The nuclei of somatic cells fused to mouse embryonic stem cells express pluripotency markers similar to early reprogramming events

The mechanisms that direct nuclear reprogramming to a pluripotent state are still not fully understood. For species in which the derivation of true induced pluripotent stem (iPS) cells has not yet been completely successful, insights into the particular mechanisms that govern pluripotency can help in determining which are the appropriate conditions to induce stable reprogramming in somatic cells. Cell fusion of a somatic cell to a pluripotent cell is known to induce expression of pluripotency markers in the somatic nucleus. We hypothesized that fusion of bovine fetal fibroblasts (bFFs) to mouse embryonic stem (mES) cells would induce expression of pluripotency markers in the bFF nucleus. We have previously established a method to produce and specifically select multinucleated cells originated from both mES cell and bFF (bi-species heterokaryon), using indirect immunofluorescence on live cells and the combined use of imaging and sorting flow cytometry. With these tools in place, we analyzed 200 heterokaryons collected at 24, 48 and 72h after fusion, as well as unfused bFFs and mES cells, and analyzed using RNA-seq. We found significant changes in bovine gene expression patterns between bFFs and heterokaryons obtained 24h after fusion. Upregulation of early pluripotency markers OCT4 and KLF4, as well as hypoxia response genes, contrasted with downregulation of cell cycle inhibitors such as SST. The cytokine IL6, known to increase survival of early embryos in vitro, was also upregulated in heterokaryons, although its role and mechanism of action is still unclear. The cell fusion model presented here can be used to characterize early changes in somatic nuclear reprogramming, and to study the effect of different conditions during reprogramming. Five samples total. Bi-species (mouse/bovine) heterokaryons were produced and sampled at 24, 48, and 72h after fusion. Samples of mouse only and bovine only cells were harvested in parallel as controls.

将体细胞核重编程至多能状态的核心调控机制目前仍未完全阐明。对于尚未成功建立真正诱导多能干细胞（induced pluripotent stem cells，iPS细胞）品系的物种而言，解析其多能性调控的特有机制，有助于确定在体细胞中诱导稳定重编程的适宜条件。已知将体细胞与多能细胞融合，可诱导体细胞核表达多能性标志物。本研究提出假说：牛胎儿成纤维细胞（bovine fetal fibroblasts，bFFs）与小鼠胚胎干细胞（mouse embryonic stem cells，mES细胞）融合后，可诱导牛胎儿成纤维细胞核表达多能性标志物。此前我们已建立一套实验方法，可通过活细胞间接免疫荧光染色结合成像与流式细胞术（flow cytometry）分选，特异性制备并筛选由两类细胞融合形成的异种异核体（heterokaryon）。借助该实验体系，我们分析了融合后24h、48h及72h收集的200个异核体样本，同时设置未融合的牛胎儿成纤维细胞与小鼠胚胎干细胞作为对照，并通过RNA测序（RNA-seq）完成转录组分析。结果显示，融合后24h的牛胎儿成纤维细胞与异核体之间，牛源基因的表达谱存在显著差异。早期多能性标志物OCT4与KLF4以及缺氧应答基因均出现上调，而细胞周期抑制剂SST则呈现下调。此外，已知可提升早期胚胎体外存活率的细胞因子IL6，在异核体中也出现上调，尽管其具体功能与作用机制仍未明确。本研究建立的细胞融合模型，可用于表征体细胞核重编程的早期变化，并探究重编程过程中不同条件的影响。本数据集共包含5个样本：分别在融合后24h、48h、72h收集的小鼠-牛异种异核体样本，以及同步收集的纯小鼠胚胎干细胞与纯牛胎儿成纤维细胞对照样本。