Volume 7 (2019): Issue 4 (December 2019)

Granulosa cells (GCs) provide the microenvironment necessary for the development of the follicle and the maturation of the oocyte. GCs are associated with reproductive system function and the maintenance of pregnancy by participating in the synthesis of steroid hormones. Many authors point to new ways of using GCs in regenerative medicine and indicate the significant plasticity of this cell population, suggesting that GCs can undergo a transdifferentiation process. Employing primary in vitro cell cultures and high-throughput transcriptome analysis via Affymetrix microarrays, this study describes groups of genes associated with enzymatic reactions. 52 genes were identified belonging to four gene ontology biological process terms (GO BP): “coenzyme biosynthetic process”, “coenzyme metabolic process”, “cofactor biosynthetic process” and “cofactor metabolic process”. All identified genes showed reduction in the level of mRNA expression during long-term in vitro cultivation. Significanthe transcriptomic profile variability was exhibited for the genes (ELOVL5, ELOVL6 and GPAM) involved in enzymatic regulation of fatty acid metabolism.

Running title: Enzymatic regulation in granulosa cells

The oviduct play a crucial role in reproductive process, through facilitating successful embryo growth and conception. Oviduct activity is orchestrated by various factors, depending on cyclic dynamics, which crucially affect the success of reproductive function. The morphological modifications of oviducts in response to the female reproductive cycle are well established. However, detailed characterization at the molecular level is still needed. The present study, employed primary in vitro cell cultures and high-throughput transcriptome analysis via an Affymetrix microarray approach, described nucleotide, ribonucleotide and ribonucleoside binding patterns at a molecular level in oviduct epithelial cells (OECs). 222 genes were targeted belonging to four gene ontology biological process terms (GO BP): “adenyl nucleotide binding”, “adenyl ribonucleotide binding”, “ribonucleotide binding”, “ribonucleoside binding”, which showed the greatest variability in the level of mRNA expression during of long-term cultivation. In this group of genes, special attention was paid to those showing the greatest variability in relation to the reference measurement, including OASL, PIM1, ACTA2 and ABCA1.

Running title: Oviductal nucleotide and nucleoside binding patterns

Stem cells possess unique properties, such as self-renewal ability or differentiation capacity into more specialized cells, which makes them particularly relevant for regenerative medicine and cellular therapies. Although embryonic stem cells (ESCs) are capable of differentiation into all cell lineages, their utilization is associated with ethical concerns since they are obtained from embryos. Furthermore, ESCs may form teratomas or cause immune rejection in the clinical setting. Therefore, an effort has been made to utilize stem cells derived from adult tissues, especially mesenchymal stem cells (MSCs). A particularly attractive source of MSCs is the human umbilical cord, which is typically discarded after birth and considered a medical waste, therefore the acquisition of the cells is not associated with any health risk for a patient. Moreover, umbilical cord-derived MSCs do not express MHCII, thus they exhibit reduced immunogenicity. MSCs have been isolated from all compartments of umbilical cord, however the Wharton’s jelly-derived MSCs (WJ-M-SCs) are the most clinically utilizable. There are two techniques of UC-MSCs isolation: the enzymatic and explant procedures. The explant method involves cell outgrowth of tissue pieces placed into plastic culture vessel after mechanical splitting, whereas the enzymatic technique involves minced tissue digestion in an enzymatic solution. In vitro culture conditions of the isolated cells are highly variable among the researchers, however the most commonly performed molecular assays are homogenous and include: RT-qPCR, flow cytometry and immunocytochemistry.

Running title: Human umbilical cord stem cells isolation, cultivation and genetic profiling

Macrophages, detected as CD68+ cells, are considered to have marked contribution to aorto-coronary grafts disease. The purpose of this study was to find any ultrastructural differences in CD68+ cells between arterial and venous aorto-coronary grafts.

The surplus segments of radial artery (RA) and saphenous vein (SV) were obtained from 50 patients with the mean age of 63.4±9.2 years who undergo elective coronary artery bypass grafting (CABG). The vascular segments were analyzed by means of both light (to assess number and distribution of macrophages within their walls) and transmission electron microscopy (to evaluate ultrastructure of CD68+ cells in the vessel layers).

Histological analysis revealed that not only more macrophages (median (25th; 75th percentile)) were found on the transverse sections of veins (95 (67; 135)) than arteries (66 (43; 108)) (p<0.05) but also at least of 50% of them were found in the tunica intima and tunica media in SV while only 30% in RA. TEM studies showed that biological activity of macrophages depended on CD68+ location and was irrespective of the vessel type. Those found in the tunica intima and tunica media presented ultrastructure typical for active cells rich in numerous lysosomes, well developed rough endoplasmic reticulum and Golgi apparatus whereas adventitial macrophages for unreactive residual cells.

Ultrastructural characteristics of both forms of macrophages infiltrating wall of aorto-coronary grafts is similar irrespective of the vessel type. More active cells in the inner layers of the venous conduits may contribute to their inferior outcomes compared to the arteries.

Running title: Macrophages and aorto-coronary grafts

Granulosa cells (GCs) are important component of the follicle, a principal functional unit of the ovary. They undergo highly dynamic changes during folliculogenesis and play a vital role in oocyte’s maturation. Recently, it has been shown that GCs also exhibit stem cell properties, since they express OCT-4, Nanog, Sox-2, which are markers of pluripotency, as well as several mesenchymal stem cell markers, such as CD29, CD44, CD90, CD105, CD117 or CD166. In addition, GCs are able to differentiate towards neurogenic, chondrogenic and osteogenic lineages. Since the use of embryonic stem cells in regenerative medicine is burdened with ethical concerns and the risk of immune rejection or teratoma formation, adult stem cells are emerging as a promising alternative. GCs especially seem to provide a promising source of stem cells, since they are easily obtainable during assisted reproduction techniques. In order to better understand the genetic changes taking place in proliferating granulosa cells cultured in vitro, we isolated GCs from 40 prepubertal gilts and cultured them in vitro for 168 h. After 24, 48, 72, 96, 120, 144 and 168 h of cultivation the total RNA was extracted, reverse transcription was conducted and RT-qPCR reaction was performed. We observed that CD44, CD90 and IGF1 were upregulated after the cultivation, whereas CD105 and LIF were downregulated. Collectively, our results confirm stemness potential of porcine GCs and provide an insight into the transcriptome changes during in vitro cultivation.

Running title: Molecular stemness markers in porcine granulosa cells