Differentiation of Human Ligamentum Flavum Stem Cells Toward Nucleus Pulposus-Like Cells Induced by Coculture System and Hypoxia.

STUDY DESIGN: Human ligamentum flavum (LF)-derived stem cells (LFSCs) and nucleus pulposus cells (NPCs) were cocultured under normoxia or hypoxia. OBJECTIVE: To isolate and identify human LFSCs and determine whether they can differentiate into NPCs when cocultured with NPCs under hypoxia. SUMMARY OF BACKGROUND DATA: Mesenchymal stem cell (MSC)-based therapies have been proposed as a biological treatment for intervertebral disc degeneration. MSCs derived from various tissues are leading candidates for cell-based therapies, but such cells have not been reported in LF. METHODS: LF cells were isolated from patient samples and cultured using culture flasks coated with fibronectin, and their identity was confirmed using flow cytometry. The cells were induced to differentiate into osteoblasts, chondrocytes, and adipocytes, and their morphology, immunophenotype, cell proliferation capacity, cell cycle, and expression of stem cell-specific genes were compared with those of bone marrow-MSCs (BM-MSCs) derived from the same patients. NPCs and LFSCs were cocultured in 1-μm-pore-size insert transwell-culture systems under hypoxia (2% O2) or normoxia. CD24 expression was measured by flow cytometry and confocal microscopy assay. On day 14, reverse transcription-polymerase chain reaction was used for comparing the expression of chondrogenic genes (Sox-9, collagen-II, aggrecan) and novel marker genes (KRT19, CA12, FOXF1, HIF-1α) between the 2 groups. RESULTS: LFSCs were obtained using the fibronectin differential-adhesion assay. The morphology of LFSCs was altered, and their immunophenotype, multilineage induction, cell proliferation capacity, cell cycle, and stem cell-specific gene expression were closely related-but not identical-to BM-MSCs, CD24 expression was highly significant in the differentiated LFSCs. RT/Real-time polymerase chain reaction revealed that compared with LFSCs grown under normoxia, hypoxia-treated LFSCs expressed higher levels of Sox-9, collagen-II, aggrecan, KRT19, CA12, and HIF-1α genes except FOXF1. CONCLUSION: Stem cells were identified in human LF, and LFSCs cocultured with NPCs were successfully differentiated into NP-like cells under hypoxia. This potentially provides new cell candidates for cell-based regenerative medicine and tissue engineering. LEVEL OF EVIDENCE: N/A.