Fluorescence-Activated Cell Sorting Is More Potent to Fish Intervertebral Disk Progenitor Cells Than Magnetic and Beads-Based Methods

Abstract

Low back pain related to intervertebral disk (IVD) degeneration has a major socioeconomic impact on our aging society. Therefore, stem cell therapy to activate self-repair of the IVD remains an exciting treatment strategy. In this respect, tissue-specific progenitors may play a crucial role in IVD regeneration, as these cells are perfectly adapted to this niche. Such a rare progenitor cell population residing in the nucleus pulposus (NP) (NP progenitor cells [NPPCs]) was found positive for the angiopoietin-1 receptor (Tie2+), and was demonstrated to possess self-renewal capacity and in vitro multipotency. Here, we compared three sorting protocols; that is, fluorescence-activated cell sorting (FACS), magnetic-activated cell sorting (MACS), and a mesh-based labelfree cell sorting system (pluriSelect), with respect to cell yield, potential to form colonies (colony-forming units), and in vitro functional differentiation assays for tripotency. The aim of this study was to demonstrate the efficiency of three widespread cell sorting methods for picking rare cells (<5%) and how these isolated cells then behave in downstream functional differentiation in adipogenesis, osteogenesis, and chondrogenesis. The cell yields among the isolation methods differed widely, with FACS presenting the highest yield (5.0%-4.0%), followed by MACS (1.6%-2.9%) and pluriSelect (1.1%-1.0%). The number of colonies formed was not significantly different between Tie2+ and Tie2-NPPCs. Only FACS was able to separate into two functionally different populations that showed trilineage multipotency, while MACS and pluriSelect failed to maintain a clear separation between Tie2+ and Tie2-populations in differentiation assays. To conclude, the isolation of NPPCs was possible with all three sorting methods, while FACS was the preferred technique for separation of functional Tie2+ cells.

The effects of 3D culture on the expansion and maintenance of nucleus pulposus progenitor cell multipotency

“…To verify the multipotent potential of the NPCs that we were expanding, we decided to perform a tri-lineage differentiation of those cells into adipogenic, osteogenic, and chondrogenic differentiation as previously described. 26,48 Osteogenic and adipogenic differentiation of NPCs were performed at 20% oxygen as difficulties to obtain mineralization and adipogenesis at low oxygen levels are well documented in the literature. [49][50][51][52][53][54] Culturing in adipogenic medium following a second phase expansion culture with FGF-2 was showing a great formation of lipid droplets in our control condition (hBMSCs) but also in both NPCs samples (2D and 3D).…”

“…Tie2 + cells were sorted using FACS as previously described. 8,9,26 Freshly isolated NPCs (N = 3) were incubated with an anti-human reported. 26 The Mouse IgG 1 PE-conjugated Antibody (#IC002P, clone 11 711, R&D systems) was used as isotype control to set the gate for sorting.…”

Towards Tissue-Specific Stem Cell Therapy for the Intervertebral Disc: PPARδ Agonist Increases the Yield of Human Nucleus Pulposus Progenitor Cells in Expansion

“…The NP progenitor cell (NPPC) from NP tissue might be the successor of notochordal cells to maintain homeostasis in NP tissue [8][9][10]. The main characteristics of these NPPCs are their self-renewal ability, their capability to form Colony-Forming Unit-spheroids (CFU-s) in semi-solid media, and their positivity for cell surface marker Angiopoietin Surgeries 2021, 2 93 receptor-1 (aka Tie2) [8][9][10]. The NPPCs (Tie2+ NPCs) are multipotential, but not the fully differentiated NPCs (Tie2-NPCs) [8][9][10].…”

“…The main characteristics of these NPPCs are their self-renewal ability, their capability to form Colony-Forming Unit-spheroids (CFU-s) in semi-solid media, and their positivity for cell surface marker Angiopoietin Surgeries 2021, 2 93 receptor-1 (aka Tie2) [8][9][10]. The NPPCs (Tie2+ NPCs) are multipotential, but not the fully differentiated NPCs (Tie2-NPCs) [8][9][10]. Besides the absence of Tie2, differentiated NPCs can be distinguished from multipotent NPPCs by their lower clonogenic ability [8][9][10].…”

“…The NPPCs (Tie2+ NPCs) are multipotential, but not the fully differentiated NPCs (Tie2-NPCs) [8][9][10]. Besides the absence of Tie2, differentiated NPCs can be distinguished from multipotent NPPCs by their lower clonogenic ability [8][9][10].…”