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1314 - Integration-free Cell Reprogramming Rejuvenates Human Synovial Fluid-derived Mesenchymal Stem Cells

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ORS 2019 Annual Meeting

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Integration-free Cell Reprogramming Rejuvenates Human Synovial Fluid-derived Mesenchymal Stem Cells Hongli Jiao, Brian E. Walczak, Ming-Song Lee, Wan-Ju Li University of Wisconsin-Madison, Madison, WI jiao@ortho.wisc.edu Disclosures: Hongli Jiao (N), Brian E. Walczak (N), Ming-Song Lee (N), Wan-Ju Li (N). INTRODUCTION: Mesenchymal stem cells (MSCs) hold promise as a therapeutic agent for regenerative medicine. However, their applications are often limited by properties of the cell, which are largely dependent on age and health status of a donor. Somatic cell reprogramming capable of altering the epigenetics of a cell may be used to overcome these limitations. METHODS: The study was approved by the Institutional Review Board at the University of Wisconsin-Madison, and informed consents were obtained from all study subjects. Human synovial fluid-derived mesenchymal stem cells (SF-MSCs) were reprogrammed through the non-viral, integration-free episomal approach into induced pluripotent stem cells (iPSCs), which were then subsequently differentiated into MSCs to establish iPSC-MSC lines. Activities of iPSC-MSCs and their parental SF-MSCs were compared to determine if reprogramming is able to alter MSC properties. All quantitative data of assays that analyze three donors’ cells were presented as the mean ± standard deviation. A Student’s t-test or one-way analysis of variance (ANOVA) with post-hoc Tukey’s test was used for statistical comparison. A P-value <0.05 was considered statistically significant. RESULTS: Our results showed that SF-MSCs were successfully reprogrammed into iPSCs (Fig. A-C) and both SF-MSCs and iPSC-MSCs exhibited similar fibroblast-like morphology (Fig. D). Compared to their parental SF-MSCs, iPSC-MSCs exhibited a significantly higher proliferation rate (Fig. E). Clustered heatmap of RNA sequencing (RNA-Seq) data showing differentially expressed genes (DEGs) among three cell types revealed that iPSC-MSCs were more similar to SF-MSCs than iPSCs (Fig. F). All cell types highly expressed CD90 but lacked CD45. CD105 and CD73 were both absent on iPSCs. Similar to SF-MSCs, almost all iPSC-MSCs were positive for CD73. CD105 was more strongly expressed on SF-MSCs than iPSC-MSCs (Fig. G). Compared to SF-MSCs, the osteogenic and chondrogenic potential of iPSC-MSCs was upregulated while their adipogenic potential was downregulated (Fig. H). The number of senescence-associated β-galactosidase (SA-β-gal) positive cells in SF-MSCs was significantly higher than that in iPSC-MSCs (Fig. I). We compared the expression of markers of the p53 and p16 pathways between SF-MSCs and iPSC-MSCs and found higher expression levels of p53 and p21 and lower expression levels of CDK1 in SF-MSCs than those in iPSC-MSCs. There was no significance difference in the expression of p16 between these two cell types (Fig. J, K). We found that cellular reprogramming led to an increase in the expression of telomerase reverse transcriptase (TERT) and telomerase activity, which in turn lengthened telomeres in iPSCs and iPSC-MSCs. The β-Catenin level in iPSCs was also found significantly higher than that that in SF-MSCs and iPSC-MSCs with the expression level higher in iPSC-MSCs than that in SF-MSCs (Fig. L). Knockdown of β-catenin reduced the expression of Tert, Tert activity, and telomere length (Fig. M), suggesting that Wnt signaling played a critical role in the process of cell rejuvenation. DISCUSSION: In this study, we demonstrate that cellular reprogramming is able to rejuvenate SF-MSCs through altering cellular and molecular characteristics of aging, including cellular senescence, telomere erosion, and changes in global gene expression, to increase proliferation and differentiation potential of the cell. SIGNIFICANCE: Our findings suggest that integration-free iPSC-MSCs may be an alternative cell choice to overcome the challenge of cellular aging associated with age and health status of a donor and/or induced by culture expansion for orthopedic regenerative medicine. This study also provides insight into a mechanism that governs cellular aging of MSCs.

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