Understanding cone photoreceptor dystrophies : from animal models to engineered patient-derived retinal tissues

Vision is considered the most important sense, taking on about 80% of the perceptions we receive in our everyday life. Cone-photoreceptors are responsible for high-resolution central vision and color discrimination, and their degeneration is frequently the cause of vision loss in retinal degenerative diseases (RDs). RDs are a heterogeneous group of diseases affecting millions of people worldwide, which at the moment are mostly without any therapeutic option. Animal models are extremely useful in studying the retina's development or degeneration and understanding the molecular mechanisms in inherited genetic disease affecting photoreceptors. Modeling human developmental and degenerative diseases can be particularly challenging, especially in the case of rare and complex diseases where no exhaustive animal models are available. Generation of sustainable human disease models that allow in-depth analysis of the molecular mechanism is one of the big challenges nowadays. Stem cell technology holds great potential in disease modeling and represents a new powerful tool for generating scalable and animal-free models that can more accurately illustrate clinical phenotypes of complex human diseases.  We developed a protocol to differentiate pluripotent stem cells (PSCs) into retinal sheets (RSs), which are polarized, multi-layered tissues containing cone photoreceptors and expressing outer segment (OS), connecting cilium (CC), and nuclear specific markers. Using both mouse and stem cells-based humanized models, we first investigate the role of BMI1 in mature photoreceptors. The Polycomb group protein Bmi1 is known for its neuroprotective functions by controlling senescence and apoptosis and is expressed in both retinal progenitor and neurons, but little is known about its specific role in the adult retina. It has been recently linked to late-onset neurodegenerative disorders, and it could have a role in the pathology of late-onset RDs, such as Age-related Macular Degeneration (AMD). We showed that cone photoreceptors and bipolar neurons are generated normally but then undergo rapid degeneration in Bmi1-/- mice through Rip3-associated necroptosis. Degeneration was associated with chromatin compaction anomalies, activation of tandem-repeats, and oxidative stress. Furthermore, we show that BMI1 is preferentially expressed in cones at heterochromatic foci in the human retina. Its inactivation in human embryonic stem cells (hESCs) impaired cone terminal differentiation and resulted in chromatin compaction anomalies, activation of tandem-repeats, and P53 induction. These findings provide a mechanism explaining how Bmi1 deficiency leads to cone degeneration and reveal conserved biological functions and differences for Bmi1 in photoreceptor biology between mouse and man. Using an induced Pluripotent Stem Cells (iPSCs) based human model, we then investigate the degenerative process in patients with ciliopathies, a group of heterogeneous genetic diseases affecting proteins involved in primary cilium structure and function frequently accompanied by retinal degeneration.  We generate iPSC-derived retinal sheets from patients affected by two ciliopathies, Meckel-Gruber (MKS) and Bardet-Biedl syndromes (BBS). Ciliopathic photoreceptors displayed significant common alterations in the expression of hundreds of developmental genes. Moreover, they showed several anomalies in the formation and maintenance of cilia, the mother centriole's positioning, the activation of a stress response to misfolded proteins, genomic instabilities, and DNA damage accumulation. This study reveals how combining cell reprogramming and organogenesis technologies with next-generation sequencing enables the elucidation of molecular and cellular mechanisms involved in human retinal degenerative and developmental disorders. The same approach, combining photoreceptor sheet differentiation and wide-genome expression profile, could be applied to model many genetic, developmental, and degenerative diseases affecting photoreceptors. It may help elucidate the molecular mechanisms underlining these diseases, drug screening of compounds with potential therapeutic effects, and predict drug side effects.

Keyphrases: Ciliopathies, MKS, BBS, BMI1, iPSC, Stem cells, Retina, Photoreceptors, Neurodegeneration