SpringerProtocols: Abstract: 6. Experimental Models to Analyze Differentiation Functions of Cultured Keratinocytes In Vitro and In Vivo

6. Experimental Models to Analyze Differentiation Functions of Cultured Keratinocytes In Vitro and In Vivo

By: Nicole Maas-Szabowski², Norbert E. Fusenig², Hans-Jürgen Stark²

Abstract

Full Text

Download PDF (297K)

In this chapter, we present technical details for the generation of in vitro skin equivalents consisting of collagen gels with incorporated fibroblasts covered by proliferating and differentiating keratinocytes. Epithelial-mesenchymal interactions are clearly manifest in these skin equivalents. Therefore, they have proven to be suitable experimental tools for a broad range of applications, e.g., for studies on the the paracrine regulation of keratinocyte differentiation and proliferation. On the other hand, in vivo assays cannot be abandoned totally, in particular, when such properties as malignant growth potential, disturbed differentiation control in carcinogenesis, and impact on angiogenesis are concerned. For that reason, we additionally describe xenotransplantation techniques to graft human keratinocytes and skin equivalents, respectively, onto the dorsal muscle fascia of thymus-aplastic mice.

Affiliation(s): (2) Division of Carcinogenesis and Differentiation, German Cancer Research Center, Heidelberg, Germany

Book Title: Epidermal Cells: Methods and Protocols

Series: Methods in Molecular Biology | Volume: 289 | Pub. Date: Oct-20-2004 | Page Range: 47-60 | DOI: 10.1385/1-59259-830-7:047

Subject: Cell Biology

Key Words: Organotypic cocultures - skin equivalents - keratinocyte differentiation - epithelial-mesenchymal interaction - surface transplantation assay - tumorigenicity test

References

Download References

1.	Fusenig, N. E. (1994) Epithelial-mesenchymal interactions regulate keratinocyte growth and differentiation in vitro, in The Keratinocyte Handbook (Leigh, I., Lane, B., and Watt, F., eds.), Cambridge University Press, Cambridge, UK, pp. 71–94.

2.	Stark, H. J., Baur, M., Breitkreutz, D., Mirancea, N., and Fusenig, N. E. (1999) Organotypic keratinocyte cocultures in defined medium with regular epidermal morphogenesis and differentiation. J. Invest. Dermatol. 112, 681–691.

3.	Maas-Szabowski, N. and Fusenig, N. E. (1996) Interleukin 1 induced growth factor expression in postmitotic and resting fibroblasts. J. Invest. Dermatol. 107, 849–855.

4.	Maas-Szabowski, N., Stark, H. J., and Fusenig, N. E. (2000) Keratinocyte growth regulation in defined organotypic cultures through IL-1-induced KGF expression in resting fibroblasts. J. Invest. Dermatol. 114, 1075–1084.

5.	Rheinwald, J. G. and Green, H. (1975) Serial cultivation of strains of human epidermal keratinocytes: the formation of keratinizing colonies from single cells. Cell 6, 331–344.

6.	Garlick, J. A. and Taichman, L. B. (1994) Fate of human keratinocytes during reepithelialization in an organotypic culture model. Lab. Invest. 70(6), 916–924.

7.	Nuss Parker, J., Zhao, W., Askins, K. J., Broker, T. R., and Chow, L. T. (1997) Mutational analyses of differentiation-dependent human papillomavirus type 18 enhance elements in epithelial raft cultures of neonatal foreskin keratinocytes. Cell Growth Differ. 8, 751–762.

8.	Ponec, M., Gibbs, S., Weerheim, A., Kempenaar, J., Mulder, A., and Mommaas, A. M. (1997) Epithelial growth factor and temperature regulate keratinocyte differentiation. Arch. Dermatol. Res. 289, 317–326.

9.	Bajou, K., Noel, A., Gerard, R. D., Masson, V., Brunner, N., Holst-Hansen, C., et al. (1998) Absence of host plasminogen activator inhibitor 1 prevents cancer invasion and vascularization. Nat. Med. 4(8), 923–928.

10.	Fusenig, N. E., Breitkreutz, D., Dzarlieva, R. I., Boukamp, P., Bohnert, A., and Tilgen, W. (1983) Growth and differentiation of transformed keratinocytes from mouse and human skin in vitro and in vivo. J. Invest. Dermatol. 81, 168–175.

11.	Breitkreutz, D., Schoop, V. M., Mirancea, N., Baur, M., Stark, H. J., and Fusenig, N. E. (1998) Epidermal differentiation and basement membrane formation by HaCaT cells in surface transplants. Eur. J. Cell Biol. 75, 273–286.

12.	Skobe, M., Rockwell, P., Goldstein, N., Vosseler, S., and Fusenig, N. E. (1997) Halting angiogenesis suppresses carcinoma cell invasion. Nat. Med. 3, 1222–1227.

13.	Madison, K. C., Swartzendruber, C. D., Wertz, P. W., and Downing, D. T. (1998) Lamellar granule extrusion and stratum corneum intercellular lamellae in murine keratinocyte cultures. J. Invest. Dermatol. 90, 110–116.

14.	Grinnell, F., Toda, K. I., and Lamke-Seymour, C. (1987) Reconstitution of human epidermis in vitro is accompanied by transient activation. Exp. Cell Res. 172, 439–449.

15.	Herzhoff, K., Sollberg, S., Huerkamp, C., Krieg, T., and Eckes, B. (1999) Fibroblast expression of collagen integrin receptors alpha1beta1 and alpha2beta1 is not changed in systemic scleroderma. Br. J. Dermatol. 141, 218–223.

16.	Bell, E., Ehrlich, H. P., Buttle, D. J., and Nakatsuji, T. (1981) Living tissue formed in vivo and accepted as skin-equivalent tissue of full thickness. Science 211, 1052–1054.

17.	Mackenzie, I. C. and Fusenig, N. E. (1983) Regeneration of organized epithelial structure. J. Invest. Dermatol. 81, 189–194.

18.	Dawson, R. A., Goberdhan, N. J., Freedlander, E.,and MacNeil, S. (1996) Influence of extracellular matrix proteins on human keratinocyte attachment, proliferation and transfer to a dermal wound model. Burns 22, 93–100.

19.	Supp, D. M., Supp, A. P., Bell, S. M., and Boyce, S. T. (2000) Enhanced vascularization of cultured skin substitutes genetically modified to overexpress vascular endothelial growth factor 1. J. Invest. Dermatol. 114, 5–13.

20.	Takahashi, Y. and Nogawa, H. (1991) Branching morphogenesis of mouse salivary epithelium in basement membrane-like substratum separated from mesenchyme by the membrane filter. Development 111, 327–335.

21.	Zheng, J. and Vaheri, A. (1995) Human skin fibroblasts induce anchorage-independent growth of HPV-16-DNA-immortalized cervical epithelial cells. Int. J. Cancer 61, 658–665.

22.	Coulomb, B., Dubertret, L., Merrill, C., Touraine, R., and Bell, E. (1984) The collagen lattice: A model for studying epidermalization in vitro. Br. J. Dermatol. 114, 91–101.

23.	Smola, H., Thiekötter, G., and Fusenig, N. E. (1993) Mutual induction of growth factor gene expression by epidermal-dermal cell interaction. J. Cell Biol. 122, 417–429.

24.	Limat, A., Hunziker, T., Boillat, C., Bayreuther, K., and Noser, F. (1989) Post-mitotic human dermal fibroblasts efficiently support the growth of human follicular keratinocytes. J. Invest. Dermatol. 92, 758–762.

25.	Waelti, E. R., Inaebnit, S. P., Rast, H. P., Hunziker, T., Limat, A., Braathen, L., and Wiesmann, U. (1992) Coculture of human keratinocytes on post-mitotic human dermal fibroblast feeder cells: production of large amounts of interleukin 6. J. Invest. Dermatol. 98, 805–808.

26.	Smola, H., Thiekötter, G., Baur, M., Stark, H. J., Breitkreutz, D., and Fusenig, N. E. (1994) Organotypic and epidermal-dermal co-cultures of normal human keratinocytes and dermal cells: regulation of transforming growth factor α, β1 and β2, mRNA levels. Toxicol. In Vitro 8, 641–650.

27.	Bumann, J., Santo-Holtje, L., Loffler, H., Bamberg, M., and Rodemann, H. P. (1995) Radiation-induced alterations of the proliferation dynamics of human skin fibroblasts after repeated irradiation in the subtherapeutic dose range. Strahlenther. Oncol. 171, 35–41.

28.	Parenteau, N. L., Nolte, C. M., Bilbo, P., Rosenberg, M., Wilkins, L. M., Johnson E. W., et al. (1991) Epidermis generated in vitro: practical considerations and applications. J. Cell Biochem. 45, 245–251.

29.	Smola, H., Stark, H. J., Thiekötter, G., Mirancea, N., Krieg, T., and Fusenig, N. E. (1998) Dynamics of basement membrane formation by keratinocyte-fibroblast interactions in organotypic skin culture. Exp. Cell Res. 239, 399–410.

30.	Maas-Szabowski, N., Stark, H. J., and Fusenig, N. E. (2002) Cell interaction and epithelial differentiation, in Culture of Epithelial Cells, 2nd ed. (Freshney, I. R., and Freshney, M. G., eds.), Wiley-Liss, Inc., New York, pp. 31–63.

31.	Gratzner, H. G. (1982) Monoclonal antibody to 5-bromo-and 5-iododeoxyuridine: a new reagent for detection of DNA replication. Science 218, 474–475.

32.	Cattoretti, G., Becker, M. H., Key, G., Duchrow, M., Schluter, C., Galle, J., and Gerdes, J. (1992) Monoclonal antibodies against recombinant parts of the Ki-67 antigen (MIB 1 and MIB 3) detect proliferating cells in microwave-processed formalin-fixed paraffin sections. J. Pathol. 168, 357–363.

33.	Maas-Szabowski, N., Shimotoyodome, A., and Fusenig, N. E. (1999) Keratinocyte growth regulation in fibroblast cocultures via a double paracrine mechanism. J. Cell Sci. 112, 1843–1853.

34.	Szabowski, A., Maas-Szabowski, N., Andrecht, S., Kolbus, A., Schorpp-Kistner, M., Fusenig, N. E., et al. (2000) c-Jun and JunB antagonistically control cytokine-regulated mesenchymal-epidermal interaction in skin. Cell 103, 745–755.

35.	Breitkreutz, D., Bohnert, A., Herzmann, E., Bowden, P. E., Boukamp, P., and Fusenig, N. E. (1984) Differentiation specific functions in cultured and transplanted mouse keratinocytes: Environmental influences on ultrastructure and keratin expression. Differentiation 26, 154–169.

36.	Mackenzie, I., Rittman, G., Bohnert, A., Breitkreutz, D., and Fusenig, N. E. (1993) Influence of connective tissues on the in vitro growth and differentiation of murine epidermis. Epithelial Cell Biol. 2, 107–119.

37.	Kaur, P., and Carter, W. G. (1992) Integrin expression and differentiation in transformed human epidermal cells is regulated by fibroblasts. J. Cell Sci. 103, 755–763.

38.	Noser, F. K. and Limat, A. (1987) Organotypic culture of outer root sheath cells from human hair follicles using a new culture device. In Vitro Cell Devel. Biol. 23, 541–545.

39.	Detmar, M., Schaart, F. M., Blume, U., and Orfanos, C. E. (1993) Culture of hair matrix and follicular keratinocytes. J. Invest. Dermatol. 101, 130–134.

40.	Kopan, R. and Fuchs, E. (1989) The use of retinoic acid to probe the relation between hyperproliferation-associated keratins and cell proliferation in normal and malignant epidermal cells. J. Cell Biol. 109(1), 295–307.

41.	Chen, Y. Q., Mauviel, A., Ryynanen, J., Sollberg, S., and Uitto, J. (1994) Type VII collagen gene expression by human skin fibroblasts and keratinocytes in culture: influence of donor age and cytokine responses. J. Invest. Dermatol. 102, 205–209.

42.	Gregoire, L., Munkareh, A., Rabah, R., Morris, R. T, and Lancaster, W. D. (1998) Organotypic culture of human ovarian surface epithelial cells: a potential model for ovarian carcinogenesis. In Vitro Cell Dev. Biol. Anim. 34, 636–639.

43.	Tomakidi, P., Breitkreutz, D., Fusenig, N. E., Zoller, J., Kohl, A., and Komposch, G. (1998) Establishment of oral mucosa phenotype in vitro in correlation to epithelial anchorage. Cell Tissue Res. 298, 355–366.

44.	Schoop, V. M., Mirancea, N., and Fusenig, N. E. (1999) Epidermal organization and differentiation of HaCaT keratinocytes in organotypic cucultures with human dermal fibroblasts. J. Invest. Dermatol. 112, 343–353.

45.	Maas-Szabowski, N., Starker, A., and Fusenig, N. E. (2003) Epidermal tissue regeneration and stromal interaction in HaCaT cells is initiated by TGF-α. J. Cell Sci. 116, 2937–2948.

46.	Vaccariello, M., Javaherian, A., Wang, Y., Fusenig, N. E., and Garlick, J. A. (1999) Cell interactions control the fate of malignant keratinocytes in an organotypic model of early neoplasia. J. Invest. Dermatol. 113, 384–391.

47.	Cerezo, A., Stark, H. J., Moshir, S., and Boukamp, P. (2003) Constitutive overexpression of human telomerase reverse transcriptase but not c-myc blocks terminal differentiation in human HaCaT skin keratinocytes. J. Invest. Dermatol. 121, 110–119.

48.	Regnier, M., Staquet, M. J., Schmitt, D., and Schmidt, R. (1997) Integration of Langerhans cells into a pigmented reconstructed human epidermis. J. Invest. Dermatol. 109, 510–512.

49.	Laning, J. C., DeLuca, J. E., and Hardin-Young, J. (1999) Effects of immunoregulatory cytokines on the immunogenic potential of the cellular components of a bilayered living skin equivalent. Tissue Eng. 5, 171–181.

50.	Black, A. F, Berthold, F., L’Heureux, N., Germain, L., and Auger, F A. (1998) In vitro reconstruction of a capillary-like network in a tissue-engineered skin equivalent. FASEB J. 12, 1311–1340.

51.	Kopan, R., Traska, G., and Fuchs, E. (1987) Retinoids as important regulators of terminal differentiation: examining keratin expression in individual epidermal cells at various stages of keratinization. J. Cell Biol. 105, 427–440.

52.	Chen, C. S. J., Lavker, R. M., Rodeck, U., Risse, B., and Jensen, P. (1995) Use of a serum-free epidermal culture model to show deleterious effects of epidermal growth factor on morphogenesis and differentiation. J. Invest. Dermatol. 104, 107–112.

53.	Asselineau, D. and Prunieras, M. (1984) Reconstruction of simplified skin: control of fabrication. Br. J. Dermatol. 111, 219–222.

54.	Mauch, C., Kozlowska, E., Eckes, B., and Krieg, T. (1992) Altered regulation of collagen metabolism in scleroderma fibroblasts growth within three-dimensional collagen gels. Exp. Dermatol. 1, 185–190.

55.	Atula, S., Grenman, R., and Syrjänen, S. (1997) Fibroblasts can modulate the phenotype of malignant epithelial cells in vitro. Exp. Cell Res. 235, 180–187.

56.	Fusenig, N. E., Skobe, M., Vosseler, S., Hansen, M., Lederle, W., Airola, K., et al. (2002) Tissue models to study tumor-stroma interactions, in Proteases and Their Inhibitors in Cancer Metastasis (Muschel, R. J. and Foidard, J. M., eds.), Kluwer Academic Publishers, Dordrecht, The Netherlands, pp. 205–223.

57.	Fusenig, N. E., Breitkreutz, D., Lueder, M., Boukamp, P., and Worst, P. K. M. (1981) Keratinization and structural organization in epidermal cell cultures, in International Cell Biology 1980–1981 (Schwaiger, H. G., ed.), Springer Verlag, Berlin, Germany, pp. 1004–1014.

58.	Breitkreutz, D., Stark, H. J., Mirancea, N., Tomakidi, P., Steinbauer, H., and Fusenig, N. E. (1997) Integrin and basement membrane normalization in mouse grafts of human keratinocytes—implications for epithelial homeostasis. Differentiation 61, 195–209.

59.	Meuth, M. and Green, H. (1974) Induction of a deoxycytidineless state in cultured mammalian cells by bromodeoxyuridine. Cell 2, 109–112.

Comments (Loading...)

Post comment/View all comments