A PIANO (Proper, Insufficient, Aberrant, and NO reprogramming) response to the Yamanaka factors in the initial stages of human iPSC reprogramming

Yamanaka reprogramming is revolutionary but inefficient, slow and stochastic. The underlying molecular events for these mixed outcomes of iPSC reprogramming is still unclear. Previous studies about transcriptional responses to reprogramming overlooked human reprogramming, and are compromised by the fact that only a rare population proceeds towards pluripotency and a significant amount of the collected transcriptional data may not represent the positive reprogramming. We recently developed a concept of reprogramome, which allows one to study the early transcriptional responses to the Yamanaka factors in the perspective of reprogramming legitimacy of a gene response to reprogramming. Using RNA-seq, this study scored 579 genes successfully reprogrammed within 48 hours, indicating the potency of the reprogramming factors. This report also tallied 438 genes reprogrammed significantly but insufficiently up to 72 hours, indicating a positive drive with some inadequacy of the Yamanaka factors. In addition, 953 member genes within the reprogramome were transcriptionally irresponsive to reprogramming, showing the inability of the reprogramming factors to directly act on these genes. Furthermore, there were 305 genes undergoing six types of aberrant reprogramming: over, wrong, and unwanted up- or down- reprogramming, revealing significant negative impacts of the Yamanaka factors. The mixed findings about the initial transcriptional responses to the reprogramming factors shed new insights into the robustness as well as limitations of the Yamanaka factors. Human embryonic stem cell (n=3) expression were compared to human fibroblasts (n=4) to define the reprogramome. Human fibroblasts BJ cells with and without transduction with lentiviral GFP were RNA-sequenced, the their transcriptomes were compared with the human fibroblasts transduced with the Yamanaka factors, OCT4, SOX2, KLF4 and c-MYC. Different patterns of transcirptional responses to reprogramming were identified with the perspective of reprogramming legitimacy.

山中伸弥重编程（Yamanaka reprogramming）是一项革命性技术，但存在效率低下、进程缓慢且随机性强的缺陷。目前，诱导多能干细胞（iPSC）重编程出现这些复杂结局的潜在分子机制仍未阐明。既往针对重编程转录应答的研究均未涉及人类重编程，且受限于以下事实：仅极少数细胞能够向多能性方向推进，且采集的大量转录组数据或许无法反映正向重编程过程。我们近期提出了重编程组（reprogramome）的概念，该概念可基于基因重编程应答的合法性视角，探究细胞对山中伸弥因子的早期转录应答。本研究通过RNA测序（RNA-seq）筛选出579个在48小时内成功完成重编程的基因，印证了重编程因子的诱导效能。本研究同时统计得到438个在72小时内出现显著但未完全重编程的基因，提示山中伸弥因子虽可产生正向驱动作用，但存在一定缺陷。此外，重编程组内共有953个基因在转录层面未对重编程产生应答，表明重编程因子无法直接作用于此类基因。进一步研究发现，另有305个基因发生了6类异常重编程：过度重编程、错误重编程以及非必需的上调或下调重编程，这揭示了山中伸弥因子存在显著的负面作用。上述关于重编程因子早期转录应答的多样化研究结果，为阐明山中伸弥因子的作用稳定性与局限性提供了全新视角。本研究通过比对3例人类胚胎干细胞（n=3）与4例人类成纤维细胞（n=4）的基因表达谱，完成了重编程组的定义。本研究对转导了慢病毒绿色荧光蛋白（lentiviral GFP）以及未转导的人类BJ成纤维细胞进行了RNA测序，并将其转录组与转导了山中伸弥因子（OCT4、SOX2、KLF4及c-MYC）的人类成纤维细胞转录组进行比对。基于重编程合法性视角，本研究明确了重编程应答的不同转录模式。