背景:最近,Tie2/TEK受体酪氨酸激酶(Tie2或syn。血管生成素-1受体)阳性的髓核祖细胞在人类中被检测到,牛,和老鼠。这些细胞显示出显著的多谱系分化能力和与椎间盘(IVD)变性的直接相关性,因此是再生策略的令人感兴趣的靶标。然而,关于这些Tie2+髓核细胞(NPCs)的存在和功能仍然存在争议,部分原因是难以识别和隔离。
目标:这里,我们提出了一个全面的协议,用于从人类中分选Tie2+NPC,犬,牛,和鼠IVD组织。我们描述了增强的扩增条件和优化的基于荧光激活细胞分选的方法来分选和分析Tie2+NPC。
方法:我们提出了流式细胞术方案,以分离上述物种的Tie2细胞群。此外,我们描述了在分离过程中防止IVD细胞群体中Tie2+NPC丢失的关键陷阱。提出了跨物种Tie2的跨物种系统发育分析。
结果:我们的方案对于标记和分离Tie2+NPC是有效的。总的流式细胞术程序需要大约9小时,细胞分离4到16小时,细胞扩增可能需要长达数周的时间,取决于应用程序,年龄,疾病状态,和物种。TEK基因的系统发育分析显示物种之间具有很强的同源性。
结论:目前Tie2+细胞的鉴定可以在牛,犬,鼠标,和人类标本。所提出的流式细胞术方案可以成功地分选这些多能细胞。基于Tie2+表达的分离细胞的生物学功能需要通过功能测定如体外分化来确认。体外培养条件下Tie2+组分的维持及其可能的增殖是未来研究的主题。
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.
OBJECTIVE: 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.