Characterization of distinct states of human naive pluripotency

The extent of naive characteriztics of recently reported naive human pluripotent stem cells (hPSCs) obtained in different naive-permissive media, is unclear. Moreover, these naive hPSCs were mainly derived by conversion from primed hPSCs or by direct derivation from human embryos rather than by somatic cell reprogramming. Here, we derived genetically matched human naive hPSCs by direct reprogramming of fibroblasts as well as primed-to-naive conversion using different naive conditions (NHSM, RSeT, 5iLAF and t2iLGöY). Comprehensive characterization showed that naive hPSCs obtained in these different conditions represent a spectrum of naive characteriztics irrespective of whether they were derived by conversion or reprograming. Importantly, only t2iLGöY hPSCs displayed a similar transcriptome to human cells from the inner cell mass, karyotypic stability and require re-priming for trilineage differentiation. Furthermore, our analyses identified KLF4 as a key reprogramming factor which enables conversion of primed hPSCs into naive t2iLGöY hPSCs. These findings underscore the role that reprogramming factors can play for the derivation of bona fide naive hPSCs and provide a molecular and functional reference for all the analysed conditions, which will help accelerate the downstream applications of naive hiPSCs.

目前，针对不同原始态容许培养体系中获得的近期报道的原始态人多能干细胞（naive human pluripotent stem cells, hPSCs），其原始态特征的完整覆盖范围尚不明确。此外，此类原始态hPSCs主要通过将始发态hPSCs转化或直接从人胚胎中分离获取的方式获得，而非通过体细胞重编程技术构建。本研究通过对成纤维细胞进行直接重编程，以及利用NHSM、RSeT、5iLAF及t2iLGöY等多种原始态培养条件实现始发态向原始态的转化，成功构建了遗传背景匹配的原始态hPSCs。全面的特征分析表明，无论通过转化还是重编程方式获得，不同培养条件下的原始态hPSCs均呈现出一系列连续分布的原始态特征谱。值得注意的是，仅t2iLGöY体系来源的hPSCs，其转录组特征与人类内细胞团细胞高度相似，且具备核型稳定性，同时需要进行重致敏方可完成三胚层分化。此外，本研究通过分析证实KLF4是介导始发态hPSCs向原始态t2iLGöY hPSCs转化的关键重编程因子。本研究结果凸显了重编程因子在构建合格原始态hPSCs过程中的关键作用，并为所有分析的培养条件提供了分子与功能层面的参考依据，这将有助于推动原始态诱导多能干细胞（naive human induced pluripotent stem cells, hiPSCs）的下游应用进程。