From fibroblast to blastocyst: the magic ingredients
When the lab of Shinya Yamanaka revealed that viral introduction of Oct3/4, Sox2, Klf4 and c-Myc (OSKM) into adult skin cells could transform them into induced pluripotent stem cells (iPSCs), the world quickly took notice at the prospect of using iPSC-based-therapies to potentially treat human disease [1-3]. Although cell-based therapies have not lived up to the initial hype, nuclear somatic reprogramming remains an invaluable tool for modelling human development and disease .
It was previously shown that reprogramming of fibroblasts with the transcription factors Gata3, Eomes, Tfap2c, and Myc (GETM)  or Ets2  leads to the generation of functional induced trophoblast stem cells (iTSCs). iTSCs, together with iPSCs and induced XEN extraembryonic endoderm stem cells (iXENs) are the in vitro equivalent cell types of the blastocyst (the inner cell mass (ICM), primitive endoderm (PrE) and trophectoderm (TE)). An exhaustive series of experiments from Benchetrit et al. have now gone one step further and shown that a specific combination of factors, termed GETMS (GETM together with Esrrb), can directly reprogram fibroblasts into iTSCs, iPSCs and iXENs .
Using a reporter knockin mouse model referred to as BYKE, which marks PSCs and TSCs by Nanog-2A-EGFP and Elf5-2A-EYFP-NLS respectively, MEFs were isolated and reprogrammed into either iPSCs (Oct4, Sox2, Klf4 and Myc: OSKM) or iTSCs by GETM using lentivirus transduction. Screening of a plethora of factors expressed at early developmental stages revealed that Esrrb, in combination with GETM, is able to shift TSC to an PSC fate. Reprogramming with GETMS (GETM+Esrrb) under different culture conditions revealed that these 5 transgenes can overcome the lineage barrier between embryonic and extra-embryonic cells to induce iPSC and iTSC lineages. Additionally, in a secondary MEF system (GETMS reprogrammed iPSCs injected into blastocysts to derive secondary MEFs) allowing the isolation of lines expressing different stoichiometries of GETMS, further addition of Esrrb and Eomes induced an iPSC and iTSC fate, respectively. This and transduction of secondary MEFs under different culture conditions suggested that the levels of Esrrb or Eomes dictate the iPSC or iTSC fate during GETMS reprogramming.
To functionally test GETMS-derived cells, GETMS-tdTomato-expressing iPSCs and GETMS-EGFP expressing iTSCs were injected into blastocysts and whole-transcriptome analysis compared to ESCs and blastocyst-derived TSCs (bdTSC) was carried out. This revealed that GETMS-iPSCs and -iTSCs can contribute to embryo and placenta development. Lastly, again comparing GETMS-iPSCs and -iTSCs to MEF, ESC, iPSC, and bdTSC controls at early time points, RNA-Seq and ATAC-Seq suggested GETMS, like OSKM, target and induce chromatin changes at specific embryogenic loci. Interestingly, by comparing GTMS and GETMS, a role for Esrrb was proposed in inducing a ‘XEN-like’ signature where isolated GETMS-iXENs consistently expressed higher levels of Oct4 and Sall4. Previous work has shown that a XEN-like state occurs during reprogramming prior to induction of pluripotency [8&9].
In summary, this work highlights that a specific combination of factors is capable of producing the in vitro equivalent of the three blastocyst cell types and that the spatial and temporal expression of Esrrb and Eomes may be key to determining embryonic cell fate at the earliest time point.
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- Benchetrit, H. et al. Direct Induction of the Three Pre-implantation Blastocyst Cell Types from Fibroblasts. Cell Stem Cell, 25, (2019).
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- Li, X. et al. Direct Reprogramming of Fibroblasts via a Chemically Induced XEN-like State. Cell Stem Cell, 21, (2017).