A novel mouse stem cell established in combination of Activin A and LIF. A novel mouse stem cell established in combination of Activin A and LIF

We reported the ALSCs derivation from E3.5 blastocysts and E6.5 epiblasts cultured in basic N2B27 medium supplemented with Acitivin A and LIF. Activin A subtitutes NODAL signaling in vivo and is an important factor for self-renewal. LIF is extracted from feeder and have an impact on sustaining pluripotency of ESCs. We combined the two factors and finding AL medium could sustaining a novel mouse embryonic stem cells. Furthermore, EpiSCs are also discovered that able to convert to rESCs by culturing in AL medium for several passages, which illustrates that AL medium not only support establishment of pluripotent stem cells from embryos in vivo but also could convert EpiSCs to rESCs. Then, based on multiple analyses of pluripotency, we performed RNA-seq of ALSCs to exploring the differences of ALSCs compared to EpiSCs and ESCs respctively. RNA-seq data showed ALSCs is different with EpiSCs or ESCs. Teratoma test proved ALSCs could contribute to three germ layers in vivo and embryonic body analysis testify ALSCs could differentiate to three germ layers like cells in vitro. Overall design: 3 replicates of mRNA of ALSCs. The control group named AFSCs and have been submitted in GSE130060.

本研究报道了ALSCs的建系方法：将E3.5囊胚与E6.5上胚层置于添加了激活素A（Acitivin A）与白血病抑制因子（LIF）的基础N2B27培养基（basic N2B27 medium）中进行培养。激活素A可在体内替代NODAL信号通路，是维持细胞自我更新的关键因子；白血病抑制因子（LIF）源自饲养层细胞，对维持胚胎干细胞（ESCs）的多能性具有重要作用。本研究将上述两种因子组合使用后发现，AL培养基可支持一种新型小鼠胚胎干细胞系的建立。此外，研究发现将上胚层干细胞（EpiSCs）在AL培养基中连续传代培养数代后，可将其转化为rESCs，这表明AL培养基不仅可直接从体内胚胎中建立多能干细胞系，还可将EpiSCs重编程为rESCs。随后，基于多维度的多能性分析，我们对ALSCs进行了RNA测序（RNA-seq），以分别探究ALSCs与EpiSCs、胚胎干细胞（ESCs）之间的转录组差异。RNA测序结果显示，ALSCs的表达谱与EpiSCs及ESCs均存在显著差异。畸胎瘤实验证实，ALSCs在体内可分化为三个胚层的组织细胞；拟胚体实验则进一步验证，ALSCs在体外同样能够分化为三个胚层的细胞类群。实验整体设计：ALSCs的mRNA样本共设置3个生物学重复；对照组为AFSCs，其相关测序数据已提交至GSE130060数据集。