Volume 9 (2021): Issue 1 (March 2021)

Granulosa cells play an important role in follicle development, maturation, and atresia. They are a cellular source of the two most important ovarian steroids, namely, estradiol and progesterone and are also crucial for bidirectional communication with the oocyte, thus being involved in the regulation of its growth, development and function. Growing body of evidence suggests that granulosa cells cultured in vitro display stemness and transdifferentiation potential. Together with the fact that they can be easily collected during IVF procedures, these properties of GCs may be of particular interest for both regenerative medicine and transplantology. Establishment of in vitro cell culture and its thorough characterization, including molecular, is crucial for future potential utilization of human granulosa cells in design of engineered tissue grafts or cell-based therapies, in particular targeted at female infertility. Nevertheless, the transcriptomic alterations which may occur during in vitro culture of granulosa cells are still largely uncharacterized. The aim of this study was to examine expression changes of three genes encoding histone demethylases which serve as transcription coactivators in short term in vitro cell culture of human granulosa cells. The study groups consisted of 14 patients, aged 18–40 years undergoing in vitro fertilization (IVF). Expression level assessment was performed after 24 h, 48 h, 72 h, 96 h, 120 h, 144 h and 168 h of in vitro primary cell culture utilizing RT-qPCR technique. Upregulation of PHF2 expression in all time points of the culture was observed, whereas the tendency of JHDM1D and PHF8 was mainly to decrease in expression level. Further study on a larger population would be required in order to confirm the presented tendencies.

Running title: Expression pattern of selected histone demethylases in human granulosa cells

Human cumulus cells (CCs) play a key role in the regulation of ovarian follicle maturation and oocyte fertilization. They influence the oocyte development by transferring the various molecules via the specific gap junction proteins, also known as the connexins, which provide a direct transmembrane connection between the oocyte and CCs. The human CCs were obtained in the patients diagnosed with infertility, who underwent the procedure of the controlled ovarian stimulation, and the following in vitro fertilization to elucidate the possible involvement of the CCs in the regulation of the fertilization and oocyte aging. Collected samples were long-term cultured and harvested after 7, 15, and 30 days of cultivation. Afterward, we assessed the relative expression of the following apoptosis regulatory genes - BAX, CASP9, and TP53 - using the RT-qPCR method. We noted a decrease in the expression of all above-mentioned genes in the samples harvested after 15 and 30 days, in reference to 7 days in vitro cultured CCs. In summary, our results provide precious insight into the dynamics of changes and confirm the continuous expression of the proapoptotic genes – BAX, CASP9, and TP53 in the long-term cultured CCs.

Running title: Apoptotic gene expression in the human cumulus cells

The awareness of the widespread influence of hypertension on various organ systems is ever increasing. Changes associated with this disease can be observed in the heart, brain, kidneys, but also the organ of vision. These usual microvascular changes are defined as hypertensive retinopathy. During a funduscopic examination, abnormalities such as narrowing of arterioles, symptoms of arteriole and vein intersection, cotton wool spots, intra-retinal exudates, retinal haemorrhages, and in severe cases even swelling of the optic disc and macula. This review presents an overview of the changes at the fundus of the eye that may occur in patients with hypertension, as well as problems with the classification of hypertensive retinopathy over the years, and the development of diagnostic methods in ophthalmology and fundoscopic imaging.

Running title: The history of hypertensive retinopathy research

Telomerase activity is highly correlated to the proliferation capacity and immortality of cells. To evaluate the possibility of continuous culture, myoblasts were isolated from the Pectoralis thoracicus muscle of newborn turkeys and maintained in 2D (adherence based) and suspension cultures. Furthermore, adherent myoblasts were differentiated into myotubes. Telomerase activity was evaluated in all types of obtained cultures. The expression of telomerase related genes, including TERT1, TERT2, dyskerin, as well as myogenesis related genes, including myogenin, MyoD, MRF1 and MRF5 were measured. Telomerase bands were detected in both adherent and suspended cells, but they were not detected in samples from rat muscle. Myotube differentiation caused a significant reduction in the expression of TERT1, TERT2 and Dyskerin, while MyoD, Myogenin and MRF4 were upregulated in myotubes vs. myoblasts. Long-term culture of suspended myoblasts caused a significant increase in TERT1 levels, with no significant change in expression of myogenesis related genes. Overall, the results show that myoblasts are able to grow in suspension without losing their myogenic properties. Furthermore, upregulation of TERT1 indicates continued proliferation of myoblasts and generation of enough daughter cells necessary for in vitro meat production.

Running title: Telomerase activity and myogenic properties of cultured Turkey satellite cells

Heart failure (HF) is one of the main causes of death worldwide. Recent studies reported altered DNA methylation in failing human hearts. This may suggest a role of DNA methylation, most frequently implicated in epigenetic control, in the development of heart failure. Here, employing RT-qPCR, we characterized transcript levels for main DNA methyltransferases (DNMTs), DNMT1, DNMT3A, and DNMT3B, mediate DNA methylation, and they have different functions that complement each other during methylation. All analyzes were performed at different stages of porcine myocardial cell primary culture. In the present study we demonstrated increasing transcript expression levels for all analyzed genes during in vitro cultivation. The changes for DNMT1 and DNMT3A seem to be particularly important, where statistically significant changes were observed.

Running title: DNMTs role in cardiac muscle cell culture

Stress granules (SGs) are cytoplasmic structures found in eukaryotic cells, from yeast to human cells. They are made up of proteins, RNA and small ribosome subunits (40S). They arise as a result of the rapid shutdown of active protein biosynthesis in the cell, which is the result of the appearance of a stress factor. The mechanism of regulation of protein biosynthesis in response to stress takes place at two control nodes: (1) phosphorylation of the α subunit of the eIF2 factor as a result of the action of stress-recognizing kinases or by modulation of the mTOR pathway activity, which regulates the initiation of protein biosynthesis by the formation of a complex within the so-called cap structure. The protein arrest causes aggregation of the translation process components and other cell components (other proteins or mRNA molecules) into SGs. A lot of data indicates the active participation of SGs in metabolic processes, their control role over pro- and anti-apoptotic processes as well as in the development of cancer, neurodegenerative diseases and their defensive role in viral infections.

Running title: Stress granules in the cell

Cell encapsulation utilizing biodegradable material has promising outcomes for tissue engineering. From a long time ago, alginate has been generally utilized for drug delivery, cell transplantation and as a scaffold in biomedical applications. The aim of this study was the comparison of cell viability in the presence of two polymerizing ions: Ba²⁺ and Ca²⁺ to improvement the quality of alginate scaffold. For this purpose, WJMSCs after three passage were encapsulated in alginate scaffold in the presence of Ba²⁺ and ca²⁺. Cell viability was evaluated by WST-8 assay kit after 24, 48 and 72 hours. The results showed that encapsulated cells in the presence of Ca²⁺ had more viability than Ba²⁺. It was also found that using the WST-8 assay kit is a convenient and fast method for evaluation the viability of cells. It can be claimed that Calcl2 polymerizing solution provides more favorable conditions for cell viability compared to Bacl2 solution.

Running title: Assessing the viability of stem cells by WST-8 assay kit