Department of Otolaryngology, Stanford University School of Medicine, Stanford, CA.
Int Forum Allergy Rhinol. 2013 Oct;3(10):841-7. doi: 10.1002/alr.21205. Epub 2013 Jul 30.
New epithelial cells are generated through the proliferation and differentiation of resident progenitor cells in the nasal cavity. In several upper airway diseases, such as cystic fibrosis and chronic rhinosinusitis, self-renewing progenitor cells may be functionally defective, or compromised in their ability, to regenerate cells that maintain normal mucociliary clearance. Herein, we describe our early work to define and characterize a rare population of human nasal epithelial putative progenitors.
Single-cell suspensions of human ethmoid sinus tissues were prepared following endoscopic sinus surgery. Cell surface antibodies were analyzed as candidate markers for detecting progenitor cells. A panel of antibodies, including epithelial cell adhesion molecule (EpCAM, epithelial cells), CD45 (hematopoietic cells), nerve growth factor receptor (NGFR/CD271), intercellular adhesion molecule-1 (ICAM1/CD54), and integrin-α6 (ITGA6/CD49f) were used to resolve epithelial progenitor candidates by high-dimensional flow cytometry and the gating technique of fluorescence minus one (FMO) controls.
A rare population of approximately 0.06% of total ethmoid cells was discriminated as EpCAM(-) CD45(-) NGFR(+) ICAM1(+) by surface markers. Use of ITGA6 was excluded based on FMO control analysis. This lineage-negative population was purified to 99% homogeneity by cell sorting and analyzed by immunofluorescence microscopy. Sorted cells were subsequently confirmed to uniformly express the transcription factor p63. Upon in vitro culture, lineage-negative clonal cells were confirmed to spontaneously differentiate into epithelial lineage-positive cells.
Using the NGFR and ICAM1 cellular coordinates, we have identified a promising population of native human nasal epithelial progenitor cells that require more formal investigation for their role in upper airway regeneration.
鼻腔中的固有祖细胞通过增殖和分化产生新的上皮细胞。在几种上呼吸道疾病中，如囊性纤维化和慢性鼻-鼻窦炎，自我更新祖细胞的功能可能存在缺陷，或者在其再生维持正常黏液纤毛清除功能的细胞的能力上受到损害。在此，我们描述了我们早期定义和表征人鼻腔上皮祖细胞的工作。
在内镜鼻窦手术后，从筛窦组织中制备单细胞混悬液。细胞表面抗体被分析为检测祖细胞的候选标志物。一组抗体，包括上皮细胞黏附分子（EpCAM，上皮细胞）、CD45（造血细胞）、神经生长因子受体（NGFR/CD271）、细胞间黏附分子-1（ICAM1/CD54）和整合素-α6（ITGA6/CD49f），用于通过高维流式细胞术和荧光减一（FMO）对照的门控技术解析上皮祖细胞候选物。
大约 0.06%的总筛窦细胞被表面标志物 EpCAM(-) CD45(-) NGFR(+) ICAM1(+) 区分开来。基于 FMO 对照分析，排除了使用 ITGA6。通过细胞分选将该谱系阴性群体纯化至 99%的纯度，并通过免疫荧光显微镜分析。随后通过体外培养证实，分选细胞均均匀表达转录因子 p63。在体外培养中，谱系阴性克隆细胞被证实可以自发分化为上皮谱系阳性细胞。
使用 NGFR 和 ICAM1 细胞坐标，我们已经鉴定出一种有前途的天然人鼻腔上皮祖细胞群体，需要进一步研究其在上呼吸道再生中的作用。
