Identification of transcription factors that promote the differentiation of human pluripotent stem cells into lacrimal gland epithelium-like cells

Abstract

Dry eye disease is the most prevalent pathological condition in aging eyes. One potential therapeutic strategy is the transplantation of lacrimal glands, generated in vitro from pluripotent stem cells such as human embryonic stem cells, into patients. One of the preceding requirements is a method to differentiate human embryonic stem cells into lacrimal gland epithelium cells. As the first step for this approach, this study aims to identify a set of transcription factors whose overexpression can promote the differentiation of human embryonic stem cells into lacrimal gland epithelium-like cells. We performed microarray analyses of lacrimal glands and lacrimal glands-related organs obtained from mouse embryos and adults, and identified transcription factors enriched in lacrimal gland epithelium cells. We then transfected synthetic messenger RNAs encoding human orthologues of these transcription factors into human embryonic stem cells and examined whether the human embryonic stem cells differentiate into lacrimal gland epithelium-like cells by assessing cell morphology and marker gene expression. The microarray analysis of lacrimal glands tissues identified 16 transcription factors that were enriched in lacrimal gland epithelium cells. We focused on three of the transcription factors, because they are expressed in other glands such as salivary glands and are also known to be involved in the development of lacrimal glands. We tested the overexpression of various combinations of the three transcription factors and PAX6, which is an indispensable gene for lacrimal glands development, in human embryonic stem cells. Combining PAX6, SIX1, and FOXC1 caused significant changes in morphology, i.e., elongated cell shape and increased expression (both RNAs and proteins) of epithelial markers such as cytokeratin15, branching morphogenesis markers such as BARX2, and lacrimal glands markers such as aquaporin5 and lactoferrin. We identified a set of transcription factors enriched in lacrimal gland epithelium cells and demonstrated that the simultaneous overexpression of these transcription factors can differentiate human embryonic stem cells into lacrimal gland epithelium-like cells. This study suggests the possibility of lacrimal glands regeneration from human pluripotent stem cells.

A Ligation of the Lacrimal Excretory Duct in Mouse Induces Lacrimal Gland Inflammation with Proliferative Cells

“…Animal models for efficient tissue damage have been reported for investigation on mechanism of inflammation, regenerative potential of organs, and isolation of stem/progenitor cells from the target organs [ 8 , 25 – 29 ]. Previous studies have shown that a partial radiation to salivary glands in mice leads to proliferation of cells, which have an ability to form salispheres for cell injection therapy to restore the salivary gland functions [ 30 ].…”

Regeneration of Lacrimal Gland Function to Maintain the Health of the Ocular Surface

“…40 Overexpression of tissue-specific transcription factors in human pluripotent stem cells can successfully induce the LG epithelial cell phenotype. 41 Using such induced epithelial and mesenchymal cells, small functional LGs may be generated via epithelial-mesenchymal interactions ex vivo. These generated LGs might be similar to an accessory LG rather than the main LG, but when transplanted, may work as functional LGs.…”

Advances in Functional Restoration of the Lacrimal Glands

“…94,95 Recently, a whole gene expression analysis based on microarray technology identified a subset of transcription factors (PAX6, FOXC1, SIX1, and SIX2) that are specifically and highly expressed in the ED16.5 mouse lacrimal gland epithelium compared with developmentally related organs such as the mature lacrimal glands, the mature/embryonic harderian glands and the embryonic ocular surface epithelium. 96 These transcription factors have been implicated as part of a set of core transcription factors that define the lacrimal gland epithelial cell lineage. Specifically, the overexpression of PAX6, FOXC1, and SIX1 using transcription factor-coded synthetic mRNAs in human ESCs resulted in the differentiation of cells toward the lacrimal gland epithelial cell like phenotype accompanied by expression of a part of lacrimal gland development markers, including the immature lacrimal gland epithelial marker KRT15, the branching morphogenesis marker BARX2, and the tear secretion related markers AQP5 and lactoferrin.…”