Abstract
Induced pluripotent stem (iPS) cells can be established from somatic cells. However, there is currently no established strategy to generate corneal epithelial cells from iPS cells. In this study, we investigated whether corneal epithelial cells could be differentiated from iPS cells. We tested 2 distinct sources: human adult dermal fibroblast (HDF)-derived iPS cells (253G1) and human adult corneal limbal epithelial cells (HLEC)-derived iPS cells (L1B41). We first established iPS cells from HLEC by introducing the Yamanaka 4 factors. Corneal epithelial cells were successfully induced from the iPS cells by the stromal cell-derived inducing activity (SDIA) differentiation method, as Pax6+/K12+ corneal epithelial colonies were observed after prolonged differentiation culture (12 weeks or later) in both the L1B41 and 253G1 iPS cells following retinal pigment epithelial and lens cell induction. Interestingly, the corneal epithelial differentiation efficiency was higher in L1B41 than in 253G1. DNA methylation analysis revealed that a small proportion of differentially methylated regions still existed between L1B41 and 253G1 iPS cells even though no significant difference in methylation status was detected in the specific corneal epithelium-related genes such as K12, K3, and Pax6. The present study is the first to demonstrate a strategy for corneal epithelial cell differentiation from human iPS cells, and further suggests that the epigenomic status is associated with the propensity of iPS cells to differentiate into corneal epithelial cells.
Highlights
Like embryonic stem cells, induced pluripotent stem cells are capable of differentiating into all of the various cell lineages of an organism, and are established from somatic cells by introducing transcription factors such as Oct3/4, Sox2, and Klf4 [1,2,3]
Transfection of Yamanaka 4 factors can reprogram human adult corneal limbal epithelial cells (HLEC) Since HLECs share the same cell lineage as corneal epithelial cells, we tested whether induced pluripotent stem (iPS) cells derived from them would have a propensity to develop into corneal epithelial cells
Functional pluripotency was demonstrated in these HLEC-derived iPS cells, as the cultured L1B41 cells exhibited teratoma formation containing derivatives from all 3 germ layers, such as neural, gut, retinal pigment epithelium (RPE), and cartilage tissues, 4–8 weeks after injection into mouse testis (Fig. 1E)
Summary
Like embryonic stem cells, induced pluripotent stem (iPS) cells are capable of differentiating into all of the various cell lineages of an organism, and are established from somatic cells by introducing transcription factors such as Oct3/4, Sox, and Klf4 [1,2,3]. The corneal epithelium originates from surface ectoderm during development, similar to the epidermis or lens epithelium [4]. Their stem/progenitor cells are believed to localize in the basal epithelium of the limbus located between the cornea and conjunctiva [5,6]. If corneal epithelial stem cells are completely absent due to limbal stem cell deficiencies, the peripheral conjunctival epithelium invades inwardly and the corneal surface is enveloped by vascularized conjunctival scar tissue, resulting in corneal opacification and blindness from this severe eye disease [7,8]. Transplant therapy has been performed in patients with corneal epithelial stem cell deficiencies, most failed due to immune rejection [9]. Regenerative medicine using differentiated autologous iPS cells has been proposed as a promising alternative; no differentiation strategy has been determined far
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