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
Author: Petrus-Reurer, Sandra
Date: 2018-11-23
Location: Erna Möller Hall, Blickagången 16, Karolinska Institutet, Flemingsberg.
Time: 10:00
Department: Inst för klinisk neurovetenskap / Dept of Clinical Neuroscience
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Thesis (9.280Mb)
Abstract
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-RPEB2M-/- using CRISPR-Cas9 technology) able to evade the host adaptive immune system. Upon co-culture with T-cells under stimulatory conditions, the engineered hESC-RPEB2M-/- dampened CD8+ T-cell proliferation and when mixed with natural killer (NK) cells, a cytotoxic response was triggered. Furthermore, after transplantation of the hESC-RPEB2M-/- 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.
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-RPEB2M-/- using CRISPR-Cas9 technology) able to evade the host adaptive immune system. Upon co-culture with T-cells under stimulatory conditions, the engineered hESC-RPEB2M-/- dampened CD8+ T-cell proliferation and when mixed with natural killer (NK) cells, a cytotoxic response was triggered. Furthermore, after transplantation of the hESC-RPEB2M-/- 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.
List of papers:
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. Investigative Ophthalmology and Visual Science. 2015; 56, 2423–2430.
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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.
Fulltext (DOI)
Pubmed
View record in Web of Science®
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. Investigative Ophthalmology and Visual Science. 2017; 58 (2), 1314-1322.
Fulltext (DOI)
Pubmed
View record in Web of Science®
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; and 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]
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. Investigative Ophthalmology and Visual Science. 2015; 56, 2423–2430.
Fulltext (DOI)
Pubmed
View record in Web of Science®
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.
Fulltext (DOI)
Pubmed
View record in Web of Science®
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. Investigative Ophthalmology and Visual Science. 2017; 58 (2), 1314-1322.
Fulltext (DOI)
Pubmed
View record in Web of Science®
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; and 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]
Institution: Karolinska Institutet
Supervisor: Kvanta, Anders
Co-supervisor: Lanner, Fredrik; André, Helder; Stenfelt, Sonya
Issue date: 2018-10-25
Rights:
Publication year: 2018
ISBN: 978-91-7831-156-9
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