中文摘要：

目的探讨在成分简单的培养条件下将人胚胎干细胞（hESCs）通过3D培养的方式诱导分化为人神经视网膜各种细胞的方法。方法将不同系的hESCs用消化酶消化后，以团块形式悬浮培养在含有1﹪ Matrigel的培养液中。对于SHhES8系的hESCs在第1天加入BMP4信号通路抑制剂，然后每2天换液以逐渐降低其浓度。在分化的不同时间，通过检测全能性基因和视网膜标志基因的表达水平以确定hESCs团块的分化状态。对于H9系的hESCs，抑制Wnt信号通路以促进hESCs分化过程中神经视网膜前体细胞重要转录因子CHX10的表达。数据经GraphPad Prism 6.0c student＇s t-test分析处理。结果（1）H9系hESC经过15 d的分化，各眼区转录因子的表达水平都有大幅上调（与D0相比，D4时SIX3水平上升到720.1±238.6，P = 0.039；SIX6上升到260.0±132.5，P = 0.122；PAX6上升到2661±1353，P = 0.121；LHX2上升到480.4±27.56，P 〈 0.000；RAX上升到144.9±35.61，P = 0.016），免疫荧光检测也显示此时分化的细胞已经具备早期视网膜细胞的特征；（2）在H9系hESCs分化第7天添加IWR1e抑制Wnt信号通路，可以有效地提高CHX10的表达，使含CHX10阳性细胞的团块的比例从0﹪上升到86.6﹪（P = 0.000），表明Wnt信号通路在视网膜细胞形成中起重要的调节作用；（3）SHhES8系的hESCs通过悬浮培养，在第24天时可以检测到有CHX10阳性的视泡样结构，继续培养至83d，可以检测表达不同的神经视网膜细胞标志基因的细胞，包括CRX阳性的感光细胞或前体细胞、AP2α阳性的无长突细胞和ISLET1阳性的神经节细胞。结论不同系的hESCs对同样的诱导分化显示出不同的反应；抑制Wnt信号通路可以促使CHX10表达，实现早期视网膜细胞的分化成熟；利用无血清悬浮培养的方式，可以使hESCs分化为各种视网膜细胞和具有一定三维结构的视网膜样组织。

英文摘要：

Objective To induce human embryonic stem cells（hESCs）to various types of human neural retina cells under a simple culture condition. Methods hESCs from different lines are dissociated into cell clumps and cultured in suspension using the differentiation medium supplemented with 1﹪ Matrigel. In the differentiation of hESCs from SHhES8 line, BMP4 inhibitor will be added on Day 1 and its concentration will be diluted by medium change.The differentiated cells are identified by the expression of marker genes to determine the differentiation state. To facilitate the expression of the important eye field transcription factor（EFTF）like CHX10 in H9 hESCs during differentiation, the Wnt signaling pathway inhibitor IWR1e is added into the differentiation medium. Results1. The expression levels of EFTFs were markedly upregulated on D4 and lasted up to D15 of differentiation for hESCs of H9 line（as compared to data of D0, on D4, SIX3 was upregulated to 720.1 ± 238.6, P = 0.039; SIX6 to 260.0 ± 132.5, P = 0.1222; PAX6 to 2661 ± 1353, P = 0.1208; LHX2 to 480.4 ± 27.56, P 〈 0.0001; and RAX to 144.9 ± 35.61, P =0.0156）. Immunofluorescence staining results also show that differentiated cells express marker genes representing the early stage of retina. 2. The addition of Wnt signaling pathway inhibitor IWR1e at Day7 of differentiation in hESCs from H9 line could significantly facilitate the expression of CHX10, the percentage of aggregates with CHX10 positive cells increased to 86.6﹪ from baseline（P = 0.0002）. The finding indicates that the Wnt signaling pathway plays an important role in the differentiation of retinal cells. 3. After suspension culture of hESCs from SHhES8 line for 24 days, CHX10 positive optic vesicle-like structures could be observed. At day 83, different types of neural retina cells, include CRX positive photoreceptors or precursors, AP2α positive amacrine cells and ISLET1 positive ganglion cells, could be detected. ConclusionGreat differences exist between hESCs from different lines