Kaiser Franziska, Kubaczka Caroline, Graf Monika, Langer Nina, Langkabel Jan, Arévalo Lena, Schorle Hubert
Institute of Pathology, Department of Developmental Pathology, University Medical School, Bonn, Germany.
Institute of Pathology, Department of Developmental Pathology, University Medical School, Bonn, Germany.
Placenta. 2020 Jan 15;90:128-137. doi: 10.1016/j.placenta.2019.12.017. Epub 2019 Dec 23.
The first lineage separation in mammalian development occurs when totipotent cells of the zygote give rise to the inner cell mass and the trophectoderm. The lineages are strictly separated by an epigenetic barrier. In vitro derivatives of these lineages embryonic stem cells (ESC) and trophoblast stem cells (TSC) are used to study the requirements needed to overcome the barrier in ESC to TSC conversion approaches.
Different combinations of TSC transcription factors were induced in ESC for three days. Cells were kept in TS medium with fetal bovine serum (FBS) or the chemically defined TX medium. Obtained cells were analysed for OCT4 levels, TSC surface marker levels, expression of TSC markers and methylation status of Elf5, Oct4 and Nanog promoters. Further, long-term culture stability and in vitro and in vivo differentiation was tested.
Overexpression of Gata3, Eomes, Tfap2c, Ets2 and Cdx2 in ESC resulted in induction of TSC fate. Overexpression of Cdx2 or four factors (Gata3, Eomes, Tfap2c and Ets2) resulted in complete conversion only when cells were cultured in TX medium. The obtained induced TSC (iTSC) display characteristics of bona fide TSC in terms of marker expression and promoter methylation patterns. The generated converted cells were shown to display self-renewal and to be capable to differentiate into TSC derivatives in vitro and in vivo.
Gata3, Eomes, Tfap2c, Ets2 and Cdx2 overexpression in ESC resulted in stable iTSC fate independent of culture conditions. For four factors or Cdx2 alone, TX medium is required for complete TSC conversion.
哺乳动物发育过程中的首次谱系分离发生在受精卵的全能细胞产生内细胞团和滋养外胚层时。这些谱系通过表观遗传屏障严格分开。这些谱系的体外衍生物，即胚胎干细胞（ESC）和滋养层干细胞（TSC），被用于研究在ESC向TSC转化方法中克服该屏障所需的条件。
在ESC中诱导TSC转录因子的不同组合三天。细胞保存在含有胎牛血清（FBS）的TS培养基或化学成分明确的TX培养基中。对获得的细胞进行OCT4水平、TSC表面标志物水平、TSC标志物表达以及Elf5、Oct4和Nanog启动子甲基化状态的分析。此外，还测试了长期培养稳定性以及体外和体内分化情况。
ESC中Gata3、Eomes、Tfap2c、Ets2和Cdx2的过表达导致TSC命运的诱导。仅当细胞在TX培养基中培养时，Cdx2或四种因子（Gata3、Eomes、Tfap2c和Ets2）的过表达才导致完全转化。所获得的诱导型TSC（iTSC）在标志物表达和启动子甲基化模式方面表现出真正TSC的特征。所产生的转化细胞显示出自我更新能力，并能够在体外和体内分化为TSC衍生物。
ESC中Gata3、Eomes、Tfap2c、Ets2和Cdx2的过表达导致稳定的iTSC命运，与培养条件无关。对于单独的四种因子或Cdx2，完全转化为TSC需要TX培养基。
