Successful fishing for nucleus pulposus progenitor cells of the intervertebral disc across species

Background context: Recently, Tie2/TEK receptor tyrosine kinase (Tie2 or syn. Ang-1 angiopoetin-1 receptor) positive nucleus pulposus progenitor cells were detected in human, cattle and mouse. These show remarkable multilineage differentiation capacity and direct correlation with intervertebral disc (IVD) degeneration, and are therefore an interesting target for regenerative strategies. Nevertheless, there remains controversy on the presence and function of these Tie2+-nucleus pulposus cells (NPC), in part due to the difficulty of identification and isolation.
Purpose: Here, we present a comprehensive protocol for sorting of Tie2+ NPCs from human, canine, bovine, and murine IVD tissue. We describe enhanced conditions for expansion and an optimized fluorescence-activated cell sorting-based methodology to sort and analyze Tie2+ NPCs.
Methods: We present flow cytometry protocols to isolate the Tie2+ cell population for the aforementioned species. Moreover, we describe crucial pitfalls to prevent loss of NPCs from the IVD cell population during the isolation process. A cross-species phylogenetic analysis of Tie2/TEK across species is presented.
Results: Our protocols are efficient towards labeling and isolation of Tie2+ cells. The total flow cytometry procedure requires approximately 9 hours, cell isolation 4-16 hours, cell expansion can take up to multiple weeks, dependent on application, age, disease state, and species. Phylogenetic analysis of the TEK gene revealed a strong homology among species.
Conclusions: Current identification of Tie2+ cells could be confirmed in bovine, canine, mouse and human surgical specimens. The presented flow cytometry protocol can successfully sort these multipotent cells. Biological function of identified cells needs to be confirmed by functional assays such as in vitro differentiation. In vitro culture conditions to maintain and their possible proliferation of the Tie2+ fraction is the subject of future research.