. Natl Acad. Sci. USA 84, 2302–2306 (1987).
Janich, P. et al. Human epidermal stem cell function is regulated by circadian oscillations. Cell Stem Cell 13, 745–753 (2013).
Luis, N. M. et al. Regulation of human epidermal stem cell proliferation and senescence requires polycomb-dependent and -independent functions of Cbx4. Cell Stem Cell 9, 233–246 (2011).
Mulder, K. W. et al. Diverse epigenetic strategies interact to control epidermal differentiation. Nat. Cell Biol. 14, 753–763 (2012).
Connelly, J. T. et al. Actin and serum response factor transduce physical cues from the microenvironment to regulate epidermal stem cell fate decisions. Nat. Cell Biol. 12, 711–718 (2010).
Tan, D. W. et al. Single-cell gene expression profiling reveals functional heterogeneity of undifferentiated human epidermal cells. Development 140, 1433–1444 (2013).
Li, A., Simmons, P. J. & Kaur, P. Identification and isolation of candidate human keratinocyte stem cells based on cell surface phenotype. Proc. Natl Acad. Sci. USA 95, 3902–3907 (1998).
Lowell, S., Jones, P., Le Roux, I., Dunne, J. & Watt, F. M. Stimulation of human epidermal differentiation by δ-notch signalling at the boundaries of stem-cell clusters. Curr. Biol. 10, 491–500 (2000).
Rochat, A., Kobayashi, K. & Barrandon, Y. Location of stem cells of human hair follicles by clonal analysis. Cell 76, 1063–1073 (1994).
Gallico, G. G. III, O’Connor, N. E., Compton, C. C., Kehinde, O. & Green, H. Permanent coverage of large burn wounds with autologous cultured human epithelium. New Engl. J. Med. 311, 448–451 (1984).
Compton, C. C. et al. Skin regenerated from cultured epithelial autografts on full-thickness burn wounds from 6 days to 5 years after grafting. A light, electron microscopic and immunohistochemical study. Lab Invest. 60, 600–612 (1989).
Jones, P. H., Harper, S. & Watt, F. M. Stem cell patterning and fate in human epidermis. Cell 80, 83–93 (1995).
Fortunel, N. O. et al. Exploration of the functional hierarchy of the basal layer of human epidermis at the single-cell level using parallel clonal microcultures of keratinocytes. Exp. Dermatol. 19, 387–392 (2010).
Clayton, E. et al. A single type of progenitor cell maintains normal epidermis. Nature 446, 185–189 (2007).
Doupe, D. P., Klein, A. M., Simons, B. D. & Jones, P. H. The ordered architecture of murine ear epidermis is maintained by progenitor cells with random fate. Dev. Cell 18, 317–323 (2010).
Mascre, G. et al. Distinct contribution of stem and progenitor cells to epidermal maintenance. Nature 489, 257–262 (2012).
Lim, X. et al. Interfollicular epidermal stem cells self-renew via autocrine Wnt signaling. Science 342, 1226–1230 (2013).
Alcolea, M. P. et al. Differentiation imbalance in single oesophageal progenitor cells causes clonal immortalization and field change. Nat. Cell Biol. 16, 615–622 (2014).
Coutu, D. L. & Schroeder, T. Probing cellular processes by long-term live imaging—historic problems and current solutions. J. Cell Sci. 126, 3805–3815 (2013).
Thon, J. N., Devine, M. T., Jurak Begonja, A., Tibbitts, J. & Italiano, J. E. Jr High-content live-cell imaging assay used to establish mechanism of trastuzumab emtansine (T-DM1)-mediated inhibition of platelet production. Blood 120, 1975–1984 (2012).
Watt, F. M. Epidermal stem cells as targets for gene transfer. Hum. Gene Ther. 11, 2261–2266 (2000).
He, J. et al. How variable clones build an invariant retina. Neuron 75, 786–798 (2012).
Simpson, P. Lateral inhibition and the development of the sensory bristles of the adult peripheral nervous system of Drosophila. Development 109, 509–519 (1990).
Rangarajan, A. et al. Notch signaling is a direct determinant of keratinocyte growth arrest and entry into differentiation. EMBO J. 20, 3427–3436 (2001).
Doupe, D. P. et al. A single progenitor population switches behavior to maintain and repair esophageal epithelium. Science 337, 1091–1093 (2012).
Alcolea, M. P. & Jones, P. H. Tracking cells in their native habitat: lineage tracing in epithelial neoplasia. Nat. Rev. Cancer 13, 161–171 (2013).
Cicalese, A. et al. The tumor suppressor p53 regulates polarity of self-renewing divisions in mammary stem cells. Cell 138, 1083–1095 (2009).
van den Bogaerdt, A. J. et al. Upside-down transfer of porcine keratinocytes from a porous, synthetic dressing to experimental full-thickness wounds. Wound Repair Regen. 12, 225–234 (2004).
O’Shaughnessy, R. F., Seery, J. P., Celis, J. E., Frischauf, A. & Watt, F. M. PA-FABP, a novel marker of human epidermal transit amplifying cells revealed by 2D protein gel electrophoresis and cDNA array hybridisation. FEBS Lett. 486, 149–154 (2000).
Eckert, R. L. et al. S100 proteins in the epidermis. J. Invest. Dermatol. 123, 23–33 (2003).
Signer, R. A., Magee, J. A., Salic, A. & Morrison, S. J. Haematopoietic stem cells require a highly regulated protein synthesis rate. Nature 509, 49–54 (2014).
Liu, J., Xu, Y., Stoleru, D. & Salic, A. Imaging protein synthesis in cells and tissues with an alkyne analog of puromycin. Proc. Natl Acad. Sci. USA 109, 413–418 (2012).
Shyh-Chang, N. et al. Lin28 enhances tissue repair by reprogramming cellular metabolism. Cell 155, 778–792 (2013).
Zhang, Q., Shalaby, N. A. & Buszczak, M. Changes in rRNA transcription influence proliferation and cell fate within a stem cell lineage. Science 343, 298–301 (2014).
Buszczak, M., Signer, R. A. J. & Morrison, S. J. Cellular differences in protein synthesis regulate tissue homeostasis. Cell 159, 242–251 (2014).
Taguchi, S. et al. Overexpression of the transcription factor Yin-Yang-1 suppresses differentiation of HaCaT cells in three-dimensional cell culture. J. Invest. Dermatol. 131, 37–45 (2011).
Shaw, T. & Martin, P. Epigenetic reprogramming during wound healing: loss of polycomb-mediated silencing may enable upregulation of repair genes. EMBO Rep. 10, 881–886 (2009).
Perekatt, A. O. et al. YY1 is indispensable for Lgr5 + intestinal stem cell renewal. Proc. Natl Acad. Sci. USA 111, 7695–7700 (2014).
Xu, X. et al. Yin-yang 1 negatively regulates the differentiation-specific transcription of mouse loricrin gene in undifferentiated keratinocytes. J. Invest. Dermatol. 123, 1120–1126 (2004).
Somervaille, T. C. et al. Hierarchical maintenance of MLL myeloid leukemia stem cells employs a transcriptional program shared with embryonic rather than adult stem cells. Cell Stem Cell 4, 129–140 (2009).
Wongtawan, T., Taylor, J. E., Lawson, K. A., Wilmut, I. & Pennings, S. Histone H4K20me3 and HP1α are late heterochromatin markers in development, but present in undifferentiated embryonic stem cells. J. Cell Sci. 124, 1878–1890 (2011).
Barrandon, Y. & Green, H. Cell migration is essential for sustained growth of keratinocyte colonies: the roles of transforming growth factor-α and epidermal growth factor. Cell 50, 1131–1137 (1987).
Rheinwald, J. G. & Green, H. Epidermal growth factor and the multiplication of cultured human epidermal keratinocytes. Nature 265, 421–424 (1977).
Bellas, E. & Chen, C. S. Forms, forces, and stem cell fate. Curr. Opin. Cell Biol. 31, 92–97 (2014).
Barker, N. et al. Lgr5(+ ve) stem cells drive self-renewal in the stomach and build long-lived gastric units in vitro. Cell Stem Cell 6, 25–36 (2010).
Sato, T. et al. Single Lgr5 stem cells build crypt-villus structures in vitro without a mesenchymal niche. Nature 459, 262–265 (2009).
Chapman, S., McDermott, D. H., Shen, K., Jang, M. K. & McBride, A. A. The effect of Rho kinase inhibition on long-term keratinocyte proliferation is rapid and conditional. Stem Cell Res. Ther. 5, 60 (2014).
Chapman, S., Liu, X., Meyers, C., Schlegel, R. & McBride, A. A. Human keratinocytes are efficiently immortalized by a Rho kinase inhibitor. J. Clin. Invest. 120, 2619–2626 (2010).
Luxenburg, C. et al. Wdr1-mediated cell shape dynamics and cortical tension are essential for epidermal planar cell polarity. Nat. Cell Biol. 17, 592–604 (2015).
Lee, B. et al. Transcriptional mechanisms link epithelial plasticity to adhesion and differentiation of epidermal progenitor cells. Dev. Cell 29, 47–58 (2014).
Amelio, I. et al. miR-24 triggers epidermal differentiation by controlling actin adhesion and cell migration. J. Cell Biol. 199, 347–363 (2012).
Nanba, D. et al. Cell motion predicts human epidermal stemness. J. Cell Biol. 209, 305–315 (2015).
Lecuit, T. & Yap, A. S. E-cadherin junctions as active mechanical integrators in tissue dynamics. Nat. Cell Biol. 17, 533–539 (2015).
Zhu, A. J., Haase, I. & Watt, F. M. Signaling via β1 integrins and mitogen-activated protein kinase determines human epidermal stem cell fate in vitro. Proc. Natl Acad. Sci. USA 96, 6728–6733 (1999).
Nanba, D. et al. Actin filament dynamics impacts keratinocyte stem cell maintenance. EMBO Mol. Med. 5, 640–653 (2013).
McMullan, R. et al. Keratinocyte differentiation is regulated by the Rho and ROCK signaling pathway. Curr. Biol. 13, 2185–2189 (2003).
Iglesias-Bartolome, R. et al. mTOR inhibition prevents epithelial stem cell senescence and protects from radiation-induced mucositis. Cell Stem Cell 11, 401–414 (2012).
Wabik, A. & Jones, P. H. Switching roles: the functional plasticity of adult tissue stem cells. EMBO J. 34, 1164–1179 (2015).
Centanin, L. et al. Exclusive multipotency and preferential asymmetric divisions in post-embryonic neural stem cells of the fish retina. Development 141, 3472–3482 (2014).
Rompolas, P., Mesa, K. R. & Greco, V. Spatial organization within a niche as a determinant of stem-cell fate. Nature 502, 513–518 (2013).
Buczacki, S. J. et al. Intestinal label-retaining cells are secretory precursors expressing Lgr5. Nature 495, 65–69 (2013).
Aspinall-O’Dea, M. et al. Antibody-based detection of protein phosphorylation status to track the efficacy of novel therapies using nanogram protein quantities from stem cells and cell lines. Nat. Protoc. 10, 149–168 (2015).
Jensen, K. B., Driskell, R. R. & Watt, F. M. Assaying proliferation and differentiation capacity of stem cells using disaggregated adult mouse epidermis. Nat. Protoc. 5, 898–911 (2010).
Subramanian, A. et al. Gene set enrichment analysis: a knowledge-based approach for interpreting genome-wide expression profiles. Proc. Natl Acad. Sci. USA 102, 15545–15550 (2005).
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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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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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.
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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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DOI: https://doi.org/10.1038/ncb3282
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