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Seminario INCyL – Iván Aldavero Muñoz

Visual dystrophies leading to vision impairment are becoming a global problem as life spans increase and the global population rises exponentially. Age-related macular degeneration (AMD) is the most common cause of blindness in people over 50 years old in industrialized countries. There are currently no effective, targeted, and efficient treatment options for this neurodegenerative disease. We need accessible and reproducible in vitro human models to continue making progress in understanding these retinal diseases and in identifying potential therapeutic targets for intervention. Human induced pluripotent stem cells (iPS) offer new opportunities for modeling retinal diseases, especially thanks to their ability to differentiate to retinal pigment epithelium (RPE), which plays a critical role in maintaining visual function and retinal homeostasis1.
We aimed, first, to develop a protocol for generating pure RPE sheets from iPS with high efficiency and reproducibility able to recapitulate in vitro innate retinal functions2. We also explored developing a human post-mortem retinal tissue model which retains similar characteristics3. And secondly, we aimed to study the effects of the deletion of CRB2, a protein involved in the physiological maturation and polarization of the RPE, which are essential to establish a functional RPE cell layer4; and the deletion of VEGFA, a protein whose polarized secretion is vital for the proper functioning of the vessels that innervate the RPE and is strongly involved in the neovascular subtype of AMD5.
For this, PGP1 line, an iPS commercial line sourced from primary human fibroblasts from a control male donor with a well-defined genetic and clinical background was employed. Then, RPE cells were generated and differentiated from a CRB2 CRISPR-deleted pool of iPS cells, another cell pool with VEGFA deleted, and from control cells, electroporated with RNP complexes without sgRNA guide. This directed differentiation protocol via manipulation of the insulin growth factors (IGF), basic fibroblast growth factor (FGF-2; FGF-basic), transforming growth factor beta (TGF-β), and WNT pathways leads to increased RPE fate differentiation efficiency and enables RPE sheet generation at high purity without manual selection. This method6 produced a homogeneous, pigmented, polarized7, phagocytic, post-mitotic, and characteristically polygonal monolayer of RPE cells. We expect that this differentiation protocol could be useful for the efficient production of RPE sheets for regenerative cell therapies, as well as serve as a model for the testing of potential treatments for the AMD and other retinal diseases in which the RPE is affected. However, more data is needed to determine how key proteins like CRB2 or VEGFA impact both healthy and pathological RPE.

Publicaciones Relacionadas:

  1. Bharti, K., den Hollander, A. I., Lakkaraju, A., Sinha, D., Williams, D. S., Finnemann, S. C., … & D’Amore, P. A. (2022). Cell culture models to study retinal pigment epithelium-related pathogenesis in age-related macular degeneration. Experimental eye research222, 109170.
  2. Sharma, R., Bose, D., Montford, J., Ortolan, D., & Bharti, K. (2022). Triphasic developmentally guided protocol to generate retinal pigment epithelium from induced pluripotent stem cells. STAR protocols3(3), 101582.
  3. Sonoda, S., Spee, C., Barron, E., Ryan, S. J., Kannan, R., & Hinton, D. R. (2009). A protocol for the culture and differentiation of highly polarized human retinal pigment epithelial cells. Nature protocols4(5), 662-673.
  4. Paniagua, A. E., Segurado, A., Dolón, J. F., Esteve-Rudd, J., Velasco, A., Williams, D. S., & Lillo, C. (2021). Key role for CRB2 in the maintenance of apicobasal polarity in retinal pigment epithelial cells. Frontiers in Cell and Developmental Biology9, 701853.
  5. Lin, F. L., Wang, P. Y., Chuang, Y. F., Wang, J. H., Wong, V. H., Bui, B. V., & Liu, G. S. (2020). Gene therapy intervention in neovascular eye disease: a recent update. Molecular Therapy28(10), 2120-2138.
  6. Foltz, L. P., & Clegg, D. O. (2017). Rapid, directed differentiation of retinal pigment epithelial cells from human embryonic or induced pluripotent stem cells. JoVE (Journal of Visualized Experiments), (128), e56274.
  7. Segurado, A., Rodríguez-Carrillo, A., Castellanos, B., Hernández-Galilea, E., Velasco, A., & Lillo, C. (2022). Scribble basal polarity acquisition in RPE cells and its mislocalization in a pathological AMD-like model. Frontiers in Neuroanatomy16, 983151.