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Proteomics Characterization of Primary Human Oral Epithelial Cells Using a Novel Culture Technique for Use in Tissue Regeneration.

ABSTRACT: Oral mucosa keratinocytes are widely used in regenerative medicine. The unique cultured cell population "Epithelial-derived Pop-Up Keratinocytes (ePUKs)" was previously reported as undifferentiated cells. Gravity Assisted Cell Sorting (GACS) was used to isolate a small-sized population of undifferentiated cells enriched ePUKs. LC/MS/MS analysis was performed to define the cellular profile of ePUKs of primary human oral mucosa keratinocytes. Small sized ePUKs which showed increased expression of Dickkopf WNT signaling pathway inhibitor 1 (DKK1), serpin peptidase inhibitor, clade E (nexin, plasminogen activator inhibitor type 1), member 1 (SERPINE1), follistatin and tenascin-C were verified by Western blots. These proteins are involved in the regulation of cellular movement, hair follicle development and the maintenance of its stem cell niche. The fabrication of a tissue-engineered oral mucosa, ex vivo produced oral mucosa equivalent (EVPOME), using ePUKs showed increased abundance of these verified proteins. These findings indicate that the specific phenotype of ePUKs and their ability to influence wound healing promotion are implicated by highly expressed cellular movement regulatory proteins. Therefore, ePUKs may be a useful cell source for use in regenerative medicine.

SUBMITTER: Kato H

PROVIDER: S-EPMC4813815 | biostudies-literature | 2015

REPOSITORIES: biostudies-literature

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Proteomics Characterization of Primary Human Oral Epithelial Cells Using a Novel Culture Technique for Use in Tissue Regeneration.

Kato Hiroko H Lo Andy A Kuo Shiuhyang S Nie Song S Marcelo Cynthia L CL Lubman David M DM Feinberg Stephen E SE

MOJ proteomics & bioinformatics 20150812 4

Oral mucosa keratinocytes are widely used in regenerative medicine. The unique cultured cell population "Epithelial-derived Pop-Up Keratinocytes (ePUKs)" was previously reported as undifferentiated cells. Gravity Assisted Cell Sorting (GACS) was used to isolate a small-sized population of undifferentiated cells enriched ePUKs. LC/MS/MS analysis was performed to define the cellular profile of ePUKs of primary human oral mucosa keratinocytes. Small sized ePUKs which showed increased expression of ...[more]

PMID: 27042699

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Project description:Introduction:Carrier-free autologous mucosal epithelial cell sheets have been clinically utilized as a cell therapy for various epithelial disorders. Fabrication of a transplantable oral mucosal epithelial cell sheet without mouse feeder layers requires a higher seeding density than that of a sheet with mouse feeder layer culture; therefore, a large amount of donor mucosal tissue is needed. However, cell grafts co-cultured with mouse feeder layers are classified by the US Food and Drug Administration (FDA) as xenogeneic products. The goal of this study was to evaluate the utility of oral mucosal epithelial cells expanded by primary explant culture for the fabrication of an adequate number of transplantable epithelial cell sheets without mouse feeder layers. Methods:Small fragments derived from minced oral mucosal tissue were placed into culture dishes for primary explant culture in keratinocyte culture medium. After primary explant culture, the outgrown cells were treated with trypsin-EDTA and were seeded on a temperature-responsive cell culture insert. After subculture, the cultured cells were harvested as a confluent cell sheet from the culture vessel by temperature reduction. Results:Carrier-free human oral mucosal epithelial cell sheets were fabricated in all human cases, and autologous transplantation of the harvested cell sheets showed rapid epithelial regeneration to cover epithelial defects in a rabbit model. The explant culture method, involving the use of small fragments for primary culture, was sufficient for preparing a large number of mucosal epithelial cells without mouse feeder layers. Moreover, oral mucosal epithelial cells derived from the primary explant culture after cryopreservation allowed for the fabrication of cell sheets. Conclusions:This method for fabricating transplantable oral mucosal epithelial cell sheets is an attractive technique for regenerative medicine. It offers a patient-friendly manufacturing method in which a small amount of biopsy material from the patient represents a sufficient epithelial cell source, and a manufacturing plan for preparing cell grafts can be easily tailored.

Dataset Information

Proteomics Characterization of Primary Human Oral Epithelial Cells Using a Novel Culture Technique for Use in Tissue Regeneration.

Publications

Proteomics Characterization of Primary Human Oral Epithelial Cells Using a Novel Culture Technique for Use in Tissue Regeneration.

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