Stirred suspension bioreactors maintain naïve pluripotency of human pluripotent stem cells (hPSCs)

Although cell therapies require large numbers of quality-controlled hPSCs, existing technologies are limited in their ability to efficiently grow and scale stem cells. We report here that cell-state conversion from primed-to-naïve pluripotency enhances the biomanufacturing of hPSCs. Naïve hPSCs exhibit superior growth kinetics and aggregate formation characteristics in stirred suspension bioreactors compared to their primed counterparts. Moreover, we demonstrate the role of the bioreactor mechanical environment in the maintenance of naïve pluripotency, through transcriptomic enrichment of mechano-sensing signaling for cells in the bioreactor along with a decrease in expression of lineage-specific and primed pluripotency hallmarks. Bioreactor-cultured, naïve hPSCs maintain epigenetic hallmarks of naïve pluripotency, exhibit robust production of naïve pluripotency metabolites, and display reduced expression of primed pluripotency cell surface markers. We also show that these cells retain the ability to undergo targeted differentiation into beating cardiomyocytes, hepatocytes and neural rosettes. They additionally display faster kinetics of teratoma formation compared to their primed counterparts. Naïve bioreactor hPSCs also retain structurally stable chromosomes. Our research corroborates that converting hPSCs to the naïve state enhances hPSC manufacturing and indicates a potentially important role for the bioreactor's mechanical environment in maintaining naïve pluripotency. Overall design: RNA-seq experiment was performed on naïve human pluripotent stem cells cultured under three different conditions: 1) static, 2) static suspension, 3) stirred suspension culture. We performed three pairwise transcriptomic comparisons: 1) Transcriptomic comparison of naïve hPSCs cultured under static suspension and static culture, 2) Transcriptomic comparison of naïve hPSCs cultured under stirred suspension and static culture, 3) Transcriptomic comparison of naïve hPSCs cultured under stirred suspension and static suspension culture.

尽管细胞治疗需要大量经过质量管控的人多能干细胞（human pluripotent stem cells, hPSCs），但现有技术在高效扩增与规模化培养干细胞方面存在显著局限。本研究证实，将细胞状态从始发态（primed）多能性向幼稚态（naïve）多能性转换，可有效提升人多能干细胞的生物制造效率。
与始发态人多能干细胞相比，幼稚态人多能干细胞在搅拌式悬浮生物反应器中展现出更优异的生长动力学特性与聚集体形成能力。此外，本研究通过对生物反应器内细胞的机械感应信号通路进行转录组富集分析，并观察到谱系特异性及始发态多能性标志基因的表达下调，证实了生物反应器的机械微环境在维持幼稚态多能性中的关键调控作用。
经生物反应器培养的幼稚态人多能干细胞可维持幼稚态多能性的表观遗传特征，稳定产生幼稚态多能性相关代谢物，并显著下调始发态多能性相关细胞表面标志物的表达。本研究同时证实，此类细胞仍保留定向分化为搏动性心肌细胞、肝细胞及神经玫瑰花结结构的能力；与始发态细胞相比，其畸胎瘤形成动力学更快。此外，经生物反应器培养的幼稚态人多能干细胞还可维持染色体结构的稳定性。
本研究进一步验证，将人多能干细胞转换为幼稚态可显著提升其规模化制造效率，并提示生物反应器的机械微环境在维持幼稚态多能性中具有潜在核心作用。

整体实验设计：
本研究针对三种培养条件下的幼稚态人多能干细胞开展RNA测序（RNA-seq）实验：1）静态培养；2）静态悬浮培养；3）搅拌式悬浮培养。本研究共开展三组两两转录组比对分析：
1. 静态悬浮培养与静态培养的幼稚态人多能干细胞转录组比对；
2. 搅拌式悬浮培养与静态培养的幼稚态人多能干细胞转录组比对；
3. 搅拌式悬浮培养与静态悬浮培养的幼稚态人多能干细胞转录组比对。