Retinoic acid delays initial photoreceptor differentiation and results in a highly structured mature retinal organoid.

BACKGROUND: Human-induced pluripotent stem cell-derived retinal organoids are a valuable tool for disease modelling and therapeutic development. Many efforts have been made over the last decade to optimise protocols for the generation of organoids that correctly mimic the human retina. Most protocols use common media supplements; however, protocol-dependent variability impacts data interpretation. To date, the lack of a systematic comparison of a given protocol with or without supplements makes it difficult to determine how they influence the differentiation process and morphology of the retinal organoids. METHODS: A 2D-3D differentiation method was used to generate retinal organoids, which were cultured with or without the most commonly used media supplements, notably retinoic acid. Gene expression was assayed using qPCR analysis, protein expression using immunofluorescence studies, ultrastructure using electron microscopy and 3D morphology using confocal and biphoton microscopy of whole organoids. RESULTS: Retinoic acid delayed the initial stages of differentiation by modulating photoreceptor gene expression. At later stages, the presence of retinoic acid led to the generation of mature retinal organoids with a well-structured stratified photoreceptor layer containing a predominant rod population. By contrast, the absence of retinoic acid led to cone-rich organoids with a less organised and non-stratified photoreceptor layer. CONCLUSIONS: This study proves the importance of supplemented media for culturing retinal organoids. More importantly, we demonstrate for the first time that the role of retinoic acid goes beyond inducing a rod cell fate to enhancing the organisation of the photoreceptor layer of the mature organoid.

A Novel Chromosomal Translocation Identified due to Complex Genetic Instability in iPSC Generated for Choroideremia.

Induced pluripotent stem cells (iPSCs) have revolutionized the study of human diseases as they can renew indefinitely, undergo multi-lineage differentiation, and generate disease-specific models. However, the difficulty of working with iPSCs is that they are prone to genetic instability. Furthermore, genetically unstable iPSCs are often discarded, as they can have unforeseen consequences on pathophysiological or therapeutic read-outs. We generated iPSCs from two brothers of a previously unstudied family affected with the inherited retinal dystrophy choroideremia. We detected complex rearrangements involving chromosomes 12, 20 and/or 5 in the generated iPSCs. Suspecting an underlying chromosomal aberration, we performed karyotype analysis of the original fibroblasts, and of blood cells from additional family members. We identified a novel chromosomal translocation t(12;20)(q24.3;q11.2) segregating in this family. We determined that the translocation was balanced and did not impact subsequent retinal differentiation. We show for the first time that an undetected genetic instability in somatic cells can breed further instability upon reprogramming. Therefore, the detection of chromosomal aberrations in iPSCs should not be disregarded, as they may reveal rearrangements segregating in families. Furthermore, as such rearrangements are often associated with reproductive failure or birth defects, this in turn has important consequences for genetic counseling of family members.

Zika virus induces strong inflammatory responses and impairs homeostasis and function of the human retinal pigment epithelium.

BACKGROUND: Zika virus (ZIKV) has recently re-emerged as a pathogenic agent with epidemic capacities as was well illustrated in South America. Because of the extent of this health crisis, a number of more serious symptoms have become associated with ZIKV infection than what was initially described. In particular, neuronal and ocular disorders have been characterized, both in infants and in adults. Notably, the macula and the retina can be strongly affected by ZIKV, possibly by a direct effect of the virus. This is supported by the detection of replicative and infectious virus in lachrimal fluid in human patients and mouse models. METHODS: Here, we used an innovative, state-of-the-art iPSC-derived human retinal pigment epithelium (RPE) model to study ZIKV retinal impairment. FINDINGS: We showed that the human RPE is highly susceptible to ZIKV infection and that a ZIKV African strain was more virulent and led to a more potent epithelium disruption and stronger anti-viral response than an Asian strain, suggesting lineage differences. Moreover, ZIKV infection led to impaired membrane dynamics involved in endocytosis, organelle biogenesis and potentially secretion, key mechanisms of RPE homeostasis and function. INTERPRETATION: Taken together, our results suggest that ZIKV has a highly efficient ocular tropism, which creates a strong inflammatory environment that could have acute or chronic adverse effects. FUND: This work was funded by Retina France, REACTing and La Région Languedoc-Roussillon.

Pathogenicity of a novel missense variant associated with choroideremia and its impact on gene replacement therapy.

Choroideremia (CHM) is an inherited retinal dystrophy characterised by progressive degeneration of photoreceptors, retinal pigment epithelium (RPE) and underlying choroid. It is caused by loss-of-function mutations in CHM, which has an X-linked inheritance, and is thus an ideal candidate for gene replacement strategies. CHM encodes REP1, which plays a key role in the prenylation of Rab GTPases. We recently showed that an induced pluripotent stem cell (iPSc)-derived RPE model for CHM is fully functional and reproduces the underlying prenylation defect. This criterion can thus be used for testing the pathogenic nature of novel variants. Until recently, missense variants were not associated with CHM. Currently, at least nine such variants have been reported but only two have been shown to be pathogenic. We report here the characterisation of the third pathogenic missense CHM variant, p.Leu457Pro. Clinically, the associated phenotype is indistinguishable from that of loss-of-function mutations. By contrast, this missense variant results in wild type CHM expression levels and detectable levels of mutant protein. The prenylation status of patient-specific fibroblasts and iPSc-derived RPE is within the range observed for loss-of-function mutations, consistent with the clinical phenotype. Lastly, considering the current climate of CHM gene therapy, we assayed whether the presence of mutant REP1 could interfere with a gene replacement strategy by testing the prenylation status of patient-specific iPSc-derived RPE following AAV-mediated gene transfer. Our results show that correction of the functional defect is possible and highlight the predictive value of these models for therapy screening prior to inclusion in clinical trials.