Purpose Degenerative mechanisms of retinal neurodegenerative diseases (RND) share common cellular and molecular signalization pathways. Curative treatment does not exist and cell-based therapy, through the paracrine properties of mesenchymal stem cells (MSC), is a potential unspecific treatment for RND. This study aimed to evaluate the neuroprotective capability of human bone marrow (bm) MSC secretome and its potential to modulate retinal responses to neurodegeneration. Methods An in vitro model of spontaneous retinal neurodegeneration was used to compare three days of monocultured neuroretina (NR), NR cocultured with bmMSC, and NR cultured with bmMSC secretome. We evaluated retinal morphology markers (Lectin peanut agglutinin, rhodopsin, protein kinase C α isoform, neuronal-specific nuclear protein, glial fibrillary acidic protein, TdT-mediated dUTP nick-end labeling, and vimentin) and proteins involved in apoptosis (apoptosis-inductor factor, caspase-3), necroptosis (MLKL), and autophagy (p62). Besides, we analyzed the relative mRNA expression through qPCR of genes involved in apoptosis ( BAX, BCL2, CASP3, CASP8, CASP9 ), necroptosis ( MLKL, RIPK1, RIPK3 ), autophagy ( ATG7 , BCLIN1, LC3B , mTOR , SQSTM1 ), oxidative stress ( COX2, CYBA, CYBB, GPX6, SOD1, TXN2, TXNRD1 ) and inflammation ( IL1, IL6, IL10, TGFb1, TNFa ). Results The bmMSC secretome preserves retinal morphology, limits pro-apoptotic– and pro-necroptotic–related gene and protein expression, modulates autophagy-related genes and proteins, and stimulates the activation of antioxidant-associated genes. Conclusions The neuroprotective ability of the bmMSC secretome is associated with activation of antioxidant machinery, modulation of autophagy, and inhibition of apoptosis and necroptosis during retinal degeneration. The neuroprotective effect of bmMSC secretomes in the presence/absence of MSC looks similar. Our current results reinforce the hypothesis that the human bmMSC secretome slows retinal neurodegeneration and may be a therapeutic option for treating RND.

Retinal neurodegenerative diseases are the leading causes of visual impairment and irreversible blindness worldwide. Although the retinal response to injury remains closely similar between different retinal neurodegenerative diseases, available therapeutic alternatives are only palliative, too expensive, or very specific, such as gene therapy. In that sense, the development of broad-spectrum neuroprotective therapies seems to be an excellent option. In this regard, it is essential to identify molecular targets involved in retinal degeneration, such as cell death mechanisms. Apoptosis has been considered as the primary cell death mechanism during retinal degeneration; however, recent studies have demonstrated that the only use of anti-apoptotic drugs is not enough to confer good neuroprotection in terms of cell viability and preservation. For that reason, the interrelationship that exists between apoptosis and other cell death mechanisms needs to be characterized deeply to design future therapeutic options that simultaneously block the main cell death pathways. In that sense, the study aimed to characterize the programmed cell death (in terms of apoptosis and necroptosis) and autophagy response and modulation in retinal neurodegenerative diseases, using an in vitro model of spontaneous retinal neurodegeneration. For that purpose, we measured the mRNA relative expression through qPCR of a selected pool of genes involved in apoptosis (BAX, BCL2, CASP3, CASP8, and CASP9), necroptosis (MLKL, RIPK1, and RIPK3), and autophagy (ATG7, BCLIN1, LC3B, mTOR, and SQSTM1); besides, the immunoexpression of their encoding proteins (Casp3, MLKL, RIPK1, LC3B, and p62) were analyzed using immunohistochemistry. Our results showed an increase of pro-apoptotic and pro-necroptotic related genes and proteins during in vitro retinal neurodegeneration. Besides, we describe for the first time the modulation between programmed cell death mechanisms and autophagy in an in vitro retinal neurodegeneration model. This study reinforces the idea that cell death mechanisms are closely interconnected and provides new information about molecular signaling and autophagy along the retinal degeneration process.

Purpose: The PROM1 gene encodes the protein Prominin‐1 which plays a critical role in the morphogenesis of photoreceptors outer segments. Mutations in the PROM1 gene are related to Inherited Retinal Dystrophy (IRD) phenotypes. The c.1354dupT mutation in the PROM1 gene causes a premature stop codon. The aim of the study was to define the more accurate gene editing strategy for development of an IRD model based on iPSC‐derived photoreceptors. Methods: Firstly, the most accurate CRISPR/Cas9 machinery was determined by the web tool for genome editing CRISPOR and the DNAStar Lasergene software. gRNAs were selected, accordingly to their proximity to the mutation position and their specificity and efficiency scores. Then, silent mutations were added to the repair oligonucleotides (Alt‐R HDR Donor Oligos), by using the web tool MolBiotools, to generate a restriction site for the Ssp1enzyme. Afterwards, to confirm the in silico information, in vitro studies were performed. The U2OS cell line and the control iPSC line ([FiPS] Ctrl1‐Ep6F‐5) were transfected with each selected gRNA, their respective repair oligonucleotide, and the endonuclease Cas9‐protein, by using the Neon Transfection System. Finally, a digestion with Ssp1 were performed to specifically detect the edited genomes. Results: Several gRNAs were selected in silico. In vitro studies showed higher genome editing efficiency in two of them (40RE and 47RE). Finally, the gRNA guide 40RE and its respective repair oligonucleotide were selected. Conclusions: In‐silico and in‐vitro studies concluded that the gRNA guide 40RE was the most accurate for development of an in vitro IRD model based on iPSC‐derived photoreceptors with a mutation in the PROM1 gene. Gene editing strategy designed in silico should be validated in vitro before transfecting the target cells.