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Abstract Maintenance of NAD pools is critical for neuronal survival. The capacity to maintain NAD pools declines in neurodegenerative disease. We identify that low NMNAT2, the critical neuronal NAD producing enzyme, drives retinal susceptibility to neurodegenerative insults. As proof of concept, gene therapy over-expressing full length human NMNAT2 is neuroprotective. To pharmacologically target NMNAT2, we identify that epigallocatechin gallate (EGCG) can drive NAD production in neurons through an NMNAT2 and NMN dependent mechanism. We confirm this by pharmacological and genetic inhibition of the NAD-salvage pathway. EGCG is neuroprotective in rodent (mixed sex) and human models of retinal neurodegeneration. As EGCG has poor drug-like qualities, we use it as a tool compound to generate novel small molecules which drive neuronal NAD production and provide neuroprotection. This class of NMNAT2 targeted small molecules could have an important therapeutic impact for neurodegenerative disease following further drug development.

Abstract The extracellular matrix (ECM) governs a wide spectrum of cellular fate processes, with a particular emphasis on anoikis, an integrin‐dependent form of cell death. Currently, anoikis is defined as an intrinsic apoptosis. In contrast to traditional apoptosis and necroptosis, integrin correlates ECM signaling with intracellular signaling cascades, describing the full process of anoikis. However, anoikis is frequently overlooked in physiological and pathological processes as well as traditional in vitro research models. In this review, we summarized the role of anoikis in physiological and pathological processes, spanning embryonic development, organ development, tissue repair, inflammatory responses, cardiovascular diseases, tumor metastasis, and so on. Similarly, in the realm of stem cell research focused on the functional evolution of cells, anoikis offers a potential solution to various challenges, including in vitro cell culture models, stem cell therapy, cell transplantation, and engineering applications, which are largely based on the regulation of cell fate by anoikis. More importantly, the regulatory mechanisms of anoikis based on molecular processes and ECM signaling will provide new strategies for therapeutic interventions (drug therapy and cell‐based therapy) in disease. In summary, this review provides a systematic elaboration of anoikis, thus shedding light on its future research.

Abstract Bietti crystalline dystrophy (BCD) is an inherited retinal disease (IRD) caused by mutations in the CYP4V2 gene. It is a relatively common cause of IRD in east Asia. A number of features of this disease make it highly amenable to gene supplementation therapy. This study aims to validate a series of essential precursor in vitro experiments prior to developing a clinical gene therapy for BCD. We demonstrated that HEK293, ARPE19, and patient induced pluripotent stem cell (iPSC)-derived RPE cells transduced with AAV2 vectors encoding codon optimization of CYP4V2 (AAV2.coCYP4V2) resulted in elevated protein expression levels of CYP4V2 compared to those transduced with AAV2 vectors encoding wild type CYP4V2 (AAV2.wtCYP4V2), as assessed by immunocytochemistry and western blot. Similarly, we observed significantly increased CYP4V2 enzyme activity in cells transduced with AAV2.coCYP4V2 compared to those transduced with AAV2.wtCYP4V2. We also showed CYP4V2 expression in human RPE/choroid explants transduced with AAV2.coCYP4V2 compared to those transduced with AAV2.wtCYP4V2. These preclinical data support the further development of a gene supplementation therapy for a currently untreatable blinding condition—BCD. Codon-optimized CYP4V2 transgene was superior to wild type in terms of protein expression and enzyme activity. Ex vivo culture of human RPE cells provided an effective approach to test AAV-mediated transgene delivery.

A comprehensive analysis of the ocular networks among various tissues is necessary to understand eye physiology in health and disease. Here the authors present a multi-species single-cell transcriptomic atlas consisting of cells of the cornea, iris, ciliary body, neural retina, retinal pigmented epithelium, and choroid.

CRISPR/Cas has opened the prospect of direct gene correction therapy for some inherited retinal diseases. Previous work has demonstrated the utility of adeno-associated virus (AAV) mediated delivery to retinal cells in vivo; however, with the expanding repertoire of CRISPR/Cas endonucleases, it is not clear which of these are most efficacious for retinal editing in vivo. We sought to compare CRISPR/Cas endonuclease activity using both single and dual AAV delivery strategies for gene editing in retinal cells. Plasmids of a dual vector system with SpCas9, SaCas9, Cas12a, CjCas9 and a sgRNA targeting YFP, as well as a single vector system with SaCas9/YFP sgRNA were generated and validated in YFP-expressing HEK293A cell by flow cytometry and the T7E1 assay. Paired CRISPR/Cas endonuclease and its best performing sgRNA was then packaged into an AAV2 capsid derivative, AAV7m8, and injected intravitreally into CMV-Cre:Rosa26-YFP mice. SpCas9 and Cas12a achieved better knockout efficiency than SaCas9 and CjCas9. Moreover, no significant difference in YFP gene editing was found between single and dual CRISPR/SaCas9 vector systems. With a marked reduction of YFP-positive retinal cells, AAV7m8 delivered SpCas9 was found to have the highest knockout efficacy among all investigated endonucleases. We demonstrate that the AAV7m8-mediated delivery of CRISPR/SpCas9 construct achieves the most efficient gene modification in neurosensory retinal cells in vivo.

Dysfunction of retinal glial cells, particularly Müller cells, has been implicated in several retinal diseases. Despite their important contribution to retinal homeostasis, a specific way to differentiate retinal glial cells from human pluripotent stem cells has not yet been described. Here, we report a method to differentiate retinal glial cells from human embryonic stem cells (hESCs) through promoting the Notch signaling pathway. We first generated retinal progenitor cells (RPCs) from hESCs then promoted the Notch signaling pathway using Notch ligands, including Delta-like ligand 4 and Jagged-1. We validated glial cell differentiation with qRT-PCR, immunocytochemistry, western blots and fluorescence-activated cell sorting as we promoted Notch signaling in RPCs. We found that promoting Notch signaling in RPCs for 2 weeks led to upregulation of glial cell markers, including glial fibrillary acidic protein (GFAP), glutamine synthetase, vimentin and cellular retinaldehyde-binding protein (CRALBP). Of these markers, we found the greatest increase in expression of the pan glial cell marker, GFAP. Conversely, we also found that inhibition of Notch signaling in RPCs led to upregulation of retinal neuronal markers including cone-rod homeobox (CRX) and orthodenticle homeobox 2 (OTX2) but with little expression of GFAP. This retinal glial differentiation method will help advance the generation of stem cell disease models to study the pathogenesis of retinal diseases associated with glial dysfunction such as macular telangiectasia type 2. This method may also be useful for the development of future therapeutics such as drug screening and gene editing using patient-derived retinal glial cells.

Cellular reprogramming technology holds great potential for tissue repair and regeneration to replace cells that are lost due to diseases or injuries. In addition to the landmark discovery of induced pluripotent stem cells, advances in cellular reprogramming allow the direct lineage conversion of one somatic cell type to another using defined transcription factors. This direct reprogramming technology represents a rapid way to generate target cells in the laboratory, which can be used for transplantation and studies of biology and diseases. More importantly, recent work has demonstrated the exciting application of direct reprogramming to stimulate regeneration in vivo, providing an alternative approach to transplantation of donor cells. Here, we provide an overview of the underlying concept of using cellular reprogramming to convert cell fates and discuss the current advances in cellular reprogramming both in vitro and in vivo, with particular focuses on the neural and retinal systems. We also discuss the potential of in vivo reprogramming in regenerative medicine, the challenges and potential solutions to translate this technology to the clinic.

Background. Human induced pluripotent stem cells (iPSCs) are an attractive source of cardiomyocytes for cardiac repair and regeneration. In this study, we aim to determine whether acute electrical stimulation of human iPSCs can promote their differentiation to cardiomyocytes. Methods. Human iPSCs were differentiated to cardiac cells by forming embryoid bodies (EBs) for 5 days. EBs were then subjected to brief electrical stimulation and plated down for 14 days. Results. In iPS(Foreskin)-2 cell line, brief electrical stimulation at 65 mV/mm or 200 mV/mm for 5 min significantly increased the percentage of beating EBs present by day 14 after plating. Acute electrical stimulation also significantly increased the cardiac gene expression of ACTC1, TNNT2, MYH7, and MYL7. However, the cardiogenic effect of electrical stimulation was not reproducible in another iPS cell line, CERA007c6. Beating EBs from control and electrically stimulated groups expressed various cardiac-specific transcription factors and contractile muscle markers. Beating EBs were also shown to cycle calcium and were responsive to the chronotropic agents, isoproterenol and carbamylcholine, in a concentration-dependent manner. Conclusions. Our results demonstrate that brief electrical stimulation can promote cardiac differentiation of human iPS cells. The cardiogenic effect of brief electrical stimulation is dependent on the cell line used.