Open AccessSuccessful fishing for nucleus pulposus progenitor cells of the intervertebral disc across species
Author(s) -
Sakai Daisuke,
Schol Jordy,
Bach Frances C.,
Tekari Adel,
Sagawa Nobuho,
Nakamura Yoshihiko,
Chan Samantha C.W.,
Nakai Tomoko,
Creemers Laura B.,
Frauchiger Daniela A.,
May Rahel D.,
Grad Sibylle,
Watanabe Masahiko,
Tryfonidou Marianna A.,
Gantenbein Benjamin
Publication year - 2018
Publication title -
jor spine
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.125
0ISSN - 2572-1143
DOI - 10.1002/jsp2.1018
Subject(s) - cell sorting , flow cytometry , progenitor cell , population , microbiology and biotechnology , biology , angiopoietin receptor , receptor tyrosine kinase , stem cell , cell , nucleus , receptor , signal transduction , genetics , medicine , environmental health
Background Recently, Tie2/TEK receptor tyrosine kinase (Tie2 or syn. angiopoietin‐1 receptor) positive nucleus pulposus progenitor cells were detected in human, cattle, and mouse. These cells 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 over the presence and function of these Tie2 + nucleus pulposus cells (NPCs), 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 Tie2 + NPCs from the IVD cell population during the isolation process. A cross‐species phylogenetic analysis of Tie2 across species is presented. Results Our protocols are efficient towards labeling and isolation of Tie2 + NPCs. The total flow cytometry procedure requires approximately 9 hours, cell isolation 4 to 16 hours, cell expansion can take up to multiple weeks, dependent on the 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 specimens. The presented flow cytometry protocol can successfully sort these multipotent cells. The biological function of isolated cells based on Tie2 + expression 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.