Epidermal Organization and Differentiation of HaCaT Keratinocytes in Organotypic Coculture with Human Dermal Fibroblasts  Veronika M. Schoop, Norbert E. Fusenig  Journal of Investigative Dermatology  Volume 112, Issue 3, Pages 343-353 (March 1999) DOI: 10.1046/j.1523-1747.1999.00524.x Copyright © 1999 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 1 Reconstructed squamous HaCaT epithelia in coculture with different fibroblast numbers. Organotypic cocultures containing 1 × 105 (a), 2 × 105 (b) and 5 × 105 (c–f) fibroblasts per ml gel at seeding time. After 1 wk (day 7) and with 1 × 105 fibroblasts per ml, a thin, unpolarized epithelium has developed (a), while epithelia are thicker and better structured on gels prepared with 2 × 105 fibroblasts per ml (b) and 5 × 105 fibroblasts per ml (c). Although basal cells are polarized and the upper cells are flattened, the epithelium exhibits a disorganized, dysplastic character after 7 d (d) compared with cultures of normal keratinocytes. After 2 wk with a high number of fibroblasts (5 × 105 per ml seeding density), however, a well organized and stratified epithelium (e) has formed showing cuboidal cells in the stratum basale (B), distinct spinous (S) and granular cells (G), and a parakeratotic stratum corneum (SC). The parakeratotic layers have expanded after 3 wk on top of a still well polarized basal layer (f). On DED without viable fibroblasts (g) a thin, unpolarized epithelium has not developed further even after 18 d. In contrast, a thick, stratified and organized epithelium has formed on DED repopulated with fibroblasts (arrows) (h). Scale bars: 50 μm. Journal of Investigative Dermatology 1999 112, 343-353DOI: (10.1046/j.1523-1747.1999.00524.x) Copyright © 1999 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 2 Organotypic coculture set-up. View from above onto the opaque collagen gel overlaid by the HaCaT epithelium, after 2 wk in culture on a filter insert placed into a deep-well tray. Scale bar: 1 cm. Journal of Investigative Dermatology 1999 112, 343-353DOI: (10.1046/j.1523-1747.1999.00524.x) Copyright © 1999 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 3 Localization of differentiation markers and BM components in 1 wk old cocultures. Cultures were prepared with 5 × 105 fibroblasts per ml gel and HaCaT cells were cultivated on top in FAD and lifted at the air medium interface for 1 wk. Different markers were localized by indirect immunofluorescence microscopy using monostaining (a–d, g) and double staining (e, f, h) technique. Strong suprabasal expression of K1/10 (a) and K16 (b), while involucrin (c) and transglutaminase type I (d) are pericellularly distributed in the upper layers only. Filaggrin (e) and K2e (f) are found in scattered single cells in the uppermost layer, whereas loricrin cannot be detected at this timepoint (h). Continuous deposition of the BM components nidogen (e), collagen IV (f,g), and laminin (h) at the junctional zone between HaCaT epithelium and collagen matrix. Scale bar: 50 μm. Journal of Investigative Dermatology 1999 112, 343-353DOI: (10.1046/j.1523-1747.1999.00524.x) Copyright © 1999 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 4 Differentiation markers and BM components in 2 and 3 wk old cultures. Indirect immunofluorescence of cocultures prepared with 5 × 105 fibroblasts per ml collagen gel and cultivated in FAD for 2 wk (c, d, f, g) or 3 wk (a, b, e, h) with double immunostaining in (a), (b), (e–g). Keratins K1/10 and K16 are restricted to suprabasal layers (a, b). Involucrin (c) and transglutaminase type I (d) are distributed mainly in the suprabasal layers, but also in some basal cells (d), whereas filaggrin (e), keratin K2e (f), and loricrin (g) are confined to the stratum granulosum and the parakeratotic stratum corneum. Increased deposition of nidogen in the gel (e) and strong, continuous reaction of collagen IV (f) and laminin (g) are visible after 2 and 3 wk. Spotwise staining of collagen VII is seen only in 3 wk old cultures (h). Scale bar: 50 μm. Journal of Investigative Dermatology 1999 112, 343-353DOI: (10.1046/j.1523-1747.1999.00524.x) Copyright © 1999 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 5 Ultrastructural characteristics of differentiation and BMZ. After 1 wk (day 7), the still dysplastic epithelium displays basal keratinocytes (BK), spinous cells (S), and flattened cells in the upper layer (d, a). In some areas, the intercellular spaces between these cells are widened (d). The large nuclei (Nu) (a), often indented, are surrounded by bundles of keratin filaments (K) projecting into desmosomal plaques (d, arrows, and e). After 2 wk (b) round and stellate keratohyaline granules (KG) and numerous lipid droplets (L) appear in the stratum granulosum (SG) and in just forming cornified cells (SC). After 3 wk (c, g), a multilayered epithelium (g) is covered by cornified cells (SC) with cornified envelopes (CE, c) containing keratin bundles, lipid droplets, partially degraded cell organelles and immature lamellar bodies (f). The dermal–epidermal junction zone after 1 wk (h) shows many hemidesmosomes (arrowheads) and the beginning assembly of a lamina densa (h, arrow). The hemidesmosomes (i, arrowhead) consist of their outer and inner plaque, the latter associated with keratin filaments. After 3 wk (j) the well established lamina densa is connected with the mature hemidesmosomes (arrowhead) by anchoring filaments (short arrow) and with the collagen matrix by loops of anchoring fibrils (long arrow). Scale bars: 5000 nm (d), 1000 nm (a, b, g), 500 nm (e, h), 250 nm (c, f, i, j). Journal of Investigative Dermatology 1999 112, 343-353DOI: (10.1046/j.1523-1747.1999.00524.x) Copyright © 1999 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 6 Kinetics of HaCaT cell proliferation and localization of TUNEL reaction. In cocultures prepared with 5 × 105 fibroblasts per ml gel, nuclear incorporation of BrdU was visualized by indirect immunofluorescence (bright staining, arrows) and all nuclei were counterstained with bisbenzimide (blue) (a–c). In 1 wk old cultures, many HaCaT cells in basal and suprabasal positions were labeled with BrdU (a). Proliferation was reduced and mostly restricted to cells in basal position in 2 wk old (b) and 3 wk old (c) cultures. Fragmentation of DNA was detected by TUNEL labeling using fluorescein-conjugated nucleotide polymers showing staining (in green) in all nuclei of the parakeratotic stratum corneum after 18 d (d, dotted line indicates the border between HaCaT epithelium and collagen matrix). The same section (e) was counterstained with the DNA Hoechst dye resulting in blue staining in all cell layers (e, double labeling). Scale bars: 50 μm. Journal of Investigative Dermatology 1999 112, 343-353DOI: (10.1046/j.1523-1747.1999.00524.x) Copyright © 1999 The Society for Investigative Dermatology, Inc Terms and Conditions