RESUMO
Human pluripotent stem cells (PSCs) can be differentiated into retinal organoids with proper neural layer organization, yet the protocols are technically challenging and time consuming. We have modified a widely used differentiation protocol by switching all-trans retinoic acid with 9-cis retinal to accelerate photoreceptor differentiation and improve morphogenesis. In this report, we provide a detailed and improved protocol to generate retinal organoids from human pluripotent stem cells. For complete details on the use and execution of this protocol, please refer to Kaya et al. (2019).
Assuntos
Técnicas Citológicas , Organoides/citologia , Células-Tronco Pluripotentes/citologia , Células Fotorreceptoras Retinianas Bastonetes/citologia , Diterpenos , Humanos , RetinaldeídoRESUMO
To obtain immunogenic conjugate antigens, adipic acid dihydrazide (ADH), as a bridge, and 1-ethyl-3-(3-dimethylaminopropyl) carbodiimidehydrochloride (EDAC), as a coupling agent, were used to conjugate the purified fusion protein, clumping factor A-fibronectin binding protein ClfA A-FnBPA, and type 5 capsular polysaccharide (CP5). The conjugates were mixed with an adjuvant, and mice were immunized 3 times and challenged with Staphylococcus aureus 1 week later. Antibody titers were determined by indirect enzyme-linked immunosorbent assay (ELISA). At 14 days after the first immunization, antibodies against the purified protein and conjugate were detected; after 28 days, antibody levels increased; and a week after the third immunization, antibody levels continued to increase. However, the conjugate antibody titers were higher than those of the purified protein during the study, and no IgG antibodies against purified CP5 were detected during the entire experiment. The protection rate increased to 90% in the conjugate group, indicating that the conjugate imparts a relatively higher protective efficacy than the purified protein and purified CP5.
Afin d'obtenir des antigènes conjugués immunogéniques, du dihydrazide adipique acide (DHA), comme pont, et le 1-éthyl-3-(3-diméthylaminopropyl) carbodiimidehydhrochlorure (EDAC), comme agent liguant, ont été utilisés pour conjuguer la protéine de fusion purifiée ClfAA-FnBPA (facteur de coagulation A-protéine liant la fibronectine) et le polysaccharide capsulaire de type 5 (PC5). Les conjugués ont été mélangés avec un adjuvant, et des souris immunisées trois fois et soumis à une infection défi par Staphylococcus aureus 1 semaine plus tard. Les titres d'anticorps ont été déterminés par épreuve immuno-enzymatique indirecte (ELISA). Au jour 14 suivant la première immunisation, des anticorps dirigés contre la protéine purifiée et le conjugué ont été détectés; après 28 jours, les taux d'anticorps ont augmenté, et 1 semaine après la troisième immunisation, les taux d'anticorps ont continué d'augmenter. Toutefois, durant l'étude les titres d'anticorps envers le conjugué étaient supérieurs à ceux de la protéine purifiée, et aucun anticorps de type IgG dirigé contre le PC5 purifié ne fut détecté durant l'expérience entière. Le taux de protection augmenta jusqu'à 90 % dans le groupe du conjugué, indiquant que le conjugué entraîne une protection efficace supérieure à la protéine purifiée et au PC5 purifié.(Traduit par Docteur Serge Messier).
Assuntos
Adesinas Bacterianas/imunologia , Cápsulas Bacterianas/imunologia , Vacinas Bacterianas/imunologia , Coagulase/imunologia , Infecções Estafilocócicas/prevenção & controle , Staphylococcus aureus/imunologia , Adjuvantes Imunológicos/administração & dosagem , Animais , Anticorpos Antibacterianos/sangue , Antígenos de Bactérias/imunologia , Clonagem Molecular , Feminino , Camundongos , Camundongos Endogâmicos BALB C , Distribuição Aleatória , Infecções Estafilocócicas/microbiologiaRESUMO
Defects in the photoreceptor-specific gene aryl hydrocarbon receptor interacting protein-like 1 (Aipl1) are associated with Leber congenital amaurosis (LCA), a childhood blinding disease with early-onset retinal degeneration and vision loss. Furthermore, Aipl1 defects are characterized at the most severe end of the LCA spectrum. The rapid photoreceptor degeneration and vision loss observed in the LCA patient population are mimicked in a mouse model lacking AIPL1. Using this model, we evaluated if gene replacement therapy using recent advancements in adeno-associated viral vectors (AAV) provides advantages in preventing rapid retinal degeneration. Specifically, we demonstrated that the novel self-complementary Y733F capsid mutant AAV2/8 (sc-Y733F-AAV) provided greater preservation of photoreceptors and functional vision in Aipl1 null mice compared with single-stranded AAV2/8. The benefits of sc-Y733F-AAV were evident following viral administration during the active phase of retinal degeneration, where only sc-Y733F-AAV treatment achieved functional vision rescue. This result was likely due to higher and earlier onset of Aipl1 expression. Based on our studies, we conclude that the sc-Y733F-AAV2/8 viral vector, to date, achieves the best rescue for rapid retinal degeneration in Aipl1 null mice. Our results provide important considerations for viral vectors to be used in future gene therapy clinical trials targeting a wider severity spectrum of inherited retinal dystrophies.