Magdalena Kulus, Jakub Kulus, Maurycy Jankowski, Blanka Borowiec, Michal Jeseta, Dorota Bukowska, Klaus P. Brüssow, Bartosz Kempisty and Paweł Antosik
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.
Marta Rybska, Sandra Knap, Maurycy Jankowski, Michal Jeseta, Dorota Bukowska, Paweł Antosik, Michał Nowicki, Maciej Zabel, Bartosz Kempisty and Jędrzej M. Jaśkowski
The pig is a polyestrous animal in which the ovarian cycle lasts about 21 days and results in ovulation of 10-25 oocytes. Ovum reaches 120-150 μm in diameter, with the surrounding corona radiata providing communication with the environment. The zona pellucida is composed of glycoproteins: ZP1, ZP2, ZP3. In the course of oogenesis, RNA and protein accumulation for embryonic development occurs. Maternal mRNA is the template for protein production. Nuclear, cytoplasmic and genomic maturity condition the ability of the ovum to undergo fertilization. There are several differences in protein expression profiles observed between in vitro and in vivo conditions. Oogenesis is the process of differentiating female primary sex cells into gametes. During development gonocytes migrate from the yolk sac into the primary gonads with TGF-1, fibronectin, and laminin regulating this process. Cell cycle is blocked in dictyotene. Primary oocyte maturation is resumed before each ovulation and lasts until the next block in metaphase II. At the moment of penetration of the sperm into the ovum, the metaphase block is broken. The oocytes, surrounded by a single layer of granular cells, form the ovarian follicle. The exchange of signals between the oocyte and the cumulus cells done by gap-junctions, as well as various endo and paracrine signals. The contact between the corona radiata cells provides substances necessary for growth, through the same gap junctions. Studies on follicular cells can be used to amplify the knowledge of gene expression in these cells, in order to open way for potential clinical applications.
Maurycy Jankowski, Marta Dyszkiewicz-Konwińska, Joanna Budna, Sandra Knap, Artur Bryja, Sylwia Borys, Wiesława Kranc, Magdalena Magas, Michal Jeseta, Dorota Bukowska, Paweł Antosik, Klaus P. Brüssow, Marie Machatkova, Małgorzata Bruska, Michał Nowicki, Maciej Zabel and Bartosz Kempisty
In modern medical research, stem cells are one of the main focuses, believed to be able to provide the solution to many currently unsolvable medical cases. However, their extraordinary potential for differentiation creates much obstacles in their potential application in clinical environment, without understanding the whole array of molecular mechanisms that drive the processes associated with their development and maturation. Because of that, there is a large need for studies that concern the most basic levels of those processes. Progenitor stem cells are a favorable target, as they are relatively lineage committed, making the amount of signaling required to reach the final form much lower. Their presence in the adult organism is also an advantage in their potential use, as they can be extracted without the need for storage from the moment of pre-natal development or birth. Epithelial tissues, because of their usual location or function, exhibit extraordinary level of plasticity and proliferative potential. That fact makes them one of the top candidates for use in applications such as tissue engineering, cell based therapies, regenerative and reconstructive medicine. The potential clinical application, however, need to be based on well developed methods, in order to provide an effective treatment without causing major side effects. To achieve that goal, a large amount of research, aiming to analyze the molecular basics of proliferation and differentiation of epithelial stem cells, and stem cells in general, needs to be conducted.
Ievgeniia Kocherova, Maciej Brązert, Patrycja Sujka-Kordowska, Aneta Konwerska, Magdalena Kulus, Błażej Chermuła, Piotr Celichowski, Hanna Piotrowska-Kempisty, Paweł Antosik, Dorota Bukowska, Małgorzata Bruska, Leszek Pawelczyk, Maciej Zabel, Michał Nowicki, Bartosz Kempisty and Michal Jeseta
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
Magdalena Kulus, Małgorzata Józkowiak, Jakub Kulus, Małgorzata Popis, Blanka Borowiec, Katarzyna Stefańska, Piotr Celichowski, Mariusz J. Nawrocki, Dorota Bukowska, Klaus P. Brüssow, Bartosz Kempisty, Michal Jeseta and Paweł Antosik
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.
Magdalena Kulus, Blanka Borowiec, Małgorzata Popis, Piotr Celichowski, Michal Jeseta, Dorota Bukowska, Hanna Piotrowska-Kempisty, Małgorzata Bruska, Maciej Zabel, Michał Nowicki, Bartosz Kempisty and Paweł Antosik
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
Mariusz J. Nawrocki, Joanna Budna, Piotr Celichowski, Ronza Khozmi, Artur Bryja, Wiesława Kranc, Sylwia Borys, Sylwia Ciesiółka, Sandra Knap, Michal Jeseta, Dorota Bukowska, Paweł Antosik, Klaus P. Brüssow, Małgorzata Bruska, Michał Nowicki, Maciej Zabel and Bartosz Kempisty
The morphological and biochemical modification of oviductal epithelial cells (OECs) belongs to the compound process responsible for proper oocytes transport and successful fertilization. However, the main mechanisms which regulated this process are still not entirely known. Moreover, the OECs metabolism, which may be identified as the “cellular activity” marker, is poorly recognized. In this study we investigated the fructose and mannose metabolic pathway in porcine OECs primary long-term cultured in vitro.
In our study, we employ a primary long term in vitro culture (IVC) and microarray approach (the Affymetrix microarray were used for analysis of transcriptomic profile of OECs) for expression levels analysis.
We found that from the whole analyzed transcriptome, 1537 genes were upregulated and 995 were down regulated after 7 days of culture, 1471 genes were upregulated and 1061 were downregulated after 15 days of culture and 1329 genes were upregulated and 1203 were downregulated after 30 days of culture. Moreover, the differential expression of SORD, FPGT, PFKFB4, TPI1, MPI, ALDOC, HK2 and PFKFB3 at 24 hours, 7 day, 15 day and 30 day, was also observed.
We suggested that fructose and mannose metabolism may be important molecular bio-marker of porcine OECs capability in in vitro model. The metabolic profile is significantly accompanied by cells proliferation in vitro. The transcriptomic profile of SORD, FPGT, PFKFB4, TPI1, MPI, ALDOC, HK2 and PFKFB3 expression may be identified as “fingerprint” of fructose and mannose metabolism in OECs as well as involved in cellular in vitro developmental capacity in pigs.
Mariusz J. Nawrocki, Piotr Celichowski, Joanna Budna, Artur Bryja, Wiesława Kranc, Sylwia Ciesiółka, Sylwia Borys, Sandra Knap, Michal Jeseta, Ronza Khozmi, Dorota Bukowska, Paweł Antosik, Klaus P. Brüssow, Małgorzata Bruska, Michał Nowicki, Maciej Zabel and Bartosz Kempisty
The mammalian oocytes undergo significant biochemical and structural modifications during maturation both in vitro and in vivo. These changes involve chromatin reorganization and modification within metabolic status of cytoplasmic organelles. After oocytes’ successful maturation the substantially increased storage of RNA was observed. Moreover, the early embryo interaction with maternal endometrial tissue after fertilization is up to now considered as the main marker of proper embryo implantation and early growth. In this study, we first investigated the expression profile of genes involved in blood vessel formation and blood circulation in porcine oocytes before and after in vitro maturation.
The cumulus-oocyte complexes were collected from pubertal Landrace gilts and classified as before in vitro maturation (in Vivo) or after in vitro maturation (in Vitro). The RNA was isolated from these two experimental groups and analyzed using Affymetrix microarrays.
We found an increased expression of genes involved in ontological groups such as “blood circulation” (TPM1, ECE1, ACTA2, EPHX2, EDNRA, NPR2, MYOF, TACR3, VEGFA, GUCY1B3), “blood vessel development” (ANGPTL4, CYR61, SEMA5A, ID1, RHOB, RTN4, IHH, ANGPT2, EDNRA, TGFBR3, MYO1E, MMP14), and “blood vessels morphogenesis” (ANGPT2, as well as other common transcripts) in in Vivo group as compared to decreased expression of these genes in in Vitro group of oocytes.
It has been suggested that investigated genes undergo significant expression before in vitro maturation, when enhanced storage of large amount of RNA takes place. Creating templates for synthesis of proteins is required for formation of fully mature gametes and early embryo growth. Therefore we hypothesized that the processes of vascularization and/or angiogenesis reach a high activity in immature oocytes and are distinct from achievement of maturational stage by oocytes in pigs.
Katarzyna Stefańska, Agata Chamier-Gliszczyńska, Maurycy Jankowski, Piotr Celichowski, Magdalena Kulus, Magdalena Rojewska, Paweł Antosik, Dorota Bukowska, Małgorzata Bruska, Michał Nowicki, Bartosz Kempisty, Michal Jeseta and Jana Zakova
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.
Joanna Budna, Piotr Celichowski, Artur Bryja, Michal Jeseta, Maurycy Jankowski, Dorota Bukowska, Paweł Antosik, Andrzej Nowicki, Klaus P. Brüssow, Małgorzata Bruska, Michał Nowicki, Maciej Zabel and Bartosz Kempisty
Mammalian oocytes undergo compound processes of nuclear and cytoplasmic maturation that allow them to reach MII stage. Only fully mature, oocyte can be successfully fertilized by a single spermatozoon. Fatty acids, apart from their role in cellular metabolism, inflammation and tissue development, have positive and detrimental effects on oocyte maturation, fertilization, blastocyst cleavage rate and embryo development in mammals. Using microarrays, we have analyzed the expression changes in fatty acids- -related genes during in vitro maturation of porcine oocytes. The oocytes were recovered from ovaries of 45 pubertal crossbred Landrace gilts and subsequently subjected to BCB test. For further analyses, only granulosa cell-free BCB+ oocytes were used and divided into two groups. The first one, described as “before IVM”, was directly exposed to molecular assays, the second one, described as “after IVM”, was first in vitro matured and then subjected to a second BCB test. Oocytes, if classified as BCB+, were then passed to corresponding molecular analyses. We found significant down-regulation of genes involved in fatty acid metabolic process, such as: ACSL6, EPHX2, FADS2, PTGES, TPI1, TBXAS1, NDUFAB1, MIF, ACADSB and DECR1 in porcine oocytes analyzed after IVM, in comparison to those analyzed before IVM. In conclusion, apart from poor data available concerning analyzed genes in relation to reproductive events, significant changes in their expression point to their potential role as an oocyte developmental competence markers in pigs. Introducing molecular diagnostics of oocytes could be the prospective tool for selection of best gametes, leading to improved outcomes of in vitro fertilization.