Bovine mammary epithelial/stem cells: Possible application in the production of pharmaceutical interest proteins with non-GMO approach

Neelesh Sharma,Dong Kee Jeong 한국동물생명공학회(구 한국동물번식학회) 2017 발생공학 국제심포지엄 및 학술대회 Vol.2017 No.10

The demand of pharmaceutical proteins and continuously increasing worldwide, hence cost of production of these proteins is a key factor in the competitive market. Recombinant proteins have several benefits over proteins which are isolated from non-recombinant sources. There are several methods developed for expression of recombinant proteins in large scale. Among these mammalian cell culture based expression system is most used world wide followed by expression of recombinant proteins in live animal bioreactor. Although concept of generating therapeutic proteins in the mammalian cell cultures based system existed for more than two decades, it is still less efficient for producing cost effective therapeutics. One of the issues for lack of advancement in this field is exorbitant cost involved in downstream processing of the expressed proteins. This is largely due to lack of a cell culture system which may aid in high level expression of therapeutic protein followed by efficiently processing and secreation of the expressed protein in the culture. To achieve this, development of cheaper and less cumbersome procedures has become necessary. Cultivated mammalian cells have become the dominant system for the production of recombinant proteins for clinical applications because of their capacity for proper protein folding, assembly and post-translational modifications. Thus, the quality and efficacy of a protein can be superior when expressed in mammalian cells versus other hosts such as bacteria, plants and yeast. The mammary gland has generally been considered the organ of choice to express valuable recombinant proteins because milk is easily collected in large volumes and is the best available bioreactor. However, large scale production of recombinant proteins is still an art in spite of increased qualitative and quantitative demand for these proteins. Foreign proteins are commonly reported to be produced in transgenic milk at rates of several grams per liter. Researchers are constantly challenged to improve and optimize the existing expression systems, and also to develop novel approaches to face the demands of producing the complex proteins. Although concept of generating therapeutic proteins in the mammalian cell culture based system existed for more than two decades, it is still less efficient for producing cost effective therapeutics. We have established bovine mammary epithelial stem cells (bMESCs) using various markers such as Nanog, Oct4, Sox9, CK14, CK18, CSN2, CSN3 etc. Antibacterial pharmaceutical proteins were screened using various bio-informatic tools and then selected proteins such human lipocalin-2 (LCN2) and bovine lactoferricin for the transfection in cow bMESCs. We used different mammary specific vector systems as career for the transfer of targeted genes in bMECs. However, transfection in mammary epithelial/ stem cells is little difficult as compare to other cells as these cells have secretory properties. We successfully expressed both pharmaceutical proteins in the cow mammary epithelial stem cells and established the cell lines. Therefore, development of protocols and establishment of cow mammary epithelial/stem cell lines can be used for various studies and as non-GMO bioreactor for the production of pharmaceutical value proteins.

Autophagy of bovine mammary epithelial cell induced by intracellular Staphylococcus aureus

Na Geng,Kangping Liu,Jianwei Lu,Yuliang Xu,Xiaozhou Wang,Run Wang,Jianzhu Liu,Yongxia Liu,Bo Han 한국미생물학회 2020 The journal of microbiology Vol.58 No.4

Bovine mastitis is a common disease in the dairy industry that causes great economic losses. As the primary pathogen of contagious mastitis, Staphylococcus aureus (S. aureus) can invade bovine mammary epithelial cells, thus evading immune defenses and resulting in persistent infection. Recently, autophagy has been considered an important mechanism for host cells to clear intracellular pathogens. In the current study, autophagy caused by S. aureus was detected, and the correlation between autophagy and intracellular S. aureus survival was assessed. First, a model of intracellular S. aureus infection was established. Then, the autophagy of MAC-T cells was evaluated by confocal microscopy and western blot. Moreover, the activation of the PI3K-Akt-mTOR and ERK1/2 signaling pathways was determined by western blot. Finally, the relationship between intracellular bacteria and autophagy was analyzed by using autophagy regulators (3-methyladenine [3-MA], rapamycin [Rapa] and chloroquine [CQ]). The results showed that S. aureus caused obvious induction of autophagosome formation, transformation of LC3I/II, and degradation of p62/SQSTM1 in MAC-T cells; furthermore, the PI3K-Akt-mTOR and ERK1/2 signaling pathways were activated. The number of intracellular S. aureus increased significantly with autophagy activation by rapamycin, whereas the number decreased when the autophagy flux was inhibited by chloroquine. Therefore, this study indicated that intracellular S. aureus can induce autophagy and utilize it to survive in bovine mammary epithelial cells.

Improved development of somatic cell cloned bovine embryos by a mammary gland epithelia cells in vitro model

XiaoYing He,LiBing Ma,Xiao-ning He,Wan-tong Si,Yue-Mao Zheng 대한수의학회 2016 Journal of Veterinary Science Vol.17 No.2

Previous studies have established a bovine mammary gland epithelia cells in vitro model by the adenovirus-mediated telomerase (hTERT-bMGEs). The present study was conducted to confirm whether hTERT-bMGEs were effective target cells to improve the efficiency of transgenic expression and somatic cell nuclear transfer (SCNT). To accomplish this, a mammary-specific vector encoding human lysozyme and green fluorescent protein was used to verify the transgenic efficiency of hTERT-bMGEs, and untreated bovine mammary gland epithelial cells (bMGEs) were used as a control group. The results showed that the hTERT-bMGEs group had much higher transgenic efficiency and protein expression than the bMGEs group. Furthermore, the nontransgenic and transgenic hTERT-bMGEs were used as donor cells to evaluate the efficiency of SCNT. There were no significant differences in rates of cleavage or blastocysts or hatched blastocysts of cloned embryos from nontransgenic hTERT-bMGEs at passage 18 and 28 groups (82.8% vs. 81.9%, 28.6% vs. 24.8%, 58.6% vs. 55.3%, respectively) and the transgenic group (80.8%, 26.5% and 53.4%); however, they were significantly higher than the bMGEs group (71.2%, 12.8% and 14.8%), (p < 0.05). We confirmed that hTERT-bMGEs could serve as effective target cells for improving development of somatic cell cloned cattle embryos.

Synergistic Effect of Dexamethasone and Prolactin on VEGF Expression in Bovine Mammary Epithelial Cells via p44/p42 MAP Kinase

Nakajima, Kei-Ichi,Nakamura, Masato,Ishisaki, Akira,Kozakai, Takaharu Asian Australasian Association of Animal Productio 2009 Animal Bioscience Vol.22 No.6

Vascular endothelial growth factor (VEGF) is a key regulator of angiogenesis under various physiological and pathological conditions. We found that the VEGF isoforms VEGF120, VEGF164, and VEGF188 were expressed in the bovine mammary gland and bovine mammary epithelial cells (bMECs). Expression of VEGF in the mammary gland was significantly higher during the lactation period than during the dry period. Although dexamethasone or prolactin alone had little effect on the expression of VEGF, that in dexamethasone-treated cells was significantly induced after additional treatment with prolactin. Furthermore, the VEGF expression induced by the combination of dexamethasone and prolactin was reduced by PD98059 in a dose-dependent manner. This combination also stimulated the phosphorylation of p44/p42 MAP kinase in these cells. These results strongly suggest that the combination of dexamethasone and prolactin stimulates VEGF expression in bMECs via p44/p42 MAP kinase.

Phenylalanine and valine differentially stimulate milk protein synthetic and energy-mediated pathway in immortalized bovine mammary epithelial cells

( Jungeun Kim ),( Jeong-eun Lee ),( Jae-sung Lee ),( Jin-seung Park ),( Jun-ok Moon ),( Hong-gu Lee ) 한국축산학회(구 한국동물자원과학회) 2020 한국축산학회지 Vol.62 No.2

Studies on promoting milk protein yield by supplementation of amino acids have been globally conducted. Nevertheless, there is a lack of knowledge of what pathways affected by individual amino acid in mammary epithelial cells that produce milk in practice. Phenylalanine (PHE) and valine (VAL) are essential amino acids for dairy cows, however, researches on mammary cell levels are still lacking. Thus, the aim of this study was conducted to evaluate the effects of PHE and VAL on milk protein synthesis-related and energy-mediated cellular signaling in vitro using immortalized bovine mammary epithelial (MAC-T) cells. To investigate the effects of PHE and VAL, the following concentrations were added to treatment medium: 0, 0.3, 0.6, 0.9, 1.2, and 1.5 mM. The addition of PHE or VAL did not adversely affect cell viability compared to control group. The concentrations of cultured medium reached its maximum at 0.9 mM PHE and 0.6 mM VAL (p < 0.05). Therefore, aforementioned 2 treatments were analyzed for pro-teomics. Glucose transporter 1 and mammalian target of rapamycin mRNA expression levels were up-regulated by PHE (166% and 138%, respectively) (p < 0.05). Meanwhile, sodium-de-pendent neutral amino acids transporter type 2 (ASCT2) and β-casein were up-regulated by VAL (173% in ASCT2, 238% in and 218% in β-casein) (p < 0.05). A total of 134, 142, and 133 proteins were detected in control group, PHE treated group, and VAL treated group, respec-tively. Among significantly fold-changed proteins, proteins involved in translation initiation or energy metabolism were detected, however, expressed differentially between PHE and VAL. Thus, pathway analysis showed different stimulatory effects on energy metabolism and tran-scriptional pathways. Collectively, these results showed different stimulatory effects of PHE and VAL on protein synthesis-related and energy-mediated cellular signaling in MAC-T cells.

A Modified PiggyBac Transposon System Mediated by Exogenous mRNA to Perform Gene Delivery in Bovine Mammary Epithelial Cells

Guangdong Hu,Jing Wang,Hui Huang,Fusheng Quan,Jian Kang,Yongyan Wu,Yuanpeng Gao,Feng Su,Minghao Shao,Yong Zhang 한국생물공학회 2014 Biotechnology and Bioprocess Engineering Vol.19 No.2

Transposons are widely used for geneticengineering in various model organisms. Recently, piggyBac(PB) has been developed as a transposable and efficientgene transfer tool in mammalian cells. In the present study,we developed three types of PB transposon systemscontaining a dual plasmid system (DPS), a single plasmidsystem (SPS), and a DNA-mRNA combined system (DRPS)and characterized their basic properties in HEK293 cells. The basic elements of the donor plasmid included aselectable-reporter gene expression cassette, two loxP sitesin the same orientation, a multiple cloning site, and twochicken β-globin insulator core elements. We further identifiedthe function of the selectable-reporter and examined PBintegration sites in the human genome. Moreover, wecompared the transposition efficacy and found that SPStransposed more efficiently, as compared to DPS; integrationinto the host genome was determined by measuring PBaseactivity. Results discovered the loss of PBase activity in theDRPS, indicating that this system is much more biologicallysafe, as compared to DPS and SPS. Finally, we employedthe DRPS to successfully perform a gene delivery intobovine mammary epithelial cells (BMECs). Taken together,the information from this study will improve the flexibilityof PB transposon systems and reduce the genotoxicity ofPBase in genetic engineering.

Protective effects of 5-aminolevulinic acid on heat stress in bovine mammary epithelial cells

Islam Md Aminul,Noguchi Yoko,Taniguchi Shin,Yonekura Shinichi 아세아·태평양축산학회 2021 Animal Bioscience Vol.34 No.6

Objective: Cells have increased susceptibility to activation of apoptosis when suffering heat stress (HS). An effective supplementation strategy to mimic heat-induced apoptosis of bovine mammary epithelial cells (MECs) is necessary to maintain optimal milk production. This study aimed to investigate possible protective effects of the anti-apoptotic activity of 5-aminolevulinic acid (5-ALA) against HS-induced damage of bovine MECs. Methods: Bovine MECs were pretreated with or without 5-ALA at concentrations of 10, 100, and 500 μM for 24 h followed by HS (42.5°C for 24 h and 48 h). Cell viability was measured with 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assays. Real-time quantitative polymerase chain reaction and Western blotting were used to explore the regulation of genes associated with apoptosis, oxidative stress, and endoplasmic reticulum (ER) stress genes. Results: We found that 5-ALA induces cytoprotection via inhibition of apoptosis markers after HS-induced damage. Pretreatment of bovine MECs with 5-ALA resulted in dramatic upregulation of mRNA for nuclear factor erythroid-derived 2-like factor 2, heme oxygenase-1, and NAD(P)H quinone oxidoreductase 1, all of which are antioxidant stress genes. Moreover, 5-ALA pretreatment significantly suppressed HS-induced ER stress-associated markers, glucose-regulated protein 78, and C/EBP homologous protein expression levels. Conclusion: 5-ALA can ameliorate the ER stress in heat stressed bovine MEC via enhancing the expression of antioxidant gene. Objective: Cells have increased susceptibility to activation of apoptosis when suffering heat stress (HS). An effective supplementation strategy to mimic heat-induced apoptosis of bovine mammary epithelial cells (MECs) is necessary to maintain optimal milk production. This study aimed to investigate possible protective effects of the anti-apoptotic activity of 5-aminolevulinic acid (5-ALA) against HS-induced damage of bovine MECs.Methods: Bovine MECs were pretreated with or without 5-ALA at concentrations of 10, 100, and 500 μM for 24 h followed by HS (42.5°C for 24 h and 48 h). Cell viability was measured with 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assays. Real-time quantitative polymerase chain reaction and Western blotting were used to explore the regulation of genes associated with apoptosis, oxidative stress, and endoplasmic reticulum (ER) stress genes.Results: We found that 5-ALA induces cytoprotection via inhibition of apoptosis markers after HS-induced damage. Pretreatment of bovine MECs with 5-ALA resulted in dramatic upregulation of mRNA for nuclear factor erythroid-derived 2-like factor 2, heme oxygenase-1, and NAD(P)H quinone oxidoreductase 1, all of which are antioxidant stress genes. Moreover, 5-ALA pretreatment significantly suppressed HS-induced ER stress-associated markers, glucose-regulated protein 78, and C/EBP homologous protein expression levels.Conclusion: 5-ALA can ameliorate the ER stress in heat stressed bovine MEC via enhancing the expression of antioxidant gene.

C-C motif chemokine ligand 2 induces proliferation and prevents lipopolysaccharide-induced inflammatory responses in bovine mammary epithelial cells

Yang, Changwon,Lim, Whasun,Bae, Hyocheol,Bazer, Fuller W.,Song, Gwonhwa Elsevier 2018 Journal of dairy science Vol.101 No.5

<P>C-C motif chemokine ligand 2 (CCL2) is a small chemokine which belongs to the CC-type chemokine family, and has chemoattractant activity for recruitment of monocytes to sites of inflammation. Overexpressed CCL2 binding to its receptor C-C chemokine receptor 2 increases the risk of breast cancer in humans, but its effects on proliferation of bovine mammary epithelial cells is not known. Maintaining a high level of proliferative activity in bovine mammary epithelial cells during lactation is important for improving milk yield and can benefit the dairy industry economically. In the present study, we demonstrated that CCL2 induces proliferation of MAC-T cells, a bovine mammary epithelial cell line, and stimulates progression of the cell cycle through stimulation of expression of cyclin D1. Moreover, CCL2 activates phosphoinositide 3-kinase (PI3K)/AKT [AKT, P70-S6 kinase 1 (P70S6K), ribosomal protein S6 (S6)] and mitogen activated protein kinase (MAPK) [extracellular signal-regulated kinase-1/2 (ERK1/2), c-Jun N-terminal kinase (JNK), and P38] pathways, which are involved in proliferation of MAC-T cells, as evidenced by co-treatment of MAC-T cells with pharmacological inhibitors of cell signaling transcription factors including Wortmannin, U0126, and SP600125. The CCL2 in MAC-T cells attenuates endoplasmic reticulum stress induced by tunicamycin, suggesting that CCL2 regulates intracellular synthesis of proteins and lipids and prevents activation of apoptotic pathways initiated in response to endoplasmic reticulum stress. Furthermore, CCL2 is involved in alleviating lipopolysaccharide (LPS)-induced inflammatory responses in MAC-T cells by reducing LPS-induced expression of IL8, IL6, and nuclear factor kappa B subunit 1 (NFKB1). Collectively, CCL2 is a novel target for improving the quantity and quality of milk from cows through stimulation of proliferation on mammary epithelial cells and attenuation of LPS-induced inflammatory responses.</P>

Bee Venom Decreases LPS-Induced Inflammatory Responses in Bovine Mammary Epithelial Cells

( Chang Hee Jeong ),( Wei Nee Cheng ),( Hyojin Bae ),( Kyung Woo Lee ),( Sang Mi Han ),( Michael C. Petriello ),( Hong Gu Lee ),( Han Geuk Seo ),( Sung Gu Han ) 한국미생물생명공학회(구 한국산업미생물학회) 2017 Journal of microbiology and biotechnology Vol.27 No.10

The world dairy industry has long been challenged by bovine mastitis, an inflammatory disease, which causes economic loss due to decreased milk production and quality. Attempts have been made to prevent or treat this disease with multiple approaches, primarily through increased abuse of antibiotics, but effective natural solutions remain elusive. Bee venom (BV) contains a variety of peptides (e.g., melittin) and shows multiple bioactivities, including prevention of inflammation. Thus, in the current study, it was hypothesized that BV can reduce inflammation in bovine mammary epithelial cells (MAC-T). To examine the hypothesis, cells were treated with LPS (1 μg/ml) to induce an inflammatory response and the antiinflammatory effects of BV (2.5 and 5 μg/ml) were investigated. The cellular mechanisms of BV against LPS-induced inflammation were also investigated. Results showed that BV can attenuate expression of an inflammatory protein, COX2, and pro-inflammatory cytokines such as IL-6 and TNF-α. Activation of NF-κB, an inflammatory transcription factor, was significantly downregulated by BV in cells treated with LPS, through dephosphorylation of ERK1/2. Moreover, pretreatment of cells with BV attenuated LPS-induced production of intracellular reactive oxygen species (e.g., superoxide anion). These results support our hypothesis that BV can decrease LPS-induced inflammatory responses in bovine mammary epithelial cells through inhibition of oxidative stress, NF-κB, ERK1/2, and COX-2 signaling.

Optimizing hormonal and amino acid combinations for enhanced cell proliferation and cell cycle progression in bovine mammary epithelial cells

Kwon Hyuk Cheol,Jung Hyun Su,Kim Do Hyun,한종현,Han Seo Gu,금동현,Hong Seong Joon,한성구 아세아·태평양축산학회 2023 Animal Bioscience Vol.36 No.11

Objective: The number of bovine mammary epithelial cells (BMECs) is closely associated with the quantity of milk production in dairy cows; however, the optimal levels and the combined effects of hormones and essential amino acids (EAAs) on cell proliferation are not completely understood. Thus, the purpose of this study was to determine the optimal combination of individual hormones and EAAs for cell proliferation and related signaling pathways in BMECs. Methods: Immortalized BMECs (MAC-T) were treated with six hormones (insulin, cortisol, progesterone, estrone, 17β-estradiol, and epidermal growth factor) and ten EAAs (arginine, histidine, leucine, isoleucine, threonine, tryptophan, lysine, methionine, phenylalanine, and valine) for 24 h. Results: Cells were cultured in a medium containing 10% fetal bovine serum (FBS) as FBS supplemented at a concentration of 10% to 50% showed a comparable increase in cell proliferation rate. The optimized combination of four hormones (insulin, cortisol, progesterone, and 17β-estradiol) and 20% of a mixture of ten EAAs led to the highest cell proliferation rate, which led to a significant increase in cell cycle progression at the S and G2/M phases, in the protein levels of proliferating cell nuclear antigen and cyclin B1, cell nucleus staining, and in cell numbers. Conclusion: The optimal combination of hormones and EAAs increased BMEC proliferation by enhancing cell cycle progression in the S and G/2M phases. Our findings indicate that optimizing hormone and amino acid levels has the potential to enhance milk production, both in cell culture settings by promoting increased cell numbers, and in dairy cows by regulating feed intake.