Safety and Regenerative Properties of Immortalized Human Mesenchymal Stromal Cell Secretome

The secretome of mesenchymal stromal cells (MSCs) can efficiently stimulate regeneration and therefore is a tempting remedy for “cell-free cellular therapy”. However, the usage of primary MSC cultures as secretome-producers for translation studies has obvious obstacles, including rapid aging of MSC cultures, the need for a large number of verified donors and donor-to-donor variability of secretome content. MSCs immortalization allows to overcome those limitations and to obtain secretome-producing cultures with prolonged lifetime. However, the efficacy and safety of such secretomes are critical issues, which limit their usage as therapeutic agents. In this study we have tested in large detail how the immortalization of MSC cultures affects the content, biological activity and safety of their secretome. MSCs immortalization via overexpression of human TERT gene does not significantly alter the qualitative and quantitative composition of their secretome or its activity according to the results of proteomic analysis, ELISA, qPCR and functional tests in vitro. Moreover, we have demonstrated that the secretome of immortalized MSCs does not contain detectable amounts of telomerase and does not possess any transforming activity. Altogether, our data suggest that immortalized MSC cultures may become a reliable source for obtaining standardized active secretome in large-scale quantities for clinical use. Overall design: To assess the potential ability of iMSC secretome to alter expression of pro- and anti-oncogenes in target cells, we analyzed the transcriptome of primary human dermal fibroblasts treated with the secretome of pMSCs and iMSCs. For this purpose, we used cultures of primary human dermal fibroblasts of 5-8 passages of 80-90% confluent. In experimental groups growth medium was replaced with the non-concentrated secretome of pMSCs or iMSCs. In the negative control group, cells were cultured in complete fibroblast growth medium, and in the positive control groups mutagen solutions (3mM sodium nitrite or 0.01% DMS in complete fibroblast growth medium) were used [32,33]. In the DMS group, 6 hours after the start of the experiment, the DMS solution was replaced with the complete fibroblast growth medium. Test solutions were renewed every 3 days, and 7 days after the start of the experiment, the fibroblast cultures were lysed for total RNA isolation as described above. About 800 ng of total RNA (for each group) was utilized for library construction employing the N??Next® Poly(?) mRN? Magneti? Is?lati?n M?dul? alongside the N??Next® Ultra II™ Dir??ti?nal RN? Libr?ry Pr?? Kit for Illumin? (N?w ?ngl?nd Bi?l?bs), following the manufacturer's guidelines. Libraries integrity and quality were evaluated using the Bi??nalyz?r 2100 (?gil?nt), and confirmed by quantitative PCR. Sequencing was conducted on an Illumin? N?v?S?q 6000 platform with 61 b? p?ir?d-end reads. Subsequent data processing and analysis were performed using the S??R aligner, Subr??d, and D?S?q2 software tools. The resulting gene lists were annotated using the g:Profiler resource.

间充质基质细胞（mesenchymal stromal cells, MSCs）的分泌组（secretome）可高效刺激组织再生，因此成为“无细胞细胞疗法”的极具吸引力的治疗候选方案。然而，以原代MSC培养物作为分泌组生产源开展转化研究存在显著障碍：包括MSC培养物快速衰老、需依赖大量经过验证的供体，以及不同供体来源的分泌组内容物存在显著异质性。MSC永生化技术可克服上述局限，获得具有更长存活周期的分泌组生产培养物。但此类分泌组的疗效与安全性仍是限制其作为治疗制剂应用的核心问题。本研究系统探究了MSC培养物永生化对其分泌组的组成、生物学活性与安全性的影响。
通过过表达人端粒酶逆转录酶（human TERT）基因实现的MSC永生化，经蛋白质组学分析、ELISA、qPCR及体外功能实验验证，并未显著改变其分泌组的质与量组成，亦未影响其生物活性。此外，本研究证实永生化MSC的分泌组中未检出端粒酶，亦无任何转化活性。综上，本研究数据表明，永生化MSC培养物可成为规模化获取标准化活性分泌组以用于临床的可靠来源。
整体实验设计：为评估永生化MSC（iMSCs）分泌组调控靶细胞中原癌基因与抑癌基因表达的潜在能力，本研究分析了经原代MSC（pMSCs）及iMSCs分泌组处理的原代人真皮成纤维细胞的转录组。实验采用传代5-8代、汇合度80%-90%的原代人真皮成纤维细胞培养物。实验组将生长培养基替换为未浓缩的pMSCs或iMSCs分泌组；阴性对照组细胞使用完全成纤维细胞生长培养基培养；阳性对照组则使用含诱变剂的完全成纤维细胞生长培养基（3mM亚硝酸钠或0.01%二甲基亚砜（DMS））[32,33]。在DMS组中，实验开始6小时后将DMS溶液更换为完全成纤维细胞生长培养基。每3天更换一次测试培养基，实验开始7天后裂解成纤维细胞培养物以提取总RNA，方法如前所述。
每组取约800ng总RNA用于文库构建，采用NEBnext® Poly(A) mRNA Magnetic Isolation Module配合NEBnext® Ultra II™ Directional RNA Library Prep Kit for Illumina（New England Biolabs），严格遵循制造商的操作指南。使用Agilent 2100生物分析仪评估文库的完整性与质量，并通过定量PCR验证。测序在Illumina NovaSeq 6000平台上进行，采用61bp双端读长。后续数据处理与分析采用STAR aligner、Subread及DESeq2软件工具完成。最终得到的基因列表通过g:Profiler资源进行注释。