Advertisement

Monitoring the Cycling Activity of Cultured Human Keratinocytes Using a CFSE-Based Dye Tracking Approach

Protocol
First Online:
Part of the Methods in Molecular Biology book series (MIMB, volume 989)

Abstract

The development of methods and tools suitable for functional analysis of keratinocytes placed in an in vitro context is of great importance for characterizing properties associated with their normal state, for detecting abnormalities related to pathological states, or for studying the effects of extrinsic factors. In the present chapter, we describe the use of the intracellular fluorescent dye carboxyfluorescein succinimidyl ester (CFSE) to monitor cell division in mass cultures of normal human keratinocytes. We detail the preparation of CFSE-labeled keratinocyte samples and the identification by flow cytometry of cell subpopulations exhibiting different cycling rates in a mitogenic culture context. In addition, we show that the CFSE-based division-tracking approach enables the monitoring of keratinocyte responsiveness to growth modulators, which is here exemplified by the cell-cycling inhibition mediated by the growth factor TGF-β1. Finally, we show that keratinocyte subpopulations, separated according to their mitotic history using CFSE fluorescence tracking, can be sorted by flow cytometry and used for further functional characterization, including determination of clone-forming efficiency.

Key words

CFSE Keratinocytes Cell division tracking Mitotic history Proliferation assay Flow cytometry TGF-β1 
This is a preview of subscription content, log in to check access.

Notes

Acknowledgments

The authors wish to thank AFM-Myobank (La Pitié-Salpêtrière Hospital, Paris) for providing biopsies of normal adult human skin. This research project was financially supported by CEA and ANR-CESA024. Loubna Chadli benefited from doctoral fellowships from CEA (direction of life sciences, International Research Training for Excellence in Life Science (IRTELIS) program), and from the Association pour la Recherche sur le Cancer (ARC).

References

  1. 1.
    Chadli L, Martin MT, Fortunel NO (2011) Investigating human keratinocyte stem cell identity. Eur J Dermatol 21(Suppl 2):4–11PubMedGoogle Scholar
  2. 2.
    Kaur P, Potten CS (2011) The interfollicular epidermal stem cell saga: sensationalism versus reality check. Exp Dermatol 20:697–702PubMedCrossRefGoogle Scholar
  3. 3.
    Li A, Simmons PJ, Kaur P (1998) Identification and isolation of candidate human keratinocyte stem cells based on cell surface phenotype. Proc Natl Acad Sci USA 95:3902–3907PubMedCrossRefGoogle Scholar
  4. 4.
    Fortunel NO, Cadio E, Vaigot P, Chadli L, Moratille S, Bouet S, Roméo PH, Martin MT (2010) 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 19:387–392PubMedCrossRefGoogle Scholar
  5. 5.
    Fortunel NO, Chadli L, Bourreau E, Cadio E, Vaigot P, Marie M, Deshayes N, Rathman-Josserand M, Leclaire J, Martin MT (2011) Cellular adhesion on collagen: a simple method to select human basal keratinocytes which preserves their high growth capacity. Eur J Dermatol 21(Suppl 2):12–20PubMedGoogle Scholar
  6. 6.
    Larderet G, Fortunel NO, Vaigot P, Cegalerba M, Maltère P, Zobiri O, Gidrol X, Waksman G, Martin MT (2006) Human side population keratinocytes exhibit long-term proliferative potential and a specific gene expression profile and can form a pluristratified epidermis. Stem Cells 24:965–974PubMedCrossRefGoogle Scholar
  7. 7.
    Fortunel NO, Hatzfeld JA, Rosemary PA, Ferraris C, Monier MN, Haydont V, Longuet J, Brethon B, Lim B, Castiel I, Schmidt R, Hatzfeld A (2003) Long-term expansion of human functional epidermal precursor cells: promotion of extensive amplification by low TGF-beta1 concentrations. J Cell Sci 116:4043–4052PubMedCrossRefGoogle Scholar
  8. 8.
    Barrandon Y, Green H (1987) Three clonal types of keratinocyte with different capacities for multiplication. Proc Natl Acad Sci USA 84:2302–2306PubMedCrossRefGoogle Scholar
  9. 9.
    Ueckert JE, Nebe von Caron G, Bos AP, ter Steeg PF (1997) Flow cytometric analysis of Lactobacillus plantarum to monitor lag times, cell division and injury. Lett Appl Microbiol 25:295–299PubMedCrossRefGoogle Scholar
  10. 10.
    Khil LY, Kim JY, Yoon JB, Kim JM, Keum WK, Kim ST, Yoon Y, Yoon MY, Moon CK, Lee JH, Ha J, Kim SS, Kang I (1997) Insulin has a limited effect on the cell cycle progression in 3T3 L1 fibroblasts. Mol Cells 7:742–748PubMedGoogle Scholar
  11. 11.
    De Boer RJ, Ganusov VV, Milutinović D, Hodgkin PD, Perelson AS (2006) Estimating lymphocyte division and death rates from CFSE data. Bull Math Biol 68:1011–1031PubMedCrossRefGoogle Scholar
  12. 12.
    Hawkins ED, Hommel M, Turner ML, Battye FL, Markham JF, Hodgkin PD (2007) Measuring lymphocyte proliferation, survival and differentiation using CFSE time-series data. Nat Protoc 2:2057–2067PubMedCrossRefGoogle Scholar
  13. 13.
    Batard P, Monier M-N, Fortunel N, Ducos K, Sansilvestri-Morel P, Phan T, Hatzfeld A, Hatzfeld JA (2000) TGF-(beta)1 maintains hematopoietic immaturity by a reversible negative control of cell cycle and induces CD34 antigen up-modulation. J Cell Sci 113:383–390PubMedGoogle Scholar
  14. 14.
    Bennaceur-Griscelli A, Pondarré C, Schiavon V, Vainchenker W, Coulombel L (2001) Stromal cells retard the differentiation of CD34(+)CD38(low/neg) human primitive progenitors exposed to cytokines independent of their mitotic history. Blood 97:435–441PubMedCrossRefGoogle Scholar
  15. 15.
    Ganusov VV, Pilyugin SS, de Boer RJ, Murali-Krishna K, Ahmed R, Antia R (2005) Quantifying cell turnover using CFSE data. J Immunol Methods 298:183–200PubMedCrossRefGoogle Scholar
  16. 16.
    Ko KH, Odell R, Nordon RE (2007) Analysis of cell differentiation by division tracking cytometry. Cytometry A 71:773–782PubMedGoogle Scholar
  17. 17.
    Luzyanina T, Mrusek S, Edwards JT, Roose D, Ehl S, Bocharov G (2007) Computational analysis of CFSE proliferation assay. J Math Biol 54:57–89PubMedCrossRefGoogle Scholar
  18. 18.
    Yates A, Chan C, Strid J, Moon S, Callard R, George AJ, Stark J (2007) Reconstruction of cell population dynamics using CFSE. BMC Bioinformatics 8:196PubMedCrossRefGoogle Scholar
  19. 19.
    Banks HT, Sutton KL, Thompson WC, Bocharov G, Roose D, Schenkel T, Meyerhans A (2011) Estimation of cell proliferation dynamics using CFSE data. Bull Math Biol 73:116–150PubMedCrossRefGoogle Scholar
  20. 20.
    Fortunel NO, Vaigot P, Cadio E, Martin MT (2010) Functional investigations of keratinocyte stem cells and progenitors at a single-cell level using multiparallel clonal microcultures. Methods Mol Biol 585:13–23PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media New York 2013

Authors and Affiliations

  1. 1.Laboratory of Genomics and Radiobiology of Keratinopoiesis (LGRK)Institute of Cellular and Molecular Radiobiology (iRCM), Alternative Energies and Atomic Energy Commission (CEA)EvryFrance

Personalised recommendations

Cite protocol

Buy options