A passage-dependent network for estimating the in vitro senescence of mesenchymal stromal/stem cells using microarray, bulk and single cell RNA sequencing

Yong Yang; Wencheng Zhang; Xicheng Wang; Jingxian Yang; Yangyang Cui; Haimeng Song; Weiping Li; Wei Li; Le Wu; Yao Du; Zhiying He; Jun Shi; Jiangnan Zhang

doi:10.3389/fcell.2023.998666

A passage-dependent network for estimating the in vitro senescence of mesenchymal stromal/stem cells using microarray, bulk and single cell RNA sequencing

Front Cell Dev Biol. 2023 Feb 7:11:998666. doi: 10.3389/fcell.2023.998666. eCollection 2023.

Authors

Yong Yang¹, Wencheng Zhang^{2

3

4}, Xicheng Wang^{2

3

4}, Jingxian Yang⁵, Yangyang Cui^{2

3

4

6}, Haimeng Song^{2

3

4}, Weiping Li⁷, Wei Li⁸, Le Wu⁸, Yao Du¹, Zhiying He^{2

3

4}, Jun Shi¹, Jiangnan Zhang¹

Affiliations

¹ Department of General Surgery, The First Affiliated Hospital of Nanchang University, Nanchang, Jiangxi, China.
² Institute for Regenerative Medicine, Shanghai East Hospital, School of Life Sciences and Technology, Tongji University, Shanghai, China.
³ Shanghai Engineering Research Center of Stem Cells Translational Medicine, Shanghai, China.
⁴ Shanghai Institute of Stem Cell Research and Clinical Translation, Shanghai, China.
⁵ Department of Anesthesiology, The First Affiliated Hospital of Nanchang University, Nanchang, Jiangxi, China.
⁶ Postgraduate Training Base of Shanghai East Hospital, Jinzhou Medical University, Jinzhou, Liaoning, China.
⁷ Department of Gastrointestinal Surgery, The First People's Hospital of Taicang City, Taicang Affiliated Hospital of Soochow University, Taicang, Jiangsu, China.
⁸ Department of General Surgery, Fuzhou Dongxiang District People's Hospital, Fuzhou, Jiangxi, China.

Abstract

Long-term in vitro culture of human mesenchymal stem cells (MSCs) leads to cell lifespan shortening and growth stagnation due to cell senescence. Here, using sequencing data generated in the public domain, we have established a specific regulatory network of "transcription factor (TF)-microRNA (miRNA)-Target" to provide key molecules for evaluating the passage-dependent replicative senescence of mesenchymal stem cells for the quality control and status evaluation of mesenchymal stem cells prepared by different procedures. Short time-series expression miner (STEM) analysis was performed on the RNA-seq and miRNA-seq databases of mesenchymal stem cells from various passages to reveal the dynamic passage-related changes of miRNAs and mRNAs. Potential miRNA targets were predicted using seven miRNA target prediction databases, including TargetScan, miRTarBase, miRDB, miRWalk, RNA22, RNAinter, and TargetMiner. Then use the TransmiR v2.0 database to obtain experimental-supported transcription factor for regulating the selected miRNA. More than ten sequencing data related to mesenchymal stem cells or mesenchymal stem cells reprogramming were used to validate key miRNAs and mRNAs. And gene set variation analysis (GSVA) was performed to calculate the passage-dependent signature. The results showed that during the passage of mesenchymal stem cells, a total of 29 miRNAs were gradually downregulated and 210 mRNA were gradually upregulated. Enrichment analysis showed that the 29 miRNAs acted as multipotent regulatory factors of stem cells and participated in a variety of signaling pathways, including TGF-beta, HIPPO and oxygen related pathways. 210 mRNAs were involved in cell senescence. According to the target prediction results, the targets of these key miRNAs and mRNAs intersect to form a regulatory network of "TF-miRNA-Target" related to replicative senescence of cultured mesenchymal stem cells, across 35 transcription factor, 7 miRNAs (has-mir-454-3p, has-mir-196b-5p, has-mir-130b-5p, has-mir-1271-5p, has-let-7i-5p, has-let-7a-5p, and has-let-7b-5p) and 7 predicted targets (PRUNE2, DIO2, CPA4, PRKAA2, DMD, DDAH1, and GATA6). This network was further validated by analyzing datasets from a variety of mesenchymal stem cells subculture and lineage reprogramming studies, as well as qPCR analysis of early passages mesenchymal stem cells versus mesenchymal stem cells with senescence morphologies (SA-β-Gal⁺). The "TF-miRNA-Target" regulatory network constructed in this study reveals the functional mechanism of miRNAs in promoting the senescence of MSCs during in vitro expansion and provides indicators for monitoring the quality of functional mesenchymal stem cells during the preparation and clinical application.

Keywords: extracellular matrix (ECM); in vitro expansion; mesenchymal stem cells (MSCs); microRNAs (miRNAs); regulatory network construction; replicative senescence.

Grants and funding

This work was funded by Major Program of National Key Research and Development Project (2020YFA0112600, 2019YFA0801502), National Natural Science Foundation of China (82173019, 82270638, 82203741), Shanghai Pujiang Program (21PJD059), the Project of Shanghai Science and Technology Commission (19140902900, 22ZR1451100, 22Y11908500), Program of Shanghai Academic/Technology Research Leader (20XD1434000), Peak Disciplines (Type IV) of Institutions of Higher Learning in Shanghai, Jiangxi Provincial Natural Science Foundation (20212ACB206033), and Shanghai Engineering Research Center of Stem Cells Translational Medicine (20DZ2255100).