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Published (Version of record)CC BY-NC-ND V4.0, Open Access
Published (Version of record)CC BY-NC-ND V4.0, Open Access
Journal article
Principles of signaling pathway modulation for enhancing human naive pluripotency induction
Cell stem cell, Vol.28(9), pp.1549-1565.e12
02/Sep/2021
PMID: 33915080
Abstract
Isolating human MEK/ERK signaling-independent pluripotent stem cells (PSCs) with naive pluripotency characteristics while maintaining differentiation competence and (epi)genetic integrity remains challenging. Here, we engineer reporter systems that allow the screening for defined conditions that induce molecular and functional features of human naive pluripotency. Synergistic inhibition of WNT/β-CATENIN, protein kinase C (PKC), and SRC signaling consolidates the induction of teratoma-competent naive human PSCs, with the capacity to differentiate into trophoblast stem cells (TSCs) and extraembryonic naive endodermal (nEND) cells in vitro. Divergent signaling and transcriptional requirements for boosting naive pluripotency were found between mouse and human. P53 depletion in naive hPSCs increased their contribution to mouse-human cross-species chimeric embryos upon priming and differentiation. Finally, MEK/ERK inhibition can be substituted with the inhibition of NOTCH/RBPj, which induces alternative naive-like hPSCs with a diminished risk for deleterious global DNA hypomethylation. Our findings set a framework for defining the signaling foundations of human naive pluripotency.
Details
- Title
- Principles of signaling pathway modulation for enhancing human naive pluripotency induction
- Creators
- Jonathan Bayerl (Corresponding Author) - 972WIS_INST___111Muneef Ayyash - 972WIS_INST___111Tom Shani - 972WIS_INST___111Yair Shlomo Manor - 972WIS_INST___111Ohad Gafni - 972WIS_INST___111Rada Massarwa - 972WIS_INST___111Yael Kalma - Tel Aviv Sourasky Medical CenterAlejandro Aguilera-Castrejon - 972WIS_INST___111Mirie Zerbib - 972WIS_INST___111Hadar Amir - Tel Aviv UniversityDaoud Sheban - 972WIS_INST___111Shay Geula - 972WIS_INST___111Nofar Mor - 972WIS_INST___111Leehee Weinberger - 972WIS_INST___111Segev Naveh Tassa - 972WIS_INST___111Vladislav Krupalnik - 972WIS_INST___111Bernardo Oldak - 972WIS_INST___111Nir Livnat - 972WIS_INST___111Shadi Tarazi - 972WIS_INST___111Emilie Wildschutz - 972WIS_INST___111Shadi Tawil - 972WIS_INST___111Shahd Ashouokhi - 972WIS_INST___111Lior Lasman - 972WIS_INST___111Varda Rotter - 972WIS_INST___122Suhair Hanna - Rambam Health Care CampusDalit Ben-Yosef (Corresponding Author) - Tel Aviv Sourasky Medical CenterNoa Novershtern - 972WIS_INST___111Sergey Viukov - 972WIS_INST___111Jacob H Hanna (Corresponding Author) - 972WIS_INST___111
- Resource Type
- Journal article
- Publication Details
- Cell stem cell, Vol.28(9), pp.1549-1565.e12; 02/Sep/2021
- Number of pages
- 30
- Publisher
- Elsevier Inc
- Language
- English
- DOI
- https://doi.org/10.1016/j.stem.2021.04.001
- PMID
- 33915080
- Grant note
- This work was funded by Pascal and Ilana Mantoux, Nella and Leon Benoziyo Center for Neurological Diseases, David and Fela Shapell Family Center for Genetic Disorders, Kekst Family Institute for Medical Genetics, Helen and Martin Kimmel Institute for Stem Cell Research, Flight Attendant Medical Research Institute (FAMRI), European Research Council (ERC-CoG-2016 726497-CellNaivety), Israel Science Foundation (ISF), Minerva, Israel Cancer Research Fund (ICRF), and BSF. We thank D. Trono and J. Pontis for TE analysis, N. Benvenisty and S. Bar for RNA-seq-based karyotyping and SNP analysis, R. Jaenisch for cell lines, A. Meissner for DNMT1 TET-OFF reporter cell line and constructs, A. Smith for H9-NK2 reset cell line, and Q. Ying for mouse βCAT mutants. Author contributions : J.B. and M.A. conducted screens and optimizations for HENSM and conducted molecular and differentiation characterizations of naive cells. T.S. conducted RNA-seq, ChIP-seq, and ATAC-seq library preparation and bioinformatic analysis; generated RBPJ KO lines; and established 0HENSM conditions. J.B. conducted all chimaera section and microscopy analysis. M.A. conducted WNT pathway signaling and mutant work. Y.S.M. generated KLF and TFAP2C KO lines and analysis. R.M. supervised microscopy analysis of cross-species chimeras. O.G. and J.B. generated p53 mutant hiPSC lines. J.B. conducted micro-injections with help from M.Z., S. Tawil, and O.G. Y.K., H.A., J.H.H., and D.B.-Y. derived lines from human blastocysts. L.W. assisted in HENSM optimizations and PGC differentiation. A.A.-C. assisted in microscopy imaging. S.G. generated METTL3 mutant lines and mouse Klf17 knockout. S.N.T. generated human extra-embryonic endoderm cells. N.N. supervised all bioinformatic analysis and conducted WGBS analysis. S.V. generated human TSC lines and all other mutant and reporter human cell lines. V.R. advised on P53 mutants. S.H. conducted teratoma analysis. L.L., E.W., S. Tarazi, N.L., B.O., N.M., D.S., S.A., and V.K. assisted in immunostaining, RT-PCR, karyotyping, and western blot throughout this study. J.H.H conceived the idea for this project and participated in HENSM condition optimizations. J.H.H., M.A., J.B., and N.N wrote the manuscript with input from all other authors.
- Record Identifier
- 993280841203596
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