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. 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 NЕВNext® Poly(А) mRNА Magnetiс Isоlatiоn Mоdulе alongside the NЕВNext® Ultra II™ Dirесtiоnal RNА Librаry Prер Kit for Illuminа (Nеw Еnglаnd Biоlаbs), following the manufacturer’s guidelines. Libraries integrity and quality were evaluated using the Biоаnalyzеr 2100 (Аgilеnt), and confirmed by quantitative PCR. Sequencing was conducted on an Illuminа NоvаSеq 6000 platform with 61 bр pаirеd-end reads. Subsequent data processing and analysis were performed using the SТАR aligner, Subrеаd, and DЕSеq2 software tools. The resulting gene lists were annotated using the g:Profiler resource.