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Heart failure remains a major cause of death worldwide. There is a need to establish new management options as current treatment is frequently suboptimal. Clinical approaches based on autologous stem cell transplant is potentially a good alternative. The heart was long considered an organ unable to regenerate and renew. However, several reports imply that it may possess modest intrinsic regenerative potential. To allow for detailed characterization of cell cultures, whole transcriptome profiling was performed after 0, 7, 15, and 30 days of in vitro cell cultures (IVC) from the right atrial appendage and right atrial wall utilizing microarray technology. In total, 4239 differentially expressed genes (DEGs) with ratio > abs |2| and adjusted p-value ≤ 0.05 for the right atrial wall and 4662 DEGs for the right atrial appendage were identified. It was shown that a subset of DEGs, which have demonstrated some regulation of expression levels with the duration of the cell culture, were enriched in the following GO BP (Gene Ontology Biological Process) terms: “stem cell population maintenance” and “stem cell proliferation”. The results were validated by RT-qPCR. The establishment and detailed characterization of in vitro culture of myocardial cells may be important for future applications of these cells in heart regeneration processes.

Maintaining of female fertility is strictly dependent on proper hormonal regulation. Granulosa cells (GCs) are components of ovarian follicles, and they are important in paracrine regulation of the ovary. Preovulatory follicle GCs are responsible for production of estrogens to the ovary microenvironment and lead to the LH surge. Proper functioning of GCs is necessary to ensure appropriate conditions for oocyte development, maturation, ovulation and its release to the oviduct. Long-term in vitro culture of GCs show significant stem-like characteristics. Understanding the molecular processes underlying GCs differentiation towards different cell lineages may reveal other possible stem cell markers. A transcriptomic analysis of short-term primary in vitro cultured GCs, which were isolated from porcine preovulatory follicles was the major focus of the study. The ontological groups herby considered are associated with endodermal and epithelial tissues. Results were and compare to freshly isolated GC cells. 6 the most reduced expression: HSD17B1, DAPL1, NEBL, MAL2, DAB1, ITM2A were chosen for analysis. These genes have been response for processes associated with GCs development and differentiation towards endodermal and epithelial tissues, which make them important for further consideration.

Stem cells are an important subject of research, and are increasingly used in the treatment of various diseases. Due to the development of advanced in vitro techniques, they have become an integral part of modern medicine. The sources of human stem cells are primarily bone marrow and adipose tissue, although non – embryonic stem cells are also scattered throughout the body. Notably, recent research has focused on stem cells found in the oral cavity, both in the dental pulp and oral mucosa. Furthermore, isolation of stem cells from umbilical cord blood is also becoming increasingly popular, while wharton’s jelly and amniotic fluid also seem to be an interesting source of stem cells. The safety and efficacy of stem cells use can be established by animal studies, which are a key element of preclinical research. Mouse, rat and pig models allow for testing of stem cell therapies. Recent studies primarily use mesenchymal stem cells such as mouse – adipose derived mesenchymal stem cells and mouse and rat hematopoietic stem cells. Great hope for future therapies is the use of bioengineering to program cells into induced stem cells, which have the biggest ability for differentiation and transdifferentiation, which carries no risk of teratogenesis. Stem cells are used in many areas of medicine, especially in regenerative medicine, with a growing interest in orthopedics and in the treatment of heart failure. Mesenchymal stem cells are the most used stem cell type, which despite their limited ability to differentiate, give great therapeutic results, mainly due to their immunomodulating effect. Recent studies have even shown that the use of mesenchymal stem cells may be useful in the treatment of COVID-19. Moreover, Research on the use of mesenchymal stem cells in the treatment of Crohn’s disease, acute-graft-versus-host disease and type I diabetes are also promising. The aim of the current review is to present and systematize current knowledge about stem cells, their use and related in vitro research. Running title: Research and use of human stem cells

The mature human placenta and umbilical cord are rich sources of perinatal mesenchymal stem cells (MSCs). Both cell populations have similar characteristics and cellular properties. Each population can differentiate into multiple mesenchymal subpopulations and retain their self-renewal capacities. Perinatal stem cells can be isolated from tissues obtained from the planned cesarean sections and vaginal deliveries. Their isolation is relatively easy, making them readily available for implementation in various in vitro studies and clinical trials. Their differentiation abilities could be used in advanced regenerative medicine protocols to form new bone, cartilage, or tendons. Moreover, their unique anti-inflammatory and immunomodulatory properties have been implemented in the experimental treatment of multiple autoimmune and degenerative diseases. Numerous phase I/II clinical trials confirmed the safety of perinatal MSCs injections and infusions, albeit the efficacy of those cellular therapies should be investigated in the subsequent large-scale randomized trials. Running title: Clinical applications of the perinatal mesenchymal stem cells

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

The interest in stem cell research continuously increased over the last decades, becoming one of the most important trends in the 21st century medicine. Stem cell-based therapies have a potential to become a solution for a range of currently untreatable diseases, such as spinal cord injuries, type I diabetes, Parkinson’s disease, heart disease, stroke, and osteoarthritis. Hence, this study, based on canine material, aims to investigate the molecular basis of adipose-derived stem cell (ASC) differentiation into chondrocytes, to serve as a transcriptomic reference for further research aiming to introduce ASC into treatment of bone and cartilage related diseases, such as osteoarthritis in veterinary medicine. Adipose tissue samples were harvested from a canine specimen subjected to a routine ovariohysterecromy procedure at an associated veterinary clinic. The material was treated for ASC isolation and chondrogenic differentiation. RNA samples were isolated at day 1 of culture, day 30 of culture in unsupplemented culture media, and day 30 of culture in chondrogenic differentiation media. The resulting RNA was analyzed using RNAseq assays, with the results validated by RT-qPCR. Between differentiated chondrocytes, early and late cultures, most up- and down-regulated genes in each comparison were selected for further analysis., there are several genes (e.g., MMP12, MPEG1, CHI3L1, and CD36) that could be identified as new markers of chondrogenesis and the influence of long-term culture conditions on ASCs. The results of the study prove the usefulness of the in vitro culture model, providing further molecular insight into the processes associated with ASC culture and differentiation. Furthermore, the knowledge obtained could be used as a molecular reference for future in vivo and clinical studies.

ADSCs are readily accessible and widely available. Isolated through a minimally invasive procedure from adipose depots, they can be found at diverse body location, where they served various functions, including energy homeostasis. They can be obtained upon surgeries from otherwise waste tissues, like after excision of fat tissue or liposuction. In addition, due to the possibility to isolate many ADSCs, in vitro proliferation can be performed in a short time period, resulting in cells showing more predictable results[1]. For this study ADSCs were obtained from waste material following routing sterilization procedures of dogs. This study aimed to analyse the expression of MSC specific markers before and after in vitro differentiation of ASCs. Three positive and three negative markers were analysed, CD105, CD73, CD90, CD34, CD14 and CD45. There were significant differences detected in the expression of all of the genes, with most of them exhibiting notable downregulation. The only exception, CD14 showed major upregulation after the process of differentiation. These changes confirm the success of differentiation, as well as suggest that this process significantly lowers the stem-like ability of ASCs. This knowledge should serve as a reference for further molecular and clinical studies, possibly aiding the understanding of the internal mechanisms governing the differentiation and stemness of ASCs, to enable their widespread and safe application in regenerative medicine. Running title: Mesenchymal markers during ASC osteogenic differentiation

The human granulosa cells (GCs) surround the oocyte and form the ovarian follicle’s proper architecture. These sub-populations include mural granulosa cells, antral granulosa cells, and cumulus granulosa cells. Their main functions are to support the oocyte’s growth (cumulus granulosa cells) and estradiol production (mural granulosa cells). After ovulation, the granulosa cells transform into the luteal cells of the corpus luteum and produce progesterone. Our study investigated the expression profile of three genes: TGFB1, CD105, and FSP1 during a 7-day in vitro culture. The analysis was conducted using the RT-qPCR technique. Changes in the expression of CD105 and FSP1 could be observed during the 7-day in vitro culture. In the case of TGFB, the expression remained at a similar level, with no statistically significant differences observed. Running title: Expression of TGFB1, CD105 and FSP1 in granulosa cells

Genes influencing oocyte maturation may be valuable for predicting their developmental potential, as well as discerning the mechanistic pathways regulating oocyte development. In the presented research microarray gene expression analysis of immature and in vitro matured porcine oocytes was performed. Two groups of oocytes were compared in the study: before (3 × n = 50) and after in vitro maturation (3 × n = 50). The selection of viable oocytes was performed using the brilliant cresyl blue (BCB) test. Furthermore, microarrays and RT-qPCR was used to analyze the transcriptome of the oocytes before and after IVM. The study focused on the genes undergoing differential expression in two gene-ontology groups: “Cellular response to hormone stimulus” and “Cellular response to unfolded protein”, which contain genes that may directly or indirectly be involved in signal transduction during oocyte maturation. Examination of all the genes of interest showed a lower level of their expression after IVM. From the total number of genes in these gene ontologies ten of the highest change in expression were identified: FOS, ID2, BTG2, CYR61, ESR1, AR, TACR3, CCND2, EGR2 and TGFBR3. The successful maturation of the oocytes was additionally confirmed with the use of lipid droplet assay. The genes were briefly described and related to the literature sources, to investigate their potential roles in the process of oocyte maturation. The results of the study may serve as a basic molecular reference for further research aimed at improving the methods of oocyte in vitro maturation, which plays an important role in the procedures of assisted reproduction.

The primary function of ovarian granulosa cells (GCs) is the support of oocytes during maturation and development. Molecular analyses of granulosa cell-associated processes, leading to improvement of understanding of the cell cycle events during the formation of ovarian follicles (folliculogenesis), may be key to improve the in vitro fertilization procedures. Primary in vitro culture of porcine GCs was employed to examine the changes in the transcriptomic profile of genes belonging to “cell cycle”, “cell division”, “cell cycle process”, “cell cycle phase transition”, “cell cycle G1/S phase transition”, “cell cycle G2/M phase transition” and “cell cycle checkpoint” ontology groups. During the analysis, microarrays were employed to study the transcriptome of GCs, analyzing the total RNA of cells from specific periods of in vitro cultures. This research was based on material obtained from 40 landrace gilts of similar weight, age and the same living conditions. RNA was isolated at specific timeframes: before the culture was established (0 h) and after 48 h, 96 h and 144 h in vitro. Out of 133 differentially expressed genes, we chose the 10 most up-regulated (SFRP2, PDPN, PDE3A, FGFR2, PLK2, THBS1, ETS1, LIF, ANXA1, TGFB1) and the 10 most downregulated (IGF1, NCAPD2, CABLES1, H1FOO, NEK2, PPAT, TXNIP, NUP210, RGS2 and CCNE2). Some of these genes known to play key roles in the regulation of correct cell cycle passage (up-regulated SFRP2, PDE3A, PLK2, LIF and down-regulated CCNE2, TXNIP, NEK2). The data obtained provide a potential reference for studies on the process of mammalian folliculogenesis, as well as suggests possible new genetic markers for cell cycle progress in in vitro cultured porcine granulosa cells.

Simple Summary The extracellular matrix (ECM) is involved in many physiological processes that occur in the ovary and affect reproduction in animals and humans. The ECM has been shown to significantly affect folliculogenesis, ovulation, and corpus luteum formation. This is mainly due to the involvement of ECM in intercellular signaling. In the present study, we report the gene expression profile of porcine granulosa cells during their primary in vitro culture. The genes presented are related to ECM formation but also to cadherins and integrins that influence intercellular dialogue. During the study, it was shown that most of the genes were upregulated. A detailed understanding of the expression of genes such as POSTN, CHI3L1, CAV-1, IRS1, DCN in in vitro culture of granulosa cells may provide a basis for further studies on the molecular mechanisms occurring within the ovary. Knowledge of ECM-related gene expression within granulosa cells can also be used to study the recently discovered stemness of these cells. Moreover, the presented data may serve for the development of assisted reproduction techniques, which, especially in vitro, are becoming increasingly common. Abstract The extracellular matrix (ECM) in granulosa cells is functionally very important, and it is involved in many processes related to ovarian follicle growth and ovulation. The aim of this study was to describe the expression profile of genes within granulosa cells that are associated with extracellular matrix formation, intercellular signaling, and cell–cell fusion. The material for this study was ovaries of sexually mature pigs obtained from a commercial slaughterhouse. Laboratory-derived granulosa cells (GCs) from ovarian follicles were cultured in a primary in vitro culture model. The extracted genetic material (0, 48, 96, and 144 h) were subjected to microarray expression analysis. Among 81 genes, 66 showed increased expression and only 15 showed decreased expression were assigned to 7 gene ontology groups “extracellular matrix binding”, “extracellular matrix structural constituent”, “binding, bridging”, “cadherin binding”, “cell adhesion molecule binding”, “collagen binding” and “cadherin binding involved in cell-cell adhesion”. The 10 genes with the highest expression (POSTN, ITGA2, FN1, LAMB1, ITGB3, CHI3L1, PCOLCE2, CAV1, DCN, COL14A1) and 10 of the most down-regulated (SPP1, IRS1, CNTLN, TMPO, PAICS, ANK2, ADAM23, ABI3BP, DNAJB1, IGF1) were selected for further analysis. The results were validated by RT-qPCR. The current results may serve as preliminary data for further analyses using in vitro granulosa cell cultures in assisted reproduction technologies, studies of pathological processes in the ovary as well as in the use of the stemness potential of GCs.

Modern science is becoming increasingly committed to environmentally friendly solutions, mitigating the impact of the developing human civilisation on the environment. One of the leading fields aimed at sustainable agriculture is in vitro meat production. Cellular agriculture aims to provide a source of animal-free meat products, which would decrease worldwide nutritional dependency on animal husbandry, thereby reducing the significant impact of this industry on Earth’s climate. However, while some studies successfully produced lab-based meat on a small scale, scalability of this approach requires significant optimisation of the methodology in order to ensure its viability on an industrial scale. One of the methodological promises of in vitro meat production is the application of cell suspension-based bioreactors. Hence, this study focused on a complex transcriptomic comparison of adherent undifferentiated, differentiated and suspension-cultured myosatellite cells, aiming to determine the effects of different culture methods on their transcriptome. Modern next-generation sequencing (RNAseq) was used to determine the levels of transcripts in the cultures’ cell samples. Then, differential expression and pathway analyses were performed using bionformatical methods. The significantly regulated pathways included: EIF2, mTOR, GP6, integrin and HIFα signalling. Differential regulation of gene expression, as well as significant enrichment and modulation of pathway activity, suggest that suspension culture potentially promotes the ex vivo-associated loss of physiological characteristics and gain of plasticity. Therefore, it seems that suspension cultures, often considered the desired method for in vitro meat production, require further investigation to fully elucidate their effect on myosatellite cells and, therefore, possibly enable their easier scalability to ensure suitability for industrial application.

The oviduct is a key organ responsible for ultimate oocytes maturation, transport of gametes, sperm capacitation, fertilization, as well as early embryo development. Its innermost layer, oviductal epithelium, represents a highly dynamic structure which undergoes changes in response to different physiological and pathological processes. Previously, the expression profile of genes involved in several important processes in porcine oviductal epithelial cells (OECs) during long-term primary in vitro culture. The present study further characterizes the porcine OECs model using Affymetrix microarray assay and it analyzes gene expression changes observed on the 7th, 15th and 30th day of culture. 25 genes belonging to “coenzyme metabolic process”, “cofactor biosynthetic process” and “cofactor metabolic process” GO BP terms were differentially expressed in culture. The most up-regulated genes were ALDH1L2, P2RX7, PANK1, ACSS2, SCD, AASS and PDK3. In contrast, several genes appeared to be significantly down-regulated, e.g. ACSL4 and HAAO. Considering the biological roles of the most regulated genes, it can be concluded that these changes may indicate the increased metabolic and proliferation activity of studied cells in primary in vitro culture. Running title: Cofactor and coenzyme metabolism in porcine oviductal epithelial cells

Alterations in cells depend on their genetic material, its activation and translation of the products. The genes responsible for the cell cycle processes and apoptosis of porcine oviductal cells have been presented in our study. The processes occurring in the reproductive system of females are extremely complex and require in-depth knowledge. Thanks to in vitro studies on the fallopian tube epithelium cells, we can get closer to understanding the biochemical and morphological changes occurring in mammalian organisms. Our research was conducted on fallopian tubes obtained from commercially bred pigs and its aim was to assess the expression profile of genes responsible for the most important processes of cellular life. Cell cultures were carried out for 30 days, with the obtained cells subjected to molecular analysis. We have shown significant regulation of “cell death” and “cell cycle” genes, some of which are related to the reproductive system. The alterations in transcriptomic profile and mutual relations between the genes were analyzed and related to the literature findings. The knowledge gained could help in identifying new potential markers of the in vitro occurrence of processes described by the ontology groups of interest. Running title: pig, oocytes, microarray assays, in vitro maturation (IVM)

Oxygen metabolism is crucial in establishing successful pregnancy, since excessive amount of reactive oxygen species (ROS) may exert deleterious effects on the developing embryo. There are several defense mechanisms against oxidative stress in the female reproductive tract, including production of antioxidant enzymes by oviductal epithelial cells (OECs). Undoubtedly, OECs play major part in female fertility and may also serve as an in vitro model of the oviduct. Therefore, the aim of this study was to investigate the expression of genes involved in oxygen metabolism. We have isolated OECs from oviducts of crossbred gilts (n=45) and maintained their in vitro culture for 30 days, collecting their RNA at days 1, 7, 15 and 30. The gene expression was determined with the use of Affymetrix® Porcine Gene 1.1 ST Array Strip. Our results revealed 166 differentially expressed genes belonging to four ontology groups: „cellular response to oxidative stress”, “cellular response to oxygen-containing compound”, “cellular response to oxygen levels” and “cellular response to reactive oxygen species”, most of which are also involved in other major processes in the organism. However, our findings provide a valuable insight into porcine reproductive biology and may be utilized in optimization of assisted reproduction techniques. Running title: Genes involved in oxygen metabolism in oviductal epithelial cells

Among many factors, the epithelium lining the oviductal lumenis very important for the development of the oocyte and its subsequent fertilization. The oviductal epithelium is characterized by the presence of ciliary cells, supporting the movement of cumulus-oocyte complexes towards the uterus. By interacting with the semen, the epithelium of the fallopian tube makes the sperm acquire the ability to fertilize. So far, the exact molecular mechanisms of these changes have not been known. Hence, understanding the metabolism of oviduct epithelial cells and the level of expression of individual groups of genes seems to be a way to deepen the knowledge about the broadly understood reproduction. In our research, we decided to culture oviductal epithelial cells (OECs) in vitro for a long period of time. After 24h, 7, 15 and 30 days, the OECs were harvested, with their RNA isolated. Transcriptomic changes were analyzed using microarrays. The “cellular response to lipid” group was represented by the following genes: MUC1, CYP24A1, KLF4, IL24, SNAI2, CXCL10, PPARD, TNC, ABCA10, while the genes belonging to the “cellular lipid metabolic processes” were: LIPG, ARSK, ACADL, FADS3, P2RX7, ACSS2, PPARD, KITLG, SPTLC3, ERBB3, KLF4, CRABP2. Additionally, PPARD and ACADL were members of the “fatty acid beta-oxidation” ontology group. Our study describes genes that are not directly related to fertility processes. However, significant changes in their expression in in vitro cultured OECs may indicate their usefulness as markers of OECs’ physiological processes. Running title: Fatty acids changes in porcine oviductal epithelial cells in in vitro cultivation

Oxygen metabolism has an important role in the normal functioning of reproductive system, as well as the pathogenesis of female infertility. Oxidative stress seems to be responsible for the initiation or development of reproductive organ diseases, including polycystic ovary syndrome, endometriosis, preeclampsia, etc. Given the important role of maintaining balance between the production of ROS and antioxidant defence in the proper functioning of reproductive system, in the present study we aimed to analyse the expression of genes related to oxygen metabolism in porcine oviductal epithelial cells during long-term in vitro culture. The oviducts were collected from 45 crossbred gilts at the age of approximately nine months that displayed at least two regular oestrous cycles. The oviductal endothelial cells were isolated by enzymatic digestion to establish long-term primary cultures. Gene expression changes between 7, 15 and 30 daysof culturewere analysed with the use ofwhole transcriptome profiling by Affymetrix microarrays. The most of the “cellular response to oxidative stress” genes were upregulated. However, we did not observe any main trend in changes within the “cellular response to oxygen-containing compound” ontology group, where the gene expression levels were changed in various manner. Running title: Oxygen metabolism in porcine oviductal epithelial cells

Morphological and biochemical changes in the cells surrounding the oocyte seem to be extremely important in an effective fertilization process. Thanks to advanced cell culture techniques, as well as biochemical and bioinformatics analyses, we can partly imitate the phenomena occurring in the living organism. Previous studies showed a possibility of short – and long – term OEC in vitro cultivation, during which these cells have shown to have significant proliferation and expression of genes responsible for differentiation. Our research was aimed at maintaining a culture of porcine oviduct epithelial cells and analyzing their gene expression profile. The study employed cross-bred gilts at the age of about 9 months, obtained from commercial herds. With the use of Affymetrix® Porcine Gene 1.1 ST Array Strip, we have examined the expression of 12257 transcripts. Genes with fold change higher than abs (2) and with corrected p-value lower than 0.05 were considered as differentially expressed. We chose 20 genes with the most marked expression (10 up – regulated, 10 down – regulated) for further investigation in the context of literature sources. These genes belonged to three ontological groups: “cell cycle process”, “cell division” and “cell proliferation”. The results obtained from these studies may be the basis for further molecular analyses.

Correct maturation of the oocyte is crucial for further fertilization and embryogenesis. It comprises of both nuclear and cytoplasmic maturation, during which the proteins, nutrients and mRNAs are assembled. Cumulus cells are connected with the oocyte via gap-junctions, which enable bi-directional transfer of molecules, forming cumulus-oocyte complex (COC). The expression pattern in CCs is thought to resemble the genes expressed in the oocyte. The CCs surrounding the gamete of high developmental competence have an increased expression of apoptotic markers. Therefore, our aim in this study was to determine whether any apoptosis-related genes are upregulated in porcine oocytes before or after IVM. We isolated COCs from 45 pubertal crossbred gilts, performed brilliant cresyl blue (BCB) staining and analyzed the gene expression pattern in oocytes before and after IVM with the use of microarray analysis. The results include 419 differentially expressed transcripts, 25 of which belong to „regulation of apoptosis” and „regulation of cell death” GO BP terms. This set of genes includes BCLAF1, EIF2AK3, KLF10, MIF, MAP3K1, NOTCH2, TXNIP and APP, all of which have been upregulated in immature porcine oocytes. Our results suggest that they play part in porcine oocyte maturation and could be used as potential markers of female gamete’s developmental competence. This knowledge could serve as a basis to improve ART in pigs.

Exocrine pancreatic tumors in cats and dogs are very uncommon, with exocrine pancreatic adenocarcinoma described as a particularly rare, malignant tumor in a small animals. It is usually derived from pancreatic ducts, as well as glandular tissue. Very often the disease has a subclinical course and is diagnosed only during post – mortem examination. Adenocarcinoma metastasize to both the surrounding and distant organs. The etiology of the cancer progress in the pancreas remains unknown. Pancreatic pseudocyst (pseudocystis pancreatis) is a very rare disease in domestic animals. Only a few cases of pancreatic pseudocyst in dogs and cats are described in the available literature. On the other hand, in humans it is a common complication of pancreatitis. The pancreatic pseudocyst can be a result of pancreatitis, as well as be a secondary condition to acute pancreatitis, trauma or chronic inflammation. It is a capsule made of fibrous or granulation tissue, containing sterile pancreatic juice. The pathogenesis of pancreatic pseudocyst is still unknown. Clinical signs are not specific and may resemble symptoms of pancreatitis. The ultrasound examination shows a cyst in the pancreas. This paper describes the case of pancreatic adenocarcinoma in a cat that occurred simultaneously with the pancreatic pseudocyst. Currently, literature indicates the necessity to differentiate the above cancer from cystic lesion. Due to the simultaneous occurrence of both pathologies, extremely rare diagnosis and spectacular picture of changes, it was decided to present this case.

Simple Summary Wound healing and vascularization mechanisms are key steps in the complex morphological process of tissue reconstruction. Additionally, these processes in the oral cavity are more rapid than in the skin and result in less scar formation. Epithelial cells and fibroblasts play an important role in the process of wound healing. In our study, we focused on fibroblasts and monitored changes in gene expression during their in vitro culture. Based on the analysis, we distinguished three groups of processes that play important roles in tissue regeneration: response to wounding, wound healing and vascularization. We identified genes that were involved in all three processes. These genes could be selected as tissue specific repair markers for oral fibroblasts. Abstract The mechanisms of wound healing and vascularization are crucial steps of the complex morphological process of tissue reconstruction. In addition to epithelial cells, fibroblasts play an important role in this process. They are characterized by dynamic proliferation and they form the stroma for epithelial cells. In this study, we have used primary cultures of oral fibroblasts, obtained from porcine buccal mucosa. Cells were maintained long-term in in vitro conditions, in order to investigate the expression profile of the molecular markers involved in wound healing and vascularization. Based on the Affymetrix assays, we have observed three ontological groups of markers as wound healing group, response to wounding group and vascularization group, represented by different genes characterized by their expression profile during long-term primary in vitro culture (IVC) of porcine oral fibroblasts. Following the analysis of gene expression in three previously identified groups of genes, we have identified that transforming growth factor beta 1 (TGFB1), ITGB3, PDPN, and ETS1 are involved in all three processes, suggesting that these genes could be recognized as markers of repair specific for oral fibroblasts within the porcine mucosal tissue.

Even though chemotherapy and immunotherapy emerged to limit continual and unregulated proliferation of cancer cells, currently available therapeutic agents are associated with high toxicity levels and low success rates. Additionally, ongoing multi-targeted therapies are limited only for few carcinogenesis pathways, due to continually emerging and evolving mutations of proto-oncogenes and tumor-suppressive genes. CRISPR/Cas9, as a specific gene-editing tool, is used to correct causative mutations with minimal toxicity, but is also employed as an adjuvant to immunotherapy to achieve a more robust immunological response. Some of the most critical limitations of the CRISPR/Cas9 technology include off-target mutations, resulting in nonspecific restrictions of DNA upstream of the Protospacer Adjacent Motifs (PAM), ethical agreements, and the lack of a scientific consensus aiming at risk evaluation. Currently, CRISPR/Cas9 is tested on animal models to enhance genome editing specificity and induce a stronger anti-tumor response. Moreover, ongoing clinical trials use the CRISPR/Cas9 system in immune cells to modify genomes in a target-specific manner. Recently, error-free in vitro systems have been engineered to overcome limitations of this gene-editing system. The aim of the article is to present the knowledge concerning the use of CRISPR Cas9 technique in targeting treatment-resistant cancers. Additionally, the use of CRISPR/Cas9 is aided as an emerging supplementation of immunotherapy, currently used in experimental oncology. Demonstrating further, applications and advances of the CRISPR/Cas9 technique are presented in animal models and human clinical trials. Concluding, an overview of the limitations of the gene-editing tool is proffered.

Gingivae, as the part of periodontium, are involved in tooth support and possess the ability to heal rapidly, without scar formation. Recently, dental tissues have been identified as a potential source of mesenchymal stem cells (MSCs) and several populations of MSCs were isolated from the orofacial region, including gingival mesenchymal stem cells (GMSCs). GMSCs exhibit robust immunomodulatory and differentiation potential and are easily obtainable, which make them promising candidates for cellular therapies. Apart from being tested for application in immunologic- and inflammatory-related disorders and various tissue regeneration, GMSCs promise to be a valuable tool in cancer treatment, especially in tongue squamous cell carcinoma (TSCC) with the use of targeted therapy, since GMSCs are able to selectively migrate towards the cancerous cells both in vitro and in vivo. In addition to their ability to uptake and release anti-neoplastic drugs, GMSCs may be transduced with apoptosis-inducing factors and used for cancer growth inhibition. Moreover, GMSCs, as most mammalian cells, secrete exosomes, which are a subset of extracellular vesicles with a diameter of 40–160 nm, containing DNA, RNA, lipids, metabolites, and proteins. Such GMSCs-derived exosomes may be useful therapeutic tool in cell-free therapy, as well as their culture medium. GMSCs exhibit molecular and stem-cell properties that make them well suited in preclinical and clinical studies.

Polyspermia is an adverse phenomenon during mammalian fertilization when more than one sperm fuses with a single oocyte. The egg cell is prepared to prevent polyspermia by, among other ways, producing cortical granules (CGs), which are specialized intracellular structures containing enzymes that aim to harden the zona pellucida and block the fusion of subsequent sperm. This work focused on exploring the expression profile of genes that may be associated with cortical reactions, and evaluated the distribution of CGs in immature oocytes and the peripheral density of CGs in mature oocytes. Oocytes were isolated and then processed for in vitro maturation (IVM). Transcriptomic analysis of genes belonging to five ontological groups has been conducted. Six genes showed increased expression after IVM (ARHGEF2, MAP1B, CXCL12, FN1, DAB2, and SOX9), while the majority of genes decreased expression after IVM. Using CG distribution analysis in immature oocytes, movement towards the cortical zone of the oocyte during meiotic competence acquisition was observed. CGs peripheral density decreased with the rise in meiotic competence during the IVM process. The current results reveal important new insights into the in vitro maturation of oocytes. Our results may serve as a basis for further studies to investigate the cortical reaction of oocytes.

Neovascularization and angiogenesis are vital processes in the repair of damaged tissue, creating new blood vessel networks and increasing oxygen and nutrient supply for regeneration. The importance of Adipose-derived Mesenchymal Stem Cells (ASCs) contained in the adipose tissue surrounding blood vessel networks to these processes remains unknown and the exact mechanisms responsible for directing adipogenic cell fate remain to be discovered. As adipose tissue contains a heterogenous population of partially differentiated cells of adipocyte lineage; tissue repair, angiogenesis and neovascularization may be closely linked to the function of ASCs in a complex relationship. This review aims to investigate the link between ASCs and angiogenesis/neovascularization, with references to current studies. The molecular mechanisms of these processes, as well as ASC differentiation and proliferation are described in detail. ASCs may differentiate into endothelial cells during neovascularization; however, recent clinical trials have suggested that ASCs may also stimulate angiogenesis and neovascularization indirectly through the release of paracrine factors.

The correct oviductal development and morphogenesis of its epithelium are crucial factors influencing female fertility. Oviduct is involved in maintaining an optimal environment for gametes and preimplantation embryo development; secretory oviductal epithelial cells (OECs) synthesize components of oviductal fluid. Oviductal epithelium also participates in sperm binding and its hyperactivation. For better understanding of the genetic bases that underlay porcine oviductal development, OECs were isolated from porcine oviducts and established long-term primary culture. A microarray approach was utilized to determine the differentially expressed genes during specific time periods. Cells were harvested on day 7, 15 and 30 of in vitro primary culture and their RNA was isolated. Gene expression was analyzed and statistical analysis was performed. 48 differentially expressed genes belonging to “tube morphogenesis”, “tube development”, “morphogenesis of an epithelium”, “morphogenesis of branching structure” and “morphogenesis of branching epithelium” GO BP terms were selected, of which 10 most upregulated include BMP4, ARG1, SLIT2, FGFR1, DAB2, TNC, EPAS1, HHEX, ITGB3 and LOX. The results help to shed light on the porcine oviductal development and its epithelial morphogenesis, and show that after long-term culture the OECs still proliferate and maintain their tube forming properties.

An oviduct is an essential organ for gamete transport, oocyte maturation, fertilization, spermatozoon capacitation and early embryo development. The epithelium plays an important role in oviduct functioning. The products of secretory cells provide an optimal environment and influence gamete activities and embryonic development. The oviduct physiology changes during the female cycle, thus, the ratio of the secreted molecules in the oviduct fluid differs between phases. In this study, a differential gene expression in porcine oviduct epithelial cells was examined during the long-term primary in vitro culture. The microarray expression analysis revealed 2552 genes, 1537 of which were upregulated and 995 were downregulated after 7 days of culture, with subsequent changes in expression during 30 day-long culture. The obtained genes were classified into 8 GO BP terms, connected with angiogenesis and circulatory system development, extracted by DAVID software. Among all genes, 10 most up-regulated and 10 most down-regulated genes were selected for further investigation. Interactions between genes were indicated by STRING software and REACTOME FIViz application to the Cytoscape 3.6.0 software. Most of the genes belonged to more than one ontology group. Although studied genes are mostly responsible for angiogenesis and circulatory system development, they can also be found to be expressed in processes connected with fertilization and early embryo development. The latter function is focused on more, considering the fact that these genes were expressed in epithelial cells of the fallopian tube which is largely responsible for reproductive processes.

Ovarian cysts remain to be one of the most common and serious problems in reproduction of farm animals, as well as humans. Apart from causing the fall in reproductive potential of the ovaries, occupying the place in which folliculogenesis and oogenesis occur, they also cause hormone imbalances, by preventing corpus luteum formation, hence lowering the amount of steroid hormone production. While singular cysts rarely affect fertility, hormone fluctuations that are associated with their presence promotes their multiplication, which usually has more adverse effects. While the cysts are easily detectable in humans, possessing distinct echography while examined by ultrasound, multiple factors prevent widespread use of effective detection methods among large herds of farm animals. Because of lack of noticeable symptoms of early stages of such malignancies, they rarely get detected before the animal stops to exhibit symptoms of heat. That causes scientific research to be focused on not only methods of detection, but also the ways to negate the effects of ovarian cysts and bring the affected specimen back to reproductive potential. Despite that, high costs of diagnosis and treatment, cause them to be uncommon on commercial farms. As lack of fertility eliminates animals from breeding purposed herds, ovarian cysts persist as a cause of large losses of the animal husbandry business. Continuous research, focused on natural examples of ovarian cysts should be conducted, in order to improve methods of detection, prevention, treatment and recovery from the effects of ovarian cysts.

The mucous membrane is composed of two layers. The layer of stratified squamous epithelium and the underlying layer of the connective tissue. The epithelium is composed of keratinocytes that are in different stages of differentiation, depending on their localization. In our research, after isolation of primary in vitro cultured buccal pouch mucosal cells, we observed keratinocytes in various stages of differentiation and fibroblasts. These cells, depending on the ionic dynamics, may be subject to different morphological and biochemical transformations. Understanding the expression profile of the normal oral mucosal tissue is important for further research into the effects of biomaterials on the mucosal cells, their growth, proliferation, and differentiation. The porcine buccal pouch mucosal cells were used in this study. The oral mucosa was separated surgically and isolated enzymatically. The cells were in vitro cultured for 30 days, and after each step of in vitro culture (7 days, 15 days, 30 days), samples were collected for isolation of total RNA. The gene expression profile was measured using Affymetrix microarray assays. In results, we observed genes belonging to two ontology groups: cation homeostasis and cation transport. These genes were up-regulated after 7 days of in vitro culture as compared to down-regulation after 15 and 30 days of in vitro culture. These results suggested that dynamic growth, proliferation and cell adhesion are more intense in the first 7 days of in vitro culture. We also observed, for the first time, the expression of ATP13A3 in porcine oral mucosal cells.

The efficiency of the process of obtaining mature oocytes, and then of porcine embryos in vitro depends on many factors and requires meeting many conditions. These include selection of morphologically appropriate oocytes, selection of appropriate medium components, as well as a number of abiotic factors (appropriate microenvironment during in vitro culture). Oocytes were taken from 45 pubertal crossbred Landrace gilts. The BCB test was carried out. BCB + oocytes were divided into two groups: “before IVM” and “after IVM”. “Before IVM” oocytes were subjected to molecular analyzes immediately after collection, while “after IVM” oocytes underwent in vitro maturation and then the second BCB test. Oocytes that remained BCB+ after the second test were used for molecular analyzes using Affymetrix expression microarrays. A group of genes responsible for response to organic substance and response to abiotic stimulus, which underwent significant changes (decrease) was discovered after oocyte in vitro maturation. Genes such as MM, PLDP, SERPINH, MYOF, DHX9, HSPA5, VCP, KIT, SERPINH1, PLD1, and VCP showed the largest decrease after the culture period. The levels of these genes were therefore elevated in oocytes before the in vitro maturation process. In conclusion, a number of organic and abiotic factors have an impact on the process of the oocyte in vitro maturation. The presented results confirm the literature data in which the low efficiency of obtaining mature oocytes in in vitro conditions is mentioned, which further impacts the amount of viable embryos obtained.

The oral mucosa is a compound tissue composed of several cells types, including fibroblasts and keratinocytes, that are characterized by different morphology, as well as biochemical and metabolomic properties. The oral mucosal cells are the most important factors mediated between transport and drugs delivery. The changes in cellular ion homeostasis may significantly affect the bioavailability of administrated drugs and their transport across the mucous membrane. Therefore we investigated the expression profile of genes involved in ion transport and homeostasis in porcine buccal pouch mucosal cells. The oral mucosa was separated surgically and isolated enzymatically. The cells were examined during long-term in vitro culture (IVC). The cultured cells were collected at 7, 15 and 30 days of IVC and subsequently transferred to RNA isolation and next, the gene expression profile was measured using Affymetrix microarray assays. In the results, we can extract genes belonging to four ontology groups: “ion homeostasis”, “ion transport”, “metal ion transport”, and “inorganic ion homeostasis”. For TGFB1 and CCL2, we observed up-regulation after 7 days of IVC, down-regulation after 15 days of IVC and upregulation again after 30 days of IVC. The ATP13A3, ATP1B1, CCL8, LYN, STEAP1, PDPN, PTGS2, and SLC5A3genes showed high activity after day 7 of IVC, and in the days 15 and 30 of IVC showed low activity. We showed an expression profile of genes associated with the effects of ion influence on the porcine normal oral mucosal cell development in IVC. These studies may be the starting point for further research into oral diseases and will allow for the comparison of the gene expression profile of normal and disease altered cells.

Pyometra is the most common gynecological disease in female dogs. It usually occurs in middle age female dogs, usually about two months after the completion of heat. This disease is the accumulation of purulent fluid inside the uterus. Etiology of pyometra is not fully understood. It is assumed, that pyometra is a result of hormonal disorders in the endometrium combined with bacterial superinfection. The diagnosis is based on the interview, clinical examination, additional laboratory tests and ultrasound or x-ray of the abdomen. There are two treatments: ovariohysterectomy and conservative treatment with pharmacological agents for example prostaglandin, aglepriston, antibiotics with a broad spectrum of action. Currently conducted molecular studies have a large influence on the development of the present knowledge on the pathogenesis and course of pyometra, whose conclusions may be used to change the current therapeutic protocols.

The superfamily of transforming growth factors β (TGF-β) consists of cytokines that are crucial in regulating the organism’s biological functions and includes three isoforms of TGF-β protein, Anti-Müllerian Hormone (AMH), inhibin A and B, activins, 20 bone morphogenetic proteins (BMP1-20) and 9 growth factors (GDF1-9). Their signal transduction pathway involves three types of membrane receptors that exhibit a serine/threonine kinase activity, as well as the Smad proteins. After ligand binding, the Smad proteins are phosphorylated and translocated to the nucleus, where they interact with transcription factors and affect gene expression. TGF-β family members are involved in cell growth and differentiation, as well as chemo-taxis and apoptosis, and play an important role during an inflammation. Defects in TGF-β proteins or in their signalling pathway underlie many severe diseases, such as systemic lupus, systemic scleroderma, bronchial asthma, atherosclerosis, hyperthyroidism or cancer. These factors are also crucial in mammal reproductive functions, as they are involved in folliculogenesis, steroidogenesis, ovulation, maternal-embryo interaction, embryo development and uterine decidualization. Their defects result in issues with fertility. This review focuses on the relevance of TGF-β family members in a mammal reproduction with an emphasis on three TGF-β isoforms, inhibins A and B, GDF-9 and their signal transduction pathway.

The morphological and biochemical modification of oviductal epithelial cells (OECs) belongs to the group of compound processes responsible for proper oocyte transport and successful fertilization. The cellular interactions between cumulus-oocyte complexes (COCs) and oviductal epithelial cells (OECs) are crucial for this unique mechanism. In the present study we have analyzed angiogenesis and blood vessel development processes at transcript levels. By employing microarrays, four ontological groups associated with these mechanisms have been described. Differentially expressed genes belonging to the “angiogenesis”, “blood circulation”, “blood vessel development” and “blood vessel morphogenesis” GO BP terms were investigated as a potential markers for the creation of new blood vessels in cells under in vitro primary culture conditions.

Constant advances in medicine, both human and veterinary, lead to continuous discovery of new drugs and treatments. Recently, the aspect of stem cell use in regenerative medicine has been very popular. There are still too few clinical trials on animals that could precisely estimate the therapeutic efficacy of cell therapy. However, stem cells are a source of extraordinary potential for multiplication and differentiation which, if used properly, can prove to be an effective mean of treatment of numerous diseases that are currently considered untreatable. The purpose of review is the characterization and clinical use of stem cells in mostly occurring diseases. Particular attention has been given to the issue of mesenchymal stromal cells, which so far have been most widely used in clinical practice. Current research into stem cells has allowed scientists to discover many different types of these cells, describe their characteristics and divide them into groups, with the most important being embryonic stem cells and somatic (adult) stem cells. Adult stem cells, due to their availability and lack of ethical problems, are used in veterinary practice. Different types of mesenchymal stem cells are distinguished, based on their origin. Adipose tissue derived stem cells and stromal vascular fraction find the widest clinical application. In veterinary medicine, stem cells therapies are most commonly used in the case of horse orthopedic injuries and in diseases of various origin in dogs and cats. While further research is needed to confirm the effectiveness of cell therapies, they have much potential to find plenty of potential applications in future medicine.

A wide variety of mechanisms controlling oligomerization are observed. The dynamic nature of protein oligomerization is important for bioactivity control. The oocyte must undergo a series of changes to become a mature form before it can fully participate in the processes associated with its function as a female gamete. The growth of oocytes in the follicular environment is accompanied by surrounding somatic cumulus (CCs) and granulosa cells (GCs). It has been shown that oocytes tested before and after in vitro maturation (IVM) differ significantly in the transcriptomic and proteomic profiles. The aim of this study was to determine new proteomic markers for the oligomerization of porcine oocyte proteins that are associated with cell maturation competence. The Affymetrix microarray assay was performed to examine the gene expression profile associated with protein oligomerization in oocytes before and after IVM. In total, 12258 different transcriptomes were analyzed, of which 419 genes with lower expression in oocytes after IVM. We found 9 genes: GJA1, VCP, JUP, MIF, MAP3K1, INSR, ANGPTL4, EIF2AK3, DECR1, which were significantly down-regulated in oocytes after IVM (in vitro group) compared to oocytes analyzed before IVM (in vivo group). The higher expression of genes involved in the oligomerization of the protein before IVM indicates that they can be recognized as important markers of biological activation of proteins necessary for the further growth and development of pig embryos.