Development of lacrimal gland organoids from iPSC derived multizonal ocular cells

Abstract

Lacrimal gland plays a vital role in maintaining the health and function of the ocular surface. Dysfunction of the gland leads to disruption of ocular surface homeostasis and can lead to severe outcomes. Approaches evolving through regenerative medicine have recently gained importance to restore the function of the gland. Using human induced pluripotent stem cells (iPSCs), we generated functional in vitro lacrimal gland organoids by adopting the multi zonal ocular differentiation approach. We differentiated human iPSCs and confirmed commitment to neuro ectodermal lineage. Then we identified emergence of mesenchymal and epithelial lacrimal gland progenitor cells by the third week of differentiation. Differentiated progenitors underwent branching morphogenesis in the following weeks, typical of lacrimal gland development. We were able to confirm the presence of lacrimal gland specific acinar, ductal, and myoepithelial cells and structures during weeks 4–7. Further on, we demonstrated the role of miR-205 in regulation of the lacrimal gland organoid development by monitoring miR-205 and FGF10 mRNA levels throughout the differentiation process. In addition, we assessed the functionality of the organoids using the β-Hexosaminidase assay, confirming the secretory function of lacrimal organoids. Finally, metabolomics analysis revealed a shift from amino acid metabolism to lipid metabolism in differentiated organoids. These functional, tear proteins secreting human lacrimal gland organoids harbor a great potential for the improvement of existing treatment options of lacrimal gland dysfunction and can serve as a platform to study human lacrimal gland development and morphogenesis.

Generation of Anterior Segment of the Eye Cells from hiPSCs in Microfluidic Platforms

“…Recently, we reported on the differentiation of hiPSCs into multi‐zonal ocular cells (MZOCs), which led to the generation of lacrimal gland organoids in ≈ 35 days. [ 14 ] Following a similar protocol, we first differentiated hiPSCs into anterior neuroectodermal cells within 48 h. Subsequently, an eye‐field differentiation (ED) medium was used to induce growth into eye‐field stem cells. Over the next five days, the differentiation medium was changed to an ocular differentiation (OD) medium for two weeks to induce multi‐zonal ocular structure cells ( Figure 1 A).…”

“…According to the literature, these multi-zonal structures can be further differentiated into specialized cell types, such as limbal stem cells, cornea epithelial cells, periocular mesenchymal cells, and lacrimal glands. [10,11,14,16,27] Cells were characterized by immunostaining, and it was observed that multi-zonal cells expressed ocular progenitor markers such as PAX6 and P63 at day 21 (Figure 1B). Expression of P63 and PAX6 in the culture indicated the generation of a primitive epithelial surface like that found in human limbal cells.…”

“…Several in vitro models have been generated to mimic the corneal, conjunctival, limbal progenitor cells, ocular lens, and lacrimal gland using hiPSCs have recently been reported. [9][10][11][12][13][14] In a recent study, self-formed multi-zonal eye-like organoids were generated using hiPSCs, recapitulating whole-eye development. [10,15,16] They identified different parts of the eye including ocular surface ectoderm, neuro-retina, retinal pigment epithelium, lens, and more recently, adult corneal epithelial stem cells and lacrimal gland.…”

OrganoLabeling: Quick and accurate annotation tool for organoid images

“…Organoids provide in vitro venues to investigate molecular and mechanistic studies on target organ including realistic models for diseases and drug testing. Advances in stem cell research, bioengineering and organoid technologies lead the generation of diverse portfolio of organoids including brain, intestine, liver, kidney, stomach, lung, thyroid and lacrimal gland [1][2][3][4][5][6][7][8][9][10][11]. Brain organoids (BOs) resemble embryonic development of the cerebellum and can be employed in understanding neurodegenerative diseases and brain research [12].…”