Single-cell RNA sequencing on human osteoblasts

Here, we used single-cell RNA sequencing (scRNA-seq) to provide a high-resolution cellular taxonomy of freshly isolated primary human osteoblasts. Based on the gene expression patterns and cell lineage reconstruction, we identified three distinct clusters including preosteoblasts, mature osteoblasts, and an undetermined rare osteoblast subpopulation. Trajectory inference analysis suggested that the undetermined cluster may include osteoblast precursor cells which regulate the osteoblastogenesis process by giving rise to pre and mature osteoblasts. Investigation of the biological processes and signaling pathways enriched in each subpopulation revealed that in addition to bone formation, pre and undetermined osteoblasts may regulate both angiogenesis and hemopoiesis. We applied a widely used protocol for human osteoblast isolation to obtain the alkaline phosphatase (ALPL)high/CD45low cells from the femur head of one human subject with osteoarthritis through FACS. In total, 9,801 single cells were encapsulated for cDNA synthesis and barcoded using the 10x Genomics Chromium system, followed by library construction and sequencing. After the qua¬lity control, we obtained 8,557 ALPLhigh/CD45low cells, with an average of 1,503 genes detected per cell.