Notably, EpiSCs are refractory to PGC induction, unlike embryonic day 5.5 (E5.5)–6.5 epiblast. (Hayashi et al., 2011; Murakami et al., 2016; Ohinata et al., 2009). Naive ESCs are also unresponsive to germ cell inductive stimuli, unless they are transitioned for 24–48 h into a population termed epiblast-like cells (EpiLCs) (Hayashi et al., 2011; Nakaki et al., 2013). EpiLCs are molecularly as well as functionally distinct from both naive ESCs and EpiSCs (Buecker et al., 2014; Hayashi et al., 2011; Kalkan et al., 2017; Smith, 2017). They are enriched in formative phase cells related to pre-streak epiblast but are heterogeneous and persist only transiently (Hayashi et al., 2011).
In mice, the formative phase of pluripotency is definitively distinguished from naive and primed phases by competence for germline specification (Hayashi et al., 2011; Ohinata et al., 2009).
エピブラストの培養条件を変えて、 AloX cells、EpiSCs maintained in either activin and FGF (AF) 、EpiSCs maintained in either activin, FGF, and XAV939 (AFX)などの細胞タイプを誘導して、それぞれの細胞の遺伝子発現の違いを図示している。
qRT-PCR analysis of marker gene expression relative to ESCs in 2iL ( =1) in AloX cells and EpiSCs maintained in either activin and FGF (AF) or activin, FGF, and XAV939 (AFX),
エピブラストから、どのような条件でESに戻れるかの知見もいまだ、流動的である事が書かれている。 All cells died or differentiated within a few days, demonstrating complete extinction of ESC identity. This finding is in marked contrast to other reports of “intermediate” pluripotent states, which readily revert to ESCs (D’Aniello et al., 2016; Neagu et al., 2020; Rathjen et al., 1999).
ESやICM cellsほどではないが、FS細胞、primary formative epiblast cellsも、キメラに寄与できることが示されている。 FS cells and primary formative epiblast cells can contribute to blastocyst chimeras, although with lower efficiency than ESCs or ICM cells.
Chimera Colonization ・・・ Chimera formation conceivably might entail reversion of FS cells to naive status in the blastocyst. We therefore inspected embryos 24 h after injection. FS cells were localized to the ICM, but immunostaining showed that in contrast to host naive epiblast or introduced ESCs, FS cells did not express the naive pluripotency specific transcription factor Klf4 and retained the formative marker Oct6 (Figure 3F). Therefore, FS cells maintain formative identity within the blastocyst environment.
Chimera formation by FS cells derived from post-implantation epiblast challenges the conclusion from classic embryo-embryo chimera studies that epiblast cells lose colonization ability entirely by E5.5 (Gardner and Brook, 1997; Gardner et al., 1985). We revisited those experiments by using a fluorescent reporter to allow sensitive detection of contributions. We dissected epiblasts from cavitated E5.5 and pre-streak E6.0–6.25 transgenic embryos expressing membrane-bound tdTomato (mTmG). Epiblasts were dissociated using Accutase with addition of ROCKi to improve viability and 10 cells injected per blastocyst. We detected tdTomato-positive cells in 11 out of 91 embryos recovered at E9.5 (Figures 3G, 3H, and S3H–S3L). Contributions were typically sparse and, interestingly, were most frequently in the yolk sac mesoderm and amnion. In three chimeras, however, colonization was widespread in the embryo proper (Figures 3G, 3H, and S3H). We did not detect any contribution from streak stage (E6.5–7.0) epiblast cells (Figure S3L). These observations establish that FS cells and primary formative epiblast cells can contribute to blastocyst chimeras, although with lower efficiency than ESCs or ICM cells.