Generation of retinal organoids from human induced pluripotent stem cells (hiPSCs)

Glaucoma is a neurodegenerative disease characterized by vision loss, due to retinal ganglion cell (RGC) death. The RGCs are the first retinal neurons generated during development from multipotent retinal progenitor cells (RPCs), followed by other retinal cells type, i.e. bipolar, horizontal and amacrine cells, Muller glial cells, and cone/rod photoreceptors. Since the precise mechanism underlying neurodegeneration in glaucoma is not fully understood, retinal organoids (ROs) derived from human pluripotent stem cells (h-IPSCs) could be a valuable tool to investigate the early steps involved in the glaucomatous cascade. ROs, also known as "mini-retinas," follow the in vivo steps of retinogenesis by providing an in vivo human model to study the development and behavior of RGCs, hence the harmful effects of this neurodegenerative disease. Herein, the h-IPSCs were differentiated towards ROs, according to Amélie Slembrouck-Brec et al. protocol. The retinogenesis was evaluated over time by analyzing the expression of several markers, such as NOGGIN, DKK1, LHX2, PAX6, and BRN3a. Notably, the gene expression of BRN3a, a specific RGC marker, increased from day 28 to day 56 of the differentiation protocol. In this regard, the real-time PCR proves that separation with MACS sorting for CD90/THY-1 at day 56 was successful. Although these preliminary results have led to a satisfactory result, further investigations will be necessary to evaluate, through more specific markers, how many of the CD90-positive cells are also RGCs.