Optic nerve damage as a result of trauma, ischemia, glaucoma or other forms of optic neuropathy disease, leads to disconnection between the eye and brain and death of retinal ganglion cells (RGCs), causing permanent loss of vision. Therapeutic options for treating optic neuropathy are limited and represent a significant unmet medical need. Development of a regenerative strategy for replacement of lost RGCs lies at the core of the future cell-based therapy for these conditions. Successful long-term restoration of visual function depends on the type of cells for transplantation. Primary RGCs of neonatal mice are now reported to have the potential for serving such a purpose.
Optic nerve damage as a result of trauma, ischemia, glaucoma or other forms of optic neuropathy disease, leads to disconnection between the eye and brain and death of retinal ganglion cells (RGCs), causing permanent loss of vision. Therapeutic options for treating optic neuropathy are limited and represent a significant unmet medical need. Development of a regenerative strategy for replacement of lost RGCs lies at the core of the future cell-based therapy for these conditions. Successful long-term restoration of visual function depends on the type of cells for transplantation. Primary RGCs of neonatal mice are now reported to have the potential for serving such a purpose.
目的：建立能驱动GFP在视网膜神经节细胞(retinal ganglion cell，RGC)中特异性表达的小鼠胚胎干细胞系。方法：通过同源重组的方式建立Brn3b-GFP敲入的小鼠胚胎干细胞系(Brn3b-GFP ESC)，利用3D培养将其诱导成视网膜类器官检测GFP表达的细胞特异性，再用流式细胞分选富集GFP阳性RGC，采用玻璃体腔注射的方式将GFP阳性RGC移植到健康小鼠和NMDA损伤模型小鼠眼中探索该细胞的应用价值。结果：Brn3b-GFP ESC经3D视网膜诱导培养后在RGC中特异性表达GFP，将这些GFP阳性RGC移植到两种小鼠中2周后能在所有视网膜内观察到GFP阳性细胞存活，且均能观察到有供体RGC整合到宿主视网膜RGC层。结论：本研究建立了RGC特异的报告基因干细胞系Brn3b-GFP ESC，通过将该细胞系诱导成视网膜类器官进而获得的GFP阳性RGC移植后能够整合进宿主视网膜。该细胞系的建立将为青光眼及相关疾病提供重要的研究手段和工具。
Objective: This study was designed to establish a mouse embryonic stem cell line that can drive GFP expression specifically in retinal ganglion cells (RGCs). Methods: In this study, we established a Brn3b-GFP knock-in embryonic stem cell line (Brn3b-GFP ESC) by homologous recombination. By 3D culture, we induced these cells into retinal organoids to investigate the cell-specificity of GFP expression. GFP-positive RGCs were then enriched by flow cytometry and transplanted by intravitreal injection into the eyes of healthy mice and NMDA injury model mice to explore the feasibility of a potential clinical application. Results: GFP was specifically expressed in RGCs following induction of Brn3b-GFP ESCs into 3D retinal organoids. Two weeks after these GFP-positive RGCs were transplanted into the control and injured mice, GFP-positive cells were observed in all transplanted retinas, and donor RGCs were seen to integrate into the RGC layer of the host retina. Conclusion: This study has established a retinal ganglion cell-specific reporter stem cell line Brn3b-GFP ESC. The GFP-positive RGCs obtained by inducing the cell line into retinal organoids can be integrated into the host retina after transplantation. The establishment of such a cell line will provide an important research tool for glaucoma and related diseases.
