Reconstituted Skin from Murine Embryonic Stem Cells

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Reconstituted Skin from Murine Embryonic Stem Cells Christelle Coraux, Caroline Hilmi, Matthieu Rouleau, Anne Spadafora, Jocelyne Hinnrasky, Jean-Paul Ortonne, Christian Dani, Daniel Aberdam Current Biology Volume 13, Issue 10, Pages 849-853 (May 2003) DOI: 10.1016/S0960-9822(03)00296-3

Figure 1 Keratinocyte Differentiation of Mouse ES Cells (A) Immunofluorescent staining of K14 at (a, c, and e) day 8 and (b, d, and f) day 15 of differentiated ES cells cultured on a (a–f) HNF-secreted matrix in the (a and b) absence or the (c and d) presence of BMP-4 or (e and f) L-ascorbic acid. The scale bar represents 11 μm. (B) Quantification of K14-positive cells at day 15 by flow cytometry analysis on ES cells treated as in (A). The relative percentage of positive cells is shown in brackets. (C) Detection of keratinocyte-specific gene expression by RT-PCR at various stages of differentiation. Total RNA was extracted from either undifferentiated ES cells cultured on gelatin in the presence of LIF (D0) or ES cells cultured during 8 days on HNF-secreted matrix in the presence of L-ascorbic acid (D8). The sizes of the amplified products are: actin control, 318 bp; lama3, 695 bp; K14, 401 bp; and K10, 482 bp. Current Biology 2003 13, 849-853DOI: (10.1016/S0960-9822(03)00296-3)

Figure 2 Histological Aspect of ES-Derived Reconstituted Skin (A) Histological aspect of in vivo mouse E17.5 embryonic skin (left) and ES-derived reconstituted skin (right). Hematoxylin/eosin histological staining shows the different layers of the pluristratified epidermis, with keratohyalin granules in the stratum granulosum (arrowheads). The scale bar represents 5.5 μm. (B) An RT-PCR experiment to demonstrate the absence of human cells within the cells seeded on HA membranes for organotypic culture. Total RNA was extracted from either HNF (lane 1), undifferentiated ES cells (lane 2), or ES cells cultured for 8 days on a HNF-secreted matrix in the presence of L-ascorbic acid (lane 3). Sizes of the amplified products are: 28S control, 211 bp; human-specific GAPDH, 193 bp. Current Biology 2003 13, 849-853DOI: (10.1016/S0960-9822(03)00296-3)

Figure 3 Immunofluorescence Analysis of the Reconstituted Skin (A–J) The basal layer expresses (A) K14 (red), whereas the suprabasal layers express (B) K10 (red). The arrows delineate the basement membrane. The stratum granulosum and corneum express (C) corneodesmosin (red) and (D) filaggrin (green). The adhesion ligand laminin-5 is deposited in the (E) BM, and cells of the basal layer express (F) α6 and (G) β4 integrin subunits. The dermal-epidermal junction is immunostained for (A) laminin-1 (green), (B) type IV collagen (green), (H) type VII collagen, (I) fibronectin, and (J) nidogen, which is also detected in the cells under the BM. Nuclei are stained blue in (C) and (D). The scale bar represents 5.5 μm in (J) and 11 μm in (A)–(I). Current Biology 2003 13, 849-853DOI: (10.1016/S0960-9822(03)00296-3)

Figure 4 Electron Micrographs of the ES-Derived Reconstituted Skin (A) The reconstituted epidermis displays basal keratinocytes connected with desmosomes (white arrowheads and insert) laying on an intact BM. Hemidesmosomes are visible all along the BMZ (black arrows). (B) High magnification of hemidesmosome (HD) intracellularly connected to keratin filaments (ke). From the hemidesmosome, anchoring filaments (Af) cross the lamina lucida (LL) of the BM to connect to anchoring fibrils (AF) in the lamina densa. The magnification in (A) is ×3,000, the magnification in the insert in (A) is ×30,000, and the magnification in (B) is ×50,000. Current Biology 2003 13, 849-853DOI: (10.1016/S0960-9822(03)00296-3)