Generation of induced pluripotent stem cells (UCLi024-A) from a patient with argininosuccinate lyase deficiency carrying a homozygous c.437G > A (p.Arg146Gln) mutation.

Argininosuccinic aciduria (ASA) is a rare inherited metabolic disease caused by argininosuccinate lyase (ASL) deficiency. Patients with ASA present with hyperammonaemia due to an impaired urea cycle pathway in the liver, and systemic disease with epileptic encephalopathy, chronic liver disease, and arterial hypertension. A human induced pluripotent stem cell (iPSC) line from the fibroblasts of a patient with ASA with homozygous pathogenic c.437G > A mutation of hASL was generated. Characterization of the cell line demonstrated pluripotency, differentiation potential and normal karyotype. This cell line, called UCLi024-A, can be utilized for in vitro disease modelling of ASA, and design of novel therapeutics.

Molecular pathology of Usher 1B patient-derived retinal organoids at single cell resolution.

Usher syndrome-associated retinitis pigmentosa (RP) causes progressive retinal degeneration, which has no cure. The pathomechanism of Usher type 1B (USH1B)-RP caused by MYO7A mutation remains elusive because of the lack of faithful animal models and limited knowledge of MYO7A function. Here, we analyzed 3D retinal organoids generated from USH1B patient-derived induced pluripotent stem cells. Increased differential gene expression occurred over time without excessive photoreceptor cell death in USH1B organoids compared with controls. Dysregulated genes were enriched first for mitochondrial functions and then proteasomal ubiquitin-dependent protein catabolic processes and RNA splicing. Single-cell RNA sequencing revealed MYO7A expression in rod photoreceptor and Müller glial cells corresponding to upregulation of stress responses in NRL+ rods and apoptotic signaling pathways in VIM+ Müller cells, pointing to the defensive mechanisms that mitigate photoreceptor cell death. This first human model for USH1B-RP provides a representation of patient retina in vivo relevant for development of therapeutic strategies.

Generation of 3D retinal tissue from human pluripotent stem cells using a directed small molecule-based serum-free microwell platform.

Retinal degenerative diseases are a leading cause of blindness worldwide with debilitating life-long consequences for the affected individuals. Cell therapy is considered a potential future clinical intervention to restore and preserve sight by replacing lost photoreceptors and/or retinal pigment epithelium. Development of protocols to generate retinal tissue from human pluripotent stem cells (hPSC), reliably and at scale, can provide a platform to generate photoreceptors for cell therapy and to model retinal disease in vitro. Here, we describe an improved differentiation platform to generate retinal organoids from hPSC at scale and free from time-consuming manual microdissection steps. The scale up was achieved using an agarose mould platform enabling generation of uniform self-assembled 3D spheres from dissociated hPSC in microwells. Subsequent retinal differentiation was efficiently achieved via a stepwise differentiation protocol using a number of small molecules. To facilitate clinical translation, xeno-free approaches were developed by substituting Matrigel™ and foetal bovine serum with recombinant laminin and human platelet lysate, respectively. Generated retinal organoids exhibited important features reminiscent of retinal tissue including correct site-specific localisation of proteins involved in phototransduction.