Corneal stromal keratocyte modeling from human cornea organoids

About This Grant

Scarring of the stroma, the central layer of the cornea, due to Injury or infection, and severe stromal thinning due to keratoconus, cause corneal blindness worldwide. Cornea transplantation is often the only cure for many of these conditions, but limited supply of donor tissue is a major concern. Cell therapy for the stroma, still under development, primarily uses limbal stem cells from donor corneas. Therefore, there is a critical need for treatment of the stroma that will be independent of donor tissues. To address this need, we recently developed human cornea organoids (HCOs) that mimic the fetal cornea. Generated from induced pluripotent stem cells (iPSC) the HCOs present a breakthrough model system of co-differentiating stromal keratocyte, epithelial and endothelial cells in a more cornea-like 3-dimensional extracellular matrix (ECM). Our single cell RNA sequence (scRNA-seq) study shows that HCOs harbor stromal progenitor and fetal corneal keratocyte-like cells which may have the desired long-lasting functionality in the cornea. In addition, we found that HCOs express ECM genes and produce a stroma rich in collagen type III, fibronectin, and other ECM components known to make up the immature corneal stroma. Additionally, as the TGF-𝛽 network is the master regulator of healthy and fibrotic ECM, this network must be specifically regulated in HCOs to maintain its regenerative ECM quality. Therefore, we hypothesize that the HCO is a novel stromal therapy resource and a corneal surrogate for identifying TGF-𝜷 signals that promote healthy but limit fibrotic ECM production. Our preliminary data shows that a mix of stem, progenitor and differentiating cells can be rapidly extracted from the HCO stroma without further monolayer culturing. Injected into decellularized donor corneas the HCO-stromal cells can integrate in the tissue. Second, the extracted ECM from HCOs promote migration in cell culture and corneal wound healing in the mouse. Importantly, as the HCO expresses all major receptors and transcription factors, it is feasible to investigate the TGF-𝛽 network in this model. Aim 1 will determine if extracted HCO-stromal cells will integrate and function in a decellularized donor cornea. Aim 2 will test whether the HCO-stromal cells or the extracted HCO-ECM promote scarless wound healing in the mouse. Aim 3 will identify specific TGF-𝛽 signals that underpin the immature ECM of HCOs. Our findings will develop the HCO as a promising donor tissue-free source of regenerative biomaterial and cells and identify regenerative TGF-𝛽 signals for future treatments of corneal scarring.