FACS reveals more Pluripotent Intervertebral Disc Progenitor Cells compared to MACS and pluriSelect

Abstract

INTRODUCTION: Nucleus Pulposus Progenitor Cells (NPPCs), positive for the angiopoietin-1 receptor (Tie2), were demonstrated in human, mouse, canine and bovine NP tissue [1,2,3]. Tie2+ NPPCs possess a multi-lineage differentiation potential, and regeneration potential is attributed to them. However, the isolation of Tie2+ NPPCs can be cumbersome. Hence, three isolation methods were compared. METHODS: Bovine NP cells were isolated from 10-14-month-old animals. Cell sorting was performed with an antibody against Tie2 (bs-1300R, Bioss) using FACS, magnetic-activated cell sorting (MACS) and pluriSelect, a size-based sorting method. Outcomes were evaluated by cell yield of Tie2+ cells, the ability of sorted cells to form colonies and tri-lineage differentiation assays. RESULTS & DISCUSSION: FACS resulted in the highest Tie2+ cell yield (5.0 ± 4.0%) followed by MACS (1.6 ± 2.9%) and pluriSelect (1.1 ± 1.4%). Colony forming ability did not differ between Tie2+ and Tie2- cells for any isolation method. However, Tie2+ cells obtained by MACS tended to have more colonies than FACS and pluriSelect. Osteogenic and adipogenic differentiation of Tie2+ and Tie2- cells did not result in a clear distinction for MACS and pluriSelect; Tie2+ FACS-sorted cells demonstrated superior osteogenic and adipogenic differentiation over Tie2- cells. Also for chondrogenesis, the Tie2+ FACS-sorted Tie2+ NPPCs tended to produce more proteoglycan versus Tie2- NPPCs, whereas for MACS and pluriSelect no difference was found. CONCLUSIONS: Based on the parameters tested, isolation of NPPC is possible with all three methods. However, cell yields differed widely. FACS although most invasive, appears to be the most specific sorting method for these Tie2 + cells among the tested methods as Tie2 + cells do not demonstrate osteogenic and adipogenic differentiation. As for cell yield MACS seems to reveal the most, possibly this is due to inclusion of cells expressing Tie2 less strongly.