Circulating MicroRNA Expression in Two Murine Models of Retinal Neovascularization

Choroidal neovascularization (CNV) is a pathologic process where blood vessels invade the retina, and is one of the hallmarks of neovascular age-related macular degeneration (nAMD). Emerging evidence suggests that epigenetic mechanisms such as microRNA regulation of gene expression, are relevant to nAMD and provide an exciting avenue of research. In this study, we performed a literature search of nAMD-related dysregulation of blood derived circulating microRNA (cmiRNA), and found that the results in between studies are largely irreconcilable. Only four cmiRNA were dysregulated in the same direction in two studies, two cmiRNA were dysregulated in opposite directions, and the majority of cmiRNA were never replicated by an independent study. We investigated cmiRNA expression in two established murine models of retinal neovascularization and inflammation: laser-induced CNV and oxygen-induced retinopathy (OIR). We theorized that two models with different external triggers and the same retinal endpoint would help identify cmiRNA which are robustly associated with retinal nAMD hallmarks. In the laser-induced CNV model, cmiRNA dysregulation was investigated in three successive experiments, and the top ten cmiRNA candidates were identified. These cmiRNA were further tested in the OIR model. Overall, two cmiRNA were dysregulated in the same direction in both models, implying that they were dysregulated in conjunction with retinal neovascularization and inflammation. Surprisingly, five cmiRNA were dysregulated in opposite directions in the murine models. We theorize that this finding indicates that cmiRNA dysregulation is strongly influenced by a disease trigger, which could be conducive for a future diagnostic application

Retinal organoid differentiation, characterization, and adaptation as a model for Retinitis Pigmentosa

Retinal organoids (ROs) are a 3-dimensional cell culture model system, which mimic the cellular composition and histoarchitecture of native retinal tissue. ROs are differentiated from human induced pluripotent stem cells (iPSC), thereby allowing in vitro investigations into 3D human tissue, comprised of a range of organotypic cell types. In this study, ROs were investigated in the context of two distinct queries: RO differentiation and development was improved, and their suitability as a model system for inherited retinal diseases with photoreceptor degeneration, was evaluated. The first query addresses four distinct projects (i-iv). (i) Three previously published RO differentiation protocols were compared, and the method which produced the highest quantity and best quality of ROs was identified. (ii) Previous reports have suggested that nonstationary culture improves RO differentiation, so the influence of an orbital shaker on RO development was evaluated. Immunocytochemistry and RNA sequencing revealed that nonstationary culture did not significantly impact RO development or maturation. (iii) A technique was developed, which improved RO photoreceptor outer segment retention during processing. (iv) A challenge which was resolved in this study, is the lack of insight into RO development past 11 months. The mRNA expression, protein expression, and histoarchitecture of up to 2-year-old ROs was analyzed, which revealed the preservation of retinal histoarchitecture. In general, photoreceptor marker expression decreased over time, but photoreceptors with pristine morphological development were still present in 2-year-old ROs. Interestingly, Mueller cell marker expression increased over time. In the second part of this study, two genetically distinct causes for retinitis pigmentosa were investigated. First, ROs were differentiated from patient iPSC with autosomal dominant mutations in the Retinitis Pigmentosa 1 Axonemal Microtubule Associated gene (adRP1), which causes adult-onset RP with degeneration of rod photoreceptors. Investigations into 4-, 5-, and 12-month-old adRP1 ROs showed normal photoreceptor development. In contrast, rod photoreceptor marker expression was significantly reduced in 1.5-year-old adRP1 ROs, indicating that long-term culture of ROs may be suitable to model adult-onset RP. Finally, CRISPR/Cas9-mediated gene editing was used to produce RP1 knockout and isogenic control iPSC, which were then differentiated to ROs. The characterization of RP1 knockout ROs is currently underway. In summary, this project highlights the versatility of ROs as a retinal model

Success and resilience factors of rural enterprises in Mexico: RENDRUS case

Objective: To identify the success and business resilience factors of rural companies, through an analysis of rural companies in Mexico, in order to contribute to the differentiation of these concepts. Methodology: The companies of the National Network for Sustainable Rural Development were used as a case study, who were asked key questions to identify their factors of success and business resilience. Results: The main success and resilience factors of these companies were identified; Permanent job creation turned out to be the main success factor and punctuality in order delivery was the most identified resilience factor. Implications: Identifying and recognizing the difference between the two factors can help companies and their leaders or executives understand and adjust their strategies based on their objectives: to be successful or to be resilient. Conclusions: There is a difference between a success factor and a business resilience factor; a success factor is a way of standing out among similar companies, while a business resilience factor is used to face adversities and continue over time.Objective: To identify the success and resilience factors of rural enterprises in Mexico in order to contribute to the theoretical and empirical differentiation of these concepts. Design/Methodology/Approach: We used the National Network for Sustainable Rural Development Companies as a case study to formulate key questions to identify their success and business resilience factors. Results: Permanent job creation turned out to be the main success factor, and timely delivery of the product was the main resilience factor. Study Limitations/Implications: Recognizing the difference between success and resilience factors can help rural enterprises, their leaders, and decision-makers in the rural sector to understand and adjust their operating strategies based on their objectives. Findings/Conclusions: A success factor is a way to stand out among similar rural enterprises, while a business resilience factor serves to face adversity and continue over time

Transient Retention of Photoreceptor Outer Segments in Matrigel-Embedded Retinal Organoids

Retinal organoids (ROs) are three-dimensional retinal tissues, which are differentiated in vitro from induced pluripotent stem cells (iPSC), ultimately forming all main retinal cell types under defined culture conditions. ROs show several highly specialized retinal features, including the outgrowth of photoreceptor outer segments (OSs). In vivo, the photoreceptor OSs are enveloped and maintained by protrusions of retinal pigment epithelium (RPE) cells, the so-called apical microvilli, while ROs fail to recapitulate this critical interaction in culture development. Here, we define specific co-culture conditions aiming to compensate for the missing physical proximity of RPE and OSs in RO development. Accordingly, functional RPE cells and ROs were differentiated simultaneously from the same iPSC clone, the former resulting in byproduct RPE or bRPE cells. While some co-culture approaches indicated a temporary functional interaction between bRPE and RO photoreceptors, they did not improve the photoreceptor histoarchitecture. In contrast, embedding ROs in a basement membrane extract without bRPE cells showed a robust improvement in the rate of photoreceptor OS retention. RO embedding is a quick and easy method that greatly enhances the preservation of photoreceptor OSs, an important structure for modelling retinal diseases with the involvement of photoreceptors

A Circulating MicroRNA Profile in a Laser-Induced Mouse Model of Choroidal Neovascularization

Funding: This research was funded by the Deutsche Forschungsgemeinschaft (GR5065/1-1). Author Contributions: Conceptualization, F.G. and B.H.F.W.; Data curation, T.S.; Formal analysis, P.B., M.K., A.A., and T.S.; Funding acquisition, C.K. and F.G.; Investigation, M.K. and B.H.F.W.; Methodology, C.K. and A.A.;Project administration, B.H.F.W.; Resources, M.K., A.A., T.L., and F.G.; Software, C.K. and T.S.; Supervision, T.L., F.G., and B.H.F.W.; Validation, P.B.; Visualization, C.K.; Writing—original draft, C.K. and P.B.; Writing—review & editing, B.H.F.W. All authors have read and agreed to the published version of the manuscript.Peer reviewedPublisher PD

La comprensión lectora en alumnos de 3er grado de primaria

Licenciatura en Educació

Retinal organoid differentiation methods determine organoid cellular composition

Aim: We aimed to compare the quantity and quality of aging retinal organoids generated by applying three distinct differentiation protocols for human-derived induced pluripotent stem cells (hiPSC).Methods: hiPSC were differentiated to retinal organoids using a 3D technique (Method 1) and a 3D-2D-3D technique (Method 2), the latter modified by the addition of BMP4 (Method 3). To investigate the retinal organoid quantity, we counted the number of retinal domains, precursors to organoids during differentiation. The retinal organoid quality was evaluated by immunostaining for markers of different retinal cell types in whole cryosections after days 85, 120, and 200 in culture.Results: Method 3 produced strikingly more retinal domains per differentiation (65 ± 27) than Methods 1 (12.3 ± 11.2) and 2 (6.3 ± 6.7). Furthermore, retinal organoids differentiated with Method 3 contained significantly more CRX-positive photoreceptors and BRN3A-positive ganglion cells after 85 days in culture, compared to Methods 1 and 2. After 200 days in culture, the retinal organoids differentiated with Method 3 showed proper maturation, as demonstrated by the expression of mature rod and cone photoreceptor markers.Conclusion: This study demonstrates that the retinal organoid differentiation method can significantly impact the cellular composition of retinal organoids at various time points of development

An Eye on Age-Related Macular Degeneration: The Role of MicroRNAs in Disease Pathology

Age-related macular degeneration (AMD) is the primary cause of blindness in developed countries, and is the third leading cause worldwide. Emerging evidence suggests that beside environmental and genetic factors, epigenetic mechanisms, such as microRNA (miRNA) regulation of gene expression, are relevant to AMD providing an exciting new avenue for research and therapy. MiRNAs are short, non-coding RNAs thought to be imperative for coping with cellular stress. Numerous studies have analyzed miRNA dysregulation in AMD patients, although with varying outcomes. Four studies which profiled dysregulated circulating miRNAs in AMD yielded unique sets, and there is only minimal overlap in ocular miRNA profiling of AMD. Mouse models of AMD, including oxygen-induced retinopathy and laser-induced choroidal neovascularization, showed similarities to some extent with miRNA patterns in AMD. For example, miR-146a is an extensively researched miRNA thought to modulate inflammation, and was found to be upregulated in AMD mice and cellular systems, but also in human AMD retinae and vitreous humor. Similarly, mir-17, miR-125b and miR-155 were dysregulated in multiple AMD mouse models as well as in human AMD plasma or retinae. These miRNAs are thought to regulate angiogenesis, apoptosis, phagocytosis, and inflammation. A promising avenue of research is the modulation of such miRNAs, as the phenotype of AMD mice could be ameliorated with antagomirs or miRNA-mimic treatment. However, before meaningful strides can be made to develop miRNAs as a diagnostic or therapeutic tool, reproducible miRNA profiles need to be established for the various clinical outcomes of AMD