June 2015
Volume 56, Issue 7
Free
ARVO Annual Meeting Abstract  |   June 2015
Induction of Lacrimal Gland Epithelial Cell Phenotype from Human ES Cells by Defined Factors
Author Affiliations & Notes
  • Masatoshi Hirayama
    Ophthalmology, Keio Univ School of Medicine, Tokyo, Japan
    Department of Systems Medicine, Keio University, School of Medicine, Tokyo, Japan
  • Tetsuya Kawakita
    Ophthalmology, Keio Univ School of Medicine, Tokyo, Japan
  • Shigeto Shimmura
    Ophthalmology, Keio Univ School of Medicine, Tokyo, Japan
  • Minoru SH Ko
    Department of Systems Medicine, Keio University, School of Medicine, Tokyo, Japan
  • Kazuo Tsubota
    Ophthalmology, Keio Univ School of Medicine, Tokyo, Japan
  • Footnotes
    Commercial Relationships Masatoshi Hirayama, None; Tetsuya Kawakita, None; Shigeto Shimmura, None; Minoru Ko, None; Kazuo Tsubota, None
  • Footnotes
    Support None
Investigative Ophthalmology & Visual Science June 2015, Vol.56, 3198. doi:
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      Masatoshi Hirayama, Tetsuya Kawakita, Shigeto Shimmura, Minoru SH Ko, Kazuo Tsubota; Induction of Lacrimal Gland Epithelial Cell Phenotype from Human ES Cells by Defined Factors . Invest. Ophthalmol. Vis. Sci. 2015;56(7 ):3198.

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      © ARVO (1962-2015); The Authors (2016-present)

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Abstract

Purpose: Tear secreted from lacrimal gland plays a multifaceted role to maintain a homeostatic microenvironment for ocular surface. Dry eye is an important public health problem, and it is expected to develop a novel therapeutic treatment for the restoration of the lacrimal gland functions. We had reported a successful fully functioning lacrimal gland replacement achieved though the transplantation of bioengineered lacrimal gland germ in adult mouse (ARVO2014). To realize the lacrimal gland regeneration, it is required to establish a differentiation method of lacrimal gland cells from clinically available cell sources such as stem cells. Here, we reported a novel method to differentiate lacrimal gland epithelial cell phenotype from human embryonic stem (ES) cells.

Methods: The care and handling of animals were performed in accordance with NIH guidelines. Protocols were approved by the Animal Care and Use Committee. We performed the microarray analysis to find the specific transcriptional factors, which expressed in mouse lacrimal gland germ epithelial cells. We made artificial human messenger RNA (mRNA) of targeted transcriptional factors. By using the lipofection method, we performed a direct induction of artificial mRNA of target transcriptional factors to human ES cells, and cultured them. The morphological changes, mRNA expressions, and protein expressions including lactoferrin (LTF) were analyzed.

Results: Some candidate transcriptional factors expressed in mouse lacrimal gland germ epithelial cells were identified by microarray analysis. We successfully made artificial mRNA of candidates. Five days after culturing following the direct induction of artificial mRNA to human ES cells, we could observe characteristic morphological changes in the differentiated cells. The mRNA expression of PAX6 (essential for lacrimal gland development), FGF10, BARX2 (for branching morphogenesis), KRT15 (lacrimal gland epithelial cell marker), AQP5 (water channel) and LTF (major secretory protein of lacrimal gland) were increased. Moreover, the protein expression of PAX6, FGF10, BARX2, KRT15, AQP5 and LTF were also detected in the differentiated cells.

Conclusions: We identified essential transcriptional factors for lacrimal gland development, and demonstrated the direct induction of lacrimal gland epithelial cell phenotype from human ES cells by a novel method using artificial mRNA.

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