Tissue Engineering, Orthopeadic Research & Mechanobiology, Department for BioMedical Research (DBMR), Medical Faculty, University of Bern, Bern, Switzerland.
Laboratory of Molecular and Cellular Screening Processes, Centre of Biotechnology of Sfax, University of Sfax, Sfax, Tunisia.
Tissue Eng Part C Methods. 2019 Oct;25(10):571-580. doi: 10.1089/ten.TEC.2018.0375. Epub 2019 Sep 9.
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 multipotency. Here, we compared three sorting protocols; that is, fluorescence-activated cell sorting (FACS), magnetic-activated cell sorting (MACS), and a mesh-based label-free cell sorting system (pluriSelect), with respect to cell yield, potential to form colonies (colony-forming units), and 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. Impact Statement Tissue-specific progenitor cells such as nucleus pulposus progenitor cells of the IVD could become an ultimate cell source for tissue engineering strategies as these cells are presumably best adapted to the tissue's microenvironment. Fluorescence-activated cell sorting seemed to outcompete magnetic-activated cell sorting and pluriSelect concerning selecting a rare cell population from IVD tissue as could be demonstrated by improved cell yield and functional differentiation assays.
与椎间盘（IVD）退行性变相关的下腰痛对我们老龄化社会的社会经济有重大影响。因此，激活 IVD 自身修复的干细胞治疗仍然是一种令人兴奋的治疗策略。在这方面，组织特异性祖细胞可能在 IVD 再生中发挥关键作用，因为这些细胞完全适应这一微环境。在髓核（NP）中发现了一种阳性表达血管生成素-1 受体（Tie2+）的罕见祖细胞群体（NP 祖细胞[NPPCs]），并且证明其具有自我更新能力和多能性。在这里，我们比较了三种分选方案；即荧光激活细胞分选（FACS）、磁激活细胞分选（MACS）和基于网格的无标记细胞分选系统（pluriSelect），就细胞产量、形成集落的潜力（集落形成单位）以及三潜能的功能分化测定而言。本研究的目的是证明三种广泛应用的细胞分选方法分选稀有细胞（<5%）的效率，以及这些分离细胞在随后的成脂、成骨和成软骨分化的下游功能分化中的行为。三种分离方法的细胞产量差异很大，FACS 的产量最高（5.0%±4.0%），其次是 MACS（1.6%±2.9%）和 pluriSelect（1.1%±1.0%）。Tie2+和 Tie2- NPPCs 形成的集落数量无显著差异。只有 FACS 能够分离出两种具有三系多能性的功能不同的群体，而 MACS 和 pluriSelect 未能在分化试验中保持 Tie2+和 Tie2-群体之间的清晰分离。总之，三种分选方法都可分离 NPPCs，而 FACS 是分离功能 Tie2+细胞的首选技术。影响说明 椎间盘的组织特异性祖细胞，如椎间盘的髓核祖细胞，可能成为组织工程策略的最终细胞来源，因为这些细胞可能最适应组织的微环境。荧光激活细胞分选似乎优于磁激活细胞分选和 pluriSelect，能够从 IVD 组织中选择稀有细胞群体，这可以通过提高细胞产量和功能分化试验来证明。
