title: Using human epithelial amnion cells in human de-epidermized dermis for skin regeneration.
abstract: BACKGROUND
  Human amniotic epithelial cells (hAECs) is a desirable reserve of stem cells. Human de-epidermized dermis (DED) retains basic tissue structure and parts of the basement membrane (BM) components at the acelluIar dermal surface, and provides a potential tool for skin regeneration.
  
  
  OBJECTIVE
  To evaluate the potential role of hAECs in skin regeneration, we used DED to perform organotypic culture of hAECs to develop organotypic skin.
  
  
  METHODS
  HAECs were isolated and cultured. Biological characteristics of hAECs were determined by immunocytochemistry and flow cytometry. To prepare DED, the epidermis was removed and then repeated freeze-thaw cycles. HAECs and fibroblast were seeded onto DED to perform the submerged culture for 3 days and then to be maintained at the air-liquid interface for 14 days to form organotypic culture. To identify whether the obtained DED retain the BM structure and components, the histological characteristics of DED and the BM were detected by immunohistochemistry. To evaluate whether the organotypic skin has similar histological characteristics with normal human skin, the marks of epidermal proliferation and differentiation and basement membrane component were detected by immunohistochemistry. Moreover, cell ultrastructure, cell-cell contact and ultrastructure of BM were examined under the transmission electron microscopy.
  
  
  RESULTS
  HAECs has stem-cell characteristics with strong pluripotent Oct-4 and embryonic marker SSEA-4 expression. DED has effectively cleansed the cell components and continuous distributions of laminin and collagen IV. The histological appearance of tissue-engineered skin in vitro has 4 to 9 continuous layers of stratified epithelium and is similar to normal human skin in morphology. Immunohistochemical studies revealed that proliferation and differentiation markers such as Ki67, CK19, CK14, CK10, filaggrin but not CK18 expressed similar pattern characteristics to normal human epidermis. In addition, Periodic acid-Schiff stain showed that a uniform red staining strip located at the epidermal-dermal junction. BM component proteins (type IV collagen and laminin) and cell adhesion protein (desmoglein) were detected by immunohistochemistry in organotypic skin. Ultrastructurally, desmosomes, hemidesmosomes and BM zone (BMZ) were observed in organotypic skin.
  
  
  CONCLUSIONS
  Our studies indicate that the hAECs is a promising stem cell source for tissue-engineered skin, and DED with hAECs is a potential application prospects in regenerative medicine.
authors.affiliation: Department of Dermatology, The First Hospital of China Medical University, Shenyang, Liaoning 110001, PR China; Department of Dermatology, Affiliated Hospital of Guiyang Medical College, Guiyang, Guizhou 550001, PR China.
authors.forename: Lei-Wei
authors.initials: LW
authors.lastname: Jiang
authors.affiliation: Department of Dermatology, The First Hospital of China Medical University, Shenyang, Liaoning 110001, PR China.
authors.forename: Hongduo
authors.initials: H
authors.lastname: Chen
authors.affiliation: Department of Dermatology, The First Hospital of China Medical University, Shenyang, Liaoning 110001, PR China; Department of Dermatology, Affiliated Hospital of Guiyang Medical College, Guiyang, Guizhou 550001, PR China. Electronic address: hongguanglu@hotmail.com.
authors.forename: Hongguang
authors.initials: H
authors.lastname: Lu
mesh_terms: D000650:Amnion / Q000166:cytology*; D001485:Basement Membrane / Q000378:metabolism; D015815:Cell Adhesion Molecules / Q000378:metabolism; D002454:Cell Differentiation; D049109:Cell Proliferation; D002478:Cells, Cultured; D020405:Dermis / Q000166:cytology; D000078404:Epidermal Cells; D000091344:Filaggrin Proteins; D000066449:Human Embryonic Stem Cells / Q000166:cytology / Q000378:metabolism / Q000637:transplantation; D006801:Humans; D008565:Membrane Proteins / Q000378:metabolism; D008954:Models, Biological; D009924:Organ Culture Techniques; D012038:Regeneration; D044968:Regenerative Medicine; D012879:Skin Physiological Phenomena; D017768:Skin, Artificial; D023822:Tissue Engineering / Q000379:methods*
chemical_list: D015815:Cell Adhesion Molecules; C000717249:FLG protein, human; D000091344:Filaggrin Proteins; D008565:Membrane Proteins
keywords: Human amniotic epithelial cells (hAECs); Human de-epidermized dermis (DED); Organotypic culture; Organotypic skin; Skin regeneration