Altered Gene Expression in Human Adipose Stem Cells Cultured with Fetal Bovine Serum Compared to Human Supplements. Homo sapiens

Mesenchymal stromal cells (MSCs) are promising candidates for innovative cell therapeutic applications. For clinical scale manufacturing regulatory agencies recommend to replace fetal bovine serum (FBS) commonly used in MSC expansion media as soon as equivalent alternative supplements are available. We already demonstrated that pooled blood group AB human serum (HS) and thrombin-activated platelet releasate plasma (tPRP) support the expansion of multipotent adipose tissue-derived MSCs (ASCs). Slight differences in size, growth pattern and adhesion prompted us to investigate the level of equivalence by compiling the transcriptional profiles of ASCs cultivated in these supplements. A whole genome gene expression analysis was performed and data verified by PCR and protein analyses. Microarray-based screening of 34,039 genes revealed 102 genes differentially expressed in ASCs cultured with FBS compared to HS or tPRP supplements. A significantly higher expression in FBS cultures was found for 90 genes (fold change .2). Only 12 of the 102 genes showed a lower expression in FBS compared to HS or tPRP cultures (fold change .0.5). Differences between cells cultivated in HS and tPRP were hardly evident. Supporting previous observations of reduced adhesion of cells cultivated in the human alternatives we detected a number of adhesion and extracellular matrix associated molecules expressed at lower levels in ASCs cultivated with human supplements. Confirmative assays analysing transcript or protein expression with selected genes supported these results. Likewise a number of mesodermal differentiation associated genes were higher expressed in cells grown in FBS. Quantifying adipogenic and osteogenic differentiation lacked to demonstrate a clear correlation to the supplement due to donor-sepcific variances. Our results emphasize the necessity of comparability studies as they indicate that FBS induces a culture adaptation exceeding that of ex vivo culture in human supplements and thus may contribute to the therapeutic potential. Overall design: Microarray hybridizations (Agilent: Whole Human Genome Oligo Microarray; 41k unique probe) were carried out with 5 µg Cy3 labeled cRNA and 5 µg Cy5 labeled cRNA, both prepared from each sample.

间充质基质细胞（Mesenchymal stromal cells, MSCs）是创新细胞治疗应用中极具潜力的候选细胞。针对临床规模生产，监管机构建议在获得等效替代补料后，尽快替换目前常用于MSCs扩增培养基的胎牛血清（FBS）。本团队此前已证实，混合AB型人血清（HS）与凝血酶激活的血小板释放血浆（tPRP）可支持多能脂肪来源间充质基质细胞（ASCs）的体外扩增。鉴于不同补料培养的细胞在尺寸、生长模式及黏附特性上存在细微差异，本研究通过分析不同补料培养下ASCs的转录组谱，探究其等效性水平。
本研究开展了全基因组基因表达分析，并通过聚合酶链式反应（PCR）与蛋白质分析对所得数据进行验证。针对34039个基因的微阵列筛查显示，相较于以HS或tPRP为补料培养的ASCs，FBS培养的ASCs中有102个基因存在差异表达。其中90个基因在FBS培养组中表达显著上调（倍数变化＞2），仅12个基因在FBS培养组中表达显著下调（倍数变化＜0.5）。HS与tPRP培养组的细胞间差异几乎可忽略。
本研究检测到一系列黏附相关与细胞外基质相关分子在人源补料培养的ASCs中表达水平更低，这与此前报道的人源替代补料培养的细胞黏附能力降低的结果一致。针对选定基因的转录或蛋白表达验证实验进一步支持了上述结论。同样，多个中胚层分化相关基因在FBS培养的细胞中表达水平更高。由于供体间存在特异性差异，对成脂与成骨分化的定量分析未能明确展现出与补料类型的清晰相关性。
本研究结果凸显了可比性研究的必要性，结果表明FBS诱导的培养适应性变化强于人源补料的体外培养，或可对细胞的治疗潜能产生影响。
总体实验设计：采用安捷伦（Agilent）全人类基因组寡核苷酸微阵列（含41k个独特探针）进行微阵列杂交，每个样本均制备5μg Cy3标记的cRNA与5μg Cy5标记的cRNA用于杂交。