PhD student, Karolinska Institutet
Development of a stem cell-derived therapy for age-related macular degeneration
Age-related macular degeneration (AMD) is one of the most important causes of blindness in the Western countries. The disease of unknown etiology causes the loss of the retinal pigment epithelial cell (RPE) layer, which leads to subsequent degeneration of essential retinal structures (e.g. photoreceptors) causing severe vision impairment. Geographic atrophy (GA) or the advanced dry form of the disease is responsible for 20% of the visual loss in patients with AMD and has no treatment available. Therefore, subretinal transplantation of RPE cells derived from human embryonic stem cells (hESC) emerges as a potential replacement therapy in GA, this being the focus of my research. Specifically, we aim to:
i) Derive RPE cells from hESC in the most pure and efficient manner: We have developed a protocol to efficiently differentiate RPEs from hESC in a xeno-free and defined manner, which should avoid any potential risk of rejection due to non-human proteins upon transplantation (Plaza Reyes, Petrus-Reurer et al, 2016, Stem Cell Reports). We are currently improving this process to make it less labor consuming and easier to be scaled up.
ii) Assess the behavior and integration/functionality of the derived cells in a relevant preclinical model of disease: With such model (e.g. rabbit eye) that entails surgical and imaging methods close to those applied in humans, we demonstrated that we are able to recapitulate the features of GA in a large-eyed model of disease. We also showed the importance of having a conserved retinal milieu for the donor cells to integrate, reinforcing the idea of an early stage treatment of GA patients (Petrus-Reurer, Bartuma et al, 2017, IOVS).
iii) Characterize and modulate the immunologic properties of the derived hESC-RPE to create a minimally immunogenic cell source: We are currently developing a CRISPR-Cas9 engineered cell line that lacks some of the immunologic molecules responsible for transplant rejection (e.g. HLA-I, HLA-II or both). We aim to further characterize their immunological response both in vitro and in vivo.
Finally, we hope that altogether these advances in regenerative medicine can serve as a safe, efficient and minimally invasive replacement therapy to treat patients suffering from age-related macular degeneration, as well as a platform for other similar stem-cell based therapies intending to reach the clinics.
Abstract: To analyze the morphologic effects of subretinal blebs in rabbits using real-time imaging by spectral-domain optical coherence tomography (SD-OCT), infrared-confocal scanning laser ophthalmoscopy (IR-cSLO), and blue-light fundus autofluorescence (BAF).Subretinal blebs of PBS or balanced salt solution (BSS) were induced in albino or pigmented rabbits using a transvitreal pars plana technique. Spectral-domain optical coherence tomography, IR-cSLO, and BAF were done at multiple intervals for up to 12 weeks after subretinal bleb injection. The morphologic effects were compared with histologic analysis on hematoxylin-eosin-stained sections of the neurosensory retina and on flat-mounts of phalloidin-labeled RPE.Scans of SD-OCT of the normal rabbit posterior segment revealed 11 bands including six layers of the photoreceptors. Subretinal blebs of PBS or BSS caused acute swelling of the neurosensory retina followed by gradual atrophy. Outer retinal thickness was significantly reduced with pronounced degeneration of all the photoreceptor OCT layers. En face IR-cSLO showed a hyperreflective area corresponding to the progressive photoreceptor degeneration, whereas BAF revealed both hyper- and hypofluorescent changes in the RPE layer. The in vivo results were confirmed by histology and on subretinal flatmounts demonstrating extensive photoreceptor loss and disruption of the RPE mosaic.Subretinal blebs induce pronounced photoreceptor degeneration and RPE changes in the rabbit as demonstrated by in vivo imaging using SD-OCT, IR-cSLO, and BAF.
Pub.: 20 Mar '15, Pinned: 17 Sep '17
Abstract: Human embryonic stem cell (hESC)-derived retinal pigment epithelial (RPE) cells could replace lost tissue in geographic atrophy (GA) but efficacy has yet to be demonstrated in a large-eyed model. Also, production of hESC-RPE has not yet been achieved in a xeno-free and defined manner, which is critical for clinical compliance and reduced immunogenicity. Here we describe an effective differentiation methodology using human laminin-521 matrix with xeno-free and defined medium. Differentiated cells exhibited characteristics of native RPE including morphology, pigmentation, marker expression, monolayer integrity, and polarization together with phagocytic activity. Furthermore, we established a large-eyed GA model that allowed in vivo imaging of hESC-RPE and host retina. Cells transplanted in suspension showed long-term integration and formed polarized monolayers exhibiting phagocytic and photoreceptor rescue capacity. We have developed a xeno-free and defined hESC-RPE differentiation method and present evidence of functional integration of clinically compliant hESC-RPE in a large-eyed disease model.
Pub.: 05 Jan '16, Pinned: 17 Sep '17
Abstract: Subretinal suspension transplants of human embryonic stem cell-derived retinal pigment epithelial cells (hESC-RPE) have the capacity to form functional monolayers in naive eyes. We explore hESC-RPE integration when transplanted in suspension to a large-eyed model of geographic atrophy (GA).Derivation of hESC-RPE was performed in a xeno-free and defined manner. Subretinal bleb injection of PBS or sodium iodate (NaIO3) was used to induce a GA-like phenotype. Suspensions of hESC-RPE were transplanted to the subretinal space of naive or PBS-/NaIO3-treated rabbits using a transvitreal pars plana technique. Integration of hESC-RPE was monitored by multimodal real-time imaging and by immunohistochemistry.Subretinal blebs of PBS or NaIO3 caused different degrees of outer neuroretinal degeneration, RPE hyperautofluorescence, focal RPE loss, and choroidal atrophy; that is, hallmark characteristics of GA. In nonpretreated naive eyes, hESC-RPE integrated as subretinal monolayers with preserved overlying photoreceptors, yet not in areas with outer neuroretinal degeneration and native RPE loss. When transplanted to eyes with PBS-/NaIO3-induced degeneration, hESC-RPE failed to integrate.In a large-eyed preclinical model, subretinal suspension transplants of hESC-RPE did not integrate in areas with GA-like degeneration.
Pub.: 28 Feb '17, Pinned: 17 Sep '17