Exploring mammalian preimplantation development and pluripotency

Our current understanding of how stem cells arise and transition during embryonic development has been limited by analysis tools that have lacked single-cell, whole-genome resolution. This thesis emphasizes the use of novel techniques and cutting-edge technology to better evaluate the biological underpinning of stem cell dynamics in both mouse and human. Development relies on stem cells to establish different lineage potentials from the same starting material. Stem cell populations are produced during embryogenesis at multiple stages, existing in various cellular states. These cells have unique self-renewal properties that allow them to divide without differentiating. Stem cell plasticity becomes more restricted as development progresses. A totipotent stem cell state arises after fertilization once embryo cells can generate exact copies of themselves. Totipotent cells maintain the competency for specification into both embryonic (organism) and extraembryonic (placenta and yolk sack) lineages. Once the mammalian blastocyst is formed, the embryonic lineage is maintained exclusively in epiblast (EPI) cells. Both pre- and postimplantation EPI cells are considered pluripotent stem cells, which lack the capacity for generating extraembryonic tissues but maintain full competency to develop into all embryonic germ lineages. During embryogenesis EPI cells transition through several definable pluripotent states, several of which can be maintained in vitro. This thesis focuses on utilizing better methods for evaluating how well in vitro stem cell culture systems recapitulate endogenous developmental cell types.

In Paper I we assessed pre- and postimplantation mouse embryonic stem cells and compared their allelic and transcriptional profiles with developing in vivo cell types. We were able to make unprecedented observations of X chromosome inactivation (XCI) dynamics, elucidating evidence that in vitro mouse XCI does not follow the perceived dogma that preimplantation stem cells express two fully active X chromosomes. By assessing the full length of each X chromosome with allelic resolution we found that XCI is initiated heterogeneously in preimplantation female stem cells with an observable elongated transition between stem cell states. In Paper II we screen two states of human pluripotent stem cells and preimplantation human embryos to define cell surface markers that attempts to effectively separate preimplantation from postimplantation epiblast. The markers provide a sorting method for state conversions. Paper III and IV complement one another in their intent to define the limits of mouse totipotency using transcriptomics and implementation of functional aggregation assays that effectively evaluate lineage specification and commitment. We determined when the first lineage segregation is defined and used an assortment of molecular tools to evaluate embryonic and extraembryonic contribution. This establishes a benchmark for defining totipotency.

Together the findings presented in this thesis add significant contribution toward an improved understanding of mammalian embryonic development and stem cell biology.

List of scientific papers

I. Chen G, SCHELL JP**, Benitez JA, Petropoulos S,Yilmaz M, Reinius B, Alekseenko Z, Shi L, Hedlund E, Lanner F, Sandberg R, Deng Q. (2016). Single-cell analyses of X Chromosome inactivation dynamics and pluripotency during differentiation. Genome Research. 26(10):1342-1354. *First author, **Second author.
https://doi.org/10.1101/gr.201954.115

II. Collier AJ*, Panula SP*, SCHELL JP**, Chovanec P, Plaza Reyes A, Petropoulos S, Corcoran AE, Walker R, Douagi I, Lanner F, Rugg-Gunn PJ. (2017). Comprehensive Cell Surface Protein Profiling Identifies Specific Markers of Human Naive and Primed Pluripotent States. Cell Stem Cell. 20(6):874-890.e7. *First author, **Second author.
https://doi.org/10.1016/j.stem.2017.02.014

III. Posfai E, Petropoulos S, de Barros F, SCHELL JP, Jurisica I, Sandberg R, Lanner F, Rossant J. (2017). Position- and Hippo signalling-dependent plasticity during lineage segregation in the early mouse embryo. Elife. 6:e22906.
https://doi.org/10.7554/eLife.22906

IV. Posfai E*, SCHELL JP*, Adrian Janiszewski*,Isidora R, Murray A, Bradshaw B, Pardon T, Bakkali M, Talon I, Geest N, Kumar P, To S, Petropoulos S, Jurisicova A, Pasque V, Lanner F, Rossant J. (2020). Defining Totipotency Using Criteria of Increasing Stringency. bioRxiv. *First author. [Manuscript]
https://doi.org/10.1101/2020.03.02.972893