Chemical & Biological Integrative Research Center, Korea Institute of Science and Technology, Seoul 02792, Korea.
KHU-KIST Department of Converging Science and Technology, Graduate School, Kyung Hee University, Seoul 02447, Korea.
Int J Mol Sci. 2022 Jun 10;23(12):6527. doi: 10.3390/ijms23126527.
Co-culture system, in which two or more distinct cell types are cultured together, is advantageous in that it can mimic the environment of the in vivo niche of the cells. In this study, we presented a strategy to analyze the secretome of a specific cell type under the co-culture condition in serum-supplemented media. For the cell-specific secretome analysis, we expressed the mouse mutant methionyl-tRNA synthetase for the incorporation of the non-canonical amino acid, azidonorleucine into the newly synthesized proteins in cells of which the secretome is targeted. The azidonorleucine-tagged secretome could be enriched, based on click chemistry, and distinguished from any other contaminating proteins, either from the cell culture media or the other cells co-cultured with the cells of interest. In order to have more reliable true-positive identifications of cell-specific secretory bodies, we established criteria to exclude any identified human peptide matched to bovine proteins. As a result, we identified a maximum of 719 secreted proteins in the secretome analysis under this co-culture condition. Last, we applied this platform to profile the secretome of mesenchymal stem cells and predicted its therapeutic potential on osteoarthritis based on secretome analysis.
共培养系统是将两种或多种不同类型的细胞一起培养，这种方法的优势在于它可以模拟细胞体内微环境。在本研究中，我们提出了一种在有血清补充的培养基中分析共培养条件下特定细胞类型的分泌组的策略。为了进行细胞特异性分泌组分析，我们在目的细胞中表达了突变的甲硫氨酰-tRNA 合成酶，以将非典型氨基酸叠氮亮氨酸掺入新合成的蛋白质中。基于点击化学，可以富集叠氮亮氨酸标记的分泌组，并将其与细胞培养基或与感兴趣的细胞共培养的其他细胞中的任何其他污染蛋白区分开来。为了更可靠地鉴定细胞特异性分泌小体的真正阳性，我们建立了排除任何与人蛋白匹配的牛蛋白的鉴定标准。结果，在这种共培养条件下的分泌组分析中，我们最多鉴定到了 719 种分泌蛋白。最后，我们将该平台应用于间充质干细胞的分泌组分析，并基于分泌组分析预测其对骨关节炎的治疗潜力。
