Generation of Retinal Organoid-Retinal Pigment Epithelium Cocultures

The discovery of human induced pluripotent stem cell (hiPSC)-derived retinal organoids (ROs) provided a new avenue for retinal research to scientists worldwide. ROs are a unique model system composed of main neuroretinal cell types with a similar structure to the native tissue. However, they have notable drawbacks, including underdeveloped photoreceptor outer segments (OSs) and the lack of physiological contact between photoreceptors and retinal pigment epithelium (RPE) cells. Consequently, the applications of ROs as a model for disorders affecting photoreceptor-RPE system, such as retinitis pigmentosa (RP), are restricted. The objective of this thesis was to discover effective methods to overcome this constraint. The RO differentiation protocol was modified to produce RPE cells (bRPE) as a byproduct. This involved extended culturing of the hiPSCs that had not matured into ROs, using nicotinamide to promote RPE development. The adapted protocol was tested in multiple differentiations of three hiPSC lines derived from healthy donors. The bRPE cells exhibited typical RPE morphology and characteristic protein expression in immunofluorescent stainings. In qRT-PCR analysis, three RPE markers were significantly upregulated compared to undifferentiated hiPSCs. Based on the results, it can be concluded that the modified differentiation protocol successfully and consistently produced RPE cells. This thesis evaluated two approaches to coculture ROs with bRPE cells, intending to promote physical contact between photoreceptors and bRPE. Two coculture experiments were conducted in suspension while testing the first method. Many bRPE cells detached from the RO surface within a few days despite close contact being established between them. A second approach involved culturing under adherent conditions. In this experiment, the bRPE cells were grown as a monolayer on transwell filter inserts. ROs temporarily adhered to the bRPE but detached during harvest. Still, cocultured bRPE stained positive for rhodopsin, a photoreceptor OS marker, providing evidence of a functional connection between the two. Although our experiments did not show an improved OS structure in cocultures, they confirmed a physiological interaction between ROs and bRPE. This provides a solid foundation for future RO-RPE coculture experiments. Finally, this study investigated hiPSC clones treated with CRISPR/Cas9 to induce novel mutations in the Retinitis Pigmentosa 1 Axonemal Microtubule Associated (RP1) gene. Mutations in the RP1 gene often lead to RP, a genetic condition linked to the degeneration of photoreceptors. One hiPSC clone was found to carry a novel homozygous mutation, which is believed to result in the RP1-knockout phenotype. Future differentiation of ROs from this hiPSC clone may provide a framework for studying the functions of the RP1 protein and RP pathogenesis

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

Serum Insulin-like Growth Factor-Binding Protein-2 as a Prognostic Factor for COVID-19 Severity

Insulin-like growth factor-binding protein (IGFBP)-2 is a regulator of anabolic pathways, which become inactivated in severe illness. Here, we measured the serum IGFBP-2 levels of COVID-19 patients with moderate and severe disease as well as healthy controls to identify the associations of serum IGFBP-2 levels with disease severity. Patients with severe COVID-19 had higher serum IGFBP-2 levels than those with moderate disease and healthy controls, who had similar levels. Non-survivors of COVID-19 tended to have elevated serum IGFBP-2 levels compared to survivors. Increased serum IGFBP-2 levels were observed in patients requiring dialysis and vasopressor therapy. Serum IGFBP-2 was positively correlated with procalcitonin in both patient groups. Bacterial co-infection in severe COVID-19 patients did not influence serum IGFBP-2 levels. Patients with liver cirrhosis and obesity, showing increased and decreased serum IGFBP-2 levels, respectively, were excluded from the study. The present analysis showed that higher serum IGFBP-2 levels are associated with increased disease severity in COVID-19 patients. The similarity in serum IGFBP-2 levels between patients with moderate COVID-19 and healthy controls suggests that elevated IGFBP-2 is associated with critical illness rather than SARS-CoV-2 infection itself

навчальний посібник

Конституційне право України: прагматичний курс : навч. посіб. / М. В. Афанасьєва, Ю. Ю. Бальцій, Ю. Д. Батан [та ін.] : за заг. ред. М. В. Афанасьєвої, А. А. Єзерова ; тех. ред. Ю. Д. Батан. - Одеса : Юридична література, 2017. - 256 с.Видання навчального посібника "Конституційне право України: прагматичний курс" спрямоване, передусім, врахувати потребу студентів у лаконічному та водночас комплексному викладенні навчального матеріалу, який би відповідав тематиці програми вступних випробувань з конституційного права України та відображав новітні конституційно-правові перетворення. Цінність даного посібника для вступників полягає в тому, що його структура дозволяє якнайкраще підготуватися до тестування, оскільки включає саме ті теми та питання, які включені до програми вступних випробувань. Крім того, прагматичний курс цінний для студентів 2-го курсу як опорний конспект лекцій при підготовці до практичних занять, а аткож до іспиту з конституційного права України. Навчальний посібник стане корисним при підготовці до державного екзаменаційного іспиту з конституційного права України для студентів 4-го курсу