Development of an easy method for constructing three dimensional cultured skin using epidermal stem cell and amnion membrane

2010 Fiscal Year Final Research Report

• Project/Area Number: 20591347
• Research Category: Grant-in-Aid for Scientific Research (C)
• Allocation Type: Single-year Grants
• Section: 一般
• Research Field: Dermatology
• Research Institution: Ehime University
• Principal Investigator: KAMEDA Kenji Ehime University, 総合科学研究支援センター, 講師 (60363264)
• Co-Investigator(Kenkyū-buntansha): SHIRAKATA Yuji 愛媛大学, 大学院・医学系研究科, 講師 (50226320)
• Project Period (FY): 2008 – 2010
• Keywords: 皮膚炎症 / 再生学

Research Abstract

In order to develop an easy method for constructing a three-dimensional cultured skin, we focused on epidermal stem cells and amnion membrane. We analyzed the condition for side population cells as stem cells, and clarified that ten microgram per ml of Hoechst dye, 60 min treatment at 37 degree are best for side population staining. Then we examined side population cell rate among different passaged cells, and demonstrated that side population cell ratio decreased along with passaging. Next we analyzed about amnion membrane. Amnion membrane was overlaid on type I collagen gel embedded with fibroblasts; then, normal human keratinocytes were seeded on the epithelial side of the amnion membrane. When the keratinocytes reached confluence, the three-dimensional cultured skin was lifted to the air-liquid interface and cultured for up to 3 weeks. Samples were harvested at different times and manipulated for morphological, immunohistochemical, and ultrastructural investigation. In the three-dimensional cultured skin with amnion, the epidermis was better stratified, with more compact, polarized, columnar basal cells, and the expression of differentiation and proliferation markers was more similar to that of normal human skin than was that of the three-dimensional cultured skin without amnion membrane. A more continuous basement membrane and better-developed hemidesmosomes were found in the three-dimensional cultured skin with amnion. It is possible to construct a better cultured skin using amnion membrane.

Research Products

• [Journal Article] E2 Polyubiquitin-conjugating enzyme Ubc13 in keratinocytes is essential for epidermal integrity. (2010)
  • Author(s): Sayama K, Yamamoto M, Shirakata Y, Hanakawa Y, Hirakawa S, Dai X, Tohyama M, Tokumaru S, Shin MS, Sakurai H, Akira S, Hashimoto K.
  • Journal Title: J Biol Chem. 285(39) Pages: 30042-30049
  • Peer Reviewed
• [Journal Article] Inflammatory mediator TAK1 regulates hair follicle morphogenesis and anagen induction shown by using keratinocyte-specific TAK1-deficient mice. (2010)
  • Author(s): Sayama K, Kajiya K, Sugawara K, Sato S, Hirakawa S, Shirakata Y, Hanakawa Y, Dai X, Ishimatsu-Tsuji Y, Metzger D, Chambon P, Akira S, Paus R, Kishimoto J, Hashimoto K.
  • Journal Title: PLoS One. 23;5(6) Pages: e11275
  • Peer Reviewed
• [Journal Article] Regulation of epidermal keratinocytes by growth factors. (2010)
  • Author(s): Shirakata Y.
  • Journal Title: J Dermatol Sci. 59(2) Pages: 73-80
  • Peer Reviewed
• [Journal Article] Auto- and cross-induction by betacellulin in epidermal keratinocytes. (2010)
  • Author(s): Shirakata Y, Tokumaru S, Sayama K, Hashimoto K.
  • Journal Title: J Dermatol Sci. 58(2) Pages: 162-164
  • Peer Reviewed
• [Journal Article] Possible Roles of IL-27 in the Pathogenesis of Psoriasis. (2010)
  • Author(s): Shibata S, Tada Y, Kanda N, Nashiro K, Kamata M, Karakawa M, Miyagaki T, Kai H, Saeki H, Shirakata Y Tamaki K, Sato S.
  • Journal Title: J Invest Dermatol. 130(4) Pages: 1034-1039
  • Peer Reviewed
• [Journal Article] Living Skin Equivalents Constructed Using Human Amnions as a Matrix. (2009)
  • Author(s): Yang L, Shirakata Y, Tokumaru S, Xiuju D, Tohyama M, Hanakawa Y, Hirakawa S, Sayama K, Hashimoto K
  • Journal Title: J Dermatol Sci. 56 Pages: 188-195
  • Peer Reviewed
• [Journal Article] IL-17 and IL-22 mediate IL-20 subfamily cytokine production in cultured keratinocytes via increased IL-22 receptor expression. (2009)
  • Author(s): Tohyama M, Hanakawa Y, Shirakata Y, Dai X, Yang L, Hirakawa S, Tokumaru S, Okazaki H, Sayama K, Hashimoto K.
  • Journal Title: Eur J Immunol. 39 Pages: 2279-2288
  • Peer Reviewed
• [Journal Article] UVB wavelength dependency of antimicrobial peptide induction for innate immunity in normal human keratinocytes. (2009)
  • Author(s): Torii K, Maeda A, Saito C, Furuhashi T, Shintani Y, Shirakata Y, Morita A.
  • Journal Title: J Dermatol Sci. 56 Pages: 217-219
  • Peer Reviewed
• [Journal Article] Alteration of TLR3 pathways by glucocorticoids may be responsible for immunosusceptibility of human corneal epithelial cells to viral infections. (2009)
  • Author(s): Hara Y, Shiraishi A, Kobayashi T, Kadota Y, Shirakata Y, Hashimoto K, Ohashi Y.
  • Journal Title: Mol Vis. 15 Pages: 937-948
  • Peer Reviewed
• [Journal Article] The NF-kB, p38 MAPK and STAT1 pathways differentially regulate the dsRNA-mediated innate immune responses of epidermal keratinocytes. (2008)
  • Author(s): Dai X, Sayama K, Tohyama M, Shirakata Y, Yang L, Hirakawa S, Tokumaru S, Hashimoto K.
  • Journal Title: Int Immunol. 20 Pages: 901-909
  • Peer Reviewed
• [Journal Article] PPARgamma is an important transcription factor in 1alpha,25-dihydroxyvitamin D3-induced involucrin expression. (2008)
  • Author(s): Dai X, Sayama K, Shirakata Y, Tokumaru S, Yang L, Tohyama M, Hirakawa S, Hanakawa Y, Hashimoto K.
  • Journal Title: J Dermatol Sci 50 Pages: 53-60
  • Peer Reviewed
• [Presentation] Deletion of SOCS3 in the epidermis causes impaired skin wound healing in vivo. (2010)
  • Author(s): Shirakata Y, Yang L, Tsuda T, Tohyama M, Miyawaki S, Kameda K, Sayama K, Yoshimura A, Hashimoto K.
  • Organizer: The 40^h Annual Meeting of the European Society for Dermatological Research
  • Place of Presentation: Helsinki, Finland
  • Year and Date: 20100908-20100911
• [Presentation] Interferon alpha enhances IL-22 receptor expression on epidermal keratinocytes. (2010)
  • Author(s): Tohyama M, Yang L, Tsuda T, Miyawaki S, Shirakata Y, Sayama K, Hashimoto K.
  • Organizer: The 40^h Annual Meeting of the European Society for Dermatological Research
  • Place of Presentation: Helsinki, Finland
  • Year and Date: 20100908-20100911
• [Presentation] Simple method of constructing living skin equivalent using human amnion as dermal matrix. (2009)
  • Author(s): Shirakata Y, Yang L, Sayama K, Hashimoto K.
  • Organizer: The 39^<th> Annual Meeting of European Society for Dermatological Research
  • Place of Presentation: Budapest, Hungary
  • Year and Date: 20090910-20090912
• [Presentation] Development of a new skin equivalent using de-epithelialized amnion membrane. (2009)
  • Author(s): Shirakata Y, Hashimoto K.
  • Organizer: The 4^<th> Joint Meeting of Japanese Dermatological Association and Australasian College of Dermatologists
  • Place of Presentation: Sapporo
  • Year and Date: 20090710-20090712
• [Presentation] Inflammatory mediator TAK1 regulates hair follicle cycling. (2009)
  • Author(s): Sayama K, Shirakata Y, Ishimatsu-Tsuji Y, Kajiya K, Hirakawa S, Sugawara K, Chambon P, Akira S, Paus R, Kishimoto J, Hashimoto K.
  • Organizer: The 69^<th> Annual Meeting of Society for Investigative Dermatology
  • Place of Presentation: Montreal, Canada
  • Year and Date: 20090506-20090509
• [Remarks] ホームページ等