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This galley proof is being listed electronically before publishing the final manuscript (It's not final version).

Single-cell RNA sequencing reveals heterogeneity of adipose tissue-derived mesenchymal stem cells under chondrogenic induction
Jeewan Chun1,2,# (Graduate student), Ji-Hoi Moon 1,# (Professor), Kyu Hwan Kwack1 (Professor), Eun-Young Jang1,2 (Graduate student), Saebyeol Lee3 (Graduate student), Hak Kyun Kim 3,# (Professor), Jae-Hyung Lee 1,*,# (Professor)
1Department of Oral Microbiology, College of Dentistry and 2Department of Dentistry, Graduate School, Kyung Hee University,
3Department of Life Science, Chung-Ang University
This study aimed to investigate how adipose tissue-derived mesenchymal stem cells (AT-MSCs) respond to chondrogenic induction in vitro using droplet-based single-cell RNA sequencing (scRNA-seq). We analyzed 37,219 high-quality transcripts from control cells and cells induced for 1 week (1W) and 2 weeks (2W). Four distinct cell clusters (0-3), which conventional bulk analyses couldn’t detect, exhibited varying proportions. Cluster 1 was prominent in control and 1W cells, whereas cluster 3, 2, and 0 exclusively dominated in control, 1W, and 2W cells, respectively. Furthermore, we discovered heterogeneous expression levels of chondrogenic markers within clusters, concealed by bulk analysis. Gene ontology (GO) enrichment analysis of differentially expressed genes unveiled cluster-specific variations in key biological processes (BP): (1) Cluster 1 exhibited upregulation of GO-BP terms related to ribosome biogenesis and translational control, crucial for maintaining stem cell properties and homeostasis; (2) Additionally, cluster 1 showed upregulation of GO-BP terms associated with mitochondrial oxidative metabolism; (3) Cluster 3 displayed upregulation of GO-BP terms related to cell proliferation; (4) Clusters 0 and 2 demonstrated similar upregulation of GO-BP terms linked to collagen fibril organization and supramolecular fiber organization. However, only cluster 0 showed a significant decrease in GO-BP terms related to ribosome production, implying a potential correlation between ribosome regulation and the differentiation status of AT-MSCs. Overall, our findings highlight heterogeneous cell clusters with varying balances between proliferation and differentiation before and after chondrogenic stimulation. This provides enhanced insights into the single-cell dynamics of AT-MCSs during chondrogenic differentiation.
Abstract, Accepted Manuscript(in press) [Submitted on September 1, 2023, Accepted on October 12, 2023]
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