De Becker Ann, Heestermans Robbe, De Brouwer Wouter, Bockstaele Kara, Maes Ken, Van Riet Ivan
Department of Hematology, Universitair Ziekenhuis Brussel (UZ Brussel), Vrije Universiteit Brussel (VUB), Brussels, Belgium.
Research Group Hematology-Immunology, Vrije Universiteit Brussel (VUB), Brussels, Belgium.
Front Bioeng Biotechnol. 2022 Oct 12;10:1008271. doi: 10.3389/fbioe.2022.1008271. eCollection 2022.
Mesenchymal stromal cells (MSCs) are non-hematopoietic cells that have a broad therapeutic potential. To obtain sufficient cells for clinical application, they must be expanded . In the initial expansion protocols described, fetal calf serum (FCS) was used as the reference growth supplement, but more recently different groups started to replace FCS with platelet lysate (PL). We investigated in this study the impact of the culture supplement on gene expression of MSCs. Human bone marrow derived MSCs were expanded in FCS and PL supplemented medium. We found that MSCs expanded in PL-containing medium (PL-MSCs) express typical MSC immunomorphological features and can migrate, as their counterparts expanded in FCS-containing medium, through a layer of endothelial cells Additionally, they show an increased proliferation rate compared to MSCs expanded in FCS medium (FCS-MSCs) RNA sequencing performed for MSCs cultured in both types of expansion medium revealed a large impact of the choice of growth supplement on gene expression: 1974 genes were at least twofold up- or downregulated. We focused on impact of genes involved in apoptosis and senescence. Our data showed that PL-MSCs express more anti-apoptotic genes and FCS-MSCs more pro-apoptotic genes. FCS-MSCs showed upregulation of senescence-related genes after four passages whereas this was rarer in PL-MSCs at the same timepoint. Since PL-MSCs show higher proliferation rates and anti-apoptotic gene expression, they might acquire features that predispose them to malignant transformation. We screened 10 MSC samples expanded in PL-based medium for the presence of tumor-associated genetic variants using a 165 gene panel and detected only 21 different genetic variants. According to our analysis, none of these were established pathogenic mutations. Our data show that differences in culture conditions such as growth supplement have a significant impact on the gene expression profile of MSCs and favor the use of PL over FCS for expansion of MSCs.
间充质基质细胞（MSCs）是具有广泛治疗潜力的非造血细胞。为了获得足够数量的细胞用于临床应用，必须对其进行扩增。在最初描述的扩增方案中，胎牛血清（FCS）被用作参考生长补充剂，但最近不同的研究小组开始用血小板裂解物（PL）替代FCS。在本研究中，我们调查了培养补充剂对MSCs基因表达的影响。将人骨髓来源的MSCs在补充有FCS和PL的培养基中进行扩增。我们发现，在含PL的培养基（PL-MSCs）中扩增的MSCs表现出典型的MSC免疫形态学特征，并且能够像在含FCS的培养基中扩增的对应细胞一样，穿过一层内皮细胞进行迁移。此外，与在FCS培养基（FCS-MSCs）中扩增 的MSCs相比，它们的增殖率有所提高。对在两种扩增培养基中培养的MSCs进行RNA测序发现，生长补充剂的选择对基因表达有很大影响：1974个基因至少上调或下调了两倍。我们重点关注了参与细胞凋亡和衰老的基因的影响。我们的数据表明，PL-MSCs表达更多的抗凋亡基因，而FCS-MSCs表达更多的促凋亡基因。FCS-MSCs在传代四次后衰老相关基因上调，而在同一时间点，PL-MSCs中这种情况较少见。由于PL-MSCs表现出更高的增殖率和抗凋亡基因表达，它们可能获得使其易发生恶性转化的特征。我们使用165个基因的检测板，对在基于PL的培养基中扩增的10个MSC样本进行了肿瘤相关基因变异的筛查，仅检测到21种不同的基因变异。根据我们的分析，这些变异均不是已确定的致病突变。我们的数据表明，诸如生长补充剂等培养条件的差异对MSCs的基因表达谱有显著影响，并且在MSCs扩增方面，PL比FCS更具优势。
