Lineage specific differentiation is influenced by state of human pluripotency [RNA-seq]. Homo sapiens

Human pluripotent stem cells (hPSCs) have been reported in naïve and primed states. However, the ability of human PSCs to generate mature cell types is the only imperative property for translational utility. Here, we reveal that the naïve state enhances self-renewal capacity while restricting lineage differentiation in vitro to neural default fate. Gene expression analyses indicate expression of multiple lineage associated transcripts in naïve hPSCs and thus failed to predict biased functional differentiation. Naïve hPSCs can be converted to primed allowing recovery of multilineage differentiation over long serial passage or immediately through suppression of OCT4 but not NANOG. To this end, we identified chemical inhibitors of OCT4 expression that acutely restore naïve hPSC differentiation. Our study identifies unique cell fate features and critical restrictions in human pluripotent states, and provides an approach to overcome these barriers that harness both efficient naïve hPSC growth whilst maintaining in vitro differentiation capacities essential for hPSC applications. Overall design: hPSC lines were transduced with shRNA lentiviruses in order to assess the effects of reducing NANOG and OCT4 gene expression on differention in the naïve state. shRNA expressing cells were sorted and then total RNA was extracted in order to perform transcriptome profiling by RNA-seq. Each experimental condition involves 2 technical replicates of 2 biological replicates (2 tech X 2 biol = 4 reads).

人多能干细胞（human pluripotent stem cells, hPSCs）目前已被报道存在初始态（naïve state）与始发态（primed state）两种状态。然而，人多能干细胞生成成熟细胞类型的能力，是其实现转化应用的唯一必要特性。本研究发现，初始态hPSCs可增强自我更新能力，但在体外将细胞谱系分化限制为神经默认命运。基因表达分析显示，初始态hPSCs中存在多种谱系相关转录本的表达，因此无法预测其存在偏向性的功能分化潜能。初始态hPSCs可被重编程为始发态：既可通过长期连续传代恢复多谱系分化能力，也可通过抑制OCT4（八聚体结合转录因子4）而非NANOG（Nanog同源盒转录因子）快速实现这一转变。为此，本研究筛选出可抑制OCT4表达的化学抑制剂，能够快速恢复初始态hPSCs的分化能力。本研究揭示了人多能干细胞不同状态下独特的细胞命运特征与关键限制条件，并提出了一种克服上述障碍的策略，可在维持初始态hPSCs高效增殖的同时，保留其体外分化能力——这一能力是人多能干细胞应用的核心必备条件。实验整体设计：将hPSC细胞系通过shRNA慢病毒载体进行转导，以探究抑制NANOG与OCT4基因表达对初始态hPSCs分化的影响。收集表达shRNA的细胞并进行分选，随后提取总RNA，通过RNA测序（RNA-seq）开展转录组分析。每个实验条件设置2次技术重复与2次生物学重复（2技术重复×2生物学重复=共4个测序样本）。