Molecular mechanism underlying Nelfa and Bcl2 mediated pluripotent to totipotent state conversion in embryonic stem cells

In mouse embryonic stem cells (ESCs), a minority of cell population displays totipotent features resembling 2-cell-stage embryos, as known as 2-cell-like (2C-like) cells. However, how ESCs transit into the 2C-like state remains largely unknown. Here we report that overexpression of negative elongation factor A (Nelfa) can partially activates the 2C-like state of ESCs in a naive ESCs maintenance medium, whereas its knockout could not block the activation of 2C-like genes, moreover, Nelfa knockout ESCs retain the capacity of germline transmission in chimeras. We also demonstrate that the co-overexpression of Nelfa and Bcl2 robustly activate the 2C-like transcripts of ESCs in naive culture condition as well as exhibit the ability of bi-potential cell fate, remarkably, we show that individual overexpression of Bcl2 successfully induces endogenous NELFA and the 2C-like state of ESCs even in Nelfa knockout ESCs. Notably, we also found that overexpression of Nelfa significantly increases the somatic cell reprogramming efficiency and exhibit a 2C-like state in induced pluripotent stem cells (iPSCs). In summary, the findings of our study provide insights into the mechanism underlying Nelfa and Bcl2 mediated totipotent state in mouse embryonic stem cells.

在小鼠胚胎干细胞（mouse embryonic stem cells, ESCs）中，仅存在少数具备与二细胞期胚胎（2-cell-stage embryos）相似全能性特征的细胞亚群，这类细胞被称为二细胞样（2-cell-like, 2C-like）细胞。然而，胚胎干细胞向二细胞样状态转变的具体分子机制目前仍未被充分阐明。本研究证实，在原始态胚胎干细胞维持培养基（naive ESCs maintenance medium）中，过表达负延伸因子A（negative elongation factor A, Nelfa）可部分诱导胚胎干细胞的二细胞样状态；而敲除（knockout）Nelfa则无法阻断二细胞样基因的激活，且Nelfa敲除的胚胎干细胞仍保留嵌合体（chimeras）中的种系传递（germline transmission）能力。此外，我们发现，在原始态培养条件下，Nelfa与Bcl2的共过表达（co-overexpression）可高效激活胚胎干细胞的二细胞样转录谱，同时赋予细胞双潜能细胞命运的潜能；尤为关键的是，即便在Nelfa敲除的胚胎干细胞中，单独过表达Bcl2也可成功诱导内源性NELFA的表达以及胚胎干细胞的二细胞样状态。值得注意的是，我们还观察到，过表达Nelfa可显著提升体细胞重编程效率（somatic cell reprogramming efficiency），并在诱导多能干细胞（induced pluripotent stem cells, iPSCs）中诱导出二细胞样状态。综上，本研究的发现为阐明负延伸因子A与Bcl2介导小鼠胚胎干细胞全能性状态的潜在分子机制提供了新的见解。