Abstract
Purpose:
Current research efforts suggest an effective treatment for retinal degeneration stemming from loss of RPE or RPE function can be achieved by replacement therapy - where iPSC derived RPE provide a valuable replacement source. To determine whether the starting tissues retain sufficient “epigenetic memory” to influence the physiology of iPSC derived RPE cells, we compared the structural and physiological properties of RPE cells generated from genetically identical but epigenetically distinct tissue sources.
Methods:
Human fetal or adult tissues from different origins were reprogrammed to iPS cells using the four Yamanka factors. Confluent monolayers of iPSC-RPE cells were grown on semi-permeable transwells and used for morphological and physiological assays: (1) Electron micrographs of RPE ultrastructure; (2) Intracellular microelectrode recording to measure cell resting membrane potential and responses to extracellular low K+ and ATP; (3) [Ca2+]i imaging to monitor intracellular free Ca2+ activity; (4) Fluid transport measurements to analyze the steady-state trans-epithelial fluid absorption; (5) Analysis of photoreceptor outer segment phagocytosis; (6) Signature gene expression analysis; (7) Immunostaining of pluripotency markers and RPE-specific markers
Results:
Different tissue sources from the same donor result in analogous differentiation efficiency and RPE functionality. Adult derived RPE have reduced gene expression of pluripotent factors compared to fetal sources. Functional assays are critical for evaluation of RPE performance. Extended post confluent time in culture following differentiation leads to a gradual reduction in RPE functional performance.
Conclusions:
The present experiments suggest that the genetic background of the donor rather than the tissue of origin may play a role in defining the differentiation propensity of the derived RPE. Adult tissue sources may be the preferred starting material for the production of clinical grade iPS derived RPE cells because of the repression of pluripotent factors following differentiation. The time in culture defines a window for clinical readiness. Lastly, the use of RPE-specific markers for immunostaining is not predictive of functional performance and is insufficient to identify suitable RPE for transplantation.
Keywords: 721 stem cells •
701 retinal pigment epithelium