Bovine Mesenchymal Stem Cells Research Articles

Cultivation of Bovine Mesenchymal Stem Cells on Plant-Based Scaffolds in a Macrofluidic Single-Use Bioreactor for Cultured Meat.

Reducing production costs, known as scaling, is a significant obstacle in the advancement of cultivated meat. The cultivation process hinges on several key components, e.g., cells, media, scaffolds, and bioreactors. This study demonstrates an innovative approach, departing from traditional stainless steel or glass bioreactors, by integrating food-grade plant-based scaffolds and thermoplastic film bioreactors. While thermoplastic films are commonly used for constructing fluidic systems, conventional welding methods are cost-prohibitive and lack rapid prototyping capabilities, thus inflating research and development expenses. The developed laser welding technique facilitates contamination-free and leakproof sealing of polyethylene films, enabling the efficient fabrication of macrofluidic systems with various designs and dimensions. By incorporating food-grade plant-based scaffolds, such as rice seeded with bovine mesenchymal stem cells, into these bioreactors, this study demonstrates sterile cell proliferation on scaffolds within macrofluidic systems. This approach not only reduces bioreactor prototyping and construction costs but also addresses the need for scalable solutions in both research and industrial settings. Integrating single-use bioreactors with minimal shear forces and incorporating macro carriers such as puffed rice may further enhance biomass production in a scaled-out model. The use of food-grade plant-based scaffolds aligns with sustainable practices in tissue engineering and cultured-meat production, emphasizing its suitability for diverse applications.

Bovine adipose tissue-derived mesenchymal stem cells self-assemble with testicular cells and integrates and modifies the structure of a testicular organoids

Mesenchymal stem cells (MSC) display self-renewal and mesodermal differentiation potentials. These characteristics make them potentially useful for in vitro derivation of gametes, which may constitute experimental therapies for human and animal reproduction. Organoids provide a spatial support and may simulate a cellular niche for in vitro studies. In this study, we aimed at evaluating the potential integration of fetal bovine MSCs derived from adipose tissue (AT-MSCs) in testicular organoids (TOs), their spatial distribution with testicular cells during TO formation and their potential for germ cell differentiation. TOs were developed using Leydig, Sertoli, and peritubular myoid cells that were previously isolated from bovine testes (n = 6). Thereafter, TOs were characterized using immunofluorescence and Q-PCR to detect testicular cell-specific markers. AT-MSCs were labeled with PKH26 and then cultured with testicular cells at a concentration of 1 × 106 cells per well in Ultra Low Attachment U-shape bottom (ULA) plates. TOs formed by testicular cells and AT-MSCs (TOs + AT-MSCs) maintained a rounded structure throughout the 28-day culture period and did not show significant differences in their diameters. Conversely, control TOs exhibited a compact structure until day 7 of culture, while on day 28 they displayed cellular extensions around their structure. Control TOs had greater (P < 0.05) diameters compared to TOs + AT-MSCs. AT-MSCs induced an increase in proportion of Leydig and peritubular myoid cells in TOs + AT-MSCs; however, did not induce changes in the overall gene expression of testicular cell-specific markers. STAR immunolabelling detected Leydig cells that migrated from the central area to the periphery and formed brunches in control TOs. However, in TOs + AT-MSCs, Leydig cells formed a compact peripheral layer. Sertoli cells immunodetected using WT1 marker were observed within the central area forming clusters of cells in TOs + AT-MSCs. The expression of COL1A associated to peritubular myoids cells was restricted to the central region in TOs + AT-MSCs. Thus, during a 28-day culture period, fetal bovine AT-MSCs integrated and modified the structure of the TOs, by restricting formation of branches, limiting the overall increase in diameters and increasing the proportions of Leydig and peritubular myoid cells. AT-MSCs also induced a reorganization of testicular cells, changing their distribution and particularly the location of Leydig cells.

Cultured meat platform developed through the structuring of edible microcarrier-derived microtissues with oleogel-based fat substitute

With the increasing global demand for meat, cultured meat technologies are emerging, offering more sustainable solutions that aim to evade a future shortage of meat. Here, we demonstrate a cultured meat platform composed of edible microcarriers and an oleogel-based fat substitute. Scalable expansion of bovine mesenchymal stem cells on edible chitosan-collagen microcarriers is optimized to generate cellularized microtissues. In parallel, an oleogel system incorporated with plant protein is developed as a fat substitute, which is comparable to beef fat in appearance and texture. Combining the cellularized microtissues with the developed fat substitute, two types of cultured meat prototypes are introduced: layered cultured meat and burger-like cultured meat. While the layered prototype benefits enhanced stiffness, the burger-like prototype has a marbling meat-like appearance and a softer texture. Overall, this platform and the established technological basis may contribute to the development of different cultured meat products and promote their commercial production.

Low Magnesium Concentration Enforces Bone Calcium Deposition Irrespective of 1,25-Dihydroxyvitamin D3 Concentration.

Efficient coordination between Mg2+ and vitamin D maintains adequate Ca2+ levels during lactation. This study explored the possible interaction between Mg2+ (0.3, 0.8, and 3 mM) and 1,25-dihydroxyvitamin D3 (1,25D; 0.05 and 5 nM) during osteogenesis using bovine mesenchymal stem cells. After 21 days, differentiated osteocytes were subjected to OsteoImage analysis, alkaline phosphatase (ALP) activity measurements, and immunocytochemistry of NT5E, ENG (endoglin), SP7 (osterix), SPP1 (osteopontin), and the BGLAP gene product osteocalcin. The mRNA expression of NT5E, THY1, ENG, SP7, BGLAP, CYP24A1, VDR, SLC41A1, SLC41A2, SLC41A3, TRPM6, TRPM7, and NIPA1 was also assessed. Reducing the Mg2+ concentration in the medium increased the accumulation of mineral hydroxyapatite and ALP activity. There was no change in the immunocytochemical localization of stem cell markers. Expression of CYP24A1 was higher in all groups receiving 5 nM 1,25D. There were tendencies for higher mRNA abundance of THY1, BGLAP, and NIPA1 in cells receiving 0.3 mM Mg2+ and 5 nM 1,25D. In conclusion, low levels of Mg2+ greatly enhanced the deposition of bone hydroxyapatite matrix. The effect of Mg2+ was not modulated by 1,25D, although the expression of certain genes (including BGLAP) tended to be increased by the combination of low Mg2+ and high 1,25D concentrations.

Mesenchymal Stem Cells in Embryo-Maternal Communication under Healthy Conditions or Viral Infections: Lessons from a Bovine Model.

Bovine mesenchymal stem cells are a relevant cell population found in the maternal reproductive tract that exhibits the immunomodulation capacity required to prevent embryo rejection. The phenotypic plasticity showed by both endometrial mesenchymal stem cells (eMSC) and embryonic trophoblast through mesenchymal to epithelial transition and epithelial to mesenchymal transition, respectively, is essential for embryo implantation. Embryonic trophoblast maintains active crosstalk via EVs and soluble proteins with eMSC and peripheral blood MSC (pbMSC) to ensure the retention of eMSC in case of pregnancy and induce the chemotaxis of pbMSC, critical for successful implantation. Early pregnancy-related proteins and angiogenic markers are detected as cargo in EVs and the soluble fraction of the embryonic trophectoderm secretome. The pattern of protein secretion in trophectoderm-EVs changes depending on their epithelial or mesenchymal phenotype and due to the uptake of MSC EVs. However, the changes in this EV-mediated communication between maternal and embryonic MSC populations infected by viruses that cause abortions in cattle are poorly understood. They are critical in the investigation of reproductive viral pathologies.

Improvement of biological and corrosion behavior of 316 L stainless steel using PDMS-Ag doped Willemite nanocomposite coating

This study aimed to improve the biological properties and corrosion resistance of 316 L stainless steel substrate using polydimethylsiloxane (PDMS)‑silver doped Willemite nanocomposite coating. For this purpose, Ag-doped Willemite nanoparticles (NPs) were synthesized by the sol-gel method initially. Then, PDMS-Ag-doped Willemite nanocomposite coatings with various nanoparticles content were coated on the substrates using an immersion technique. The antibacterial results showed that Willemite NPs containing 5 wt% Ag possessed the highest antibacterial activity than the other samples. The examination of microstructure, hydrophobicity, adhesion strength, corrosion resistance, and biological characteristics of the coatings revealed that the addition of NPs up to 3 wt% enhances the surface roughness, hydrophobicity and coating adhesion. However, at concentrations greater than 3 wt% NPs, the coating's corrosion resistance is reduced owing to the formation of larger agglomerates and induced cracks in the coating. The presence of NPs in the coating promotes the adhesion and viability of bovine mesenchymal stem cells (bMSCs).

Establishment of Bovine-Induced Pluripotent Stem Cells.

Pluripotent stem cells (PSCs) have been successfully developed in many species. However, the establishment of bovine-induced pluripotent stem cells (biPSCs) has been challenging. Here we report the generation of biPSCs from bovine mesenchymal stem cells (bMSCs) by overexpression of lysine-specific demethylase 4A (KDM4A) and the other reprogramming factors OCT4, SOX2, KLF4, cMYC, LIN28, and NANOG (KdOSKMLN). These biPSCs exhibited silenced transgene expression at passage 10, and had prolonged self-renewal capacity for over 70 passages. The biPSCs have flat, primed-like PSC colony morphology in combined media of knockout serum replacement (KSR) and mTeSR, but switched to dome-shaped, naïve-like PSC colony morphology in mTeSR medium and 2i/LIF with single cell colonization capacity. These cells have comparable proliferation rate to the reported primed- or naïve-state human PSCs, with three-germ layer differentiation capacity and normal karyotype. Transcriptome analysis revealed a high similarity of biPSCs to reported bovine embryonic stem cells (ESCs) and embryos. The naïve-like biPSCs can be incorporated into mouse embryos, with the extended capacity of integration into extra-embryonic tissues. Finally, at least 24.5% cloning efficiency could be obtained in nuclear transfer (NT) experiment using late passage biPSCs as nuclear donors. Our report represents a significant advance in the establishment of bovine PSCs.

Comparison of characteristics of mesenchymal stem cells from bovine

Mesenchymal stem cells (MSCs) are multipotent cells capable of replicating as undifferentiated cells, and thus hold therapeutic implications in field of regenerative medicine and reproductive biotechnology. In the present study, we compared the stem cell properties of bovine ear skin tissue (ESK)- and nasal mucosa (NM)-derived MSCs. Bovine ESK-MSCs and NM-MSCs were successfully isolated by collagenase digestion and maintained proliferative capacity during the 20 consecutive passages. Both ESK-MSCs and NM-MSCs showed similar morphology and expressed common cell surface markers (CD29, CD44, CD90, and CD105). Also, we compared differentiation potentials of bovine ESK-MSCs and NM-MSCs into osteogenic, adipogenic, and chondrogenic lineages through specific staining and quantitative real-time RT-PCR. As results, bovine ESK-MSCs and NM-MSCs could differentiate into mesodermal cell lineages. However, bovine ESK-MSCs and NM-MSCs exhibited difference in expression of differentiation-related specific markers. Specifically, NM-MSCs exhibited increased expression levels of osteocalcin, peroxisome proliferator-activated receptor gamma, and aggrecan compared to ESK-MSCs. Also, ESK-MSCs exhibited increased expression levels of collagen type I, II, and lipoprotein lipase compared to NM-MSCs. We suggest that the nasal mucosa of bovine could be used as a source of bovine MSCs.

Strategies for transfection of bovine mesenchymal stem cells with pBC1-anti-CD3 vector

Cells from different origins behave differently regarding the incorporation of exogenous DNA and formation of transgenic cells. Milk production of recombinant antibody may benefit from efficient transfection protocols to produce transgenic animals. In this context, the objective of this study was to verify the transfection potential of bovine mesenchymal stem cells from Wharton’s jelly (MSC-WJ) and adipose tissue (MSC-AT), comparing co-transfection protocols with vectors pBC1-anti-CD3 and pEF-NEO-GFP, using transfection reagents Lipofectamine LTX with Plus Reagent or Xfect. Skin fibroblasts (FIB) were used as the control group. Forty-eight hours after transfection, neomycin was added and cells cultured for 2 weeks. Treated cells were submitted to fluorescence microscopy, flow cytometry, and PCR evaluations. Wharton’s jelly cells were sensitive to treatments and started necrosis. In the flow cytometry assay, the median fluorescence was higher in adipocytes than fibroblasts, for both the Xfect (20.057 ± 1.620,7 and 10.601 ± 702,86, respectively, p < 0.05) and LTX (19.590 ± 113,84 and 10.518 ± 442,65, respectively, p < 0.05). These results, associated with evaluation of epifluorescence, demonstrated that adipocytes presented a better response to transfection than other cells, independent of the kit used. Performing PCR on co-transfected cells demonstrated the presence of anti-CD3, making this approach feasible for future experiments.

Heat Shock Alters Mesenchymal Stem Cell Identity and Induces Premature Senescence.

Heat stress can have a serious impact on the health of both humans and animals. A major question is how heat stress affects normal development and differentiation at both the cellular and the organism levels. Here we use an in vitro experimental system to address how heat shock treatment influences the properties of bovine mesenchymal stem cells (MSCs)—multipotent progenitor cells—which are found in most tissues. Because cattle are sensitive to harsh external temperatures, studying the effects of heat shock on MSCs provides a unique platform to address cellular stress in a physiologically relevant model organism. Following isolation and characterization of MSCs from the cow’s umbilical cord, heat shock was induced either as a pulse (1 h) or continuously (3 days), and consequent effects on MSCs were characterized. Heat shock induced extensive phenotypic changes in MSCs and dramatically curtailed their capacity to proliferate and differentiate. These changes were associated with a partial arrest in the G1/S or G2/M checkpoints. Furthermore, MSCs lost their ability to resolve the inflammatory response of RAW macrophages in coculture. A possible explanation for this loss of function is the accumulation of reactive oxygen species and malfunction of the mitochondria in the treated cells. Heat shock treatments resulted in stress-induced premature senescence, affecting the MSCs’ ability to proliferate properly for many cell passages to follow. Exposure to elevated external temperatures leads to mitochondrial damage and oxidative stress, which in turn conveys critical changes in the proliferation, differentiation, and immunomodulatory phenotype of heat-stressed MSCs. A better understanding of the effect of heat shock on humans and animals may result in important health and economic benefits.

Placental scaffolds have the ability to support adipose-derived cells differentiation into osteogenic and chondrogenic lineages.

Prudent choices of cell sources and biomaterials, as well as meticulous cultivation of the tissue microenvironment, are essential to improving outcomes of tissue engineering treatments. With the goal of providing a high-quality alternative for bone and cartilage tissue engineering, we investigated the capability of bovine placental scaffolds to support adipose-derived cell differentiation into osteogenic and chondrogenic lineages. Decellularized bovine placenta, a high-quality scaffold with practical scalability, was chosen as the biomaterial due to its rich extracellular matrix, well-developed vasculature, high availability, low cost, and simplicity of collection. Adipose-derived cells were chosen as the cell source as they are easy to isolate, nontumorigenic, and flexibly differentiable. The bovine model was chosen for its advantages in translational medicine over the mouse model. When seeded onto the scaffolds, the isolated cells adhered to the scaffolds with cell projections, established cell-scaffold communication and proliferated while maintaining cell-cell communication. Throughout a 21-day culture period, osteogenically differentiated cells secreted mineralized matrix, and calcium deposits were observed throughout the scaffold. Under chondrogenic specific differentiation conditions, the cells modified their morphology from fibroblast-like to round cells and cartilage lacunas were observed as well as the deposit of cartilaginous matrix on the placental scaffolds. This experiment provides evidence, for the first time, that bovine placental scaffolds have the potential to support bovine mesenchymal stem cell adherence and differentiation into osteogenic and chondrogenic lineages. Therefore, the constructed material could be used for bone and cartilage tissue engineering.

TATA box binding protein and ribosomal protein 4 are suitable reference genes for normalization during quantitative polymerase chain reaction study in bovine mesenchymal stem cells.

ObjectiveQuantitative polymerase chain reaction (qPCR) has been extensively used in the field of mesenchymal stem cell (MSC) research to elucidate their characteristics and clinical potential by normalization of target genes against reference genes (RGs), which are believed to be stably expressed irrespective of various experimental conditions. However, the expression of RGs is also variable depending on the experimental conditions, which may lead to false or contradictory conclusions upon normalization. Due to the current lack of information for a clear list of stable RGs in bovine MSCs, we conducted this study to identify suitable RGs in bovine MSCs.MethodsThe cycle threshold values of ten traditionally used RGs (18S ribosomal RNA [18S], beta-2-microglobulin [B2M], H2A histone family, member Z [H2A], peptidylprolyl isomerase A [PPIA], ribosomal protein 4 [RPL4], succinate dehydrogenase complex, subunit A [SDHA], beta actin [ACTB], glyceraldehyde-3-phosphate dehydrogenase [GAPDH], TATA box binding protein [TBP], and hypoxanthine phosphoribosyltrasnfrase1 [HPRT1]) in bovine bone marrow-derived MSCs (bBMMSCs) were validated for their stabilities using three types of RG evaluation algorithms (geNorm, Normfinder, and Bestkeeper). The effect of validated RGs was then verified by normalization of lineage-specific genes (fatty acid binding protein 4 [FABP4] and osteonectin [ON]) expressions during differentiations of bBMMSCs or POU class 5 homeobox 1 (OCT4) expression between bBMMSCs and dermal skins.ResultsBased on the results obtained for the three most stable RGs from geNorm (TBP, RPL4, and H2A), Normfinder (TBP, RPL4, and SDHA), and Bestkeeper (TBP, RPL4, and SDHA), it was comprehensively determined that TBP and RPL4 were the most stable RGs in bBMMSCs. However, traditional RGs were suggested to be the least stable (18S) or moderately stable (GAPDH and ACTB) in bBMMSCs. Normalization of FABP4 or ON against TBP, RPL4, and 18S presented significant differences during differentiation of bBMMSCs. However, although significantly low expression of OCT4 was detected in dermal skins compared to that in bBMMSCs when TBP and RPL4 were used in normalization, normalization against 18S exhibited no significance.ConclusionThis study proposes that TBP and RPL4 were suitable as stable RGs for qPCR study in bovine MSCs.

Bovine tongue epithelium-derived cells: A new source of bovine mesenchymal stem cells.

Mesenchymal stem cells (MSCs) possess the ability to differentiate into multiple cell lineages, and thus, confer great potential for use in regenerative medicine and biotechnology. In the present study, we attempted to isolate and characterize bovine tongue tissue epithelium-derived MSCs (boT-MSCs) and investigate the culture conditions required for long-term culturing of boT-MSCs. boT-MSCs were successfully isolated by the collagenase digestion method and their proliferative capacity was maintained for up to 20 or more passages. We observed a significant increase in the proliferation of boT-MSCs during the 20 consecutive passages under low-glucose Dulbecco’s modified Eagle’s medium culture condition among the three culture conditions. These boT-MSCs presented pluripotency markers (octamer-binding transcription factor 3/4 (Oct3/4) and sex determining region Y-box2 (Sox2)) and cell surface markers, which included CD13, CD29, CD44, CD73, CD90, CD105, CD166, and major histocompatibility complex (MHC) class I (MHC-I) but not CD11b, CD14, CD31, CD34, CD45, CD80, CD86, CD106, CD117, and MHC-II at third passage. Moreover, these boT-MSCs could differentiate into mesodermal (adipocyte, osteocyte, and chondrocyte) cell lineages. Thus, the present study suggests that the tongue of bovines could be used as a source of bovine MSCs.

Post-calving umbilical cord tissue offcut: A potential source for the isolation of bovine mesenchymal stem cells.

Background and Aim:Veterinary health care is an emergent area in animal sciences and innovative therapeutic approaches happen to be imperative in the present days. In view of the importance of cattle health and production, it is necessary to take up contemporary approach of stem cell therapy in this sector also. This study aimed to standardize an explant culture method of bovine umbilical tissue offcut to isolate mesenchymal stem cells (MSCs) because considerable efforts are required for ensuring easy accessibility and availability of MSCs in bulk quantity, as well as in establishing and characterizing the cell lines.Materials and Methods:The umbilical cord (UC) tissue matrix offcut was collected after calving. A simplified in vitro cell isolation technique was followed to collect the emerged out cells from the explants of UC. Further, we expanded these isolated cells in vitro, observed its growth kinetics, and characterized to confirm as per the criterion of bovine MSCs.Results:A considerable exponential growth rate of the UC-derived cells was noticed. In addition to their confirmation as MSCs, the cells also exhibited plastic adherent property and maintained the spindle-shaped morphology throughout the in vitro culture. The cultured cells were found positive MSC-specific surface markers CD105, CD90, and CD73 and were negative for hematopoietic cell marker CD45. Cytochemical studies revealed the ability of the cells to differentiate into osteogenic, chondrogenic, and adipogenic lineages.Conclusion:This simplified method of isolation and culture of bovine multipotent MSCs from the UC offcut collected after calving could be extrapolated for the greater availability of the cells for prospective therapeutic applications.

Optimizing adipogenic transdifferentiation of bovine mesenchymal stem cells: a prominent role of ascorbic acid in FABP4 induction

ABSTRACT Adipocyte differentiation of bovine adipose-derived stem cells (ASC) was induced by foetal bovine serum (FBS), biotin, pantothenic acid, insulin, rosiglitazone, dexamethasone and 3-isobutyl-1-methylxanthine, followed by incubation in different media to test the influence of ascorbic acid (AsA), bovine serum lipids (BSL), FBS, glucose and acetic acid on transdifferentiation into functional adipocytes. Moreover, different culture plate coatings (collagen-A, gelatin-A or poly-L-lysine) were tested. The differentiated ASC were subjected to Nile red staining, DAPI staining, immunocytochemistry and quantitative reverse transcription PCR (for NT5E, THY1, ENG, PDGFRα, FABP4, PPARγ, LPL, FAS, GLUT4). Nile red quantification showed a significant increase in the development of lipid droplets in treatments with AsA and BSL without FBS. The presence of BSL induced a prominent increase in FABP4 mRNA abundance and in FABP4 immunofluorescence signals in coincubation with AsA. The abundance of NT5E, ENG and THY1 mRNA decreased or tended to decrease in the absence of FBS, and ENG was additionally suppressed by AsA. DAPI fluorescence was higher in cells cultured in poly-L-lysine or gelatin-A coated wells. In additional experiments, the multi-lineage differentiation potential to osteoblasts was verified in medium containing ß-glycerophosphate, dexamethasone and 1,25-dihydroxyvitamin D3 using alizarin red staining. In conclusion, bovine ASC are capable of multi-lineage differentiation. Poly-L-lysine or gelatin-A coating, the absence of FBS, and the presence of BSL and AsA favour optimal transdifferentiation into adipocytes. AsA supports transdifferentiation via a unique role in FABP4 induction, but this is not linearly related to the primarily BSL-driven lipid accumulation. Abbreviations: AcA: acetic acid; AsA: ascorbic acid; ASC: adipose-derived stem cells; BSL: bovine serum lipids; DAPI: 4´,6-diamidino-2-phenylindole; DLK: delta like non-canonical notch ligand; DMEM: Dulbecco’s modified Eagle’s medium; DPBS: Dulbecco’s phosphate-buffered saline; ENG: endoglin; FABP: fatty acid binding protein; FAS: fatty acid synthase; GLUT4: glucose transporter type 4; IBMX: 3-isobutyl-1-methylxanthine; LPL: lipoprotein lipase; MSC: mesenchymal stem cells; α-MEM: α minimum essential medium; NT5E: ecto-5ʹ-nucleotidase; PDGFRα: platelet derived growth factor receptor α; PPARγ: peroxisome proliferator activated receptor γ; RPS19: ribosomal protein S19; SEM: standard error of the mean; THY1: Thy-1 cell surface antigen; TRT: treatment; TRT-Con: treatment negative control; YWHAZ: tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation protein zeta

Использование субстратов разной жесткости для направленной дифференцировки мезенхимальных стволовых клеток крупного рогатого скота

The article describes the change in the structures of the actin cytoskeleton and the parameters of focal adhesions during three major mesoderm lineage commitments (adipocytes, osteocytes and chondrocytes) of bovine mesenchymal stem cells. It was shown that the cultivation of mesenchymal stem cells on a rigid substrate is a favorable factor for osteogenesis due to an increase in the size and number of focal adhesions, as well as the number of actin cytoskeleton structures responsible for mechanical tension. Cultivation of cells on a soft substrate stimulates both adipogenesis and chondrogenesis due to restructuring the actin cytoskeleton responsible for mechanical tension, as well as reducing the size, number and location of focal adhesions. These data establish a relationship between matrix rigidity, cytoskeletal structure and preferred directions of differentiation of mesenchymal stem cells. mesenchymal stem cells, differentiation, osteogenesis, adipogenesis, focal adhesions, actin, matrigel The study was supported by the Ministry of Education and Science of the Russian Federation (State Assignment no. 075-01250-20-01).

206 Overexpression of germ cell genes DAZL, STRA8, and BOULE in bovine adipose tissue-derived mesenchymal stem cells for male germ cell derivation

Invitro gamete derivation from stem cells has potential applications as an alternative method for dissemination of elite animal genetics, production of transgenic animals, and conservation of endangered species. Mesenchymal stem cells (MSC) may be suitable candidates for invitro gamete derivation given their differentiation capacity and their potential for cell therapy. DAZL (deleted in azoospermia-like) and BOULE (also called BOLL) encode RNA-binding proteins that control differentiation of germ cells; STRA8 (stimulated by RA-8) encodes a protein required for meiosis. Considering the crucial roles of these factors, the aim of the present study was to evaluate co-overexpression of different combinations of DAZL, STRA8, and BOULE on germ cell gene expression profile in adipose tissue-derived MSC (AT-MSC). AT-MSC were harvested from abattoir-derived male bovine fetuses (n=9; 7-8 months of gestation). The optimal concentration of Lipofectamine 2000 (1, 1.5, 2, 1.5ng µL−1; Invitrogen) was analysed in AT-MSC transfected with plasmid pSIN-EF2-Puro containing DAZL, STRA8, and BOULE coding sequences (pSIN-EF2-DAZL-P2A-STRA8-T2A-BOULE-Puro). Then, AT-MSC were transfected either with plasmids containing DAZL, STRA8, and BOULE genes (pSIN-EF2-DAZL-P2A-STRA8-T2A-BOULE-Puro), DAZL and BOULE genes (pSIN-EF2-DAZL-P2A-STRA8-T2A-Puro), DAZL (pSIN-EF2-DAZL-P2A-T2A-Puro), or empty plasmid at a concentration of 1ng µL−1 of Lipofectamine. Cell samples were obtained from each plasmid treatment and analysed for expression of housekeeping genes ACTB and GAPDH and germ cell genes DAZL, PIWIl2, STRA8, and BOULE by quantitative-PCR using relative values (Quantity) through the ΔΔCT formula. AT-MSC transfected with pSIN-EF2-DAZL-P2A-STRA8-T2A-BOULE-Puro using 1ng µL−1 of Lipofectamine achieved higher (P&amp;lt;0.05) expression of DAZL (13.3-fold) compared with cells transfected with empty vector. Moreover, AT-MSC transfected with pSIN-EF2-DAZL-P2A-STRA8-T2A-BOULE-Puro had higher (P&amp;lt;0.05) levels of DAZL mRNA (3.8-fold) compared with empty vector. Messenger RNA levels of STRA8 (1.4-fold) were only detected in AT-MSC transfected with pSIN-EF2-DAZL-P2A-STRA8-T2A-Puro; PIWIl2 and BOULE were not detected in transfected or untransfected AT-MSC. In conclusion, bovine fetal AT-MSC are amenable for overexpression of germ cell markers DAZL and STRA8. Transfection with plasmid containing three germ cell genes (DAZL, STRA8, and BOULE) allowed overexpression of DAZL, whereas transfection with plasmid containing two germ cell genes (DAZL and STRA8) achieved overexpression of STRA8. This study was supported by Fondecyt grant 1191114, Government of Chile.

135 Strategies for transfection of bovine mesenchymal stem cells with pBC1-anti-CD3 vector

Cells from different origins behave differently regarding the incorporation of exogenous genetic material and the formation of transgenic cells. In this context, the objective of this study was to verify the potential of transfection of bovine mesenchymal stem cells from Wharton's jelly and adipose tissue, comparing two transfection protocols, using Lipofectamine LTX and Plus or Xfect reagents, with the integration of humanized anti-CD3. Skin fibroblasts were used as a control group. Humanized anti-CD3 is a monoclonal antibody that interacts with the CD3 molecule of the T-cell receptor, leading to the suppression of T-cells. This antibody is considered an option in the treatment of human autoimmune diseases and against the rejection of transplanted organs. Humanized anti-CD3 was used in this work for the production of bovine transgenic cells that, in the future, will be used in the development of bioreactor animals. In all steps of this study, cell types were cultured in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal calf serum (FCS) and antibiotics, in an incubator at 39°C with 5% CO2 in air with saturated humidity. All cells were plated at 5×105 into 24-well culture dishes and co-transfected with vector pBC1-anti-CD3-IRES-FEO and pEF-NEO-GFP using Lipofectamine LTX with reagent Plus or Xfect. Forty-eight hours after transfection, neomycin was added in each treatment and cells were cultured for 2 weeks. Treated cells were submitted to fluorescence microscopy, flow cytometry, and PCR evaluations. Wharton's jelly cells were sensitive to treatments and started necrosis. In the flow cytometry assay, the median fluorescence was higher in adipocytes than in fibroblasts, for both the Xfect reagent (20.057±1.620.7 and 10.601±702.86, respectively, P&amp;lt;0.05) and for LTX (19.590±113.84 and 10.518±442.65 respectively, P&amp;lt;0.05). These results, associated with the evaluation of epifluorescence, demonstrated that adipocytes presented a better response to transfection than did other cells, independent of the kit used. Performing PCR on co-transfected adipocytes and fibroblasts demonstrated the presence of anti-CD3, making this approach feasible in future experiments. Southern blotting analysis is being performed to confirm DNA integration. Financial support was provided by Fundação de Amparo à Pesquisa do Distrito Federal (FAPDF); Embrapa MP1.

Bovine fetal mesenchymal stem cells exert antiproliferative effect against mastitis causing pathogen Staphylococcus aureus

Staphylococcus aureus is the most commonly isolated pathogen from clinical bovine mastitis samples and a difficult pathogen to combat. Mesenchymal stem cells (MSC) are multipotent progenitor cells equipped with a variety of factors that inhibit bacterial growth. The aim of the present study was to evaluate the in vitro antibacterial potential against S. aureus of conditioned medium (CM) from MSC derived from fetal bovine bone marrow (BM-MSC) and adipose tissue (AT-MSC). BM-MSC, AT-MSC and fetal fibroblasts (FB) cultures were activated by infection with S. aureus. Bacterial growth was evaluated in presence of CM, concentrated CM (CCM), activated CM (ACM) and concentrated ACM (CACM) from BM-MSC, AT-MSC and FB. Gene expression of β-defensin 4A (bBD-4A), NK-lysine 1 (NK1), cathelicidin 2 (CATHL2), hepcidin (HEP) and indoleamine 2,3 dioxygenase (IDO) and protein expression of bBD-4A were determined in activated and non-activated cells. The majority of BM-MSC and AT-MSC expressed CD73, Oct4 and Nanog, and were negative for CD34. Growth of S. aureus decreased when it was exposed to CM from BM-MSC, AT-MSC and FB. Moreover, growth of S. aureus in CCM, ACM and CACM was lower compared to controls of CM from BM-MSC and AT-MSC. Activated AT-MSC increased mRNA levels of bBD4A and NK1, and protein levels of bBD4A in CM. Thus, CM from fetal bovine BM-MSC and AT-MSC has the capacity to reduce in average ~30% of S. aureus relative growth under in vitro conditions. The in vitro antibacterial effect of fetal bovine MSC may be mediated by bBD4A and NK1 activity.

Immunomodulatory and immunogenic properties of mesenchymal stem cells derived from bovine fetal bone marrow and adipose tissue

Little information is currently available on therapeutic features of bovine mesenchymal stem cells (MSCs), despite the development of large animal experimental models including cattle may open alternative strategies for investigating MSC physiology and eventual applications for regenerative therapy. The aim of the present study was to compare in vitro immunomodulatory and immunogenic potentials of bovine fetal MSCs (bfMSCs) derived from bovine fetal bone marrow (BM-MSCs) and adipose tissue (AT-MSCs). Immunomodulatory analyses in bfMSCs were performed by determination of the effect of interferon-γ (IFNγ) on mRNA levels of indoleamine 2, 3-dioxygenase (IDO), transforming growth factor β1 (TGFβ1), prostaglandin E receptor 2 (PTGER2), interleukin-6 and -10 (IL-6 and IL-10), and IDO enzymatic activity. The effect of conditioned medium from IFNγ-stimulated bfMSCs on the proliferation of alloantigen-activated peripheral blood lymphocytes (PBLs) was assessed. Immunogenicity of bfMSCs was determined by quantification of mRNA levels of major histocompatibility complex I and II (MHC-I and -II), CD80 and CD86, and the proportion of cells positive for MHC-I and -II by flowcytometry (FACS) analyses. IFNγ treatment increased IL-6, PTGER2 and IDO gene expression and activity in bfMSCs but did not affect suppressive effect on proliferation of PBLs. Lower proportion of AT-MSCs expressed MHC-I and MHC-II in comparison to BM-MSCs. In conclusion, BM-MSCs and AT-MSCs upregulated expression of immunomodulatory genes in a similar way after IFNγ stimuli. BM-MSCs and AT-MSCs in basal condition and treated with IFNγ displayed similar in vitro immunomodulatory ability. Lower expression of MHC-I and MHC-II suggest that AT-MSCs might be less immunogenic compared to BM-MSCs.