人多能干细胞(PSC)代表了研究人眼发育和疾病的强大工具。当生长在3D中时,它们可以自组装成层状组织的视网膜;然而,大小的变化,存在单个类器官的形状和组成。微环境和驱动视网膜生成的关键生长因子的时机都没有完全理解。探讨早期视网膜发育,我们开发了SIX6-GFP报告子,其能够系统优化促进光学囊泡形成的条件。我们证明早期低氧生长条件增强SIX6表达并促进眼形成。SIX6表达通过Wnt的顺序抑制和Sonichedgehog信号的激活而进一步增强。SIX6+光学囊泡显示与视网膜身份一致的RNA表达谱;然而,腹侧间脑标记也存在。为了证明视神经囊泡导致真正的“视网膜样”结构,我们生成了SIX6-GFP/POU4F2-tdTomato双报告系,标记了整个发育中的视网膜和视网膜神经节细胞,分别。额外的大脑区域,通过收集SIX6+/POU4F2-和SIX6-类器官来鉴定包括下丘脑和中脑-后脑(MBHB)区域,分别。使用RNAseq研究转录谱,我们证明SIX6-GFP和POU4F2-tdTomato报告基因为发育人类视网膜提供了可靠的读数,下丘脑,和中脑/后脑类器官。
Human pluripotent stem cells (PSCs) represent a powerful tool to investigate human eye development and disease. When grown in 3D, they can self-assemble into laminar organized retinas; however, variation in the size, shape and composition of individual organoids exists. Neither the microenvironment nor the timing of critical growth factors driving retinogenesis are fully understood. To explore early retinal development, we developed a SIX6-GFP reporter that enabled the systematic optimization of conditions that promote optic vesicle formation. We demonstrated that early hypoxic growth conditions enhanced
SIX6 expression and promoted eye formation.
SIX6 expression was further enhanced by sequential inhibition of Wnt and activation of sonic hedgehog signaling. SIX6 + optic vesicles showed RNA expression profiles that were consistent with a retinal identity; however, ventral diencephalic markers were also present. To demonstrate that optic vesicles lead to bona fide \"retina-like\" structures we generated a
SIX6-GFP/POU4F2-tdTomato dual reporter line that labeled the entire developing retina and retinal ganglion cells, respectively. Additional brain regions, including the hypothalamus and midbrain-hindbrain (MBHB) territories were identified by harvesting
SIX6 + /POU4F2- and
SIX6- organoids, respectively. Using RNAseq to study transcriptional profiles we demonstrated that SIX6-GFP and POU4F2-tdTomato reporters provided a reliable readout for developing human retina, hypothalamus, and midbrain/hindbrain organoids.