In Vitro Culture Expansion Impairs Chondrogenic Differentiation and Therapeutic Effect of Mesenchymal Stem Cells by Regulation of Unfolded Protein Response

Abstract

In vitro expansion of mesenchymal stem cells (MSCs) has been implicated in loss of multipotency, leading to impaired chondrogenic potential and eventually therapeutic effect, as reported in our previous study. However, the precise regulatory mechanism is still unclear. Here, we demonstrate that endoplasmic reticulum (ER) stress and unfolded protein response (UPR) were highly enriched in in vitro-cultured bone marrow MSCs at passage 3 (P3 BMSCs) vs. flesh P0 BMSCs by microarray analysis. Indeed, RT-PCR and western blot analysis showed significantly lower expression levels of three key UPR-related molecules, ATF4, ATF6 and XBP1 in P3 BMSCs than P0 BMSCs. Further, we found that UPR suppression by 4-phenylbutyrate (4-PBA) impaired chondrogenesis and cartilage repair in P0 BMSCs, and UPR induction by tunicamycin (TM) enhanced chondrogenic differentiation and cartilage regeneration in in vitro-cultured P3 BMSCs. Thus, the decline of chondrogenic potential of stem cells after in vitro culture and expansion may be due to the change of ER stress and UPR pathway. Funding: This study was financially supported by National key research and development program of China (2016YFB0700804), National Natural Science Fund of China (Grant No. 81760326 and 81460345), the Guangxi Scientific Research and Technological Development Foundation (Grant No. GuikeAB16450003) and the Guangxi Science and Technology Major Project (Guike AA17204085), High level innovation teams and outstanding scholars in Guangxi Universities (The third batch), the Distinguished Young Scholars Program of Guangxi Medical University, and the Key scientific research collaboration program of Guangxi Biomedical Collaborative Innovation Center (GCICB-SR-2017002). Declaration of Interest: No conflict of interests exists in this study. Ethical Approval: The experiments were approved by Animal Care and Experiment Committee of Guangxi Medical University (protocol number: 2014-12-3).