Immortalized bovine mammary epithelial cells express stem cell markers and differentiate in vitro

Hu, Han; Zheng, Nan; Gao, Han; Dai, Wenting; Zhang, Yangdong; Li, Songli; Wang, Jiaqi

doi:10.1002/cbin.10624

Cited by 13 publications

(7 citation statements)

References 43 publications

(68 reference statements)

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“…The CMEC-H cells were established as described in our previous work (Hu et al, 2016). The CMEC-H cells exhibit the typical cobblestone morphology characteristic and propagation in long-term culture.…”

Section: Cell Culturementioning

confidence: 99%

Combination of histidine, lysine, methionine, and leucine promotes β-casein synthesis via the mechanistic target of rapamycin signaling pathway in bovine mammary epithelial cells

Gao

Zhao

Zheng

et al. 2017

Journal of Dairy Science

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The ratio of different AA in the diets of cows is vital to improve milk protein yield. β-Casein is one of the important milk proteins with high nutritive value. However, the suitable ratio of essential amino acids (EAA) for the expression of β-casein in the immortalized bovine mammary epithelial cell line is not fully characterized. This study employed response surface methodology to determine the optimal ratio of His, Lys, Met, and Leu on β-casein expression level in vitro and clarified the effect of the 4 EAA on β-casein via the mechanistic target of rapamycin (mTOR) signaling pathway. A central composite design containing 5 axial points per EAA and 28 combinations of the 4 EAA was used in our study. The results of response surface methodology and the changes of the mTOR-related signaling proteins were determined by western blot. The results showed that β-casein level was significantly affected by all 4 EAA (R = 0.71). The optimum conditions for β-casein expression are found to be 5.47 mM of His, 7.48 mM of Lys, 1.17 mM of Met, and 8.21 mM of Leu (His:Lys:Met:Leu = 5:6:1:7) in the designed scope of concentration. The interaction of Leu and Met significantly affected β-casein expression (P < 0.01). The phosphorylation of mTOR (Ser), regulatory associated protein of target of rapamycin (Ser), ribosomal protein S6 kinase 1 (Thr), ribosomal protein S6 (Ser), and eukaryotic elongation factor 2 (Thr) was increased with the supplementation of either single EAA or an optimal combination of EAA. However, the phosphorylation of eukaryotic initiation factor 4E binding protein 1 (Thr) was decreased with the addition of Lys, Met, or Leu alone. Furthermore, the phosphorylation (P) of eIF2α (Ser) was decreased when Met was supplemented alone. Under the optimal mixture of 4 EAA, the phosphorylation of mechanistic target of rapamycin complex 1 signaling proteins was significantly greater than the single EAA supplementations and the expression of β-casein was 98% as high as the positive control (i.e., medium with all AA). A similar trend was found with P-ribosomal protein S6 kinase 1 and P-ribosomal protein S6. In conclusion, the extracellular concentrations of His, Lys, Met, and Leu at a ratio of 5:6:1:7 maximized β-casein expression in the immortalized bovine mammary epithelial cell line may occur via activation of the mechanistic target of rapamycin complex 1 signaling pathway.

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Section: Cell Culturementioning

confidence: 99%

Combination of histidine, lysine, methionine, and leucine promotes β-casein synthesis via the mechanistic target of rapamycin signaling pathway in bovine mammary epithelial cells

Gao

Zhao

Zheng

et al. 2017

Journal of Dairy Science

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“…In contrast, the protein level of E-cadherin was increased in Co cells co-cultured with mF fibroblasts, but not with nF fibroblasts. Like many SV40-immortalized cells [ 26 ], Co colonocytes express CSC markers. In this study, we targeted the stem cell marker Lgr5.…”

Section: Resultsmentioning

confidence: 99%

Study of the colonic epithelial-mesenchymal dialogue through establishment of two activated or not mesenchymal cell lines: Activated and resting ones differentially modulate colonocytes in co-culture

Plaisancié

Buisson²,

Fouché³

et al. 2022

PLoS ONE

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Continuous and rapid renewal of the colonic epithelium is crucial to resist the plethora of luminal deleterious agents. Subepithelial fibroblasts contribute to this turnover by regulating epithelial proliferation and differentiation. However, when intestinal homeostasis is disturbed, fibroblasts can acquire an activated phenotype and play a major role in the progression of intestinal pathologies. To evaluate the involvement of fibroblasts in the regulation of colonocytes under homeostatic or pathological conditions, we established resting and activated conditionally immortalized fibroblast cell lines (nF and mF) from mouse colonic mucosa. We then studied the epithelial-mesenchymal interactions between activated or resting fibroblasts and the normal mouse colonocytes (Co) using a co-culture model. Both fibroblastic cell lines were characterized by RT-qPCR, western blot and immunofluorescence assay. Our results showed that nF and mF cells were positive for fibroblastic markers such as vimentin and collagen 1, and negative for cytokeratin 18 and E-cadherin, attesting to their fibroblastic type. They also expressed proteins characteristic of the epithelial stem cell niche such as Grem1, CD90 or Wnt5a. Only rare nF fibroblasts were positive for α-SMA, whereas all mF fibroblasts strongly expressed this marker, supporting that mF cells were activated fibroblasts/myofibroblasts. In coculture, nF fibroblasts and Co cells strongly interacted via paracrine exchanges resulting in BMP4 production in nF fibroblasts, activation of BMP signaling in Co colonocytes, and decreased growth of colonocytes. Activated-type mF fibroblasts did not exert the same effects on Co cells, allowing colonocytes free to proliferate. In conclusion, these two colonic fibroblast lines, associated with Co cells in coculture, should allow to better understand the role of mesenchymal cells in the preservation of homeostasis and the development of intestinal pathologies.

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“…The repair process may also require the replacement of endothelial and epithelial dead cells with bone marrow-derived progenitor cells (Sharma and Jeong, 2013). Recently, Hu et al (2016) produced immortalized bovine mammary epithelial cell lines capable of self-renewal and differentiation. Capuco et al (2009) showed that infusion of xanthosine expands mammary stem and progenitor cell populations but results are still not conclusive.…”

Section: Discussionmentioning

confidence: 99%

Tolerance to bovine clinical mastitis: Total, direct, and indirect milk losses

Detilleux

2018

Journal of Dairy Science

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The objectives of this paper were to estimate direct and indirect milk losses associated with mastitis. Indirect losses, linked to indirect tolerance, are mediated by the increase in milk somatic cell count (SCC) in response to bacterial infection. Direct losses, linked to weak direct tolerance, are not mediated by the increase in SCC. So far, studies have evaluated milk loss associated with clinical mastitis without considering both components, which may lead to biased estimates of their sum; that is, the total loss in milk. A total of 43,903 test-day records on milk and SCC from 3,716 cows and 5,858 lactations were analyzed with mediation mixed models and health trajectories to estimate the amount of direct, indirect, and total milk losses after adjustment for known and potentially unmeasured (sensitivity analyses) confounding factors. Estimates were formalized under the counterfactual causal theory of causation. In this study, milk losses were mostly mediated by an increase in SCC. They were highest in the first month of lactation, when SCC were highest. Milk losses were estimated at 0.5, 0.8, and 1.1 kg/d in first, second, and third and greater parity, respectively. Two phases described how changes in milk were associated with changes in SCC: on average, one occurred before and one after the day preceding the clinical diagnosis. In both phases, changes in milk were estimated at 1 mg/d per 10 cells/mL. After adjusting for known confounders, cow effect accounted for 20.7 and 64.2% of the variation in milk in the first and second phases, respectively. This suggests that deviations from the resilient path were highest during the second phase of inflammation and that selection for cows more tolerant to mastitis is feasible. As discussed herein, epigenetic regulation of macrophage polarization may contribute to the variation in milk observed in the second phase.

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Immortalized bovine mammary epithelial cells express stem cell markers and differentiate in vitro

Cited by 13 publications

References 43 publications

Combination of histidine, lysine, methionine, and leucine promotes β-casein synthesis via the mechanistic target of rapamycin signaling pathway in bovine mammary epithelial cells

Combination of histidine, lysine, methionine, and leucine promotes β-casein synthesis via the mechanistic target of rapamycin signaling pathway in bovine mammary epithelial cells

Study of the colonic epithelial-mesenchymal dialogue through establishment of two activated or not mesenchymal cell lines: Activated and resting ones differentially modulate colonocytes in co-culture

Tolerance to bovine clinical mastitis: Total, direct, and indirect milk losses

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