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Primate naïve pluripotent stem cells stall in the G1 phase of the cell cycle and differentiate prematurely during embryo colonization

Irène Aksoy, Cloé Rognard, Anaïs Moulin, Guillaume Marcy, Etienne Masfaraud, Florence Wianny, Véronique Cortay, Angèle Bellemin-Ménard, Nathalie Doerflinger, Manon Dirheimer, Chloé Mayère, Cian Lynch, Olivier Raineteau, Thierry Joly, Colette Dehay, Manuel Serrano, Marielle Afanassieff, Pierre Savatier

Preprint posted on 28 March 2020 https://www.biorxiv.org/content/10.1101/2020.03.27.011890v1

Article now published in Stem Cell Reports at http://dx.doi.org/10.1016/j.stemcr.2020.12.004

Mouse naïve pluripotent cells can colonise a distantly related species’ embryo, but why can’t primate PSCs do the same?

Selected by Pierre Osteil

Categories: developmental biology

 

Background:

The first report on the capability of mouse embryonic stem cells (mESCs), derived from a blastocyst, to contribute to the development of an embryo when injected into the blastocyst (Day 3.5 post coitus) 1 opened a new era for functional genomics. Indeed, since then scientists can genetically engineer mESCs and create chimeras. If the modified ESCs contribute to the germ line, the next generation of animals will be 100% transgenic. From there, many projects originated with a similar aim of achieving a comparable outcome with other mammalian species, such as monkey, our closest relative. But for decades research has faced an important challenge: generating chimeras only works efficiently in mouse but not in other mammals.  Interestingly, pluripotent stem cells can also be derived from mouse epiblast, termed Epiblast stem cells or EpiSC 4,5. However, these are incapable of contributing to the mouse embryo. This revealed the broad spectrum of pluripotency:  the self-renewal status of pluripotent stem cells (PSCs) was coined “Naïve” when a PSC can contribute to the embryo to full term development or “Primed” for those that cannot 6.

From there, a new goal was set: make non-rodent mammalian PSCs “naïve” again!

So, a wide range of protocols emerged to reprogram primate PSCs into a state close to that of the mESC 7,8,17,9–16. Gene expression, colony morphologies as well as epigenetic features were used to grant them the status of naïve.

But are they really naïve? To tackle this question, Aksoy & colleagues decided to revisit different naïve conversion protocols, then see if the reprogramming was sufficient to acquire chimerism potential.

 

The results:

The rabbit embryo: an unnoticed model in the developmental and stem cell field

One number is striking: 2956 rabbit embryos have been used in total for chimera injection.  Additionally, the rabbit embryo gastrulates as a flat disk, such as primate. The material abundance and the physical similarity of the rabbit embryos make this species a very adequate model for interspecies chimeras. On the other hand, only 36 cynomolgus monkey embryos were used, which, despite being a great achievement, demonstrates the challenges for large scale study on non-human primates.

First, Aksoy & colleagues injected mESCs into rabbit embryos from serum/LIF (n=238) and 2i/LIF (n=19). 98% of these embryos contained Serum/LIF GFP+ cells expressing NANOG and SOX2 but not SOX17, suggesting the cells are not incorporating the primitive endoderm layer but only the epiblast. This demonstrates that the rabbit embryo can be used for interspecies chimera studies.

 

Naïve non-human primate cells are not able to form chimeras

Then, they reprogrammed the primed Rhesus monkey PSCs using 7 different protocols that have been reported for successful conversion of the naïve cells. Together with the primed culture condition as a control, Rhesus PSCs were injected into 2385 embryos in total (so an average of about 300 embryos per condition). Four conditions showed successful incorporation after 3 days of culture: TL2i9, 4i/L/b16, T2iLGoY 15and LCDM (EPS)17. These conditions reprogrammed the rhPSC to a state comparable to that of the E6/E7 cynomolgus epiblast according to the transcriptome analysis (see the PCA below). Despite incorporation, they were not able to survive and divide after 3 days in the embryo. TL2i cells show the highest survival rate of 57% and seem to display an increased cell number at day 2 despite the premature expression of Gata6, a primitive endoderm marker. Even ROCK inhibitor did not rescue the survival. Similar results were obtained with human iPSCs grown in TL2i.

 

The low contribution of primate PSCs might be due to the failure to proliferate when in single cell suspension.

After obtaining these results, Aksoy & colleagues decided to answer the question of whether this incapability of incorporation into the blastocyst was due to evolutionary distance. So, they injected rhesus TL2i and human t2iLGoY into cynomolgus embryos (7 and 15 respectively) while 5 were injected with mESCs. Similar results as with the rabbit embryos were found suggesting the evolutionary distance between rabbit and rhesus monkey is not the key factor here, since mESCs can contribute.

Then, they investigated DNA replication in a condition similar to that of injection into the embryo (here DNA replication after single cell dissociation). mESCs do not show any changes in DNA replication. But for the monkey ESCs the story is quite different. First, DNA replication is significantly slower in rhESCs, but by 1 hour after dissociation proliferative cells are almost inexistent (see FACS plots) suggesting that the non-human PSCs do not replicate their DNA while in single-cell suspension. When reprogrammed into naïve condition (4i/L/b, TL2i and t2iLGoY) cells had an increased DNA replication rate but this was not maintained after dissociation. This was confirmed in a chimera set up, where only 4 cells out of 29 still replicates 24hours after injection.

 

Non-human primate naïve cells stop proliferating in G1.

FUCCI mESCs and rhESCs were generated. The team observed that the distribution is quite different with an increased G1 phase in rhesus cells of 43% compared to 18% in mESCs. After conversion of the rhPSCs into naïve condition cells and their injection into rabbit embryos almost all the cells (78% in 4i/L/b and 100% in TL2i) are stuck in G1, suggesting growth arrest.

 

Conclusion:

The reprogramming of the rhPSCs into the so-called naïve state does not restore chimerism potential which is likely due to the culture conditions not supporting cell cycle progression after dissociation prior to blastocyst injection.

 

My take on this preprint:

I decided to cover this article as I did my PhD in this lab, so I am quite passionate by the questions tackled by my former team. The power of the research conducted herein lies in the ability to study multiple mammalian species together: mouse, rabbit, monkey and human. This is a first-of-its-kind study trying to solve the question of whether multiple species’ naïve pluripotent cells are able to colonise a blastocyst. But this is not the case suggesting we might need to redefine what is called “naïve” pluripotency. The large number of embryos used in this study is making a strong case for the need to reinvestigate pluripotency of non-human primate embryos.

 

4 questions to the authors: 

1- At the end of the first result paragraph: “After gastrulation, mESCs were able to contribute only to the neuroectoderm, but not to other ectodermal structures, or to the mesoderm and endoderm of rabbit gastrula”. I found this particularly interesting. To me it means that mESCs, despite the fact that they survive and divide in rabbit embryos, are excluded from the primitive streak and for me “fail” to gastrulate. They thrive in the epiblast that becomes neurectoderm. What is your opinion on this result?

2- It doesn’t seem that you describe how you removed the autofluorescence from the imaging. “To overcome this limitation, we systematically used an anti- GFP antibody.” I don’t understand what you have done here? On supp figure 1 you show some imaging with the antibody without staining, but how did you manage to remove the autofluorescence?

3- Wouldn’t you agree that the TL2i is the best strategy so far for interspecies chimera potential?  On this note, on p12 you said “At 3 DIV, 0% (n = 6),0% (n = 4), and 50% (n = 2)” figure 5D shows 80%, 80%, and 20% of HuN cells positive for SOX2, NANOG and GATA6”. If it is true, TL2i leads to more contribution to the epiblast lineage compare t2iLGoY (33% of cells into the primitive endoderm) which is supported by the PCA showing TL2i overlap with epiblast cells.

4- One last question: Do you think your rabbit, monkey and human PSC are naïve?

 

References:

  1. Bradley, A., Evans, M., Kaufman, M. H. & Robertson, E. Formation of germ-line chimaeras from embryo-derived teratocarcinoma cell lines. Nature 309, 255–256 (1984).
  2. Tachibana, M. et al. Generation of chimeric rhesus monkeys. Cell 148, 285–295 (2012).
  3. Li, P. et al. Germline competent embryonic stem cells derived from rat blastocysts. Cell 135, 1299–310 (2008).
  4. Brons, I. G. et al. Derivation of pluripotent epiblast stem cells from mammalian embryos. Nature 448, 191–195 (2007).
  5. Tesar, P. J. et al. New cell lines from mouse epiblast share defining features with human embryonic stem cells. Nature 448, 196–199 (2007).
  6. Nichols, J. & Smith, A. Naive and primed pluripotent states. Cell Stem Cell 4, 487–92 (2009).
  7. Gafni, O. et al. Derivation of novel human ground state naive pluripotent stem cells. Nature 504, 282–286 (2013).
  8. Gao, X. et al. Establishment of porcine and human expanded potential stem cells. Nat. Cell Biol. 21, 687–699 (2019).
  9. Chen, H. et al. Reinforcement of STAT3 activity reprogrammes human embryonic stem cells to naive-like pluripotency. Nat. Commun. 6, 7095 (2015).
  10. Takashima, Y. et al. Resetting Transcription Factor Control Circuitry toward Ground-State Pluripotency in Human. Cell 162, 452–453 (2015).
  11. Chan, Y. S. et al. Induction of a human pluripotent state with distinct regulatory circuitry that resembles preimplantation epiblast. Cell Stem Cell 13, 663–675 (2013).
  12. Theunissen, T. W. et al. Systematic identification of culture conditions for induction and maintenance of naive human pluripotency. Cell Stem Cell 15, 471–487 (2014).
  13. Ohtsuka, S., Nishikawa-Torikai, S. & Niwa, H. E-cadherin promotes incorporation of mouse epiblast stem cells into normal development. PLoS One 7, e45220 (2012).
  14. Guo, G. et al. Naive Pluripotent Stem Cells Derived Directly from Isolated Cells of the Human Inner Cell Mass. Stem Cell Reports 6, 437–446 (2016).
  15. Guo, G. et al. Epigenetic resetting of human pluripotency. Development 145, (2018).
  16. Fang, R. et al. Generation of naive induced pluripotent stem cells from rhesus monkey fibroblasts. Cell Stem Cell 15, 488–497 (2014).
  17. Yang, J. et al. Establishment of mouse expanded potential stem cells. Nature 550, 393–397 (2017).

 

 

Posted on: 4 June 2020

doi: https://doi.org/10.1242/prelights.21571

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Author's response

Irene Aksoy shared

  • At the end of the first result paragraph: “After gastrulation, mESCs were able to contribute only to the neuroectoderm, but not to other ectodermal structures, or to the mesoderm and endoderm of rabbit gastrula”. I found this particularly interesting. To me it means that mESCs, despite the fact that they survive and divide in rabbit embryos, are excluded from the primitive streak and for me “fail” to gastrulate. They thrive in the epiblast that becomes neurectoderm. What is your opinion on this result?

We show that mESCs are able to very efficiently colonize the rabbit pre-implantation embryo, with nearly 100% of rabbit embryos harboring mESCs expressing pluripotency markers. This data indicates that mESCs colonize the epiblast of a distantly related species as efficiently as the mouse embryo. However, we also observed that the chimerism efficiency decreases with development of the rabbit embryo and is restricted to the neuroectodermal lineage. This is certainly due to the fact that unlike the three-dimensional egg-cylinder shape of rodent embryos during gastrulation, rabbit embryos develop into a flattened disc at the surface of the conceptus like primate embryos. These mechanical constraints might explain why at late developmental stages the mESCs fail to remain competent at colonizing the rabbit embryo, together with differences in terms of signaling pathways, transcriptional networks involved in both species.

 

  • It doesn’t seem that you describe how you removed the autofluorescence from the imaging. “To overcome this limitation, we systematically used an anti- GFP antibody.” I don’t understand what you have done here? On supp figure 1 you show some imaging with the antibody without staining, but how did you manage to remove the autofluorescence?

The autofluorescence of the embryo is a very well-known phenomenon. Here we show that performing immunostaining against the GFP protein is essential to identify true GFP signal as it will increase the signal. In suppl figure 1, we performed immunostaining and took pictures with a confocal microscope. By combining both techniques we get rid of autofluorescence and observe only true signal. This is even more important if your GFP signal is weak and if you analyze post-implantation stage embryos. GFP is not an exception, red autofluorescence is even more important when we work with post-implantation stage embryos, so if we work for example with dsRed or mCherry positive cells, it is even more important to perform immunostaining to highlight the true signal.

 

  • Wouldn’t you agree that the TL2i is the best strategy so far for interspecies chimera potential?  On this note, on p12 you said “At 3 DIV, 0% (n = 6),0% (n = 4), and 50% (n = 2)” figure 5D shows 80%, 80%, and 20% of HuN cells positive for SOX2, NANOG and GATA6”. If it is true, TL2i leads to more contribution to the epiblast lineage compare t2iLGoY (33% of cells into the primitive endoderm) which is supported by the PCA showing TL2i overlap with epiblast cells. 

For the first part of your question, when we started this project our goal was to use one or two of the protocols that were able to convert human and non-human primate cells to naïve pluripotency and apply those to the non-human primate ESCs that we have in the lab. Our ultimate goal being to generate monkey chimeric embryos to study early embryonic development and corticogenesis in non-human primates. We first tested these cell lines in rabbit as we have access to a large number of rabbit embryos. However, we were very surprised by the inefficiency of these first protocols. So, we decided to test more protocols in case one of the reprogramming cocktail will be more efficient to convert our cells to chimeric competent cells. But again, for none of them we observed an efficient colonization of the host embryo.

We thought, as many in the field, that this could be due to the phylogenetic distance between monkey and rabbit. However, when we injected mESCs into rabbit embryos we observed a high colonization efficiency, despite their divergence that is almost equidistant between primate and rabbit (between 77 and 88 million years for rodents vs glires and 85 to 97 million years between primates and rodents+glires) indicating that if a cell is naïve it can colonize the epiblast of a distantly related species. This is one of the most interesting data of the manuscript. For two protocols (TL2i and t2iLGöY) we tested their ability to colonize the monkey embryo, but again we couldn’t get high chimerism, which definitely ceiled the fact that the cells couldn’t efficiently colonize the embryo, not because of the phylogenetic distance, but certainly because they haven’t acquired efficient chimeric competency.

For the second part, we do not think that TL2i cells are the “best” cells for rabbit embryo colonization. EPS and t2iLGöY cells are as efficient as TL2i, and 4i/L/b cells are also close. We have to take into account all the experiments we have performed, including immunostainings, EdU, FUCCI assays. However, as you mention we do observe subtle differences between the cell lines, which is quite interesting and that we currently study.

 

  • One last question: Do you think your rabbit, monkey and human PSC are naïve?

The answer depends on whether you include or not chimeric competency as a specific characteristic of naïve PSC. In terms of expression of pluripotency markers, transcriptome reconfiguration and methylation status, then Yes the reprogrammed cells are naïve. If we include chimeric competency, then Not so much. This is the best that have been observed so far in terms of primate naïve reprogramming protocols and these cells are a great tool to study naïve pluripotency in other species than mouse. More efforts should be put on improving the status of the cells to check the chimeric competency box.

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