Peng Keli, Li Xu, Wu Congyu, Wang Yuna, Yu Jian, Zhang Jinxin, Gao Qian, Zhang Wenhao, Zhang Qian, Fan Yong, Yu Yang, Shuai Ling
State Key Laboratory of Medicinal Chemical Biology and College of Pharmacy, Nankai University, Tianjin 300350, China.
Department of Neurological Surgery, Feinberg School of Medicine, Northwestern University, Chicago 60611, USA.
iScience. 2019 Jan 25;11:508-518. doi: 10.1016/j.isci.2018.12.014. Epub 2019 Jan 8.
Owing to their single genome, haploid cells are powerful to uncover unknown genes by performing genetic screening in mammals. However, no haploid cell line from an extraembryonic lineage has been achieved yet, which limits the application of haploid cells in placental genetic screening. Here, we show that overexpression of Cdx2 can convert haploid embryonic stem cells to trophoblast stem cells (TSCs). p53 deletion reduces diploidization during the conversion and guarantees the generation of haploid-induced TSCs (haiTSCs). haiTSCs not only share the same molecular characterization with trophoderm-derived TSCs but also possess multipotency to placental lineages in various procedures. In addition, haiTSCs can maintain haploidy in the long term, assisted by periodic sorting and with reliance on FGF4 and heparin. Finally, we perform piggyBac-mediated high-throughput mutation in haiTSCs and use them in trophoblast lineage genetic screening. Deep sequencing analysis and validation experiments prove that Htra1 is a blocker for spongiotrophoblast specification.
由于单倍体细胞只有一套基因组，因此在哺乳动物中进行遗传筛选时，它们在发现未知基因方面具有强大的能力。然而，目前尚未获得来自胚外谱系的单倍体细胞系，这限制了单倍体细胞在胎盘遗传筛选中的应用。在此，我们表明Cdx2的过表达可将单倍体胚胎干细胞转化为滋养层干细胞（TSCs）。p53缺失可减少转化过程中的二倍体化，并确保产生单倍体诱导的TSCs（haiTSCs）。haiTSCs不仅与滋养层来源的TSCs具有相同的分子特征，而且在各种过程中对胎盘谱系具有多能性。此外，haiTSCs在周期性分选的辅助下，依赖FGF4和肝素，能够长期维持单倍体状态。最后，我们在haiTSCs中进行了piggyBac介导的高通量突变，并将其用于滋养层谱系遗传筛选。深度测序分析和验证实验证明，Htra1是海绵滋养层细胞特化的一个阻断因子。
