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Item Osteogenic Differentiation Potential of Mesenchymal Stem Cells Using Single Cell Multiomic Analysis(MDPI, 2023-09-26) Chen, Duojiao; Liu, Sheng; Chu, Xiaona; Reiter, Jill; Gao, Hongyu; McGuire, Patrick; Yu, Xuhong; Xuei, Xiaoling; Liu, Yichen; Wan, Jun; Fang, Fang; Liu, Yunlong; Wang, Yue; Medical and Molecular Genetics, School of MedicineMesenchymal stem cells (MSC) are multipotent stem cells that can differentiate into multiple cell types, including osteoblasts, chondrocytes, and adipocytes. Osteoblast differentiation is reduced during osteoporosis development, resulting in reduced bone formation. Further, MSC isolated from different donors possess distinct osteogenic capacity. In this study, we used single-cell multiomic analysis to profile the transcriptome and epigenome of MSC from four healthy donors. Data were obtained from ~1300 to 1600 cells for each donor. These cells were clustered into four groups, indicating that MSC from different donors have distinct chromatin accessible regulatory elements for regulating gene expression. To investigate the mechanism by which MSC undergo osteogenic differentiation, we used the chromatin accessibility data from the single-cell multiome data to identify individual-specific enhancer–promoter pairs and evaluated the expression levels and activities of the transcriptional regulators. The MSC from four donors showed distinct differentiation potential into osteoblasts. MSC of donor 1 showed the largest average motif activities, indicating that MSC from donor 1 was most likely to differentiate into osteoblasts. The results of our validation experiments were consistent with the bioinformatics prediction. We also tested the enrichment of genome-wide association study (GWAS) signals of several musculoskeletal disease traits in the patient-specific chromatin accessible regions identified in the single-cell multiome data, including osteoporosis, osteopenia, and osteoarthritis. We found that osteoarthritis-associated variants were only enriched in the regions identified from donor 4. In contrast, osteoporosis and osteopenia variants were enriched in regions from donor 1 and least enriched in donor 4. Since osteoporosis and osteopenia are related to the density of bone cells, the enrichment of variants from these traits should be correlated with the osteogenic potential of MSC. In summary, this study provides large-scale data to link regulatory elements with their target genes to study the regulatory relationships during the differentiation of mesenchymal stem cells and provide a deeper insight into the gene regulatory mechanism.Item Wnt3a-induced ST2 decellularized matrix ornamented PCL scaffold for bone tissue engineering(Tech Science Press, 2022) Wang, Xiaofang; Tu, Xiaolin; Ma, Yufei; Chen, Jie; Song, Yang; Liu, Guangliang; Anatomy, Cell Biology and Physiology, School of MedicineThe limited bioactivity of scaffold materials is an important factor that restricts the development of bone tissue engineering. Wnt3a activates the classic Wnt/β-catenin signaling pathway which effects bone growth and development by the accumulation of β-catenin in the nucleus. In this study, we fabricated 3D printed PCL scaffold with Wnt3a-induced murine bone marrow-derived stromal cell line ST2 decellularized matrix (Wnt3a-ST2-dCM-PCL) and ST2 decellularized matrix (ST2-dCM-PCL) by freeze-thaw cycle and DNase decellularization treatment which efficiently decellularized >90% DNA while preserved most protein. Compared to ST2-dCM-PCL, Wnt3a-ST2-dCM-PCL significantly enhanced newly-seeded ST2 proliferation, osteogenic differentiation and upregulated osteogenic marker genes alkaline phosphatase (Alp), Runx2, type I collagen (Col 1) and osteocalcin (Ocn) mRNA expression. After 14 days of osteogenic induction, Wnt3a-ST2-dCM-PCL promoted ST2 mineralization. These results demonstrated that Wnt3a-induced ST2 decellularized matrix improve scaffold materials’ osteoinductivity and osteoconductivity.