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

 

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?

 

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Posted on: 4 June 2020

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

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