Nanoscale Structural Characterisations of Ocular Tissues Derived from Human iPS Cells

The cells that comprise our body have specific functions and are adapted to suit the particular tissue in which they exist. Mature cells are generally known as differentiated cells because they have become tailored to their biological role. For a long time, it was accepted that once a cell had "chosen its path" and differentiated into a particular type of cell, it had embarked on an irreversible journey. However, in 2012 Professors Sir John Gurdon and Shinya Yamanaka, of Cambridge and Kyoto Universities, respectively, were awarded the Nobel Prize for their research, which showed that differentiated adult cells could be genetically reprogrammed to become less differentiated and capable of forming many different cell types. Such cells are called induced pluripotent stem cells, commonly abbreviated to iPS cells. The new research we propose originates with the discovery, made with our collaborators in Japan, that human iPS cells (hiPSCs) can be cultivated in the laboratory to grow in a manner that mimics the way cells in the human eye develop before birth.

Component tissues of the eye have interrelated developmental pathways, with the corneal and conjunctival epithelia that cover the eye's surface, the lens within the eye and the tear-producing lacrimal gland all evolving from the same cell type. Thin flat sheets of corneal and conjunctival epithelia have been generated from hiPSCs, and some of the corneal sheets have been used to restore sight in patients with vision loss. New research is now starting to show how hiPSCs can be used to form 3D organoids (miniaturised versions of an organ or selected aspects of it) of lacrimal gland and lens. These are key ocular tissues, which, respectively, help synthesise the tear film and focus light onto the retina. How accurately these organoids mimic the natural tissue, however, is yet to be fully appreciated. We now plan a series of experiments using high-specification electron microscopes and high-intensity x-ray beams (including use of the world's most powerful experimental x-ray source, the SPring8 synchrotron in Japan) to obtain a comprehensive understanding of 3D organoids of lacrimal glands and lenses generated from hiPSCs.

Vision loss has a devastating impact on an individual's life. The societal cost, too, is severe, with researchers at The London School of Economics estimating that the economic cost to the UK of sight loss stands at £25.2 billion each year, predicted to rise to £33.5 billion by 2050. Healthy lacrimal glands and lenses are essential for correct vision. Dysfunctional lacrimal glands cause severe dry eye syndrome, which affects tens of millions of people worldwide and can result in corneal ulceration and blindness if left untreated. Cataracts (cloudy lenses), moreover, are the single major reason for sight loss, especially in the elderly. Our research has the potential to have real future impact because well-characterised 3D lacrimal gland-like and lens-like organoids formed from cells of a human origin represent an excellent resource, as an alternative to experiments on animals, for scientists to devise and test new medications to treat/prevent sight-threatening diseases of the lacrimal gland and lens. In addition, the hiPSC-derived organoids, when more fully understood and further investigated, have the potential to be used in transplant surgery. In the near/mid-term future, research into organoid transplantation will likely be directed towards the treatment of lacrimal gland, rather than lens, pathology because of the availability of implantable synthetic lenses. The immediate value of properly characterised hiPSC-derived lens-like organoids is predicted to lie in their use to investigate medications to inhibit or reverse lens protein aggregation and the development of cataract in old age.

Human induced pluripotent stem cells (hiPSCs) can be cultivated to form small circular 2D colonies of cells that develop in a way that mimics whole eye development (Hayashi et al. Nature 2016;531:376-80). New advances are now starting to discover that cells can be "picked" from specific regions of these cell colonies and cultivated to form 3D organoids of the eye lens or tear-producing lacrimal gland (Hayashi et al. Nature 2022;in press). We propose to work with Professor Hayashi and his team to interrogate these constructs using 3D electron-optical imaging, correlative immunoelectron microscopy and synchrotron x-ray analyses. hiPSC-derived lacrimal gland-like and lens-like tissue organoids will be analysed by volume electron microscopy to provide an in-depth characterisation of their 3D microanatomy over large tissue volumes. Correlative immunoelectron microscopy will be employed to ascertain the distribution in 3D of key constituents of the organoids (aquaporins, connexins, crystallins, alpha-smooth muscle actin and tear film proteins) as they grow in 3D culture. We will also use purified, differentially sulphated chondroitin sulphate macromolecules to test the enhanced in vitro functional maturation of hiPSC-derived lacrimal gland-like organoids. Synchrotron x-ray science will also be used to probe the physical properties of hiPSC-derived lens-like organoids: specifically, synchrotron x-ray scattering will quantify the crystallin protein organisation as a key determinant of lens tissue transparency, with synchrotron x-ray interferometry used to determine the spatially-resolved optical status of lens-like organoids in 3D. Parallel experiments on foetal and adult human lenses and lacrimal glands supplied by authorised agencies with full ethical permission will provide information about native tissue parameters.