Spontaneous Cell Sorting of Fibroblasts and Keratinocytes Creates an Organotypic Human Skin Equivalent  C. Kathy Wang, Charlotte F. Nelson, Alice M. Brinkman, Anne C. Miller, Warren K. Hoeffler  Journal of Investigative Dermatology  Volume 114, Issue 4, Pages 674-680 (April 2000) DOI: 10.1046/j.1523-1747.2000.00938.x Copyright © 2000 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 1 The silicone chamber (CRD culture chambers, Renner) used to contain a mixed cell slurry of keratinocytes and dermal fibroblasts. The lower chamber is placed directly on SCID mouse muscle fascia, after a circular piece of mouse skin has been surgically removed, and serves as a physical barrier to invading mouse skin during wound healing. The upper chamber is placed over the lower chamber and serves to keep other material out of the wound, as well as to maintain humidity. The mixed cell slurry is added by pipette through a hole in the top of the upper chamber. Journal of Investigative Dermatology 2000 114, 674-680DOI: (10.1046/j.1523-1747.2000.00938.x) Copyright © 2000 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 2 Reconstituted human skin from the CeSSE model created in situ in silicone chambers on the backs of SCID mice. Complete separation of human keratinocytes and fibroblasts into discrete epidermal and dermal layers from a mixed cell slurry after 14 d. Scale bars in (b)-(f) indicate the extent of epidermal keratinocytes (white) and dermal fibroblasts (black). The region between the bars defines the BMZ, the normal interface of the two skin layers. (a) Implantation of human skin cells pipetted as a cell slurry into a silicone chamber implanted on the back of a SCID mouse. (b) Hematoxylin and eosin staining of a biopsy of the human CeSSE: upper epidermis stains purple, lower dermis stains a lighter violet. Flaking layers at the top of the epidermis are dead differentiated squames, as would be seen in normal skin. (c) Filaggrin antibody immunostaining of a CeSSE. Upper layer differentiated keratinocytes (arrow) express filaggrin, and therefore are stained brown. Biopsies were cryosectioned and detected with a perioxidase linked second antibody in this and subsequent figures (Experimental protocol). (d) Keratin 10 antibody immunostaining of a CeSSE. All keratinocytes express keratin 10 and are stained brown, except the single layer of basal keratinocytes along the DEJ, which remained purple. (e) Keratin 14 antibody immunostaining of a CeSSE. Only basal keratinocytes express keratin 14, and therefore stain dark brown. (f) Laminin-5 antibody immunostaining of a CeSSE. Laminin-5 is a component of hemidesmosomes expressed by basal keratinocytes along the BMZ, and therefore dark brown staining is limited to the BMZ, as indicated by arrows. Journal of Investigative Dermatology 2000 114, 674-680DOI: (10.1046/j.1523-1747.2000.00938.x) Copyright © 2000 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 3 Reconstituted human skin after 14 d immunostained for markers of human dermal fibroblasts. Scale bars on the right of each figure indicate the extent of epidermal keratinocytes (white) and dermal fibroblasts (black). The grey bar indicates the region of mouse fibroblasts. (a) Collagen VII antibody immunostaining. Collagen VII is expressed primarily by dermal fibroblasts and is localized to the upper dermis along the BMZ, and therefore brown staining is along the BMZ as indicated by arrows. (b) Vimentin antibody immunostaining. Vimentin is uniformly expressed by dermal fibroblasts, and therefore brown staining is seen in the entire dermal layer. (c) Human fibroblast specific monoclonal antibody 5B5 immunostaining. This antibody recognizes an enzyme intermediate in collagen synthesis, propyl-4-hydroxylase, and recognizes only human, but not mouse, fibroblasts. Brown staining seen in reconstituted dermis. In this particular reconstitution fibroblasts of mouse origin are present below the human dermis, but do not stain with 5B5. Journal of Investigative Dermatology 2000 114, 674-680DOI: (10.1046/j.1523-1747.2000.00938.x) Copyright © 2000 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 4 Retroviral β-galactosidase expression vector targeted to keratinocytes or fibroblasts, and used in the CeSSE model to reconstitute human skin. (a) Diagram of retroviral expression vector used to express β-galactosidase. Long-terminal repeat (LTR) contains promoter used to drive expression (bent arrow) of inserted β-galactosidase gene, and ψ+ indicates retroviral packaging sequence (Kinsella 1996). (b) Retroviral β-galactosidase expression vector was used to infect dermal fibroblasts in tissue culture, and subsequently used in the CeSSE model to reconstitute human skin. Blue staining is limited to the dermis. (c) Retroviral β-galactosidase expression vector was used to infect keratinocytes in tissue culture, and subsequently used in the CeSSE model to reconstitute human skin. Blue staining is limited to the epidermis. Journal of Investigative Dermatology 2000 114, 674-680DOI: (10.1046/j.1523-1747.2000.00938.x) Copyright © 2000 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 5 Electron micrographs of basal keratinocytes and the BMZ from normal human skin, reconstituted human skin created by the standard composite model, or reconstituted human skin created by the cell-sorting method. (a) In this normal skin sample basal keratinocytes located along the BMZ form a well-developed network of keratin intermediate filaments as shown (arrows). (b) In this composite model human skin keratinocytes were seeded directly onto devitalized dermis, with the basal keratinocytes showing no clear presence of intermediate filaments (no arrows). (c) In the CeSSE model intermediate filaments are present (arrows). (d) In the normal skin sample at higher magnification the well-developed network of intermediate filaments (arrows) connect to hemidesomosomes, electron-dense ‘‘buttons’' located along the BMZ, as seen in the regions labeled h. (e) In the composite skin sample at higher magnification no intermediate filaments are seen, but hemidesmosomes are visible, as seen in regions labeled h. (f) In the CeSSE skin sample at higher magnification intermediate filaments are seen (arrows) connecting to hemidesmosomes, as seen in regions labeled h. Scale bars: (a–c) 2.4 μm; (d–f) 0.4 μm. Journal of Investigative Dermatology 2000 114, 674-680DOI: (10.1046/j.1523-1747.2000.00938.x) Copyright © 2000 The Society for Investigative Dermatology, Inc Terms and Conditions