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. 2017 Jan 24;3:1.
doi: 10.1038/s41514-016-0001-8. eCollection 2017.

Identification of Transcription Factors That Promote the Differentiation of Human Pluripotent Stem Cells Into Lacrimal Gland Epithelium-Like Cells

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Identification of Transcription Factors That Promote the Differentiation of Human Pluripotent Stem Cells Into Lacrimal Gland Epithelium-Like Cells

Masatoshi Hirayama et al. NPJ Aging Mech Dis. .
Free PMC article

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.

Figures

Fig. 1
Fig. 1
Identification of tissue specific TFs for LGE. a Phase-contrast microscopic images of E16.5 mouse lacrimal gland (upper) and harderian gland (lower). Images of whole (left), separated epithelium (center), and separated mesenchyme (right) are shown. Scale bar 100 µm. b The gene expression comparison of mature LGE markers in the microarray analysis between E16.5 LGE and W7 LG. E16.5 embryonic day 16.5, LGE lacrimal gland epithelium, W7 LG 7-week-old mouse lacrimal gland. c The gene expression comparison of LGE markers in the microarray analysis between E16.5 LGE and W7 LG. E16.5 embryonic day 16.5, LGE lacrimal gland epithelium, W7 LG 7-week-old mouse lacrimal gland. d Hierarchical clustering analysis. The number of specific genes is shown. W7 LG 7-week-old mouse lacrimal gland, W7 HG 7-week-old mouse harderian gland, E16.5 embryonic day 16.5, LGE lacrimal gland epithelium, LGM lacrimal gland mesenchyme, HGE harderian gland epithelium, HGM harderian gland mesenchyme, LidE eyelid conjunctiva epithelium, LidM eyelid conjunctive mesenchyme. e Gene expression profiles of 16 TFs among 134 genes and PAX6 analyzed by web database tool. Foxc1, Six1, Six2, and Ctdspl express highly also in salivary glands, which are secretory glands similar to the lacrimal glands. Heat map (Green to red) represents a Z-score among organs. f Flowchart of an approach to identify premature lacrimal gland epithelium specific transcription factors
Fig. 2
Fig. 2
Establishment of a culture condition for the overexpression of transcription factors. a Immunohistological analysis of mouse E16.5 LGE in DKSFM (left), corneal epithelial medium (center), and DMEM with serum (right). Scale bar 100 µm. b Phase-contrast images of hESCs in DKSFM at day 5. Scale bar 100 µm. c Relative mRNA expression profiles in hESCs after culture in DKSFM and the control cells that also grown for the same length of time (5 days) in basal media as the experimental cells. Error bars represent mean ± standard deviation (SD) of three samples. d Immunohistochemical analysis of the cultured hESCs in DKSFM with antibodies against BARX2, KRT15, AQP5, LTF at Day 5. Scale bar 100 µm. e The analysis of expression rate of GFP and mCherry proteins by modified mRNA transfection into hESCs using FACS analysis. f PAX6, FOXC1, and SIX1 expression in cells 8 h after transfection with the synthetic modified mRNAs
Fig. 3
Fig. 3
The effect of PAX6 and FOXC1 overexpression in hESCs. a Schematic representation of time course of mRNA transfection of PAX6 and FOXC1. b Phase-contrast images of hESCs transfected with PAX6 and FOXC1 mRNAs. Scale bar 100 µm. c A phase-contrast image of PAX6 and FOXC1 transfected hESCs at Day 5 in basal medium. Scale bar 100 µm. d Relative mRNA expression levels of representative ectodermal markers (FGF5, LEFTY2), branching morphogenesis marker (BARX2), and lacrimal gland epithelial markers (KRT15, AQP5, and LTF) after transfecting PAX6 and FOXC1 mRNAs into hESCs. Quantitative PCR analyses of total RNA extracts on day 5, or the control cells also grown for the same length of time (5 days) as the experimental cells. Error bars represent mean ± SD of three samples. e Relative mRNA expression profiles of the developmental markers including lacrimal gland epithelial markers after PAX6 mRNA induction in hESCs on Day 5, or the control cells also grown for the same length of time (5 days) as the experimental cells. Error bars represent mean ± SD of three samples
Fig. 4
Fig. 4
The effect of overexpression of the combination of PAX6, FOXC1, and SIX1. a Schematic representation of mRNA transfection procedure for PAX6, FOXC1, and SIX1. b Phase-contrast images of hESCs transfected with a combination of three TFs: PAX6, FOXC1, and SIX1 at Day 2 (left) and Day 5 (center), and an enlarged image of the boxed area in the center panel (right). Scale bar 100 µm. c The expression profiles of representative markers after PAX6, FOXC1, and SIX1 induction in hESCs. Quantitative PCR analysis of total RNA extracts at Day 5, or the control cells also grown for the same length of time (5 days) as the experimental cells. Error bars represent mean ± SD of three samples. d Immunohistochemical analysis of differentiated cells with antibodies against BARX2, KRT15, AQP5, and LTF at day 5. The control cells also grown for the same length of time (5 days) as the experimental cells. Scale bar 100 µm

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