Osteogenic Differentiation Potential of Mesenchymal Stem Cells using Single Cell Multiomic Analysis

Mesenchymal 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 mul-tiomic analysis to profile the transcriptome and epigenome of MSC from four healthy donors. Da-ta were obtained from ~1,300 to 1,600 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 os-teogenic 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 dif-ferentiation potential into osteoblasts. MSC of donor one showed the largest average motif activi-ties, indicating that MSC from donor one was most likely to differentiate into osteoblasts. The re-sults of our validation experiments were consistent with the bioinformatics prediction. We also tested the enrichment of GWAS signals of several musculoskeletal disease traits in the patient-specific chromatin accessible regions identified in the single-cell multiome data, including osteo-porosis, osteopenia, and osteoarthritis. We found that osteoarthritis-associated variants were on-ly enriched in the regions identified from donor four. In contrast, osteoporosis and osteopenia variants were enriched in regions from donor one and least enriched in donor four. Since osteo-porosis 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 regula-tory relationships during the differentiation of mesenchymal stem cells and provide a deeper in-sight into the gene regulatory mechanism. Overall design: MSC from each of the four donors were collected and combined in equal numbers to create a mixed cell preparation. One million of the MSC mixed cells were centrifuged at 300 g for 5 minutes to remove culture media. The final single cell suspension was washed 3 times with PBS buffer without calcium and magnesium, plus 0.4% BSA. The single-cell suspension was then assessed for cell viability and quantity by using a he-mocytometer under a microscope. Cells were then lysed with 0.1X lysis buffer for 4 minutes to isolate nuclei, based on the protocol of Demonstrated Protocol Nuclei Isola-tion Complex Sample ATAC GEX Sequencing, CG000375 RevB (10X Genomics, Inc). Final nuclei concentration of 3000/ul was used for targeted cell recovery of 10000 cells. Following the Chromium NextGEM Multiome ATAC GEX User Guide, CG000338_RevB (10X Genomics, Inc), tagmentation of nuclei preparation was first per-formed. Thereafter, briefly, along with the single cell multiome gel beads and partition oil, the single nuclei master mixture containing tagmented single nuclei suspension was transferred onto a Next GEM Chip J, and the chip loaded to the Chromium Con-troller for GEM generation and barcoding, followed by pre-amplification PCR, ATAC library preparation, cDNA synthesis and cDNA library preparation. At each step, the quality of cDNA, ATAC library and cDNA library was examined by Bioanalyzer. The resulting ATAC and cDNA libraries were sequenced separately, cDNA library for 28bp and 91bp and ATAC library for 50bp paired-end sequencing on Illumina No-vaSeq 6000.