Establishment and characterization of a human pre-osteocyte cell line: hOsteo4-E9

Osteocytes, as the most abundant and long-lived cell type in bone tissue, have garnered significant attention due to their critical roles in bone remodeling, bone mechanobiology, bone-related diseases, and systemic homeostasis. While several well-established murine osteocyte cell lines are available, the development of a reliable human-derived osteocyte cell line remains an unmet need in the field. Here, we report the successful purification of primary bone cells from the adult human lumbar articular process and their immortalization via SV40 antigen transduction. The resulting human cell line, designated hOsteo4, exhibits high expression of early osteocyte markers (E11, DMP1), moderate levels of ALP and COL1, and low expression of late osteocyte markers (sclerostin, FGF23). Quantitative PCR analysis and in vitro osteoblastic differentiation assays indicate that hOsteo4 cells represent pre-osteocytes at the transitional stage from late osteoblasts to osteocytes. Notably, the subclone hOsteo4-E9 demonstrates robust responsiveness to fluid shear stress (FSS), characterized by dramatic morphological remodeling and upregulated focal adhesion protein expression. RNA-sequencing further reveals distinct FSS-responsive gene expression profiles when comparing human hOsteo4-E9 to the murine MLO-Y4 cell line. Collectively, hOsteo4-E9, a novel immortalized human pre-osteocyte-like cell line, represents an invaluable experimental tool that promises to advance mechanistic insights into human osteocyte biology.