Proper organogenesis relies on the orchestrated spatial and temporal presentation of graded stimuli for cell fate commitment and maturation. This represents a major obstacle to deriving complex tissue structures, like the fully developed retina, in the laboratory. Typically, in-vitro culture systems deliver uniform stimuli to induce homogenous tissue differentiation at established time points; but spatial distribution of differentiation cues relies mainly on interactions occurring within the culture environment, which are not solely adequate for proper differentiation of all retinal layers. To overcome this obstacle, we propose a novel tissue bioreactor system that allows for the compartmentalization and gradation of stimuli designed to separately induce the maturation of inner and outer retinal cell phenotypes. Our bioreactor includes an air-tight upper chamber which allows the establishment of physiologically-relevant oxygen tension gradients across the developing retina. Thus, we can recapitulate the normal physiology in which the retina is exposed to a steep gradient of oxygen tensions across the highly oxygenated outer retina, and the hypoxic inner retina. We will use this solution to model Retinoblastoma (Rb), which is one of the leading causes of childhood cancer death worldwide. Rb is not an age-related degenerative disorder, but rather a developmental disease, making it ideal for studies in a platform of retinal development. Using CRISPR/Cas9 technology, we have established RB1-knockout iPSC lines in our laboratory (RB1KO). The availability of wildtype and RB1KO cells from the same parental cell lines, lets us model somatic and germline manifestations of the disease, and study how retinoblast cells interact with normally-developing retinal tissues. This approach allows us to characterize the phenotype(s) from which Rb tumors originate, which is still a matter of debate. Similarly, using our custom bioreactor system, we can study how retinoblastoma tumors transition from oxygen-dependent growth to their most aggressive phenotype, hypoxic-adapted vitreal seeds.
Solution for: NEI 3-D Retina Organoid Challenge (3-D ROC)
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