Sestrin 2 (SESN2) is a member of the sestrin family of stress-induced proteins that negatively regulate agingassociated biological processes. This study aims to investigate the role of SESN2 in regulating the differentiation potential and senescence of mesenchymal stem cells (MSCs) derived from young and elderly donors. Bulk RNA sequencing revealed a common decline in the SESN2 mRNA levels in MSCs from elderly individuals, which was confirmed via reverse transcription-polymerase chain reaction and western blot analyses. SESN2 knockdown in MSCs from young donors resulted in phenotypic changes similar to those in MSCs from elderly donors, including an enhanced expression of senescence and adipogenic markers and diminished expression of osteogenic markers. To confirm the effect of decreased SESN2 expression on osteogenic and adipogenic differentiation, we induced Sesn2 knockdown in mouse bone marrow-derived MSCs. Sesn2 knockdown suppressed the mRNA expression of osteogenic marker genes, alkaline phosphatase activity, and matrix mineralization. Furthermore, Sesn2 knockdown enhanced mRNA expression of the adipogenic marker genes and intracellular lipid accumulation. These results suggest that a decline in SESN2 expression during aging contributes to the shift of MSC differentiation from osteogenic to adipogenic lineage.

Introduction
Materials and Methods
    1. Cell culture
    2. Sestrin 2 knockdown using siRNA
    3. Quantitative reverse transcription-polymerase chainreaction
    4. Western blot analysis
    5. Senescence-associated β-galactosidase staining
    6. Statistical analysis
Results
    1. MSCs obtained from the elderly express higherlevels of cellular senescence markers
    2. MSCs from the elderly express lower levels ofosteogenic marker genes and higher levels ofadipogenic marker genes compared to those fromthe young
    3. The expression levels of SESN2 are decreased inMSCs from the elderly
    4. Knockdown of SESN2 in young human MSCssuppresses osteogenic marker gene expressionwhile enhancing the expression of adipogenic andsenescence marker genes
    5. S esn2 knockdown inhibits osteogenic differentiationbut enhances adipogenic differentiation of mouseBMSCs
Discussion
Funding
Conflicts of Interest
Supplementary Data
www.kijob.or.kr 77Supplementary DataSupplementary data is available at http://www.kijob.or.kr only.References

• Do Yeun Kim(Department of Molecular Genetics, School of Dentistry and Dental Research Institute, Seoul National University, Seoul 08826, Republic of Korea)
• Hyun-Jung Park(Department of Molecular Genetics, School of Dentistry and Dental Research Institute, Seoul National University, Seoul 08826, Republic of Korea)
• Jeong-Hwa Baek(Department of Molecular Genetics, School of Dentistry and Dental Research Institute, Seoul National University, Seoul 08826, Republic of Korea) Corresponding author