Defining totipotency using criteria of increasing stringency
Preprint posted on 3 March 2020 https://www.biorxiv.org/content/10.1101/2020.03.02.972893v1
Developmental biology is the study of how a single cell (the zygote) generates the multitude of cell types that exist in an adult organism. The initial divisions that the mammalian embryo undertakes over the first days of development increase the number of cells and retain the ability to generate a full organism. Subsequently, at the blastocyst stage, the embryo will go on to generate the first tissue types in the embryo: the epiblast (Epi), the extraembryonic (ExE) trophectoderm (TE), and primitive endoderm (PrE). Totipotency is the ability to generate a whole organism and the extraembryonic structures required during embryo development from a single cell. Over the past years, numerous reports have been published characterizing embryonic stem cells that retain totipotency features, expanding their potential compared to regular embryonic stem cells (ESCs) that contribute to the embryo proper. However, the ability to capture totipotency in vitro hasn’t been fully achieved yet.
In this preprint, Posfai, Schell and Janiszewski re-assess the totipotency features of two recently published embryonic stem cells with expanded potential (L-EPSCs and D-EPSCs). The authors compare the transcriptome and gene regulatory networks of L-EPSCs and D-EPSCs and early pre- and post- implantation mouse embryos at a single cell level to identify the correspondence between in vitro ESCs and their in vivo counterparts. Unexpectedly for cells shown to be totipotent, EPSCs were found to be transcriptionally closer to the pre- and post-implantation epiblast. Next, the authors test the ability of ESCs to transdifferentiate into trophectoderm stem cells (TSCs) and show that no embryonic stem cell passes this test. Finally, they perform the ultimate assay to assess their developmental potential: the ability of ESCs to contribute to embryonic and extraembryonic structures in vivo. They do so by generating chimeras and analysing embryos at E4.5, E6.5 and in E12.5 placentas. The tested embryonic stem cells with expanded potential (L-EPSCs and D-EPSCs) can be found in the outer layer of the blastocyst at E4.5, but none express CDX2, the specific marker of the TE lineage. From the ESCs with expanded potential, only D-EPSCs show a small contribution to extraembryonic structures of the post-implantation embryo (Figure 1). Altogether, the analyses on this preprint indicate that EPSCs capacity to generate extraembryonic tissues is very limited, and that their transcriptional signature does not correspond to totipotent blastomeres, such as those from morulae.
Why I think this work moves the field forward
There are now robust blastoid, 2D- and 3D- gastruloid, and all sort of organoid protocols to generate structures from stem cells in vitro that recapitulate certain aspects of embryo development. These protocols combined with the capacity to culture totipotent ESCs that can give rise to the entire conceptus would allow us to recapitulate embryo development without the need for sperm and an egg, and might enable more efficient generation of chimeric animals for research and organ production for transplantation.
With this in mind, it is crucial that totipotency is defined by accurate criteria. I really like the effort of the labs in this preprint to compile and perform rigorous comparisons for reproducibility and standardization of totipotency features. Unlike most reports, this work benchmarks several stem cells for their expanded potential, allowing the direct comparison of all of them in a single manuscript. In particular, the deep transcriptional characterization of the cells, and the assessment of their in vivo contributions with marker genes, challenges the totipotent potential of expanded stem cells. The selected criteria can be used to summarize other reports in the literature where totipotency features were described (see table below).
This preprint sets the baseline for future analyses on totipotency and might be useful when revisiting previous literature where totipotency was captured in vitro.
Questions to authors
Q1. How stringent do the authors think that teratoma assays are? What is the authors’ opinion on cells having expanded potential if the stem cells generate trophoblast-derived cells in teratomas assays shown by marker expression?
Q2. The authors show that a reduced percentage of D-EPSCs can contribute to the TE/ExE ectodem in blastoids, and analysis of the transcriptional signature of blastoids is closer to E4.5 blastocysts. Do the authors think that blastoids may be closer to hatching embryos, and this is why the TE signature resembles ExE ectoderm? If that is the case, do the authors think that generating blastoids that are equivalent to the expanding blastocyst (~E3.5) might highlight an expanded in vitro potential of D-EPSCs?
Q3. Why do the authors think that cells with expanded potential can be found in outside positions more often than standard 2i/LIF ESCs but are unable to express the TE marker Cdx2?
- Q.-L. Ying, J. Wray, J. Nichols, L. Batlle-Morera, B. Doble, J. Woodgett, P. Cohen, A. Smith, The ground state of embryonic stem cell self-renewal. Nature. 453, 519–23 (2008).
- S. Tanaka, T. Kunath, A. K. Hadjantonakis, A. Nagy, J. Rossant, Promotion of trophoblast stem cell proliferation by FGF4. Science (New York, N.Y.). 282, 2072–5 (1998).
- T. Kunath, D. Arnaud, G. D. Uy, I. Okamoto, C. Chureau, Y. Yamanaka, E. Heard, R. L. Gardner, P. Avner, J. Rossant, Imprinted X-inactivation in extra-embryonic endoderm cell lines from mouse blastocysts. Development (Cambridge, England). 132, 1649–61 (2005).
- K. Takahashi, S. Yamanaka, Induction of pluripotent stem cells from mouse embryonic and adult fibroblast cultures by defined factors. Cell. 126, 663–76 (2006).
- P. J. Tesar, J. G. Chenoweth, F. A. Brook, T. J. Davies, E. P. Evans, D. L. Mack, R. L. Gardner, R. D. G. McKay, New cell lines from mouse epiblast share defining features with human embryonic stem cells. Nature. 448, 196–9 (2007).
- J. Yang, D. J. Ryan, W. Wang, J. C.-H. Tsang, G. Lan, H. Masaki, X. Gao, L. Antunes, Y. Yu, Z. Zhu, J. Wang, A. A. Kolodziejczyk, L. S. Campos, C. Wang, F. Yang, Z. Zhong, B. Fu, M. A. Eckersley-Maslin, M. Woods, Y. Tanaka, X. Chen, A. C. Wilkinson, J. Bussell, J. White, R. Ramirez-Solis, W. Reik, B. Göttgens, S. A. Teichmann, P. P. L. Tam, H. Nakauchi, X. Zou, L. Lu, P. Liu, Establishment of mouse expanded potential stem cells. Nature. 550, 393–397 (2017).
- Y. Yang, B. Liu, J. Xu, J. Wang, J. Wu, C. Shi, Y. Xu, J. Dong, C. Wang, W. Lai, J. Zhu, L. Xiong, D. Zhu, X. Li, W. Yang, T. Yamauchi, A. Sugawara, Z. Li, F. Sun, X. Li, C. Li, A. He, Y. Du, T. Wang, C. Zhao, H. Li, X. Chi, H. Zhang, Y. Liu, C. Li, S. Duo, M. Yin, H. Shen, J. C. I. Belmonte, H. Deng, Derivation of Pluripotent Stem Cells with In Vivo Embryonic and Extraembryonic Potency. Cell. 169, 243-257.e25 (2017).
- T. S. Macfarlan, W. D. Gifford, S. Driscoll, K. Lettieri, H. M. Rowe, D. Bonanomi, A. Firth, O. Singer, D. Trono, S. L. Pfaff, Embryonic stem cell potency fluctuates with endogenous retrovirus activity. Nature. 487, 57–63 (2012).
- M. Abad, L. Mosteiro, C. Pantoja, M. Cañamero, T. Rayon, I. Ors, O. Graña, D. Megías, O. Domínguez, D. Martínez, M. Manzanares, S. Ortega, M. Serrano, Reprogramming in vivo produces teratomas and iPS cells with totipotency features. Nature. 502, 340–5 (2013).
- S. M. Morgani, M. A. Canham, J. Nichols, A. A. Sharov, R. P. Migueles, M. S. H. Ko, J. M. Brickman, Totipotent embryonic stem cells arise in ground-state culture conditions. Cell reports. 3, 1945–57 (2013).
Posted on: 16 March 2020Read preprint
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