Mol Ther. 2014 Aug;22(8):1544-55.

Evidence of the survival of ectopically transplanted oral mucosal epithelial stem cells after repeated wounding of cornea.

Sugiyama H1, Yamato M1, Nishida K2, Okano T1.

1Institute of Advanced Biomedical Engineering and Science, Tokyo Women’s Medical University, Tokyo, Japan

2Department of Ophthalmology, Osaka University Graduate School of Medicine, Osaka, Japan

 

Abstract

Tissue engineering has become an essential tool in the development of regenerative medicine. We have developed cell sheet-based techniques for use in regenerative medicine that have already been successfully used in clinical applications. Native corneal epithelium is produced from limbal stem cells located in the transition zone between the cornea and the bulbar conjunctiva. Limbal stem cell deficiency (LSCD) is a severe defect of the limbal stem cells leading to vision loss due to conjunctival epithelial invasion and neovascularization. Rabbit LSCD models were treated with transplantable autologous oral mucosal epithelial cell (OEC) sheets fabricated on temperature-responsive cell culture surfaces, after which, the ocular surfaces were clear and smooth with no observable defects. The central part of the reconstructed ocular surface was scraped and wounded, after which proliferating epithelial cells covered the scraped area within a few days. The ocular surfaces were clear and smooth even after repeated scrapings and consisted of only OECs or heterogeneously mixed with corneal epithelial cells. This study demonstrates that transplanted cell sheets containing oral mucosal epithelial stem cells could reconstruct the ocular surface to maintain cornea homeostasis; moreover, they provide an ideal microenvironment to support the proliferation of remaining native limbal stem cells.

PMID: 24769908

 

Supplement

The present studies are intended to investigate the mode of action of the ectopically transplanted oral mucosal epithelial cell sheets for ocular surface reconstruction by using repeated wound healing assay.

 

 fig2

Fig. 1 Investigation of cultivated autologous oral mucosal epithelial cell (OEC) sheets transplantation into a rabbit limbal stem cell deficiency (LSCD) model. (a) Rabbit LSCD model was prepared by n-heptanol treatment after keratectomy. (b) An autologous OEC sheet was fabricated on a temperature-responsive cell culture surface with mitomycin-C–treated NIH/3T3 feeder layer and harvested by reducing temperature without enzymatic treatment. (c) OEC sheet was transplanted onto the ocular surface of a rabbit LSCD model after removing pannus. Four weeks after transplantation, the central part of reconstructed ocular surface (5 mm in diameter) was physically scraped once every 2 weeks, a total of 10 times over 24 weeks, and the scraped specimens were analyzed by real-time RT-PCR.

Regenerative medicine is attracting as a new therapy for the restoration of the lose tissues or function. Recently, Dr. Yamanaka and coworker established induced pluripotent stem cells (iPS cells) from differentiated somatic cells1. It is hoped that iPS cells have a potential to be a source for the regenerative medicine, because iPS cells are able to differentiate into the all kinds of transplantable cells from patients’ own cells. For the realization of the regenerative medicine, it is important not only to develop the cell source such as iPS cells, but also establish the way for transplantation with highly survival. Delivery method with cell suspension injection has several disadvantages, including cell loss caused by the leakage and poor survival of the injected cells. We developed cell sheet-based tissue engineering, which can give contiguous cell sheets cultivated on a temperature-responsive cell culture surfaces. This technology allows cell sheets to be transferred onto damaged organs as a structured cell sheet, have expecting the highly survival in the grafted area. Dr. Sawa and coworkers have succeeded that autologous myoblast sheets have been implanted to treat severe heart failure in clinical study2. Furthermore, Dr. Ohki and coworkers have achieved that autologous oral mucosal epithelial cell sheets are transplanted to prevent stricture formation following the removal of esophageal neoplasms by gastric endoscopy3. Therefore, we believe that cell sheet-based tissue engineering provides a new generation of regenerative medicine and contributes to the treatment of patients in all over the world.

This report focused on ocular surface reconstruction using oral mucosal epithelial cell sheets. Dr. Nishida and coworker have succeeded that autologous oral mucosal epithelial cell sheet transplantation for treating severe corneal diseases in clinical study and have a good prognosis4. For industrialization, the investigating of the safety and efficacy of the new therapy by oral mucosal epithelial cell sheet transplantation is necessary. Especially, the mode of action of a transplanted cell sheet is critical examination for the establishment of its safety and efficacy.

Native corneal epithelial layer have ability to turn over with two weeks and sustain own homeostasis. This mechanism is maintained by corneal stem cells located in limbus which is the transitional zone between cornea and conjunctiva. When the corneal stem cells are completely injured, the peripheral conjunctival epithelium invades with angiogenesis, resulting in a corneal opacificatoin leading sever visual loss, known as limbal stem cell deficiency (LSCD). Previously, keratoplasty is performed in patients with LSCD. However, this therapy has a poor favorable prognosis. Therefore, we developed autologous cultivated oral mucosal epithelial transplantation for treating a LSCD using cell sheet-based tissue engineering with temperature-responsive cell culture surfaces.

 

fig1

Fig. 2 Repeated wound-healing assay of proliferative and differentiation potential of transplanted oral mucosal epithelial cell sheets. Neovascularization and opacification were observed in the rabbit limbal stem cell deficiency (LSCD) model. An OEC sheet was transplanted onto the ocular surface after the removal of pannus (abnormal epithelium) without defects. Ocular surface was then reconstructed and confirmed to remain clear for 4 weeks, and then, the center of transplanted ocular surface was scraped to create a wound. Fluorescein staining revealed that the corneal stroma was completely reexposed. Proliferating epithelial cells covered the scraped area within a few days allowing the ocular surface to recover which was then confirmed to be stable with no evidence of fluorescein staining. Two weeks after the first scraping, the central part of healed ocular surface was physically scraped again. Epithelial cells migrated and covered the scraped area again after the second scraping. Similarly, the transplanted OEC sheet ocular surface was scraped every 2 weeks to a total of 10 times, and reepithelialization was observed after every scraping.

In this study, we performed the ocular surface reconstruction of rabbit LSCD model by transplantation of cultivated autologous oral mucosal epithelial cell sheets fabricated on a temperature-responsive culture surface. The central part of transplanted ocular surfaces was scraped every two weeks totally ten times, and subjected to gene expression analysis (Fig. 1). Re-epithelialization of the scraped area was observed within a few days in every time (Fig. 2). According to gene expression analysis, ocular surfaces were homogeneously consisted of transplanted oral mucosal epithelial cells or heterogeneously consisted of oral mucosal epithelial and corneal epithelial cells. The results of a repeated wound healing assay suggested that transplanted oral mucosal epithelial cell sheets were containing stem cells which have ability to provide differentiated epithelial cells. Furthermore, the transplanted oral mucosal epithelial cell sheets provide a circumstance for corneal epithelial cell proliferation derived from residual limbal epithelial stem cells. This study investigated an essential function of stem cell, which has an ability to provide differentiated epithelial cells for a long term by repeated wound healing assay. In this regard, we demonstrated an existence of stem cell, difference from previous study which uses putative stem cell marker expression.

 

Acknowledgments

This study was supported by the Formation of Innovation Center for Fusion of Advanced Technologies in the Special Coordination Funds for Promoting Science and Technology “Cell Sheet Tissue Engineering Center” and the Global COE program, the Multidisciplinary Education and Research Center for Regenerative Medicine, from the Ministry of Education, Culture, Sports, Science and Technology, Japan.

 

Contact

Masayuki Yamato

Institute of Advanced Biomedical Engineering and Science, Tokyo Women’s Medical University

TWIns, 8-1 Kawada-cho, Shinjuku-ku, Tokyo 162–8666, Japan.

E-mail: yamato.masayuki@twmu.ac.jp

 

 

 

 

 

References

  1. Takahashi, K., et al. Induction of pluripotent stem cells from adult human fibroblasts by defined factors. Cell 131, 861-872 (2007).
  2. Sawa, Y., et al. Tissue engineered myoblast sheets improved cardiac function sufficiently to discontinue LVAS in a patient with DCM: report of a case. Surg. Today 42, 181-184 (2012).
  3. Ohki, T., et al. Prevention of Esophageal Stricture After Endoscopic Submucosal Dissection Using Tissue-Engineered Cell Sheets. Gastroenterology (2012).
  4. Nishida, K., et al. Corneal reconstruction with tissue-engineered cell sheets composed of autologous oral mucosal epithelium. N. Engl. J. Med. 351, 1187-1196 (2004).

 

 

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