Niehage Christian, Karbanová Jana, Steenblock Charlotte, Corbeil Denis, Hoflack Bernard
Biotechnology Center (BIOTEC), Technische Universität Dresden, Dresden, Germany.
PLoS One. 2016 Aug 4;11(8):e0159824. doi: 10.1371/journal.pone.0159824. eCollection 2016.
Multipotent mesenchymal stromal cells (MSCs) are promising tools for regenerative medicine. They can be isolated from different sources based on their plastic-adherence property. The identification of reliable cell surface markers thus becomes the Holy Grail for their prospective isolation. Here, we determine the cell surface proteomes of human dental pulp-derived MSCs isolated from single donors after culture expansion in low (2%) or high (10%) serum-containing media. Cell surface proteins were tagged on intact cells using cell impermeable, cleavable sulfo-NHS-SS-biotin, which allows their enrichment by streptavidin pull-down. For the proteomic analyses, we first compared label-free methods to analyze cell surface proteomes i.e. composition, enrichment and proteomic differences, and we developed a new mathematical model to determine cell surface protein enrichment using a combinatorial gene ontology query. Using this workflow, we identified 101 cluster of differentiation (CD) markers and 286 non-CD cell surface proteins. Based on this proteome profiling, we identified 14 cell surface proteins, which varied consistently in abundance when cells were cultured under low or high serum conditions. Collectively, our analytical methods provide a basis for identifying the cell surface proteome of dental pulp stem cells isolated from single donors and its evolution during culture or differentiation. Our data provide a comprehensive cell surface proteome for the precise identification of dental pulp-derived MSC populations and their isolation for potential therapeutic intervention.
多能间充质基质细胞（MSCs）是再生医学中很有前景的工具。基于其贴壁特性，它们可以从不同来源分离出来。因此，鉴定可靠的细胞表面标志物成为其前瞻性分离的圣杯。在此，我们确定了从单个供体分离的人牙髓来源的MSCs在低（2%）或高（10%）血清培养基中培养扩增后的细胞表面蛋白质组。使用细胞不可渗透的、可裂解的磺基-NHS-SS-生物素对完整细胞上的细胞表面蛋白进行标记，这使得它们能够通过链霉亲和素下拉法进行富集。对于蛋白质组分析，我们首先比较了无标记方法来分析细胞表面蛋白质组，即组成、富集和蛋白质组差异，并开发了一种新的数学模型，使用组合基因本体查询来确定细胞表面蛋白的富集情况。使用这种工作流程，我们鉴定出101种分化簇（CD）标志物和286种非CD细胞表面蛋白。基于这种蛋白质组分析，我们鉴定出14种细胞表面蛋白，当细胞在低血清或高血清条件下培养时，它们的丰度会持续变化。总体而言，我们的分析方法为鉴定从单个供体分离的牙髓干细胞的细胞表面蛋白质组及其在培养或分化过程中的演变提供了基础。我们的数据提供了一个全面的细胞表面蛋白质组，用于精确鉴定牙髓来源的MSC群体，并将其分离用于潜在的治疗干预。
