Generating corneal organoids using human induced pluripotent stem cells

The transparent corneal tissue on the outer surface of the eyeball allows light entry and contributes to over 60% of the total refractive power of an eye. Any damage to its epithelial, stromal or endothelial cell layers can lead to visual impairment. The annular limbus surrounding the cornea harbors adult stem cells that regenerate different parts of the cornea. Cell replacement therapy using autologous or allogeneic, adult limbal grafts has been the standard of care in the treatment of patients with severe limbal stem cell deficiency (LSCD). However, in case of bilateral epithelial defects, and for the treatment of conditions affecting the stromal and endothelial cell layers, alternate stem cell sources such as the embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs) have been explored to generate various corneal cell types.

Dr Indumathi Mariappan's group at the Tej Kohli Cornea Institute has been working with human pluripotent stem cells such as the embryonic stem cells (ESCs) and derived few patient-specific induced pluripotent stem cell (iPSCs) lines for generating retinal and corneal tissues meant for regenerative applications. Towards this effort, 2 mm punch biopsies of the skin tissue of volunteers were taken to establish human dermal fibroblast (HDFs) cultures. The skin biopsies were collected from volunteers with their informed consent and the study was carried out with the approval of our institutional review board (IRB) and the institutional committee for stem cell research (IC-SCR). The HDFs were further reprogrammed into iPSCs by the ectopic expression of four stem cell genes namely, OCT4, SOX2, KLF4 and cMYC. The human iPSC lines were further characterized for their stemness, pluripotency and genomic integrity by various methods such as immunocytochemistry, reverse transcription PCR, fluorescence activated cell sorting, karyotyping and teratoma formation assays in nude mice models.

Here, we report the successful differentiation of human iPSCs into three dimensional minicorneal organoids of about 1-2 mm diameter (1/6 the size of an adult cornea), with complex cell layer organization. Such minicorneas at 10-15 weeks of maturation are comprised of a primitive epithelial cell layer on the surface, a thick neural crest derived stromal cell layer at the centre and possibly a monolayer of endothelium on the inner surface. We are currently validating these tissues for putative cornea-specific marker expression and their ability to regenerate damaged corneal tissues in animal models. In summary, we show that complex 3D corneal organoids could be generated in vitro using PSCs and they offers an unlimited tissue source for generating pure cultures of various adult ocular tissues such as the corneal epithelium, stromal keratocytes and corneal endothelial cells meant for regenerative applications.

Figure 1. Reprogramming human skin cells into induced pluripotent stem cells.
(i) Explant cultures of skin biopsies, (ii) Spindle-shaped human dermal fibroblast cells,
(iii-iv) Reprogrammed ES-like, Oct4+ induced pluripotent stem cell clone (in green).



Figure 2. Differentiation of human iPSCs into 3D microcorneal organoids.
(i) Growing iPSCs, (ii) Eye field primordial (EFP) clusters emerging in differentiation cultures, (iii) Whole eye ball like structures, with transparent corneal primordia (CP) on the surface and neuro retinal (NR) cup on the basal side. Pigmented neural crest (NC) cells marks the corneal boundary, (iv) A PAX6+ optic vesicle-like structure (in green), with a distinct optic stalk at the posterior end, (v-vi) Suspension cultures of EFPs developed retinal promordial (RP) and corneal promordial (CP) structures. All scale bars, 100 Ám.



Figure 3. Human iPSC derived corneal organoids expressing tissue-specific markers.
(a) H&E stained sections of a 15 weeks old minicornea displaying mature cornea-like features such as the thick stromal layer, lined by a stratified squamous epithelium on the apical surface. A limbus-like structure separates the cornea-like epithelium and the goblet cell enriched future conjunctiva. (b) Confocal images of tissue sections of the minicornea immunostained with antibodies against cornea-specific antigens (i) Pax6, (ii) P63, (iii) K12, (iv) Vimentin.




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