The Use of Induced Pluripotent Stem Cells as a Model for Developmental Eye Disorders

Abstract

Approximately one-third of childhood blindness is attributed to developmental eye disorders, of which 80% have a genetic cause. Eye morphogenesis is tightly regulated by a highly conserved network of transcription factors when disrupted by genetic mutations can result in severe ocular malformation. Human-induced pluripotent stem cells (hiPSCs) are an attractive tool to study early eye development as they are more physiologically relevant than animal models, can be patient-specific and their use does not elicit the ethical concerns associated with human embryonic stem cells. The generation of self-organizing hiPSC-derived optic cups is a major advancement to understanding mechanisms of ocular development and disease. Their development in vitro has been found to mirror that of the human eye and these early organoids have been used to effectively model microphthalmia caused by a VSX2 variant. hiPSC-derived optic cups, retina, and cornea organoids are powerful tools for future modeling of disease phenotypes and will enable a greater understanding of the pathophysiology of many other developmental eye disorders. These models will also provide an effective platform for identifying molecular therapeutic targets and for future clinical applications.

Keywords: VSX2; corneal hereditary endothelial dystrophy; developmental eye disorders; disease modeling; eye development; human induced pluripotent stem cells; microphthalmia; ocular maldevelopment.

Figure 1

Human-induced pluripotent stem cells (hiPSC) reprogramming. Common sources of somatic cells are reprogrammed by electroporation or lipofection. Induced overexpression of the Yamanaka factors by reprogramming methods drives hiPSC generation, visible after approximately 25 days as tightly packed colonies. Validation of hiPSCs through immunostaining and RT-PCR confirms expression patterns and levels of key pluripotency genes. Trilinear differentiation of embryoid bodies confirms the differentiation capacity of hiPSCs to all three germ layers, while karyotype analysis confirms no chromosomal abnormalities resulting from the reprogramming process.

Figure 2

In vitro optic cup differentiation with relevant genes expressed at each stage. Following the Sasai protocol, embryoid bodies are formed in the presence of ROCK inhibitor Y-27632 and cultured in a neural induction media supplemented with Wnt inhibitor IWR1e from day 0 to 12, and Wnt and SHH agonists CHIR99021 and Shh from day 15 to 18. Matrigel is added from day 2 to 18. Cells are transitioned to a retinal differentiation media supplemented with N2 and retinoic acid from day 18. According to the Mellough protocol, embryoid bodies are formed in the presence of ROCK inhibitor Y-27632 and cultured in a neural induction media supplemented with IGF-1 and B27 with decreasing knock-out serum residue (KOSR) concentrations adjusted from 20% to 15% at day 7 and from 15% to 10% at day 11. At day 18, cells are transitioned to a retinal differentiation media supplemented with retinoic acid, taurine, and triiodothyronine (T3). Following the 2D/3D differentiation technique most recently described by Capowski et al. (2019); embryoid bodies are formed from iPSCs after 2 days of culture with ROCK inhibitor Y-27632. Cells are weaned into a neural induction media containing N2 and supplemented with BMP4 from day 6 to day 16. At day 7, embryoid bodies are plated to differentiate as a 2D monolayer of cells. The eye field forms around day 10, as cells are guided towards optic cup-like structures. At day 16, cells were transitioned to retinal differentiation media supplemented with 2% B27. By ~25 days, optic cup-like structures are visible and are excised from the adherent culture for further maintenance in suspension and cultured in retinal differentiation media supplemented with FBS, taurine, retinoic acid, and a chemically defined lipid supplement.

Figure 3

Important milestones in establishing the fidelity of in vitro-generated optic vesicles to human fetal tissue (HFT) at early developmental time points. RNA-sequencing has generated transcriptomic profiles of hiPSC-derived optic vesicles and HFT. A comparison of these profiles has revealed novel genes involved in ocular development and highlighted the high molecular fidelity of in vitro optic vesicle development to human embryological development.

Figure 4

Early ocular morphogenesis. (A) Developmental pathways such as Wnt, BMP, and fibroblast growth factor (FGF) drive upregulation of eye-field transcription factors in the anterior neural plate, creating the specified region known as the “eye-field.” (B) Deepening of optic sulci and evagination of optic vesicle around 22 days post-conception. The newly formed optic vesicle ubiquitously expresses all eye-field transcription factors. (C) The action of signaling pathways determines presumptive regions in the optic vesicle characterized by unique gene expression patterns in the third and 4th weeks of gestation. (D) Interactions between the optic vesicle, surface ectoderm, and extraocular mesenchyme cause the invagination of the optic cup at approximately 5 weeks post-conception. MITF and VSX2 interactions create boundaries between retinal pigment epithelium (RPE) and neuroretina (NR) in the developing optic cups. The lens pit begins to form from the surface ectoderm. (E) In the 5th week of gestation following optic cup formation, Wnt and FGF pathways drive RPE/NR differentiation and clear definition of these regions through ciliary margin formation. The lens vesicle forms as the lens pit detach from the surface ectoderm. (F) By the 7th week of gestation, lens fibers extend to form the lens from the hollow lens vesicle. The cornea forms from the overlying surface ectoderm. NR and RPE are clearly defined and separated by the ciliary margins, while the optic nerve forms from the convergence of the optic stalk.

Figure 5

Gene regulatory networks of common eye field transcription factors associated with ocular malformation guiding cell fates during (A) eye field formation and (B) optic vesicle evagination and optic cup formation. The regulatory effect of each transcription factor on the other is illustrated in the key adjacent to each figure along with the specific developmental stage.