Skip to main content
SpringerLink
Log in

Microarray analysis of oral mucosal epithelial cell sheet

  • Original Article
  • Published:
Tissue Engineering and Regenerative Medicine Aims and scope

Abstract

Corneal epithelium regeneration using autologous oral mucosal epithelial cell sheet is a successful new approach in corneal therapy. In the present study, gene expression profiling was performed to characterize engineered cell sheets. Cell sheets were obtained by culturing isolated rabbit oral mucosal epithelial cells on a thermoresponsive cultureware (UpCell®, CellSeed Inc. Japan). H&E staining of cell sheets showed a multistratified epithelium, similar to corneal epithelium. DeltaN-p63 stained positive in the basal cells, indicating that cell sheets have renewal capacity. Microarray analysis of these cell sheets showed that only 160 genes out of 43,000 rabbit probes listed on the microarray chip were identified. We first identified the extracellular matrix group of genes and found that matrix metalloproteinase MMP-1, MMP-3 and MMP-12, known to promote angiogenesis, were down regulated, while MMP-13 and collagen type VIII alpha 1 (COL8A1), proteins involved in wound healing, were up regulated. Tissue inhibitors of metalloproteinase TIMP-1 and TIMP-3, anti-angiogenic factors, were also identified. Gap junction protein A7 (GJA7 or Connexin 45) was found up regulated, indicating that cell sheets have developed well preserved cell-cell interactions. Alcohol dehydrogenase 5 (ADH class III) and aldehyde dehydrogenase (ALDH1A1), involved in protecting the cornea against oxidative stress induced by UV radiation, were also found up regulated. In conclusion, microarray analysis has led us to identify new target molecules and their subsequent biochemical analysis indicated how the composite cell sheets are advantageous to the original isolated cells in terms of the integrity and potency of corneal epithelial grafts without any scaffolds.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Similar content being viewed by others

References

  1. SC Tseng, SY Chen, YC Shen, et al., Critical appraisal of ex vivo expansion of human limbal epithelial stem cells, Curr Mol Med, 10, 841 (2010).

    Article  PubMed  CAS  Google Scholar 

  2. C Dieckmann, R Renner, L Milkova, et al., Regenerative medicine in dermatology: biomaterials, tissue engineering, stem cells, gene transfer and beyond, Exp Dermatol, 19, 697 (2010).

    Article  PubMed  CAS  Google Scholar 

  3. M Krakauer, JD Welder, HK Pandya, et al., Adverse effects of systemic immunosuppression in keratolimbal allograft, J Ophthalmol, 2012, 576712 (2012).

    PubMed  CAS  Google Scholar 

  4. T Nakamura, K Endo, LJ Cooper, et al., The successful culture and autologous transplantation of rabbit oral mucosal epithelial cells on amniotic membrane, Invest Opthalmol Vis Sci, 44, 106 (2003).

    Article  Google Scholar 

  5. K Nishida, M Yamato, Y Hayashida, et al., Functional bioengineered corneal epithelial sheet grafts from corneal stem cells expanded ex vivo on a temperature-responsive cell culture surface, Transplantation, 15, 379 (2004).

    Article  Google Scholar 

  6. C Burillon, L Huot, V Justin, et al., Cultured autologous oral mucosal epithelial cell sheet (CAOMECS) transplantation for the treatment of corneal limbal epithelial stem cell deficiency, Invest Ophthalmol Vis Sci, 13, 1325 (2012).

    Article  Google Scholar 

  7. Y Hayashida, K Nishida, M Yamato, et al., Ocular surface reconstruction using autologous rabbit oral mucosal epithelial sheets fabricated ex vivo on a temperature-responsive culture surface, Invest Ophthalmol Vis Sci, 46, 1632 (2005).

    Article  PubMed  Google Scholar 

  8. Y Barrandon, H Green, Three clonal types of keratinocyte with different capacities for multiplication, Proc Natl Acad Sci USA, 84, 2302 (1987).

    Article  PubMed  CAS  Google Scholar 

  9. I Bortolomai, S Canevari, I Facetti, et al., Tumor initiating cells: development and critical characterization of a model derived from the A431 carcinoma cell line forming spheres in suspension, Cell Cycle, 15, 1194 (2010).

    Article  Google Scholar 

  10. SL Karsten, LC Kudo, R Jackson, et al., Global analysis of gene expression in neural progenitors reveals specific cell-cycle, signaling, and metabolic networks, Dev Biol, 261, 165 (2003).

    Article  PubMed  CAS  Google Scholar 

  11. N Li, S Singh, P Cherukuri, et al., Reciprocal intraepithelial interactions between TP63 and hedgehog signaling regulate quiescence and activation of progenitor elaboration by mammary stem cells, Stem Cells, 26, 1253 (2008).

    Article  PubMed  CAS  Google Scholar 

  12. DW Huang, BT Sherman, RA Lempicki, Bioinformatics enrichment tools: paths toward the comprehensive functional analysis of large gene lists, Nucleic Acids Res, 37, 1 (2009).

    Article  Google Scholar 

  13. DW Huang, BT Sherman, RA Lempicki, et al., Systematic and integrative analysis of large gene lists using DAVID Bioinformatics Resources, Nature Protoc, 4, 44 (2009).

    Article  CAS  Google Scholar 

  14. H Nagase, JJ Enghild, K Suzuki, et al., Stepwise activation mechanisms of the precursor of matrix metalloproteinase 3 (stromelysin) by proteinases and (4-aminophenyl) mercuric acetate, Biochemistry, 19, 5783 (1990).

    Article  Google Scholar 

  15. VP Saw, E Schmidt, I Offiah, et al., Profibrotic phenotype of conjunctival fibroblasts from mucous membrane pemphigoid, Am J Pathol, 178, 187 (2011).

    Article  PubMed  CAS  Google Scholar 

  16. T Desronvil, D Logan-Wyatt, W Abdrabou, et al., Distribution of COL8A2 and COL8A1 gene variants in Caucasian primary open angle glaucoma patients with thin central corneal thickness, Mol Vis, 29, 2185 (2010).

    Google Scholar 

  17. AK El-Hawary, E Yassin, A Khater, et al., Expression of matrix metalloproteinase-13 and Ki-67 in nonmelanoma skin cancer in Xeroderma Pigmentosum and non-xeroderma Pigmentosum, Am J Dermatopathol, 35, 45 (2013).

    Article  PubMed  Google Scholar 

  18. GM Gordon, JS Austin, AL Sklar, et al., Comprehensive gene expression profiling and functional analysis of matrix metalloproteinases and TIMPs, and identification of ADAM-10 gene expression, in a corneal model of epithelial resurfacing, J Cell Physiol, 226, 1461 (2011).

    Article  PubMed  CAS  Google Scholar 

  19. SD Galiacy, C Froment, E Mouton-Barbosa, et al., Deeper in the human cornea proteome using nanoLC-Orbitrap MS/MS: An improvement for future studies on cornea homeostasis and pathophysiology, Proteomics, 10, 81 (2011).

    Google Scholar 

  20. E Candi, A Rufini, A Terrinoni, et al., Differential roles of p63 isoforms in epidermal development: selective genetic complementation in p63 null mice, Cell Death Differ, 13, 1037 (2006).

    Article  PubMed  CAS  Google Scholar 

  21. T Matsuura, VK Kawata, H Nagoshi, et al., Regulation of proliferation and differentiation of mouse tooth germ epithelial cells by distinct isoforms of p51/p63, Arch Oral Biol, 57, 1108 (2012).

    Article  PubMed  CAS  Google Scholar 

  22. MI Koster, S Kim, AA Mills, et al., p63 is the molecular switch for initiation of an epithelial stratification program, Genes Dev, 18, 126 (2004).

    Article  PubMed  CAS  Google Scholar 

  23. AV Danilov, D Neupane, AS Nagaraja, et al., DeltaNp63alphamediated induction of epidermal growth factor receptor promotes pancreatic cancer cell growth and chemoresistance, PLoS One, 6, 26815 (2011).

    Article  Google Scholar 

  24. E Thomas, N Zeps, M Cregan, et al., 14-3-3ó (sigma) regulates proliferation and differentiation of multipotent p63-positive cells isolated from human breastmilk, Cell Cycle, 15, 278 (2011).

    Article  Google Scholar 

  25. D Suzuki, M Senoo, Increased p63 phosphorylation marks early transition of epidermal stem cells to progenitors, J Invest Dermatol, 132, 2461 (2012).

    Article  PubMed  CAS  Google Scholar 

  26. MD Westfall, AS Joyner, CE Barbieri, et al., Ultraviolet radiation induces phosphorylation and ubiquitin-mediated degradation of DeltaNp63alpha, Cell Cycle, 4, 710 (2005).

    Article  PubMed  CAS  Google Scholar 

  27. S Merjava, P Liskova, Y Sado, et al., Changes in the localization of collagens IV and VIII in corneas obtained from patients with posterior polymorphous corneal dystrophy, Exp Eye Res, 88, 945 (2009).

    Article  PubMed  CAS  Google Scholar 

  28. U Hopfer, N Fukai, H Hopfer, et al., Targeted disruption of Col8a1 and Col8a2 genes in mice leads to anterior segment abnormalities in the eye, FASEB J, 19, 1232 (2005).

    Article  PubMed  CAS  Google Scholar 

  29. C Zhang, WR Bell, OH Sundin, et al., Immunohistochemistry and electron microscopy of early-onset fuchs corneal dystrophy in three cases with the same L450W COL8A2 mutation, Trans Am Ophthalmol Soc, 104, 85 (2006).

    PubMed  Google Scholar 

  30. I Loeffler, M Liebisch, G Wolf, et al., Collagen VIII influences epithelial phenotypic changes in experimental diabetic nephropathy, Am J Physiol Renal Physiol, 303, F733 (2012).

    Article  PubMed  CAS  Google Scholar 

  31. JM Sivak, ME Fini, MMPs in the eye: emerging roles for matrix metalloproteinases in ocular physiology, Prog Retin Eye Res, 21, 1 (2002).

    Article  PubMed  CAS  Google Scholar 

  32. H Nagase, JF Woessner Jr, Matrix metalloproteinases, J Biol Chem, 274, 21491 (1999).

    Article  PubMed  CAS  Google Scholar 

  33. Z Werb, ECM and cell surface proteolysisregulating cellular ecology, Cell, 91, 439 (1997).

    Article  PubMed  CAS  Google Scholar 

  34. JF Woessner, The matrix metalloproteinase family. In: WC Parks, RP Mecham, eds. Matrix Metalloproteinases. Academic Press. San Diego, CA, 1 (1998).

    Chapter  Google Scholar 

  35. HQ Ye, M Maeda, FS Yu, et al., Differential expression of MT1-MMP (MMP-14) and collagenase III (MMP-13) genes in normal and wounded rat corneas, Invest Opthalmol Vis Sci, 41, 2894 (2010).

    Google Scholar 

  36. M.C Kenney, M Chwa, A Alba, et al., Localization of TIMP-1, TIMP-2, TIMP-3, gelatinase A and gelatinase B in pathological human corneas, Curr Eye Res, 17, 238 (1998).

    Article  PubMed  CAS  Google Scholar 

  37. R Visse, H Nagase, Matrix metalloproteinases and tissue inhibitorsof metalloproteinases: structure, function and biochemistry, Circ Res, 92, 827 (2003).

    Article  PubMed  CAS  Google Scholar 

  38. MI Cockett, G Murphy, ML Birch, et al., Matrix metalloproteinases and metastatic cancer, Biochem Soc Symp, 63, 295 (1998).

    PubMed  CAS  Google Scholar 

  39. H Sato, M Seiki, Membrane-type matrix metalloproteinases (MTMMPs) in tumor metastasis, J Biochem, 119, 209 (1996).

    Article  PubMed  CAS  Google Scholar 

  40. D Stagos, Y Chen, M Cantore, et al., Corneal aldehyde dehydrogenases: multiple functions and novel nuclear localization, Brain Res Bull, 15, 211 (2010).

    Article  Google Scholar 

  41. JV Jester, Corneal crystallins and the development of cellular transparency, Semin Cell Dev Biol, 19, 82 (2008).

    Article  PubMed  CAS  Google Scholar 

Download references

Author information

Authors and Affiliations

  1. LA BioMed Institute at Harbor UCLA Medical Center, Torrance, California, 90502, USA

    Fawzia Bardag-Gorce, Joan Oliva, Andrew Wood, Hope Niihara, Andrew Makalinao, Sean Sabino, Derek Pan, Jacquelyn Thropay, Hiroyuki Sota & Yutaka Niihara

Authors
  1. Fawzia Bardag-Gorce
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Joan Oliva
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Andrew Wood
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Hope Niihara
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Andrew Makalinao
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Sean Sabino
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Derek Pan
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. Jacquelyn Thropay
    View author publications

    You can also search for this author in PubMed Google Scholar

  9. Hiroyuki Sota
    View author publications

    You can also search for this author in PubMed Google Scholar

  10. Yutaka Niihara
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Fawzia Bardag-Gorce.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Bardag-Gorce, F., Oliva, J., Wood, A. et al. Microarray analysis of oral mucosal epithelial cell sheet. Tissue Eng Regen Med 10, 362–370 (2013). https://doi.org/10.1007/s13770-013-1103-z

Download citation

  • Received:

  • Revised:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s13770-013-1103-z

Key words

Search

Navigation