Efficient embryoid-based method to improve generation of optic vesicles from human induced pluripotent stem cells

Abstract

Animal models have provided many insights into ocular development and disease, but they remain suboptimal for understanding human oculogenesis. Eye development requires spatiotemporal gene expression patterns and disease phenotypes can differ significantly between humans and animal models, with patient-associated mutations causing embryonic lethality reported in some animal models. The emergence of human induced pluripotent stem cell (hiPSC) technology has provided a new resource for dissecting the complex nature of early eye morphogenesis through the generation of three-dimensional (3D) cellular models. By using patient-specific hiPSCs to generate in vitro optic vesicle-like models, we can enhance the understanding of early developmental eye disorders and provide a pre-clinical platform for disease modelling and therapeutics testing. A major challenge of in vitro optic vesicle generation is the low efficiency of differentiation in 3D cultures. To address this, we adapted a previously published protocol of retinal organoid differentiation to improve embryoid body formation using a microwell plate. Established morphology, upregulated transcript levels of known early eye-field transcription factors and protein expression of standard retinal progenitor markers confirmed the optic vesicle/presumptive optic cup identity of in vitro models between day 20 and 50 of culture. This adapted protocol is relevant to researchers seeking a physiologically relevant model of early human ocular development and disease with a view to replacing animal models.

Keywords: Embryoid bodies; PAX6; VSX2; eye development; iPSCs; optic vesicles; retinal differentiation.

Figure 1.. Schematic of retinal differentiation protocol from day 0 – day 35.

Cells are cultured in neural induction media (NIM) (20% KOSR) from day 0 to day 7, in NIM (15% KOSR) from day 7 to day 11, in NIM (10% KOSR) from day 11 to day 18 and in retinal differentiation media (RDM) from day 18 to day 35.

Figure 2.. Embryoid bodies formed in Aggrewell ™ plates.

(a-b) Uniform embryoid bodies formed in Aggrewell ™ plates after two days culture in mTeSR Plus with Y-27632 photographed at 2× and 10× magnification. (c) Brightfield images of differentiating optic vesicles from embryoid bodies at day 2 to laminated optic vesicles at day 35. At day 15, arrows indicate developing neuroepithelium. At day 20, arrows indicate initial lamination detected in optic vesicles. At day 35, arrows indicate complete laminar neuroepithelium comprised of retinal progenitor cells. Scale bar represents 150 μm. (d) Average diameter of embryoid bodies formed at day 2 (n=3 replicate rounds of 60 embryoid bodies). Error bars represent standard deviation. (e) Average diameter of optic vesicles measured at day 35. (n=3 replicate rounds of 60 embryoid bodies). Error bars represent standard deviation.

Figure 3.. Changes in gene expression of early eye development transcription factors.

PAX6, RAX, OTX2, VSX2, MITF and SOX2 transcript levels in optic vesicles at day 0, day 20 and day 35. Transcript levels were measured using RT-qPCR and presented as a log ₂ fold change in expression from undifferentiated cells at day 0. Expression levels normalised to housekeeping gene glyceraldehyde 3-phosphate dehydrogenase (GAPDH). (* p<0.05, ** p<0.01, *** p<0.001). Error bars represent the standard deviation between replicates (n=3).

Figure 4.. Representative images of immunohistochemistry sections of differentiating optic vesicles at day 20.

Expression of early eye-field transcription factors (a) OTX2, PAX6 and (b) RAX is expressed in neuroepithelium at day 20 in optic vesicles. Arrows indicate neuroepithelial layer seen in zoomed panels.

Figure 5.. Representative images of immunohistochemistry sections of differentiating optic vesicles at day 35.

Thick cellular layers of neuroepithelium are present in optic vesicles at day 35 expressing (a) early retinal progenitor marker VSX2 co-expressed with ocular development master regulator PAX6, and (b) retinal progenitor marker SOX2. Arrows indicate neuroepithelial layer seen in zoomed panels.

Figure 6.. Representative images of immunohistochemistry sections of differentiating optic vesicles at day 50.

Laminar neuroepithelium thickens at day 50, with differentiating photoreceptor precursor cells detected by (a) CRX expression strongest at the basal aspect but present in the neuroepithelium characterised by retinal progenitor cells expressing (b) VSX2. Retinal ganglion cell marker (b) BRN3B is expressed closer to the centre of the optic vesicle rather than in the neuroepithelial cell layer. (a) PAX6 is expressed in the neuroepithelium and towards its basal aspect, where (c) few OTX2+ cells co-localise with early rod marker Recoverin (RCVRN) also indicative of photoreceptor precursor cell differentiation.