Single-Cell RNA Sequencing Reveals Metallothionein Heterogeneity during hESC Differentiation to Definitive Endoderm [scRNA-Seq]

Differentiation of human pluripotent stem cells toward definitive endoderm (DE) is the critical first step for generating cells comprising organs such as the gut, liver, pancreas and lung. This in-vitro differentiation process generates a heterogeneous population with a proportion of cells failing to differentiate properly and maintaining expression of pluripotency factors such as Oct4. RNA-sequencing of single cells collected at four time points during a 4-day DE differentiation identified high expression of metallothionein genes in the residual Oct4-positive cells that failed to differentiate to DE. Using X-ray fluorescence microscopy and multi-isotope mass spectrometry, we discovered that high intracellular zinc level corresponds with persistent Oct4 expression and failure to differentiate. We further show that differentiation-arrested phenotype is inversely correlated with zinc concentration in the differentiation media. This study improves our understanding of in-vitro DE differentiation and provides actionable options to improve DE differentiation efficiency. RNA-sequencing of 329 single cells collected at four time points during a 4-day DE differentiation to identify mechanisms leading to cellular heterogeneity during differentiation

人多能干细胞向定型内胚层（definitive endoderm, DE）的分化，是生成肠道、肝脏、胰腺与肺等器官组成细胞的关键起始步骤。该体外分化过程会产生异质性细胞群，其中部分细胞无法正常完成分化，持续表达八聚体结合转录因子4（Oct4）等多能性因子。本研究团队对为期4天的DE分化过程中4个时间点收集的329个单细胞进行RNA测序，在未能分化为DE的残留Oct4阳性细胞中，发现金属硫蛋白基因呈高表达状态。借助X射线荧光显微镜与多同位素质谱技术，本研究团队发现细胞内高锌水平与Oct4持续表达及分化失败存在显著关联。进一步研究证实，分化阻滞表型与分化培养基中的锌浓度呈负相关关系。本研究加深了学界对体外DE分化过程的认知，并为提升DE分化效率提供了可落地的优化方案。本数据集通过对4天DE分化过程中4个时间点收集的329个单细胞进行RNA测序，旨在解析分化过程中细胞异质性的产生机制。