By: Thi Xuan Ai Pham1, Amitesh Panda1, Harunobu Kagawa2, San Kit To1, Cankat Ertekin1, Grigorios Georgolopoulos1, Sam S F A van Knippenberg1, Ryan Nicolaas Allsop1, Alexandre Bruneau3, Jonathan Sai-Hong Chui1, Lotte Vanheer1, Adrian Janiszewski1, Joel Chappell1, Michael Oberhuemer1, Raissa Songwa Tchinda1, Irene Talon1, Sherif Khodeer1, Janet Rossant4, Frederic Lluis1, Laurent David5, Nicolas Rivron2, Bradley Philip Balaton6, Vincent Pasque
A study just published in Cell Stem Cell by the Laboratory of Cellular Reprogramming and Epigenetic Regulation (Prof. Vincent Pasque) at KU Leuven discovered a method to generate human extraembryonic mesoderm (EXM) cells in vitro from naive pluripotent stem cells. The VSC was a key tool to the discovery and analysis of this exciting new cell type.
The EXM is an extra-embryonic organ that connects the human postimplantation embryo to the cytotrophoblast and gives rise to the first blood and primitive umbilical cord. The timing of extraembryonic mesoderm formation differs between human and mouse, and there could be other major differences too. Coupled with the inaccessibility of this post-implantation cell type, a human model would be ideal. The origins of EXM in humans has been discussed for almost a century, but no clear consensus has been reached.
The Pasque lab noticed an unknown, unexpected cell type generated in their trophoblast generation protocol, and with single cell RNA sequencing and comparison to many human embryo datasets, were able to identify these as belonging to the EXM. The VSC Genius Tier-2 cluster enabled these computationally challenging integrations with thousands of cells across 6 datasets.
The VSC was a key tool to the discovery and analysis of this exciting new cell type.
Single-cell ATAC-seq to examine chromatin accessibility and SCENIC analysis to predict active transcription factors identified multiple transcription factors which may be involved in EXM identity. These transcription factors are promising targets for future studies to explore the mechanisms of human embryogenesis
Additionally, the Pasque lab performed an scRNA-seq time course of 12,978 cells across 8 time points, and identified an epiblast intermediate which we suggest is the progenitor of in vitro EXM cells. Single cell RNA sequencing of multiple time points of a separate differentiation protocol for primitive endoderm, also identified these intermediate epiblast cells and EXM. Comparison with the early blastocyst model known as blastoids revealed the presence of precocious EXM as the majority of off-target cells. Understanding EXM regulation and origin may help to improve these models in the future.
Figure. Characterization of EXMCs (Marker genes expression heatmap).
Overall this project, enabled by the VSC, was able to identify an understudied human cell type present in a trophoblast differentiation protocol and provided deep characterization of the transcription, regulation, and origin of extraembryonic mesoderm. This will enable future research into this important cell type, which could help us understand the mechanisms of human embryogenesis and fix problems in human development and fertility such as miscarriages and developmental defects
Acknowledgements (by the author)
We thank Dr. Rudolf Jaenisch and Dr. Thorold W. Theunissen for the WIBR2-29M-GP26-TN9 hESC line and Dr. Catherine Verfaillie for the Sigma hiPSC line. We are grateful to the Vlaams Supercomputer Center Leuven (https://www.vscentrum.be) for computing, in particular Jan Ooghe; the VIB/KU Leuven Center for Brain and Disease Research, in particular to Kristofer Davie, Stein Aerts and the Aerts lab for help with bioinformatics. We also thank the Genomics Core Leuven (http://genomicscore.be) for high-throughput sequencing, Jean Christophe Marine’s lab for the use of Tapestation and 10X Chromium Controller, and in particular Greet Bervoets for helpful feedback. We are also grateful to Susan Schlenner and the KU Leuven FACS core team for providing the facility and especially Reena Chinaraj who helped us during flow cytometry experiments. Research in the Pasque laboratory is supported by The Research Foundation–Flanders (FWO; Odysseus Return Grant G0F7716N to V.P.; FWO grants G0C9320N and G0B4420N to V.P.), the KU Leuven Research Fund (C1 grant C14/21/19 to V.P.) and FWO PhD fellowships to A.J. (1158318N), I.T. (1S72719N), S.K.T. (1S75720N), L.V. (1S29419N), R.N.A. (11L0722N), and T.X.A.P. (11N3122N). This project has also received funding from the ANR “BOOSTIVF”. Laurent David thanks the iPSCDTC and MicroPICell core facilities (endorsed by IBiSA and Biogenouest). The Lluis lab is supported by FWO grant G073622N and KU Leuven Research Fund C14/21/115 to F.L. and FWO PhD fellowships 1S65321N to J.C. Research in the Rivron lab is supported by the European Research Council (ERC) under the European Union’s Horizon 2020 research and innovation programme (ERC-Co grant agreement No.101002317 'BLASTOID: a discovery platform for early human embryogenesis’).
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