CD24 Positive Nucleus Pulposus Cells in Adult Human Intervertebral Discs Maintain a More Notochordal Phenotype Than GD2 Positive Cells.

Abstract

Background: Notochordal cells (NCs) present in the nucleus pulposus (NP) of the developing human intervertebral disc (IVD) disappear during the first decade of life. This loss coincides with the onset of IVD degeneration, therefore these cells are hypothesized to be important in NP homeostasis. Putative NC-derived (CD24⁺) and progenitor (TIE2⁺/GD2⁺) cell sub-populations have previously been identified in the adult human NP, but their characteristics have yet to be compared. Here, we used CD24, TIE2 and GD2 to identify and then isolate discrete cell sub-populations to assess cell phenotype.

Methods: CD24, GD2 and TIE2 positivity was assessed in a cohort of human pediatric and adult NP samples across a range of ages and histological degeneration grades using immunohistochemistry and flow cytometry. FACS sorting was used to isolate different cell sub-populations (CD24⁺/GD2⁺; CD24⁺/GD2^-; CD24^-/GD2⁺; CD24^-/GD2^-). Cell phenotype was assessed using qPCR for known NC and NP markers as well as catabolic genes.

Results: CD24⁺ and GD2⁺ cells were localized in all samples, irrespective of age or degeneration grade, while TIE2⁺ cell number was consistently very low. The same positivity trend was confirmed using flow cytometry. A small CD24⁺/GD2⁺ sub-population was present and maintained marker expression with time in culture. CD24⁺ subpopulations showed a significantly higher expression of NC markers than the CD24^- subpopulations and unsorted samples, suggesting a healthier phenotype in the CD24⁺ cells. GD2 did not appear to influence gene expression.

Conclusions: This study provides a better understanding of different cell sub-populations present in the adult NP, with identification of CD24⁺/GD2⁺ cells that are maintained with aging and degeneration. Healthy, NC-like phenotypic profiles appeared reliant on CD24, rather than GD2. The study highlights the importance of studying discrete cell sub-populations, especially CD24⁺ NP cells to better understand their role in NP homeostasis.