Sandhu Mansur A, Jurek Sandra, Trappe Susanne, Kolisek Martin, Sponder Gerhard, Aschenbach Jörg R
Institute of Veterinary Physiology, Free University of Berlin, Berlin, Germany.
Cells Tissues Organs. 2017;204(1):13-24. doi: 10.1159/000472708. Epub 2017 May 12.
To establish the influence of fetal bovine serum (FBS) and bovine serum lipids (BSL) on cell differentiation marker expression, bovine adipose-derived stem cells from subcutaneous tissue were incubated for 14 days in 4 types of differentiation media containing 10% FBS and 10 µL/mL BSL (TRT-1), no FBS and 10 µL/mL of BSL (TRT-2), 10% FBS and no BSL (TRT-3), or no supplements (TRT-4). Cells were subjected to Nile red staining, immunocytochemistry (CD73, CD90, CD105, DLK1, FabP4), and quantitative real-time PCR (CD73, CD90, CD105, FabP4). The number of cells presenting FabP4 and the percentage of mature adipocytes with large lipid droplets were increased in TRT-2, accompanied by a robust increase in FabP4 mRNA abundance and a decrease in DLK1-positive cells. In preadipocytes, CD73 was present around the nucleus and translocated towards cell membranes during differentiation. Although the percentage of CD73-positive cells was not different among treatments, its mRNA abundance, immunocytochemical staining intensity, and translocation towards cell membranes were decreased when the medium contained no FBS (TRT-2 and TRT-4). All cells showed a diffuse distribution of CD90 and CD105 and remained positive for these markers irrespective of the treatment. However, the CD90 and CD105 mRNA abundance was decreased in TRT-2 and TRT-4; i.e., in media containing no FBS. The presence of FBS increased the absolute number of cell nuclei as assessed by DAPI fluorescence. Our results suggest that bovine subcutaneous preadipocytes display typical stem cell markers. The differentiation into mature adipocytes is promoted by BSL, whereas FBS endorses cell proliferation.
为确定胎牛血清（FBS）和牛血清脂质（BSL）对细胞分化标志物表达的影响，将来自皮下组织的牛脂肪来源干细胞在4种分化培养基中培养14天，这4种培养基分别含有10% FBS和10 μL/mL BSL（TRT - 1）、不含FBS但含10 μL/mL BSL（TRT - 2）、10% FBS但不含BSL（TRT - 3）或无添加物（TRT - 4）。对细胞进行尼罗红染色、免疫细胞化学检测（CD73、CD90、CD105、DLK1、FabP4）以及定量实时PCR（CD73、CD90、CD105、FabP4）。在TRT - 2组中，呈现FabP4的细胞数量以及具有大脂滴的成熟脂肪细胞百分比增加，同时FabP4 mRNA丰度显著增加，DLK1阳性细胞减少。在前脂肪细胞中，CD73在细胞核周围存在，并在分化过程中向细胞膜移位。尽管各处理组中CD73阳性细胞的百分比没有差异，但当培养基不含FBS时（TRT - 2和TRT - 4），其mRNA丰度、免疫细胞化学染色强度以及向细胞膜的移位均降低。所有细胞均显示CD90和CD105呈弥漫性分布，且无论处理如何，这些标志物均保持阳性。然而，TRT - 2和TRT - 4组（即不含FBS的培养基）中CD90和CD105 mRNA丰度降低。通过DAPI荧光评估，FBS的存在增加了细胞核的绝对数量。我们的结果表明，牛皮下前脂肪细胞表现出典型的干细胞标志物。BSL促进向成熟脂肪细胞的分化，而FBS支持细胞增殖。
