Background:Glaucoma, the number one cause of irreversible blindness, is characterized by the loss of retinal ganglion cells (RGCs), which do not regenerate in humans or mammals after cell death. Cell transplantation provides an opportunity to restore vision in glaucoma, or other optic neuropathies.Since transplanting primary RGCs from deceased donor tissues may not be feasible, stem cell-derived RGCs could provide a plausible alternative source of donor cells for transplant. Objective:We define a robust chemically defined protocol to differentiate human embryonic stem cells (hESCs) into RGC-like neurons. Methods:Human embryonic stem cell lines (H7-A81 and H9) and induced pluripotent stem cell (iPSC) were used for RGC differentiation. RGC immaturity was measured by calcium imaging against muscimol. Cell markers were detected by immunofluorescence staining and qRT-PCR. RGC-like cells were intravitreally injected to rat eye, and co-stained with RBPMS and human nuclei markers. All experiments were conducted at least three times independently. Data were analyzed by ANOVA with Tukey’s test with P value of <0.05 considered statistically significant. Results:We detected retinal progenitor markers Rx and Pax6 after 15 days of differentiation, and the expression of markers for RGC-specific differentiation (Brn3a and Brn3b), maturation (synaptophysin) and neurite growth (β-III-Tubulin) after an additional 15 days. We further examined the physiologic differentiation of these hESC-derived RGC-like progeny to those differentiated in vitro from primary rodent retinal progenitor cells (RPCs) with calcium imaging, and found that both populations demonstrate the immature RGC-like response to muscimol, a GABAA receptor agonist. By one week after transplant to the adult rat eye by intravitreal injection, the human RGC-like cells successfully migrated into the ganglion cell layer. Conclusions:Our protocol provides a novel, short, and cost-effective approach for RGC differentiation from hESCs, and may broaden the scope for cell replacement therapy in RGC-related optic neuropathies such as glaucoma.

中文翻译：

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源自 hESC 的视网膜神经节细胞的细胞移植。

背景：青光眼是导致不可逆性失明的首要原因，其特征是视网膜神经节细胞（RGC）的损失，而人类或哺乳动物细胞死亡后不会再生。细胞移植为青光眼或其他视神经病变患者恢复视力提供了机会。由于从已故供体组织移植原代 RGC 可能不可行，因此干细胞衍生的 RGC 可以为移植提供一个可行的替代供体细胞来源。目的：我们定义了一个强大的化学定义方案，用于将人胚胎干细胞 (hESC) 分化为 RGC 样神经元。方法：使用人胚胎干细胞系（H7-A81和H9）和诱导多能干细胞（iPSC）进行RGC分化。RGC 不成熟度通过针对蝇蕈醇的钙成像来测量。通过免疫荧光染色和qRT-PCR检测细胞标志物。将 RGC 样细胞玻璃体内注射到大鼠眼睛中，并用 RBPMS 和人类细胞核标记物进行共染色。所有实验至少独立进行三次。数据通过 ANOVA 和 Tukey 检验进行分析，P 值<0.05 被认为有统计学意义。结果：分化 15 天后，我们检测到视网膜祖细胞标记物 Rx 和 Pax6，并在另外 15 天后检测到 RGC 特异性分化标记物（Brn3a 和 Brn3b）、成熟标记物（突触素）和神经突生长标记物（β-III-微管蛋白）的表达。天。我们进一步通过钙成像检查了这些 hESC 衍生的 RGC 样后代与在体外从原代啮齿类视网膜祖细胞 (RPC) 分化的后代的生理分化，发现这两个群体都表现出对蝇蕈醇（一种 GABAA）的不成熟 RGC 样反应。受体激动剂。通过玻璃体内注射移植到成年大鼠眼睛一周后，人类 RGC 样细胞成功迁移到神经节细胞层。结论：我们的方案为 RGC 与 hESC 的分化提供了一种新颖、简短且经济高效的方法，并可能扩大 RGC 相关视神经病变（如青光眼）的细胞替代治疗的范围。