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
Department for Orthopaedic Surgery Tokai University School of Medicine Isehara Japan.
Center for Regenerative Medicine Tokai University School of Medicine Isehara Japan.
JOR Spine. 2018 Jun 27;1(2):e1018. doi: 10.1002/jsp2.1018. eCollection 2018 Jun.
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.
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.
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.
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.
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.
最近，在人类、牛和小鼠中检测到了Tie2/TEK受体酪氨酸激酶（Tie2或同属血管生成素-1受体）阳性的髓核祖细胞。这些细胞具有显著的多向分化能力，且与椎间盘退变直接相关，因此是再生策略中一个有趣的靶点。然而，这些Tie2髓核细胞（NPCs）的存在和功能仍存在争议，部分原因是其鉴定和分离存在困难。
在此，我们提出了一种从人类、犬类、牛和鼠类椎间盘组织中分选Tie2 NPCs的综合方案。我们描述了用于扩增的优化条件以及基于荧光激活细胞分选的优化方法，用于分选和分析Tie2 NPCs。
我们展示了用于分离上述物种Tie2细胞群体的流式细胞术方案。此外，我们描述了在分离过程中防止Tie2 NPCs从椎间盘细胞群体中丢失的关键陷阱。还展示了跨物种的Tie2系统发育分析。
我们的方案对于Tie2 NPCs的标记和分离是有效的。流式细胞术的整个过程大约需要9小时，细胞分离需要4至16小时，细胞扩增可能需要数周时间，这取决于应用、年龄、疾病状态和物种。TEK基因的系统发育分析显示物种间具有很强的同源性。
目前在牛、犬、小鼠和人类标本中能够证实对Tie2细胞的鉴定。所展示的流式细胞术方案能够成功分选这些多能细胞。基于Tie2表达的分离细胞的生物学功能需要通过体外分化等功能测定来证实。维持分离的Tie2细胞部分及其可能的增殖的体外培养条件是未来研究的主题。
