Effects of Intravitreal Injection of Human CD34+ Bone Marrow Stem Cells in a Murine Model of Diabetic Retinopathy

Human CD34+ stem cells are mobilized from bone marrow to sites of tissue ischemia and play an important role in tissue revascularization. This study used a murine model to test the hypothesis that intravitreal injection of human CD34+ stem cells harvested from bone marrow (BMSCs) can have protective effects in eyes with diabetic retinopathy. Streptozotocin-induced diabetic mice (C57BL/6J) were used as a model for diabetic retinopathy. Subcutaneous implantation of Alzet pump, loaded with Tacrolimus and Rapamycin, 5 days prior to intravitreal injection provided continuous systemic immunosuppression for the study duration to avoid rejection of human cells. Human CD34+ BMSCs were harvested from the mononuclear cell fraction of bone marrow from a healthy donor using magnetic beads. The CD34+ cells were labeled with enhanced green fluorescent protein (EGFP) using a lentiviral vector. The right eye of each mouse received an intravitreal injection of 50,000 EGFP-labeled CD34+ BMSCs or phosphate buffered saline (PBS). Simultaneous multimodal in vivo retinal imaging system consisting of fluorescent scanning laser ophthalmoscopy (enabling fluorescein angiography), optical coherence tomography (OCT) and OCT angiography was used to confirm the development of diabetic retinopathy and study the in vivo migration of the EGFP-labeled CD34+ BMSCs in the vitreous and retina following intravitreal injection. After imaging, the mice were euthanized, and the eyes were removed for immunohistochemistry. In addition, microarray analysis of the retina and retinal flat mount analysis of retinal vasculature were performed. The development of retinal microvascular changes consistent with diabetic retinopathy was visualized using fluorescein angiography and OCT angiography between 5 and 6 months after induction of diabetes in all diabetic mice. These retinal microvascular changes include areas of capillary nonperfusion and late leakage of fluorescein dye. Multimodal in vivo imaging and immunohistochemistry identified EGFP-labeled cells in the superficial retina and along retinal vasculature at 1 and 4 weeks following intravitreal cell injection. Microarray analysis showed changes in expression of 162 murine retinal genes following intravitreal CD34+ BMSC injection when compared to PBS-injected control. The major molecular pathways affected by intravitreal CD34+ BMSC injection in the murine retina included pathways implicated in the pathogenesis of diabetic retinopathy including Toll-like receptor, MAP kinase, oxidative stress, cellular development, assembly and organization pathways. At 4 weeks following intravitreal injection, retinal flat mount analysis showed preservation of the retinal vasculature in eyes injected with CD34+ BMSCs when compared to PBS-injected control. The study findings support the hypothesis that intravitreal injection of human CD34+ BMSCs results in retinal homing and integration of these human cells with preservation of the retinal vasculature in murine eyes with diabetic retinopathy. This study used a murine model to test the hypothesis that intravitreal injection of human CD34+ stem cells harvested from bone marrow (BMSCs) can have protective effects in eyes with diabetic retinopathy. Streptozotocin-induced diabetic mice (C57BL/6J) were used as a model for diabetic retinopathy.

人类CD34+干细胞（Human CD34+ stem cells）可从骨髓募集至组织缺血部位，在组织血管重建过程中发挥关键作用。本研究采用小鼠模型验证下述假说：从骨髓获取的人类CD34+干细胞（BMSCs）玻璃体内注射给药，对糖尿病视网膜病变患眼具有保护作用。
本研究选用链脲佐菌素（Streptozotocin）诱导的C57BL/6J糖尿病小鼠作为糖尿病视网膜病变模型。在玻璃体内注射前5天，皮下植入装载他克莫司（Tacrolimus）与雷帕霉素（Rapamycin）的Alzet泵（Alzet pump），可在整个研究周期内提供持续的全身免疫抑制，以避免人类细胞被宿主排斥。
人类CD34+骨髓来源干细胞（BMSCs）从健康供者骨髓的单个核细胞组分中，通过磁珠分选技术获取。使用慢病毒载体将增强型绿色荧光蛋白（enhanced green fluorescent protein, EGFP）标记CD34+细胞。每只小鼠的右眼接受玻璃体内注射：50,000个经EGFP标记的CD34+ BMSCs，或磷酸盐缓冲液（phosphate buffered saline, PBS）。
本研究采用集成荧光扫描激光检眼镜（可实现荧光素血管造影）、光学相干断层扫描（optical coherence tomography, OCT）及OCT血管造影的多模态活体视网膜成像系统，一方面确认糖尿病视网膜病变的发生发展，另一方面研究玻璃体内注射后，EGFP标记的CD34+ BMSCs在玻璃体及视网膜内的活体迁移情况。
成像实验结束后，对小鼠实施安乐死并摘取眼球，进行免疫组织化学检测。此外，本研究还对视网膜组织开展微阵列分析，并对视网膜血管进行视网膜铺片分析。
糖尿病诱导后5至6个月，所有糖尿病小鼠均出现符合糖尿病视网膜病变特征的视网膜微血管改变，这些改变可通过荧光素血管造影与OCT血管造影可视化呈现，具体包括毛细血管无灌注区及荧光素染料晚期渗漏。
多模态活体成像与免疫组织化学检测结果显示，在细胞注射后1周和4周，可在视网膜浅层及视网膜血管沿线检测到EGFP标记的细胞。与PBS注射对照组相比，玻璃体内注射CD34+ BMSCs后，小鼠视网膜组织中共检测到162个差异表达的小鼠视网膜基因。
玻璃体内注射CD34+ BMSCs所影响的主要分子通路，均与糖尿病视网膜病变的发病机制相关，包括Toll样受体通路、丝裂原活化蛋白激酶（MAP kinase）通路、氧化应激通路，以及细胞发育、组装与组织相关通路。
玻璃体内注射后4周，视网膜铺片分析结果显示：与PBS注射对照组相比，接受CD34+ BMSCs注射的患眼视网膜血管结构得以保留。
本研究结果支持前述假说：玻璃体内注射人类CD34+ BMSCs可实现细胞的视网膜归巢与整合，并在糖尿病视网膜病变小鼠模型中保留视网膜血管结构。
本研究采用小鼠模型验证下述假说：从骨髓获取的人类CD34+干细胞（BMSCs）玻璃体内注射给药，对糖尿病视网膜病变患眼具有保护作用。本研究选用链脲佐菌素诱导的C57BL/6J糖尿病小鼠作为糖尿病视网膜病变模型。