Xeno-Free Derivation of Retinal Pigmented Epithelium from Human Pluripotent Stem Cells
Age-related macular degeneration (AMD) is the leading cause of blindness in the elderly and is characterized by the death of the retinal pigmented epithelium (RPE), the cell layer located behind the retina. The RPE maintains the health of the primary cells responsible for vision, the photoreceptors. As AMD progresses, the RPE degrades, which causes the death of the photoreceptors and a debilitating loss of sight. Human embryonic stem cells (hESCs) can generate a limitless source of RPE for cellular therapies, therefore efforts to derive RPE from human embryonic stem cells (hESCs) to graft into AMD patients are under development. This thesis addresses two of the obstacles that hinder the production of these therapeutical cells. First, to manufacture cells for clinical use, it is desirable for procedures to be performed under defined conditions free of non-human reagents (xeno-free). Therefore, a novel, biomimetic, xeno-free RGD-containing copolymer designed for cell culture was investigated and found to support healthy hESC cultures and permit their differentiation into functional RPE. These stem cells and hESC-RPE demonstrate similar gene expression, protein localization and phagocytic function as cultures grown on a xenogeneic substrate. The second obstacle pertains to the intense time requirement needed to differentiate hESCs into RPE. Therefore, we recapitulated the signaling events during early embryonic development to expedite the production of hESC-RPE from 1-3 months to 14 days. However, this protocol employs full length recombinant growth factors and animal derivatives. These components represent a challenge in employing these cells as therapies due to the risk of exposure to non-human immunogens. Preliminary work to replace the xenogeneic substrate and recombinant growth factors with small molecules is presented. These pilot studies demonstrate that a xeno-free substrate and dual-Smad inhibition via small molecules can rapidly and efficiently differentiate hESCs into a population of cells expressing a pigmentation marker. These studies contribute to the development of defined, xeno-free methods for cellular manufacture which can further the translation of stem cell therapies to the clinic.