Toward Defining the Regenerative Potential of Olfactory Mucosa: Establishment of Schwann Cell-Free Adult Canine Olfactory Ensheathing Cell Preparations Suitable for Transplantation
Toward Defining the Regenerative Potential of Olfactory Mucosa: Establishment of Schwann Cell-Free Adult Canine Olfactory Ensheathing Cell Preparations Suitable for Transplantation
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DOI：
10.3727/096368912x656108
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发表时间：
2013-02
影响因子：
3.3
通讯作者：
S. Ziege;W. Baumgärtner;K. Wewetzer
中科院分区：
文献类型：
--
作者：
S. Ziege;W. Baumgärtner;K. Wewetzer
嗅粘膜（OM）源性嗅鞘细胞（OECs）是基于自体细胞移植治疗神经系统损伤的有吸引力的候选者。然而，定义OM衍生的OEC的再生能力受到以下事实的阻碍：用于移植的细胞培养物可能含有大量污染的三叉神经许旺细胞，这些细胞通过共享OEC标记物的体外表达而逃避识别。因此，本研究的目的是量化OEC制剂中的污染雪旺细胞，并制定一个具体的消耗协议。基于观察到新鲜分离但未培养的OECs和Schwann细胞显示HNK-1和p75 NTR的差异表达，使用磁激活细胞分选（MACS）从含有HNK-1/p75 NTR阴性OECs的原代细胞悬浮液中去除髓鞘形成（HNK-1阳性）和非髓鞘形成（p75 NTR阳性）Schwann细胞。在培养过程中OEC中p75 NTR表达的上调允许其随后基于MACS从成纤维细胞分离。在MACS耗尽之前对新鲜解离的OM的免疫荧光分析揭示，代表OEC和许旺细胞两者的全部CNPase-阳性细胞的21%和所有CNPase-阳性细胞的56%表达许旺细胞抗原HNK-1或p75 NTR，表明在培养之前新鲜解离的OM含有与OEC一样多的许旺细胞，而嗅球（OB）原代细胞悬浮液显示较低水平的雪旺细胞污染。有趣的是，与OM-OECs，OB-OECs和作为对照的腓神经（FN）雪旺细胞共培养的新生大鼠背根神经节（DRG）神经元的轴突生长在OECs的存在下显著高于雪旺细胞。第一份关于OEC制剂中雪旺细胞的鉴定和特异性耗竭的报告为将来充分确定鼻粘膜OEC的再生潜力提供了坚实的基础。
Olfactory mucosa (OM)-derived olfactory ensheathing cells (OECs) are attractive candidates for autologous cell transplantation-based therapy of nervous system injury. However, defining the regenerative capacity of OM-derived OECs is impeded by the fact that cell cultures used for transplantation may contain significant amounts of contaminating trigeminal nerve Schwann cells that escape identification by sharing in vitro expression of OEC markers. The aim of the present study, therefore, was to quantify contaminating Schwann cells in OEC preparations and to develop a protocol for their specific depletion. Based on the observation that freshly dissociated, but not cultured, OECs and Schwann cells display differential expression of HNK-1 and p75NTR, magnet-activated cell sorting (MACS) was used to deplete myelinating (HNK-1-positive) and nonmyelinating (p75NTR-positive) Schwann cells from primary cell suspensions containing HNK-1-/p75NTR-negative OECs. Upregulation of p75NTR expression in OECs during culturing allowed their subsequent MACS-based separation from fibroblasts. Immunofluorescence analysis of freshly dissociated OM prior to MACS depletion revealed that 21% of the total and 56% of all CNPase-positive cells, representing both OECs and Schwann cells, expressed the Schwann cell antigens HNK-1 or p75NTR, indicating that freshly dissociated OM prior to culturing contained as many Schwann cells as OECs, while olfactory bulb (OB) primary cell suspensions revealed lower levels of Schwann cell contamination. Interestingly, neurite growth of neonatal rat dorsal root ganglion (DRG) neurons cocultured with OM-OECs, OB-OECs, and fibular nerve (FN) Schwann cells used as control was significantly higher in the presence of OECs than of Schwann cells. The first report on identification and specific depletion of Schwann cells from OEC preparations provides a solid basis for future efforts to fully define the regenerative potential of nasal mucosa OECs.