Dunja Lukovic, Ana Artero Castro, Ana Belen Garcia Delgado, María de los Angeles Martín Bernal, Noelia Luna Pelaez, Andrea Díez Lloret, Rocío Perez Espejo, Kunka Kamenarova, Laura Fernández Sánchez, Nicolás Cuenca, Marta Cortón, Avila Fernandez, Anni Sorkio, Heli Skottman, Carmen Ayuso, Slaven Erceg, Shomi S. Bhattacharya |
Scientific Reports | Human iPSC derived disease model of MERTK-associated retinitis pigmentosa | 11 August 2015 | https://doi.org/10.1038/srep12910
Abstract
Retinitis pigmentosa (RP) represents a genetically heterogeneous group of retinal dystrophies affecting mainly the rod photoreceptors and in some instances also the retinal pigment epithelium (RPE) cells of the retina. Clinical symptoms and disease progression leading to moderate to severe loss of vision are well established and despite significant progress in the identification of causative genes, the disease pathology remains unclear. Lack of this understanding has so far hindered development of effective therapies. Here we report successful generation of human induced pluripotent stem cells (iPSC) from skin fibroblasts of a patient harboring a novel Ser331Cysfs*5 mutation in the MERTK gene. The patient was diagnosed with an early onset and severe form of autosomal recessive RP (arRP). Upon differentiation of these iPSC towards RPE, patient-specific RPE cells exhibited defective phagocytosis, a characteristic phenotype of MERTK deficiency observed in human patients and animal models. Thus we have created a faithful cellular model of arRP incorporating the human genetic background which will allow us to investigate in detail the disease mechanism, explore screening of a variety of therapeutic compounds/reagents and design either combined cell and gene- based therapies or independent approaches.
Introduction
Retinitis pigmentosa (RP; OMIM 268000) with a prevalence of 1 in 3,500 individuals is the most common form of hereditary retinal disorder affecting the working age group. RP is characterized by progressive dysfunction and death of mainly the rod photoreceptor cells (PR) of the retina however in some cases retinal pigment epithelium (RPE) cells are also involved, often resulting in permanent blindness. So far 54 genes have been implicated in this disease coding for proteins involved in a myriad of functions such as phototransduction signaling cascade, retinoid cycle, cell-cell adhesion or the cytoskeleton1. The disease is inherited in all there Mendelian forms, the autosomal recessive (arRP) being the most common with over 50% of cases. Largely due to the high genetic heterogeneity and unavailability of disease tissue, pathology of the disease remains elusive.
Patient-derived induced pluripotent stem cells (iPSCs) provide an unprecedented opportunity to recapitulate disease pathogenicity without the need for genetic manipulation and creation of gene targeted animal models. Human iPSCs, similar to embryonic stem cells (ESC), can be expanded indefinitely in vitro and differentiated into any type of mature cell in the human body, without the ethical and immunogenicity issues associated with ESC2. These cells are also valuable for developing therapeutic strategies, drug toxicity screens and development of disease models, in addition to providing a source for cell transplantation therapy. RPE cells and photoreceptors (PR) have been successfully generated from iPSCs (iPSC-RPE and iPSC-PR respectively) by various groups in stepwise differentiation protocols mimicking retinal development by introducing Wnt signaling inhibitors (DKK1), Nodal antagonist Lefty A, Notch pathway inhibitor (DAPT-gamma secretase inhibitor), or IGF-13,4,5. In contrast, only RPE cells have been generated spontaneously in overgrown iPSC/ESC cultures without the addition of exogenous factors, since derivatives of neuroectoderm appear by default in non-induced cultures6,7. Generated RPE cells in these studies display a fully mature phenotype and physiological activity in vitro such as phagocytosis, secretion of vascular endothelial growth factor (VEGF) and pigment epithelium-derived factor (PEDF) and epithelial barrier formation. Cellular models of hereditary retinal dystrophies have been successfully created in vitro in Best disease and RP where patients’ fibroblasts were reprogrammed to iPSC and then converted to RPE8,9 or photoreceptor cells10, expressing the disease phenotype. iPSC- derived RPE (iPSC-RPE) cells have also been shown to have a protective effect when injected sub-retinaly into the Royal College of Surgeons (RCS) rats11 and RPE65-defective mice12. Moreover, iPSCs have met clinical-grade requirements13 as a source of RPE grafts and have recently been injected in patients affected by the exudative form (wet-type) of age-related macular degeneration (AMD)14. It has been argued that in this form of AMD the dysfunction and loss of RPE cells is the main cause of visual impairment in the elderly.
Mer tyrosine kinase receptor (MERTK) belongs to the Tyro3/Axl/Mer (TAM) receptor tyrosine kinase family of proteins distinguished by a conserved intracellular kinase domain and extracellular adhesion molecule-like domain. TAM receptors regulate a variety of processes such as cell proliferation/survival, adhesion, migration, inflammatory response, in a cell- microenvironment- and ligand- specific manner15. In previous studies MERTK was found to be disrupted in RCS rats16,17, a classic model for retinal degeneration inherited as an autosomal recessive trait and found to cause early-onset retinitis pigmentosa in patients18. RPE cells fail to phagocytize the shed outer segment (OS) material of PR, a circadian activity performed by RPE cells which serves to renew the damaged lipid and protein components of light exposed PR, while new membranous discs are formed (disc biogenesis) and inserted in the basal part of the OS. As a result, RCS rats exhibit OS associated debris accumulation in the subretinal space, abnormal OS length, eventually leading to the onset of PR degeneration by the P20 stage. Usually complete degeneration occurs by P60. Similar phenotype is observed in merkd mice19 indicating that the RPE phagocytic defect is the underlying molecular mechanism of disease in humans carrying MERTK mutations. Indeed, the reduced retinal thickness and debris detected in the sub-retinal space in patients harboring the MERTK –splice-site -mutation resembles the observed phenotype in the RCS rat20. The distinctive clinical presentation of RP is the only disease manifestation of patients harboring MERTK mutations without any systemic disease or defects of phagocytosis by macrophages, indicating a specialized function of this protein in the RPE cells. In contrast to the detailed clinical understanding of the disease, the mechanism by which MERTK acts during the phagocytosis remains partially unveiled. Outer segments are known to bind to the integrin receptor αvβ521 followed by focal adhesion kinase (FAK) activation in the apical membrane of RPE22 while the MERTK activation occurs via Gas6/Protein S, TUB, TULP1 ligand binding23,24. The latter is thought to activate autophosphorylation at tyrosine Y-749, Y-753 and Y-754 in the tyrosine kinase domain, which in turn activates the molecular cascade targeting actin or non-muscle myosin II to coordinate the cytoskeletal rearrangements necessary for phagocytic ingestion25.
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