Time-Series Single Cell RNA Sequencing of Inner Ear Organoid Development

We report the application of single cell RNA sequencing for determining cell type and quantity in developing inner ear organoids derived from human pluripotent stem cells. By obtaining samples from multiple time points throughout differentiation, we developed a roadmap at single cell resolution of the multiple cell lineages which give rise to in vitro inner ear tissue as well as 'off-target' tissue types. We find that the cells in the day 0 sample contained primarily a homogenous population of pluripotent stem cells. On day 3, after treatment with BMP4, we find that the cells have differentiated into a split between surface ectoderm, the progenitor of the inner ear tissue, and neuroectoderm. On day 6 and 8, we observed the emergence of cranial placode tissue, both anterior and posterior. Then, after treatment starting on day 8 with a WNT agonist, CHIR, we observed the maturation of inner ear tissue into hair cells and neurons, starting on day 18 and increasing through the last time point collected, day 36. This study provides an atlas of inner ear organoid differentiation from human pluripotent stem cells. Overall design: We examined 10 time points during the differentiation of inner ear organoids by single cell RNA-sequencing. We also collected samples for comparison at days 10 and 13 which did not receive CHIR treatment.

本研究报道了单细胞RNA测序（single cell RNA sequencing）在鉴定源自人类多能干细胞（human pluripotent stem cells）的发育中耳类器官的细胞类型与数量中的应用。通过收集分化进程中多个时间点的样本，我们构建了单细胞分辨率下的细胞谱系图谱，覆盖了体外内耳组织以及脱靶组织类型的多种细胞谱系。研究显示，第0天的样本中主要为均一的多能干细胞群体。在经骨形态发生蛋白4（BMP4）处理后的第3天，细胞分化为两个分支：分别为内耳组织的祖细胞——表面外胚层，以及神经外胚层。在第6天与第8天，我们观察到前部与后部颅基板组织的出现。随后，自第8天起采用WNT激动剂CHIR进行处理，我们观察到内耳组织逐步成熟为毛细胞与神经元，该成熟过程始于第18天，并在直至最终收集的第36天的所有时间点中持续推进。本研究构建了人类多能干细胞来源的内耳类器官分化图谱。整体实验设计：我们通过单细胞RNA测序分析了内耳类器官分化过程中的10个时间点样本；此外还收集了第10天与第13天未接受CHIR处理的样本作为对照。