Human embryonic stem cells for retinal repair : preclinical in vitro and in vivo studies for the treatment of age-related macular degeneration with human embryonic stem cell-derived retinal pigment epithelial cells

Age-related macular degeneration (AMD) is the major cause of vision loss in the industrialized countries in people above sixty years of age. The dry advanced form of the disease, also termed as geographic atrophy (GA), is characterized by the progressive death of retinal pigment epithelial cells (RPE) and consequent loss of the adjacent photoreceptor (PR) layer, leading to an impaired visual function. Since AMD has a multifactorial cause, including both genetic and epigenetic factors, a potential treatment for retinal regeneration relies on the generation of either autologous or allogeneic RPE and PR cells from human pluripotent stem cells (hPSC) in vitro. The overall aim of this thesis was to develop both in vitro and in vivo methods and models to move forward a stem-cell based replacement therapy for patients suffering from dry advanced forms of AMD. Specifically, we first developed a spontaneous, xeno-free and defined protocol to derive RPE from human embryonic stem cells (hESC-RPE) that acquired specific morphological and functional characteristics of native RPE. Additionally, we developed a large-eyed model (rabbit eye) with relevant pre-clinical imaging and surgical advantages when compared to other more commonly used rodent models. In fact, both the subretinal injections of PBS or the chemical NaIO3 created a retinal degeneration phenotype very similar to the lesion present in GA patients with RPE damage and PR loss. A next logical step was to evaluate the behavior of the hESC-RPE in such models of degeneration. From these studies, we first showed that hESC-RPE can rescue the neuroretina from further damage induced at the moment of subretinal injection, and second, that hESC-RPE are not able to integrate in areas of profound retinal degeneration caused by a 7-day pre-injection of either PBS or NaIO3, therefore supporting the idea of an early treatment. The use of allogeneic hESC as a transplantable source comes together with the forthcoming rejection of the donor cells. We then sought to create universal cells that lack HLA-I (hESC-RPE using CRISPR-Cas9 technology) able to evade the host adaptive immune system. Upon co-culture with T-cells under stimulatory conditions, the engineered hESC-RPE dampened CD8+ T-cell proliferation and when mixed with natural killer (NK) cells, a cytotoxic response was triggered. Furthermore, after transplantation of the hESC-RPE in the rabbit xenogeneic model, early stage rejection was reduced and the appearance of anti-human antibodies rejection associated with late rejection was delayed. Altogether, the studies described in this thesis show evidence that allogeneic replacement therapy using subretinal injection of hESC-RPE in suspension can be a successful treatment if (i) the derived cells retain native RPE cell properties; (ii) the cells are transplanted early enough so the subretinal milieu supports their integration; and (iii) the cells can be engineered so that they can evade the host immune system and consequent graft rejection. SCIENTIFIC PAPERS INCLUDED IN THE THESIS I. Bartuma, H. , Petrus-Reurer, S. , Aronsson, M. , Westman, S. , André H. , and Kvanta, A. In vivo imaging of subretinal bleb-induced outer retinal degeneration. Invest. Ophthalmol. Vis. Sci. 2015; 56, 2423–2430 DOI: 10.1167/iovs.14-16208 II. Plaza Reyes, A. , Petrus-Reurer, S. , Antonsson, L. , Stenfelt, S. , Bartuma, H. , Panula, S. , Mader, T. , Douagi, I. , André, H. , Hovatta, O. , Lanner, F. , and Kvanta, A. Xeno-free and defined human embryonic stem cell-derived retinal pigment epithelial cells functionally integrate in a large-eyed preclinical model. Stem Cell Reports. 2016; 6:9–17 DOI: 0.1016/j.stemcr. 2015.11. 008 III. Petrus-Reurer, S. , Bartuma, H. , Aronsson, M. , Westman, S. , Fredrik, L. , André H. , and Kvanta, A. Integration of subretinal suspension transplants of human embryonic stem cell-derived retinal pigment epithelial cells in a large-eyed model of geographic atrophy. Invest. Ophthalmol. Vis. Sci. 2017; 58 (2), 1314-1322 DOI: 10.1167/iovs.16-20738 IV. Petrus-Reurer, S. , Winblad, N. , Gorchs, L. , Chrobok, M. , Wagner, A.K. , Lardner, E. , Bartuma, H. , Aronsson, M. , Westman, S. , Alici, E. , Kaipe, H. , Lanner, F. , Kvanta, A. Generation and characterization of human embryonic stem cell-derived retinal pigment epithelial cells lacking major histocompatibility complex-1 and transplantation into a large-eyed preclinical model. Manuscript SCIENTIFIC PAPERS NOT INCLUDED IN THE THESIS I. Petrus-Reurer, S. , Bartuma, H. , Aronsson, M. , Westman, S. , Lanner, F. , Kvanta, A. Subretinal Transplantation of Human Embryonic Stem Cell Derived Retinal Pigment Epithelial Cells into a Large-Eyed Model of Geographic Atrophy. Journal of Visualized Experiments 2018; 131 DOI: 10.3791/56702 Co-first author Co-senior author Corresponding author