Lisa Moncrieff, I. Kocherova, A. Bryja, W. Kranc, Joanna Perek, P. Celichowski, M. Kulus, B. Kempisty, P. Mozdziak, M. Ješeta
Abstract The growth and maturation of the oocyte is a dynamic process which requires a variable supply of hormones, growth factors and energy. These needs are met partially by the surrounding somatic cells and the cumulus-oocyte complex, which communicate bi-directionally via gap junctions. Identifying and analyzing protein expression in the oocyte can provide insight in its development and growth. Further, like bone marrow stem cells, if relevant marker genes are found in oocytes, there is a potential for the oocyte to be manipulated into becoming hemopoietic stem cells. In this study, porcine oocytes were isolated and subjected to microarray analysis to compare the oocyte gene expression in vivo and in vitro maturation (IVM). Genes identified belonged to both ‘hemopoietic or lymphoid organ development’(GO:0048534) and ‘immune system development’ (GO:0002520), and the markers can be used to identify several activities such as cell migration, neurogenesis and proliferation. The following are the identified genes and all were downregulated after IVM to varying degrees: ID2, VEGFA, TGFBR3, INHBA, CDK6, BCL11A, MYO1E, ITGB1, EGR1, NOTCH2, SPTA1, KIT and TPD52. Our results should provide new markers to further investigate oocyte development and growth regulation. Running title: Markers of hemopoietic organ development
{"title":"New molecular markers involved in immune system homeostasis and hemopoietic organ development are differentially regulated during oocytes in vitro maturation","authors":"Lisa Moncrieff, I. Kocherova, A. Bryja, W. Kranc, Joanna Perek, P. Celichowski, M. Kulus, B. Kempisty, P. Mozdziak, M. Ješeta","doi":"10.2478/acb-2020-0004","DOIUrl":"https://doi.org/10.2478/acb-2020-0004","url":null,"abstract":"Abstract The growth and maturation of the oocyte is a dynamic process which requires a variable supply of hormones, growth factors and energy. These needs are met partially by the surrounding somatic cells and the cumulus-oocyte complex, which communicate bi-directionally via gap junctions. Identifying and analyzing protein expression in the oocyte can provide insight in its development and growth. Further, like bone marrow stem cells, if relevant marker genes are found in oocytes, there is a potential for the oocyte to be manipulated into becoming hemopoietic stem cells. In this study, porcine oocytes were isolated and subjected to microarray analysis to compare the oocyte gene expression in vivo and in vitro maturation (IVM). Genes identified belonged to both ‘hemopoietic or lymphoid organ development’(GO:0048534) and ‘immune system development’ (GO:0002520), and the markers can be used to identify several activities such as cell migration, neurogenesis and proliferation. The following are the identified genes and all were downregulated after IVM to varying degrees: ID2, VEGFA, TGFBR3, INHBA, CDK6, BCL11A, MYO1E, ITGB1, EGR1, NOTCH2, SPTA1, KIT and TPD52. Our results should provide new markers to further investigate oocyte development and growth regulation. Running title: Markers of hemopoietic organ development","PeriodicalId":18329,"journal":{"name":"Medical Journal of Cell Biology","volume":"8 1","pages":"35 - 43"},"PeriodicalIF":0.0,"publicationDate":"2020-03-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"46812064","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
I. Kocherova, B. Kempisty, Greg Hutchings, Lisa Moncrieff, C. Dompe, Krzysztof Janowicz, J. Petitte, J. Shibli, P. Mozdziak
Abstract In vitro models represent an alternative technique to in vivo or ex vivo studies in the drug development process. Cell-based assays are used to measure the level of proliferation and toxicity, as well as activation of signalling pathways and changes in morphology in cultivated cells. The studies conducted in vitro are aimed to estimate the newly synthesised drugs’ ability to permeate biological barriers and exert their therapeutic or cytotoxic effects. However, more than half of all studied drugs fail in the second or third phase of clinical trials due to a lack of confirmed efficacy. About a third of drugs fail because of safety issues, such as unacceptable levels of toxicity. To reduce attrition level in drug development, it is crucial to consider the implementation of translational phenotypic assays as well as to decipher various molecular mechanisms of action for new molecular entities. In this review, we summarise the existing cell-based methods most frequently used in the studies on drugs, taking into account their advantages and drawbacks. Running title: Cell-based approaches in drug development
{"title":"Cell-based approaches in drug development – a concise review","authors":"I. Kocherova, B. Kempisty, Greg Hutchings, Lisa Moncrieff, C. Dompe, Krzysztof Janowicz, J. Petitte, J. Shibli, P. Mozdziak","doi":"10.2478/acb-2020-0005","DOIUrl":"https://doi.org/10.2478/acb-2020-0005","url":null,"abstract":"Abstract In vitro models represent an alternative technique to in vivo or ex vivo studies in the drug development process. Cell-based assays are used to measure the level of proliferation and toxicity, as well as activation of signalling pathways and changes in morphology in cultivated cells. The studies conducted in vitro are aimed to estimate the newly synthesised drugs’ ability to permeate biological barriers and exert their therapeutic or cytotoxic effects. However, more than half of all studied drugs fail in the second or third phase of clinical trials due to a lack of confirmed efficacy. About a third of drugs fail because of safety issues, such as unacceptable levels of toxicity. To reduce attrition level in drug development, it is crucial to consider the implementation of translational phenotypic assays as well as to decipher various molecular mechanisms of action for new molecular entities. In this review, we summarise the existing cell-based methods most frequently used in the studies on drugs, taking into account their advantages and drawbacks. Running title: Cell-based approaches in drug development","PeriodicalId":18329,"journal":{"name":"Medical Journal of Cell Biology","volume":"8 1","pages":"44 - 49"},"PeriodicalIF":0.0,"publicationDate":"2020-03-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"44347291","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Rut Bryl, B. Borowiec, Rafael Shinoske Siroma, Nelson Pinto, M. A. Melo, J. Shibli, M. Dyszkiewicz-Konwińska
Abstract Exosomes belong to structures called extracellular vesicles (EVs). These spherical units, secreted by most eukaryotic cells, attracted significant interest among researchers in recent years. Exosomes undergo secretion from almost all types of mammalian cells, including dendritic cells, B cells, epithelial cells, mastocytes, reticulocytes, platelets, T cells, mesenchymal stem cells, adipocytes, bone marrow-derived stem cells, embryonic stem cells, fibroblasts, cardiac myocytes, endothelial cells, oligodendrocytes, astrocytes, microglia, neurons, neural stem cells, hepatocytes, lung spheroid cells, as well as tumor cells. Exosomes have several features that enable many methods of their isolation from biological material. Furthermore, physicochemical properties such as size, mass, density, or the ability to interact with specific proteins allowed for the development and advance of several effective methods. Work on exosomes’ recovery and purity made it possible to most effectively determine their isolation methods’ efficiency and accuracy. A common ground for the researchers’ interest in exosomal analyses is the role of exosomes as carriers of disease biomarkers. It has been suggested that exosomes can be used in vaccine development and other immunological-related purposes, as one of their characteristics is the ability to present antigens. Moreover, exosomes have a long half-life. As the human body does not perceive them as foreign bodies, they can penetrate cell membranes and target specific cells, making them even better candidates for the applications mentioned above. Therefore, the following review deals with the nature of exosomes, as well as various methods of their isolation and use in medicine. Running title: Current application of exosomes in medicine
{"title":"Current application of exosomes in medicine","authors":"Rut Bryl, B. Borowiec, Rafael Shinoske Siroma, Nelson Pinto, M. A. Melo, J. Shibli, M. Dyszkiewicz-Konwińska","doi":"10.2478/acb-2020-0013","DOIUrl":"https://doi.org/10.2478/acb-2020-0013","url":null,"abstract":"Abstract Exosomes belong to structures called extracellular vesicles (EVs). These spherical units, secreted by most eukaryotic cells, attracted significant interest among researchers in recent years. Exosomes undergo secretion from almost all types of mammalian cells, including dendritic cells, B cells, epithelial cells, mastocytes, reticulocytes, platelets, T cells, mesenchymal stem cells, adipocytes, bone marrow-derived stem cells, embryonic stem cells, fibroblasts, cardiac myocytes, endothelial cells, oligodendrocytes, astrocytes, microglia, neurons, neural stem cells, hepatocytes, lung spheroid cells, as well as tumor cells. Exosomes have several features that enable many methods of their isolation from biological material. Furthermore, physicochemical properties such as size, mass, density, or the ability to interact with specific proteins allowed for the development and advance of several effective methods. Work on exosomes’ recovery and purity made it possible to most effectively determine their isolation methods’ efficiency and accuracy. A common ground for the researchers’ interest in exosomal analyses is the role of exosomes as carriers of disease biomarkers. It has been suggested that exosomes can be used in vaccine development and other immunological-related purposes, as one of their characteristics is the ability to present antigens. Moreover, exosomes have a long half-life. As the human body does not perceive them as foreign bodies, they can penetrate cell membranes and target specific cells, making them even better candidates for the applications mentioned above. Therefore, the following review deals with the nature of exosomes, as well as various methods of their isolation and use in medicine. Running title: Current application of exosomes in medicine","PeriodicalId":18329,"journal":{"name":"Medical Journal of Cell Biology","volume":"8 1","pages":"101 - 111"},"PeriodicalIF":0.0,"publicationDate":"2020-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"45419075","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
M. Nawrocki, Rafał Sibiak, M. Brązert, P. Celichowski, L. Pawelczyk, B. Chermuła, C. Dompe, B. Kempisty, P. Mozdziak
Abstract 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
{"title":"Coenzyme and cofactor metabolism belongs to biochemical processes significantly regulated in human granulosa cells collected after IVF during long-term primary in vitro culture","authors":"M. Nawrocki, Rafał Sibiak, M. Brązert, P. Celichowski, L. Pawelczyk, B. Chermuła, C. Dompe, B. Kempisty, P. Mozdziak","doi":"10.2478/acb-2019-0021","DOIUrl":"https://doi.org/10.2478/acb-2019-0021","url":null,"abstract":"Abstract 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","PeriodicalId":18329,"journal":{"name":"Medical Journal of Cell Biology","volume":"7 1","pages":"152 - 160"},"PeriodicalIF":0.0,"publicationDate":"2019-12-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"46342920","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
M. Nawrocki, Rafał Sibiak, S. Kałużna, M. Brązert, P. Celichowski, L. Pawelczyk, Lisa Moncrieff, B. Kempisty, P. Mozdziak
Abstract 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
{"title":"Nucleotide, ribonucleotide and ribonucleoside binding belongs to differentially expressed genes in porcine epithelial oviductal cells during longterm primary cultivation","authors":"M. Nawrocki, Rafał Sibiak, S. Kałużna, M. Brązert, P. Celichowski, L. Pawelczyk, Lisa Moncrieff, B. Kempisty, P. Mozdziak","doi":"10.2478/acb-2019-0022","DOIUrl":"https://doi.org/10.2478/acb-2019-0022","url":null,"abstract":"Abstract 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","PeriodicalId":18329,"journal":{"name":"Medical Journal of Cell Biology","volume":"7 1","pages":"161 - 169"},"PeriodicalIF":0.0,"publicationDate":"2019-12-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"42131431","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
K. Stefańska, Rafał Sibiak, C. Dompe, Lisa Moncrieff, Greg Hutchings, Krzysztof Janowicz, B. Kempisty
Abstract 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
{"title":"Overview of methods of isolation, cultivation and genetic profiling on human umbilical cord stem cells","authors":"K. Stefańska, Rafał Sibiak, C. Dompe, Lisa Moncrieff, Greg Hutchings, Krzysztof Janowicz, B. Kempisty","doi":"10.2478/acb-2019-0023","DOIUrl":"https://doi.org/10.2478/acb-2019-0023","url":null,"abstract":"Abstract 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","PeriodicalId":18329,"journal":{"name":"Medical Journal of Cell Biology","volume":"7 1","pages":"170 - 174"},"PeriodicalIF":0.0,"publicationDate":"2019-12-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"42494391","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
K. Stefańska, Rafał Sibiak, Greg Hutchings, C. Dompe, Lisa Moncrieff, Krzysztof Janowicz, M. Ješeta, B. Kempisty, M. Machatkova, P. Mozdziak
Abstract 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
{"title":"Evidence for existence of molecular stemness markers in porcine ovarian follicular granulosa cells","authors":"K. Stefańska, Rafał Sibiak, Greg Hutchings, C. Dompe, Lisa Moncrieff, Krzysztof Janowicz, M. Ješeta, B. Kempisty, M. Machatkova, P. Mozdziak","doi":"10.2478/acb-2019-0025","DOIUrl":"https://doi.org/10.2478/acb-2019-0025","url":null,"abstract":"Abstract 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","PeriodicalId":18329,"journal":{"name":"Medical Journal of Cell Biology","volume":"7 1","pages":"183 - 188"},"PeriodicalIF":0.0,"publicationDate":"2019-12-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"43945091","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
B. Perek, K. Kowalska, A. Malińska, M. Jemielity, M. Nowicki
Abstract 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
{"title":"Ultrastructural variability of macrophages in the wall of selected aorto-coronary bypass grafts","authors":"B. Perek, K. Kowalska, A. Malińska, M. Jemielity, M. Nowicki","doi":"10.2478/acb-2019-0024","DOIUrl":"https://doi.org/10.2478/acb-2019-0024","url":null,"abstract":"Abstract 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","PeriodicalId":18329,"journal":{"name":"Medical Journal of Cell Biology","volume":"7 1","pages":"175 - 182"},"PeriodicalIF":0.0,"publicationDate":"2019-12-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"46584369","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
A. Chamier-Gliszczyńska, S. Kałużna, K. Stefańska, P. Celichowski, P. Antosik, D. Bukowska, M. Bruska, J. Žáková, M. Machatkova, M. Ješeta, M. Nowicki
Abstract The formation of mammalian oocytes begins in the ovary during fetal development. The proper development of oocytes requires close communication with surrounding somatic cells, the substances they emit allow proper maturation of oocytes. Somatic cumulus (CC) cells and oocytes form cumulus-oocyte (COC) complexes. In this study, the Affymetrix microarray analysis was used to investigate changes in gene expression occurring in oocytes before and after in vitro maturation (IVM). The aim of the study was to examine oocyte genes involved in two ontological groups, “regulation of cell migration” and “regulation of cell proliferation” discovered by the microarray method. We found a reduced expression of all 28 genes tested in the ontological groups: ID2, VEGFA, BTG2, CCND2, EDNRA, TGFBR3, GJA, LAMA2, RTN4, CDK6, IHH, MAGED1, INSR, CD9, PTGES, TXNIP, ITGB1, SMAD4, MAP3K1, NOTCH2 , IGFBP7, KLF10, KIT, TPM1, PLD1, BTG3, CD47 and MITF. We chose the most regulated genes down the IVM culture, and pointed out those belonging to two ontological groups. Increased expression of the described genes before IVM maturation may indicate the important role of these genes in the process of ovum maturation. After the maturation process, the proteins produced by them did not play such an important role. In summary, the study provides us with many genes that can serve as molecular markers of oocyte processes associated with in vitro maturation. This knowledge can be used for detailed studies on the regulation of oocyte maturation processes. Running title: Genes regulating cellular migration and proliferation in porcine oocytes
{"title":"Analysis of expression of genes responsible for regulation of cellular proliferation and migration – microarray approach based on porcine oocyte model","authors":"A. Chamier-Gliszczyńska, S. Kałużna, K. Stefańska, P. Celichowski, P. Antosik, D. Bukowska, M. Bruska, J. Žáková, M. Machatkova, M. Ješeta, M. Nowicki","doi":"10.2478/acb-2019-0007","DOIUrl":"https://doi.org/10.2478/acb-2019-0007","url":null,"abstract":"Abstract The formation of mammalian oocytes begins in the ovary during fetal development. The proper development of oocytes requires close communication with surrounding somatic cells, the substances they emit allow proper maturation of oocytes. Somatic cumulus (CC) cells and oocytes form cumulus-oocyte (COC) complexes. In this study, the Affymetrix microarray analysis was used to investigate changes in gene expression occurring in oocytes before and after in vitro maturation (IVM). The aim of the study was to examine oocyte genes involved in two ontological groups, “regulation of cell migration” and “regulation of cell proliferation” discovered by the microarray method. We found a reduced expression of all 28 genes tested in the ontological groups: ID2, VEGFA, BTG2, CCND2, EDNRA, TGFBR3, GJA, LAMA2, RTN4, CDK6, IHH, MAGED1, INSR, CD9, PTGES, TXNIP, ITGB1, SMAD4, MAP3K1, NOTCH2 , IGFBP7, KLF10, KIT, TPM1, PLD1, BTG3, CD47 and MITF. We chose the most regulated genes down the IVM culture, and pointed out those belonging to two ontological groups. Increased expression of the described genes before IVM maturation may indicate the important role of these genes in the process of ovum maturation. After the maturation process, the proteins produced by them did not play such an important role. In summary, the study provides us with many genes that can serve as molecular markers of oocyte processes associated with in vitro maturation. This knowledge can be used for detailed studies on the regulation of oocyte maturation processes. Running title: Genes regulating cellular migration and proliferation in porcine oocytes","PeriodicalId":18329,"journal":{"name":"Medical Journal of Cell Biology","volume":"7 1","pages":"48 - 57"},"PeriodicalIF":0.0,"publicationDate":"2019-10-12","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"43178946","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
M. Kulus, B. Borowiec, M. Popis, P. Celichowski, M. Ješeta, D. Bukowska, H. Piotrowska-Kempisty, M. Bruska, M. Zabel, M. Nowicki, B. Kempisty, P. Antosik
Abstract 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
{"title":"Genes encoding proteins regulating fatty acid metabolism and cellular response to lipids are differentially expressed in porcine luminal epithelium during long-term culture","authors":"M. Kulus, B. Borowiec, M. Popis, P. Celichowski, M. Ješeta, D. Bukowska, H. Piotrowska-Kempisty, M. Bruska, M. Zabel, M. Nowicki, B. Kempisty, P. Antosik","doi":"10.2478/acb-2019-0008","DOIUrl":"https://doi.org/10.2478/acb-2019-0008","url":null,"abstract":"Abstract 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","PeriodicalId":18329,"journal":{"name":"Medical Journal of Cell Biology","volume":"7 1","pages":"58 - 65"},"PeriodicalIF":0.0,"publicationDate":"2019-10-12","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"46532359","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
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