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SUMMARYSchwann cells (SCs) are the myelinating and non-myelinating glia of the peripheral nervous system (PNS) and are essential for its function. Defects in SCs are associated with many PNS disorders including diabetic peripheral neuropathy (DPN), a condition affecting millions of patients. Here we present a strategy for deriving and purifying SCs from human pluripotent stem cells (hPSCs). The scalable cultures of SCs allow basic and translational applications such as high-resolution molecular and functional characterization, developmental studies, modeling and mechanistic understanding of SC diseases and drug discovery. Our hPSC-derived SCs recapitulate the key molecular features of primary SCs and are capable of engrafting efficiently and producing myelin in injured sciatic nerves in rats. We further established an hPSC-based in vitro model of DPN that revealed the selective vulnerability of human SCs to hyperglycemia-induced cytotoxicity. We established a high-throughput screening system that identified a candidate drug that counteracts glucose-mediated cytotoxicity in SCs and normalizes glucose-induced transcriptional and metabolic abnormalities in SCs. Treatment of hyperglycemic mice with this drug candidate rescues sensory function, prevents SC death, and counteracts myelin damage in sciatic nerves suggesting considerable potential as a novel treatment for DPN.

The maturation of human pluripotent stem cell (hPSC)-derived neurons mimics the protracted timing of human brain development, extending over months and years to reach adult-like function. Prolonged in vitro maturation presents a major challenge to stem cell-based applications in modeling and treating neurological disease. We designed a high-content imaging assay based on morphological and functional readouts in hPSC-derived cortical neurons to reveal underlying pathways and to identify chemicals capable of accelerating neuronal maturation. Probing a library of 2688 bioactive drugs, we identified multiple compounds that drive neuronal maturation including inhibitors of LSD1 and DOT1L and activators of calcium-dependent transcription. A cocktail of 4 factors GSK-2879552, EPZ-5676, NMDA and Bay K 8644, which we collectively termed GENtoniK, triggered maturation across all assays tested including measures of synaptic density, electrophysiology and transcriptomics. Remarkably, GENtoniK was similarly effective in enhancing neuronal maturation in 3D cortical organoids and in spinal motoneurons, and improved aspects of cell maturation in non-neural lineages such as melanocytes and pancreatic beta cells. These results demonstrate that the maturation of multiple hPSC-derived cell types can be enhanced by simple pharmacological intervention and suggests that some of the mechanisms controlling the timing of human maturation are shared across lineages.

Experimental Autoimmune Uveitis (EAU) is driven by immune cells responding to self-antigens. Many features of this non-infectious, intraocular inflammatory disease model recapitulate the clinical phenotype of posterior uveitis affecting humans. EAU has been used reliably to study the efficacy of novel inflammatory therapeutics, their mode of action and to further investigate the mechanisms that underpin disease progression of intraocular disorders. Here, we provide a detailed protocol on EAU induction in the C57BL/6J mouse - the most widely used model organism with susceptibility to this disease. Clinical assessment of disease severity and progression will be demonstrated using fundoscopy, histological examination and fluorescein angiography. The induction procedure involves subcutaneous injection of an emulsion containing a peptide (IRBP1-20) from the ocular protein interphotoreceptor retinoid binding protein (also known as retinol binding protein 3), Complete Freund's Adjuvant (CFA) and supplemented with killed Mycobacterium tuberculosis. Injection of this viscous emulsion on the back of the neck is followed by a single intraperitoneal injection of Bordetella pertussis toxin. At the onset of symptoms (day 12-14) and under general anesthesia, fundoscopic images are taken to assess disease progression through clinical examination. These data can be directly compared with those at later timepoints and peak disease (day 20-22) with differences analyzed. At the same time, this protocol allows the investigator to assess potential differences in vessel permeability and damage using fluorescein angiography. EAU can be induced in other mouse strains - both wildtype or genetically modified - and combined with novel therapies offering flexibility for studying drug efficacy and/or disease mechanisms.

Non-infectious uveitis (NIU) is an inflammatory eye disease initiated via CD4+ T-cell activation and transmigration, resulting in focal retinal tissue damage and visual acuity disturbance. Cell adhesion molecules (CAMs) are activated during the inflammatory process to facilitate the leukocyte recruitment cascade. Our review focused on CAM-targeted therapies in experimental autoimmune uveitis (EAU) and NIU. We concluded that CAM-based therapies have demonstrated benefits for controlling EAU severity with decreases in immune cell migration, especially via ICAM-1/LFA-1 and VCAM-1/VLA-4 (integrin) pathways. P-selectin and E-selectin are more involved specifically in uveitis related to vasculitis. These therapies have potential clinical applications for the development of a more personalized and specific treatment. Localized therapies are the future direction to avoid serious systemic side effects.

Treatment of posterior eye diseases with intravitreal injections of drugs, while effective, is invasive and associated with side effects such as retinal detachment and endophthalmitis. In this work, we have formulated a model compound, rapamycin (RAP), in nanoparticle-based eye drops and evaluated the delivery of RAP to the posterior eye tissues in a healthy rabbit. We have also studied the formulation in experimental autoimmune uveitis (EAU) mouse model with retinal inflammation. Aqueous RAP eye drops were prepared using N-palmitoyl-N-monomethyl-N,N-dimethyl-N,N,N-trimethyl-6-O-glycolchitosan (Molecular Envelope Technology - MET) containing 0.23 ± 0.001% w/v RAP with viscosity, osmolarity, and pH within the ocular comfort range, and the formulation (MET-RAP) was stable in terms of drug content at both refrigeration and room temperature for one month. The MET-RAP eye drops delivered RAP to the choroid-retina with a Cmax of 145 ± 49 ng/g (tmax = 1 h). The topical application of the MET-RAP eye drops to the EAU mouse model resulted in significant disease suppression compared to controls, with activity similar to dexamethasone eye drops. The MET-RAP eye drops also resulted in a reduction of RORγt and an increase in both Foxp3 expression and IL-10 secretion, indicating a mechanism involving the inhibition of Th17 cells and the up-regulation of T-reg cells. The MET-RAP formulation delivers RAP to the posterior eye segments, and the formulation is active in EAU.