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Trophectoderm Potency is Retained Exclusively in Human Naïve Cells

Ge Guo, Giuliano Giuseppe Stirparo, Stanley Strawbridge, Jian Yang, James Clarke, Meng Amy Li, Sam Myers, Buse Nurten Özel, Jennifer Nichols, Austin Smith

Preprint posted on February 04, 2020 https://www.biorxiv.org/content/10.1101/2020.02.04.933812v1

Regretting lineage decisions: Human naïve cells and the inner cell mass from human blastocysts retain the potential to generate trophectoderm-like cells.

Selected by Sergio Menchero

Categories: developmental biology

Background & Summary:

Embryonic development is understood as a series of branching decisions taken under the control of lineage programs. These lineage bifurcations are generally unidirectional, and once a cell is committed to a lineage it cannot come back.

In mammals, after the first fate decision, cells are segregated in two lineages: trophectoderm (TE, future placenta) and inner cell mass (ICM, pluripotent population that will give rise to the embryo). In the mouse, during the specification of these first lineages there is a certain degree of plasticity (Posfai et al. 2017) but once they are established, cells from one lineage cannot contribute to the other lineage. Mouse trophoblast stem cells (mTSCs) and mouse embryonic stem cells (mESCs), the in vitro counterparts of TE and ICM respectively, retain the potential from the lineage they were derived from, although they can be interconverted to other lineages by genetic manipulations and modification of culture conditions (Garg et al. 2016).

Interestingly, naïve human ESCs (hESCs) have a transcriptional signature more similar to human morula cells, an earlier stage, than to the segregated ICM (Theunissen et al. 2016). Because at this stage the first lineages have not been segregated yet (Petropoulos et al. 2016), the putative potential of these naïve hESCs to contribute to TE fate has been a standing question in the field.

Major findings in the preprint:

  • Naïve human ESCs can upregulate TE-related markers. Naïve mESCs are cultured in the presence of a MEK inhibitor (PD) and maintain their capacity to self-renew. When naïve hESCs were cultured with PD alone, some of them differentiated and started to express TE-markers such as GATA2 and GATA3. The authors generated a GATA3 knock-in reporter and confirmed the upregulation of the reporter upon culture in PD.
  • Inhibition of Nodal and Activin pathway increases TE differentiation potential. When adding A83, an inhibitor of the Nodal and Activin pathways, to the culture media in addition to PD, the GATA3 reporter and other TE markers were activated earlier and stronger. Global transcriptomic analysis of naïve hESCs with PD+A83 showed that cells progressively acquired a TE signature similar to the TE of human embryos.
Figure 1 (Figure 2G in the preprint). RT-QPCR assay of GATA2 and GATA3 expression over time in different culture conditions.

 

  • Human ESCs with different transcriptomic signatures including primed hESCs, extended-potential hESCs (hEPSCs) and naïve hESCs behave differently upon inhibition of MEK and Nodal. It had been reported that primed hESCs and hEPSCs could differentiate toward TE in a BMP-dependent manner. However, in this preprint, the authors show that primed hESCs and hEPSCs acquire an amnion-like transcriptome upon culture with PD+A83 inhibitors instead of TE identity as naïve hESCs do. Moreover, naïve hESCs follow a BMP-independent differentiation path to differentiate to TE. This suggests that not all the human ESCs have the same default differentiation program, and differences in potentiality could affect the differentiation trajectories.
  • Perturbations in key factors of the pluripotency and TE networks affect the potential of naïve hESCs to differentiate to TE.
    • Targeted mutagenesis of the pluripotency-associated transcription factors OCT4, SOX2 and NANOG using CRISPR/Cas9 led to upregulation of TE markers, even when cells were cultured on N2B27 which does not promote TE differentiation in control conditions.
    • Forced expression of NANOG prevented the upregulation of TE markers.
    • TFAP2C and YAP mutant hESCs showed a reduced induction of TE markers as compared to control cells suggesting an important role for TE differentiation.
  • ICM from human embryos retain the potential to generate TE cells. Culture of isolated ICMs in N2B27 medium or in PD+A83 gave rise to flattened cells that expressed GATA3. Those cells surrounded a cluster of SOX2 positive cells that represent the epiblast. In those outgrowth cultures, some cells with a primitive endoderm identity, recognised by the expression of SOX17, were also present. In vivo, cells from the ICM are expected to segregate into SOX2 or SOX17 positive cells when the epiblast and the primitive endoderm are specified. However, the fact that cells with TE markers also emerge from ICM cells indicates that the human embryo retains a high degree of plasticity.
Figure 2 (Figure 7D-E in the preprint). Human ICM outgrowth culture in N2B27 + LDN (upper panel) or PD+A83+LDN (bottom panel). Immunostaining for key markers GATA3 (trophectoderm), SOX17 (primitive endoderm) and SOX2 (epiblast) are shown.

 

General comments

The scarce availability of human embryos, together with ethical issues that their usage can raise, make their in vitro counterparts a very valuable tool to understand human embryo development. The authors of this preprint use naïve hESCs to nicely demonstrate that they retain the ability to upregulate the TE transcriptional program under certain culture conditions. These findings highlight the potential of naïve hESCs and how they could help to understand the specification of the TE identity in future studies, including the activation or inactivation of specific regulatory elements.

One important aspect to investigate is whether naïve hESCs de-differentiate from an epiblast-like state or if naïve hESCs mimic a cell state prior to the first lineage decision and differences in culture conditions bias their progression to a TE-like or ICM-like identity.

Questions to the authors

  • In your experiments, you see that naïve hESCs pass through an ICM-like cell population before their differentiation to TE. If naïve hESCs have a transcriptional signature more similar to human morula cells than to the segregated ICM as it has been proposed (Theunissen et al. 2016), why do think they need to pass through an ICM-like state? If they represent a stage prior to the first lineage decision, a priori it could be independent of the specification of the ICM.
  • Have you evaluated how far can naïve hESCs differentiate into TE derivatives? Do you think the “capacitation” process can affect their potential to give rise to more differentiated tissues?
  • Analysis of scRNA-seq data from human preimplantation embryos revealed that blastomeres are transcriptionally very similar until the early blastocyst stage (Petropoulos et al. 2016). Do you think that the fact that human ICM retains TE lineage plasticity is because the lineages have been segregated more recently than in the mouse? Do you see any differences when using ICMs from early or very late blastocysts? Would you expect this plasticity to occur in other species where the TE lineage is also specified late like the pig (Ramos-Ibeas et al. 2019)?

References

Garg et al. (2016) Capturing Identity and Fate Ex Vivo: Stem Cells From the Mouse Blastocyst. Curr Top Dev Biol 120, 361-400.

Petropoulos et al. (2016) Single-Cell RNA-Seq Reveals Lineage and X Chromosome Dynamics in Human Preimplantation Embryos. Cell 165(4), 1012-26

Posfai et al. (2017) Position- And Hippo Signaling-Dependent Plasticity During Lineage Segregation in the Early Mouse Embryo. Elife 6:e22906.

Ramos-Ibeas et al. (2019) Pluripotency and X Chromosome Dynamics Revealed in Pig Pre-Gastrulating Embryos by Single Cell Analysis. Nat Commun 10(1), 500

Theunissen et al. (2016) Molecular Criteria for Defining the Naive Human Pluripotent State. Cell Stem Cell 19(4), 502-515.

 

Tags: human embryo, human naive esc, plasticity, trophectoderm

Posted on: 1st March 2020 , updated on: 2nd March 2020

doi: https://doi.org/10.1242/prelights.17446

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Author's response

Ge Guo, Giuliano Giuseppe Stirparo, Jennifer Nichols & Austin Smith shared

In your experiments, you see that naïve hESCs pass through an ICM-like cell population before their differentiation to TE. If naïve hESCs have a transcriptional signature more similar to human morula cells than to the segregated ICM as it has been proposed (Theunissen et al. 2016), why do think they need to pass through an ICM-like state? If they represent a stage prior to the first lineage decision, a priori it could be independent of the specification of the ICM.

Response

The study by Theunissen et al is focussed on retrotransposon expression. They report that that specifically in their culture conditions (5iLA) for naïve hPSC they detect expression of transposons activated in the morula as well as in the late blastocyst ICM. Of note, however, Theunissen et al acknowledge: “Based on gene expression, naive cells were most closely aligned with the late blastocyst (Figures 2C and S1D)”. We have previously documented that the global gene expression signature of naïve human PSCs is most related to the naïve epiblast in the late ICM (Stirparo et al, 2018, Figures 7 and S7). The figure below presents the correlations between different naïve cell cultures and human embryo stages based on all expressed genes (Panel A) and the expression distribution in naïve cells of genes enriched in different embryo stages (Panel B).

Panel A: Bootstrap of Spearman’s correlation (100 times, size=50) between human naïve stem cells (different lines and culture conditions) and pre-implantation stages of human embryo development (Petropoulos et al., 2016, Stirparo et al., 2018).
Panel B: Distribution of log2expression value in each naïve stem cell line/condition for genes enriched in the different stages of human embryo development (e.g. epiblast enriched genes: log2 FPKM > 2 in epiblast and log2FPKM < 2 in earlyICM, compacted MOR and 8-cell stage).

 

It could still be argued that a fraction of cells in naïve PSC cultures are in a more primitive morula-like state. However, we consistently observe conversion of more than 70% of cells in the culture to GATA3 positive status (Figure 2F,H). Furthermore, it is evident that naive cells become less similar to naïve epiblast and more similar to early ICM before acquiring a trophectoderm signature (Figures 4A-F, S4E-G).

Have you evaluated how far can naïve hESCs differentiate into TE derivatives? Do you think the “capacitation” process can affect their potential to give rise to more differentiated tissues?

Response

We have not examined trophoblast maturation. We do not consider that TE differentiation is dependent on capacitation, which refers to the formative transition to amnion, somatic and germline competence (Figure 7G).

Analysis of scRNA-seq data from human preimplantation embryos revealed that blastomeres are transcriptionally very similar until the early blastocyst stage (Petropoulos et al. 2016). Do you think that the fact that human ICM retains TE lineage plasticity is because the lineages have been segregated more recently than in the mouse? Do you see any differences when using ICMs from early or very late blastocysts? Would you expect this plasticity to occur in other species where the TE lineage is also specified late like the pig (Ramos-Ibeas et al. 2019)?

Response

Multiple studies in the mouse support a sequential lineage bifurcation model, whereby trophoblast and early ICM are fully committed in the early blastocyst before hypoblast and epiblast specification commence. The human blastocyst is formed by E5. Our findings demonstrate that human ICMs at E6 or E7, which already contain epiblast and hypoblast (Blakeley et al., 2015; Petropoulos et al, 2016; Stirparo et al, 2018; Xiang et al, 2020), can regenerate trophectoderm efficiently. It would be interesting to address whether there is a difference in efficiency between E6 and E7, but this is problematic due to the limited numbers of human embryos available. Of note, inspection of the transcriptome data in Xiang et al, 2020 reveals that both early ICM and epiblast signatures are present in individual human embryos, even at E7 (Figure 7F). The persistence of cells with early ICM identity means that developmental progression is desynchronised compared with mouse. We cannot predict the situation in other mammals, but it will be very interesting to find out.

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