Enrichment of committed human nucleus pulposus cells expressing chondroitin sulfate proteoglycans under alginate...

Intervertebral disc (IVD) degeneration is associated with a malfunction of the nucleus pulposus (NP). Alginate culturing provides a favorable microenvironment for the phenotypic maintenance of chondrocyte-like NP cells. However, NP cells are recently evidenced to present heterogeneous populations, including progenitors, fibroblastic cells and primitive NP cells. The aim of this study is to profile the phenotypic changes of distinct human NP cells populations and describe the dynamic expression of chondroitin sulfate glycosaminoglycans (CS-GAGs) in extended alginate encapsulation.

Non-degenerated (ND-NPC) and degenerated (D-NPC) NP cells were expanded in monolayers, and subject to 28-day culture in alginate after serial passaging. CS-GAG compositional expression in monolayer-/alginate-cultured NP cells was evaluated by carbohydrate electrophoresis. Cellular phenotypic changes were assessed by immunologic detection and gene expression analysis.

Relative to D-NPC, ND-NPC displayed remarkably higher expression levels of chondroitin-4-sulfate GAGs over the 28-day culture. Compared with monolayer culture, ND-NPC showed increased NP marker expression of KRT18, KRT19, and CDH2, as well as chondrocyte markers SOX9 and MIA in alginate culture. In contrast, expression of fibroblastic marker COL1A1, COL3A1, and FN1 were reduced. Interestingly, ND-NPC showed a loss of Tie2+ but gain in KRT19+/CD24+ population during alginate culture. In contrast, D-NPC showed more consistent expression levels of NP surface markers during culture.

We demonstrate for the first time that extended alginate culture selectively enriches the committed NP cells and favors chondroitin-4-sulfate proteoglycan production. These findings suggest its validity as a model to investigate IVD cell function.