Osteoprotegerin (OPG), a conventional potent inhibitor of osteoclastogenesis, is being increasingly recognized for its diverse roles beyond bone metabolism. However, the cell-autonomous role of OPG in regulating the differentiation and fate of mesenchymal progenitor cells (MPCs) remains to be fully elucidated. To address this issue, OPG-knockout (OPG-KO) human induced pluripotent stem cell-derived MPCs were generated using CRISPR/Cas9 technology. Transcriptomics revealed that OPG deficiency fundamentally alters the functional landscape of the MPCs, with a significant downregulation of the pathways related to the extracellular matrix (ECM), cell adhesion, and structural signaling. Specifically, the expression of numerous key ECM components was broadly attenuated in OPG-KO MPCs. Such molecular disruption functionally translated into severely impaired osteogenic potential, characterized by a marked transcriptional attenuation of osteogenic markers and reduced matrix mineralization at the cell level. Collectively, our findings demonstrate that OPG is essential for maintaining the structural integrity of the MPC niche by regulating the expression of ECM-related genes, thereby promoting osteoblast differentiation.

Introduction
Materials and Methods
    1. Maintenance of human induced pluripotent stemcells
    2. Differentiation of hiPSCs into mesenchymalprogenitors
    3. RNA-sequencing
    4. Quantitative real-time polymerase chain reaction
    5. Differentiation of hiPSC-MPCs into osteoblasts
    6. Alizarin Red S staining
    7. Statistical analysis
Results
    1. Differentiation of OPG-KO hiPSCs intomesenchymal progenitor cells
    2. Global transcriptomic comparison of WT- andOPG-KO MPCs
    3. Osteoprotegerin regulates extracellular matrixremodeling and cell adhesion in mesenchymalprogenitor cells
    4. Impairment of osteoblast differentiation andmineralization by OPG deficiency
Discussion
Funding
Conflicts of Interest
Supplementary Data
References

• Jujin Jeong(Department of Interdisciplinary Program of Biomedical Engineering, School of Dentistry, Chonnam National University, Gwangju 61186, Republic of Korea)
• Byeong-Cheol Park(Department of Interdisciplinary Program of Biomedical Engineering, School of Dentistry, Chonnam National University, Gwangju 61186, Republic of Korea)
• Tae-Hoon Lee(Department of Interdisciplinary Program of Biomedical Engineering, School of Dentistry, Chonnam National University, Gwangju 61186, Republic of Korea, Department of Dental Bioscience, School of Dentistry, Chonnam National University, Gwangju 61186, Republic of Korea, Department of Oral Biochemistry, Dental Science Research Institute, School of Dentistry, Chonnam National University, Gwangju 61186, Republic of Korea) Corresponding author
• Sang-Wook Park(Department of Interdisciplinary Program of Biomedical Engineering, School of Dentistry, Chonnam National University, Gwangju 61186, Republic of Korea, Department of Dental Bioscience, School of Dentistry, Chonnam National University, Gwangju 61186, Republic of Korea, Department of Oral Biochemistry, Dental Science Research Institute, School of Dentistry, Chonnam National University, Gwangju 61186, Republic of Korea) Corresponding author