Li Yan, He Xinzheng, Li Jun, Ni Fangfang, Sun Qingqing, Zhou Yan
Department of Ophthalmology, No. 113 Hospital of PLA, Ningbo, Zhejiang 315000, P.R. China.
Department of Glaucoma, Ningbo Eye Hospital, Ningbo, Zhejiang 315000, P.R. China.
Mol Med Rep. 2017 Jun;15(6):3529-3534. doi: 10.3892/mmr.2017.6481. Epub 2017 Apr 19.
Damage of retinal ganglion cells (RGCs) is the major consequence of glaucoma and regeneration of RGCs is extremely difficult once the damage has occurred. Retinal stem cells (RSCs) are considered an ideal choice for RGC regeneration. Pigmented cells from the ciliary margin (PCMs) have great retinal differentiation potential and may be an ideal RSC candidate. However, the ciliary margin is too small, so the number of cells that can be obtained is limited. Bone marrow‑derived mesenchymal stem cells (BMMSCs) are another type of stem cell that have been previously investigated for RGC regeneration. BMMSCs expand sufficiently, whereas the retinal differentiation of BMMSCs is insufficient. The aim of the present study was to investigate whether the co‑culture of PCMs and BMMSCs may combine the advantages of both cell types to establish a novel and effective stem cell source for RGC regeneration. Primary rat PCMs and BMMSCs were isolated and co‑cultured. Cell growth was observed by an inverted microscope and proliferation was monitored by an MTT assay. Cell cycle analysis was performed by using a flow cytometer, while the expression of the photoreceptor‑specific homeobox gene (cone‑rod homeobox, Crx) was determined by reverse transcription‑quantitative polymerase chain reaction and western blot analysis. In addition, retinal differentiation was confirmed by immunofluorescence staining of major markers of retinal differentiation, including rhodopsin, visual system homeobox 2 and heparin sulfate. The co‑cultured cells expanded successfully, in a similar way to BMMSCs. In addition, the expression of Crx and retinal markers were significantly upregulated following BMMSC and PCM co‑culture. The results of the present study demonstrated that the co‑culture of BMMSCs and PCMs may be used as a source of RSCs.
视网膜神经节细胞（RGCs）损伤是青光眼的主要后果，一旦损伤发生，RGCs的再生极其困难。视网膜干细胞（RSCs）被认为是RGC再生的理想选择。睫状缘色素细胞（PCMs）具有很强的视网膜分化潜能，可能是理想的RSC候选细胞。然而，睫状缘太小，因此可获得的细胞数量有限。骨髓间充质干细胞（BMMSCs）是先前已被研究用于RGC再生的另一类干细胞。BMMSCs能充分扩增，而其向视网膜的分化能力不足。本研究的目的是探讨PCMs与BMMSCs共培养是否可以结合两种细胞类型的优势，从而建立一种新型有效的用于RGC再生的干细胞来源。分离并共培养原代大鼠PCMs和BMMSCs。通过倒置显微镜观察细胞生长情况，采用MTT法监测细胞增殖。使用流式细胞仪进行细胞周期分析，同时通过逆转录-定量聚合酶链反应和蛋白质印迹分析确定光感受器特异性同源框基因（视锥-视杆同源框，Crx）的表达。此外，通过对视网膜分化主要标志物（包括视紫红质、视觉系统同源框2和硫酸乙酰肝素）的免疫荧光染色来确认视网膜分化。共培养的细胞成功扩增，其方式与BMMSCs相似。此外，BMMSC与PCM共培养后，Crx和视网膜标志物的表达显著上调。本研究结果表明，BMMSCs与PCMs共培养可作为RSCs的一种来源。
