Laundos Tiago L, Silva Joana, Assunção Marisa, Quelhas Pedro, Monteiro Cátia, Oliveira Carla, Oliveira Maria J, Pêgo Ana P, Amaral Isabel F
Instituto de Engenharia Biomédica (INEB), Universidade do Porto, Portugal.
Instituto de Investigação e Inovação em Saúde (i3S), Universidade do Porto, Portugal.
J Tissue Eng Regen Med. 2017 Aug;11(8):2227-2240. doi: 10.1002/term.2121. Epub 2016 Feb 15.
Embryonic stem (ES)-derived neural stem/progenitor cells (ES-NSPCs) constitute a promising cell source for application in cell therapies for the treatment of central nervous system disorders. In this study, a rotary orbital hydrodynamic culture system was applied to single-cell suspensions of ES-NSPCs, to obtain homogeneously-sized ES-NSPC cellular aggregates (neurospheres). Hydrodynamic culture allowed the formation of ES-NSPC neurospheres with a narrower size distribution than statically cultured neurospheres, increasing orbital speeds leading to smaller-sized neurospheres and higher neurosphere yield. Neurospheres formed under hydrodynamic conditions (72 h at 55 rpm) showed higher cell compaction and comparable percentages of viable, dead, apoptotic and proliferative cells. Further characterization of cellular aggregates provided new insights into the effect of hydrodynamic shear on ES-NSPC behaviour. Rotary neurospheres exhibited reduced protein levels of N-cadherin and β-catenin, and higher deposition of laminin (without impacting fibronectin deposition), matrix metalloproteinase-2 (MMP-2) activity and percentage of neuronal cells. In line with the increased MMP-2 activity levels found, hydrodynamically-cultured neurospheres showed higher outward migration on laminin. Moreover, when cultured in a 3D fibrin hydrogel, rotary neurospheres generated an increased percentage of neuronal cells. In conclusion, the application of a constant orbital speed to single-cell suspensions of ES-NSPCs, besides allowing the formation of homogeneously-sized neurospheres, promoted ES-NSPC differentiation and outward migration, possibly by influencing the expression of cell-cell adhesion molecules and the secretion of proteases/extracellular matrix proteins. These findings are important when establishing the culture conditions needed to obtain uniformly-sized ES-NSPC aggregates, either for use in regenerative therapies or in in vitro platforms for biomaterial development or pharmacological screening. Copyright © 2016 John Wiley & Sons, Ltd.
胚胎干细胞（ES）来源的神经干/祖细胞（ES-NSPCs）是用于中枢神经系统疾病细胞治疗的一种很有前景的细胞来源。在本研究中，将旋转轨道流体动力学培养系统应用于ES-NSPCs的单细胞悬液，以获得大小均匀的ES-NSPC细胞聚集体（神经球）。流体动力学培养使得形成的ES-NSPC神经球比静态培养的神经球具有更窄的大小分布，增加轨道速度会导致神经球尺寸更小且神经球产量更高。在流体动力学条件下（55 rpm培养72小时）形成的神经球显示出更高的细胞致密性以及活细胞、死细胞、凋亡细胞和增殖细胞的比例相当。对细胞聚集体的进一步表征为流体动力学剪切对ES-NSPC行为的影响提供了新的见解。旋转神经球显示N-钙黏蛋白和β-连环蛋白的蛋白质水平降低，层粘连蛋白沉积增加（不影响纤连蛋白沉积）、基质金属蛋白酶-2（MMP-2）活性以及神经元细胞百分比增加。与发现的MMP-2活性水平升高一致，流体动力学培养的神经球在层粘连蛋白上显示出更高的向外迁移。此外，当在三维纤维蛋白水凝胶中培养时，旋转神经球产生的神经元细胞百分比增加。总之，对ES-NSPCs的单细胞悬液施加恒定的轨道速度，除了能形成大小均匀的神经球外，还可能通过影响细胞间黏附分子的表达以及蛋白酶/细胞外基质蛋白的分泌促进ES-NSPC的分化和向外迁移。在建立获得大小均匀的ES-NSPC聚集体所需的培养条件时，这些发现很重要，这些聚集体可用于再生疗法或用于生物材料开发或药理筛选的体外平台。版权所有© 2016约翰威立父子有限公司。
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