Defining conditions to direct human pluripotent stem cells towards the glaucoma-affected trabecular meshwork cell lineage

Current therapies seek to reduce intraocular pressure (IOP), which is the most important modifiable risk factor for glaucoma. IOP elevation can occur due to decreased outflow of aqueous humour, which mainly exits the eye via a specialised tissue known as the trabecular meshwork (TM). In mammalian embryos, the TM arises from neural crest (NC) derived mesenchymal cells. Although TM cell dysfunction has a key role in the development of many forms of glaucoma, the precise molecular mechanisms involved are unknown. A principal reason for this poor understanding is the distinct lack of appropriate cellular models. Accordingly, we aim to define the conditions that direct differentiation of pluripotent stem cells to TM fate. Initial steps focus on differentiation of H9 human embryonic stem cells to NO-derived mesenchymal fate, using small-molecule SMAD inhibitors and timed Wnt activation to induce NC, followed by exposure to foetal bovine serum. Whilst subsequent regulators of TM differentiation are unknown, we have identified several molecular clues to TM differentiation. Mutations in the transcription factors FOXC1, PITX2 and PAX6 result in abnormal human TM development (anterior segment dysgenesis), and these transcription factors are regulated by BMP4 and TGF-13 in murine models. Thus, the latter steps of this protocol focus on timed exposure of mesenchymal stem cells to BMP4 and TGF-13, and other factors known to influence TM maturation, including retinoic acid and aqueous humour. Here, we present data describing optimisation of this protocol. Following optimisation, differentiation of patient-derived induced pluripotent stem (iPS) cells will enable the development of a clinically relevant model of glaucoma. Patient-specific iPS-derived TM cells will reveal molecular changes that may explain disease onset or progression, and serve as a platform for therapeutic drug j3Creening .