Abstract
The maintenance of human embryonic stem cell (hESC) self-renewal and pluripotency is governed by distinct signaling pathways, yet endogenous pluripotency-supporting pathways remain understudied despite extensive exogenous signaling research. Here, we identify a previously unrecognized role of endogenous VEGF signaling in sustaining primed hESC pluripotency. VEGF signaling is robustly activated in primed hESCs, quiescent in naïve cells, and inactivated upon differentiation. Strikingly, targeted VEGFR inhibition (pharmacological, soluble decoy receptors [sFLT1/sKDR], or CRISPR-mediated VEGFR1/2 knockout) in primed hESCs disrupts self-renewal and induces trophoblast-like differentiation. Mechanistically, VEGFR inhibition activates the BMP pathway and down-regulates NANOG, which directly binds and represses select BMP components and trophoblast lineage-specific genes. Functionally, BMP inhibition partially and NANOG overexpression substantially rescue the phenotype induced by VEGF signaling ablation. Collectively, our work uncovers a pivotal VEGF-dependent network maintaining primed pluripotency, providing valuable insights into integrated pluripotency and lineage regulation by signaling cascades and transcription factors.
Similar content being viewed by others
Data availability
RNA sequencing data were deposited in the Genome Sequence Archive in the National Genomics Data Center, Beijing Institute of Genomics (China National Center for Bioinformation) of the Chinese Academy of Sciences under accession code HRA010308. Source data are provided with this paper.
References
Thomson, J. A. et al. Embryonic stem cell lines derived from human blastocysts. Science 282, 1145–1147 (1998).
Martin, G. R. Isolation of a pluripotent cell line from early mouse embryos cultured in medium conditioned by teratocarcinoma stem cells. Proc. Natl. Acad. Sci. USA 78, 7634–7638 (1981).
Gilbert, J. M. et al. A controlled prospective trial of adjuvant razoxane in resectable colorectal cancer. Recent Results Cancer Res. 79, 48–58 (1981).
Nichols, J. & Smith, A. Naive and primed pluripotent states. Cell Stem Cell 4, 487–492 (2009).
Brons, I. G. M. et al. Derivation of pluripotent epiblast stem cells from mammalian embryos. Nature 448, 191–195 (2007).
Rossant, J. Stem cells and early lineage development. Cell 132, 527–531 (2008).
Boyer, L. A. et al. Core transcriptional regulatory circuitry in human embryonic stem cells. Cell 122, 947–956 (2005).
Whyte, W. A. et al. Master transcription factors and mediator establish super-enhancers at key cell identity genes. Cell 153, 307–319 (2013).
Suzuki, A. et al. Nanog binds to Smad1 and blocks bone morphogenetic protein-induced differentiation of embryonic stem cells. Proc. Natl. Acad. Sci. USA 103, 10294–10299 (2006).
Hyslop, L. et al. Downregulation of NANOG induces differentiation of human embryonic stem cells to extraembryonic lineages. Stem Cells 23, 1035–1043 (2005).
Vallier, L. et al. Activin/Nodal signalling maintains pluripotency by controlling Nanog expression. Development 136, 1339–1349 (2009).
Wang, Z., Oron, E., Nelson, B., Razis, S. & Ivanova, N. Distinct lineage specification roles for NANOG, OCT4, and SOX2 in human embryonic stem cells. Cell Stem Cell 10, 440–454 (2012).
Zaehres, H. et al. High-efficiency RNA interference in human embryonic stem cells. Stem Cells 23, 299–305 (2005).
Fong, H. L., Hohenstein, K. A. & Donovan, P. J. Regulation of self-renewal and pluripotency by Sox2 in human embryonic stem cells. Stem Cells 26, 1931–1938 (2008).
Bendall, S. C. et al. IGF and FGF cooperatively establish the regulatory stem cell niche of pluripotent human cells in vitro. Nature 448, 1015–1021 (2007).
Beattie, G. M. et al. Activin A maintains pluripotency of human embryonic stem cells in the absence of feeder layers. Stem Cells 23, 489–495 (2005).
Greber, B., Lehrach, H. & Adjaye, J. Fibroblast growth factor 2 modulates transforming growth factor β signaling in mouse embryonic fibroblasts and human ESCs (hESCs) to support hESC self-renewal. Stem Cells 25, 455–464 (2007).
Levenstein, M. E. et al. Basic fibroblast growth factor support of human embryonic stem cell self-renewal. Stem Cells 24, 568–574 (2006).
Xu, R. H. et al. NANOG is a direct target of TGFβ/Activin-mediated SMAD signaling in human ESCs. Cell Stem Cell 3, 196–206 (2008).
Jiang, J. M. & Ng, H. H. TGFβ and SMADs talk to NANOG in human embryonic stem cells. Cell Stem Cell 3, 127–128 (2008).
Kang, H. B. et al. Basic fibroblast growth factor activates ERK and induces c-Fos in human embryonic stem cell line MizhES1. Stem Cells Dev. 14, 395–401 (2005).
Ho, L. et al. ELABELA is an endogenous growth factor that sustains hESC self-renewal via the PI3K/AKT pathway. Cell Stem Cell 17, 435–447 (2015).
Apte, R. S., Chen, D. S. & Ferrara, N. VEGF in signaling and disease: beyond discovery and development. Cell 176, 1248–1264 (2019).
Patel, S. A. et al. Molecular mechanisms and future implications of VEGF/VEGFR in cancer therapy. Clin. Cancer Res. 29, 30–39 (2023).
Xin, C., Zhu, C., Jin, Y. & Li, H. Discovering the role of VEGF signaling pathway in mesendodermal induction of human embryonic stem cells. Biochem Biophys. Res. Commun. 553, 58–64 (2021).
Randolph, L. N., Bao, X., Oddo, M. & Lian, X. L. Sex-dependent VEGF expression underlies variations in human pluripotent stem cell to endothelial progenitor differentiation. Sci. Rep. 9, 16696 (2019).
Zhao, H., Li, M., Ouyang, Q., Lin, G. & Hu, L. VEGF promotes endothelial cell differentiation from human embryonic stem cells mainly through PKC-ɛ/η pathway. Stem Cells Dev. 29, 90–99 (2020).
Chen, G. et al. Blocking autocrine VEGF signaling by sunitinib, an anti-cancer drug, promotes embryonic stem cell self-renewal and somatic cell reprogramming. Cell Res. 24, 1121–1136 (2014).
Zhu, Z. X. et al. PHB associates with the HIRA complex to control an epigenetic-metabolic circuit in human ESCs. Cell Stem Cell 20, 274–289.e7 (2017).
Theunissen, T. W. et al. Systematic identification of culture conditions for induction and maintenance of naive human pluripotency. Cell Stem Cell 15, 471–487 (2014).
Fischer, L. A., Khan, S. A. & Theunissen, T. W. Induction of Human Naïve pluripotency using 5i/L/A medium. Methods Mol. Biol. 2416, 13–28 (2022).
Liu, Z.-L., Chen, H.-H., Zheng, L.-L., Sun, L.-P. & Shi, L. Angiogenic signaling pathways and anti-angiogenic therapy for cancer. Signal Transduct. Target Ther. 8, 198 (2023).
Mandegar, M. A. et al. CRISPR interference efficiently induces specific and reversible gene silencing in human iPSCs. Cell Stem Cell 18, 541–553 (2016).
Amita, M. et al. Complete and unidirectional conversion of human embryonic stem cells to trophoblast by BMP4. Proc. Natl. Acad. Sci. USA 110, E1212–E1221 (2013).
Shao, M. et al. NANOG governs cell metabolism and redox homeostasis in human naïve embryonic stem cells. EMBO Rep. 26, 6292 (2025).
Ernst, J. & Kellis, M. ChromHMM: automating chromatin-state discovery and characterization. Nat. Methods 9, 215–216 (2012).
Ernst, J. & Kellis, M. Chromatin-state discovery and genome annotation with ChromHMM. Nat. Protoc. 12, 2478–2492 (2017).
Lee, C. Q. E. et al. What is trophoblast? A combination of criteria define human first-trimester trophoblast. Stem Cell Rep. 6, 257–272 (2016).
Okae, H. et al. Derivation of Human Trophoblast Stem Cells. Cell Stem Cell 22, 50–63 (2018).
Seetharam, A. S. et al. The product of BMP-directed differentiation protocols for human primed pluripotent stem cells is placental trophoblast and not amnion. Stem Cell Rep. 17, 1289–1302 (2022).
Xiang, L. et al. A developmental landscape of 3D-cultured human pre-gastrulation embryos. Nature 577, 537–542 (2020).
Bergmann, S. et al. Spatial profiling of early primate gastrulation in utero. Nature 609, 136–143 (2022).
Li, J. et al. MEK/ERK signaling contributes to the maintenance of human embryonic stem cell self-renewal. Differentiation 75, 299–307 (2007).
D’Amour, K. A. et al. Efficient differentiation of human embryonic stem cells to definitive endoderm. Nat. Biotechnol. 23, 1534–1541 (2005).
Xu, R. H. et al. BMP4 initiates human embryonic stem cell differentiation to trophoblast. Nat. Biotechnol. 20, 1261–1264 (2002).
Bernardo, A. S. et al. BRACHYURY and CDX2 mediate BMP-induced differentiation of human and mouse pluripotent stem cells into embryonic and extraembryonic lineages. Cell Stem Cell 9, 144–155 (2011).
Yu, P. Z., Pan, G. J., Yu, J. Y. & Thomson, J. A. FGF2 sustains NANOG and switches the outcome of BMP4-induced human embryonic stem cell differentiation. Cell Stem Cell 8, 326–334 (2011).
Horii, M. et al. Human pluripotent stem cells as a model of trophoblast differentiation in both normal development and disease. Proc. Natl. Acad. Sci. USA 113, E3882–E3891 (2016).
Roberts, R. M., Ezashi, T., Sheridan, M. A. & Yang, Y. Specification of trophoblast from embryonic stem cells exposed to BMP4. Biol. Reprod. 99, 212–224 (2018).
Io, S. et al. Capturing human trophoblast development with naive pluripotent stem cells in vitro. Cell Stem Cell 28, 1023–1039.e1013 (2021).
Guo, G. et al. Human naive epiblast cells possess unrestricted lineage potential. Cell Stem Cell 28, 1040–1056.e6 (2021).
Wei, Y. et al. Efficient derivation of human trophoblast stem cells from primed pluripotent stem cells. Sci. Adv. 7, eabf4416 (2021).
Soncin, F. et al. Derivation of functional trophoblast stem cells from primed human pluripotent stem cells. Stem Cell Rep. 17, 1303–1317 (2022).
Jang, Y. J., Kim, M., Lee, B. K. & Kim, J. Induction of human trophoblast stem-like cells from primed pluripotent stem cells. Proc. Natl. Acad. Sci. USA 119, e2115709119 (2022).
Viukov, S. et al. Human primed and naïve PSCs are both able to differentiate into trophoblast stem cells. Stem Cell Rep. 17, 2484–2500 (2022).
Hammer, A. et al. Amnion epithelial cells, in contrast to trophoblast cells, express all classical HLA class I molecules together with HLA-G. Am. J. Reprod. Immunol. 37, 161–171 (1997).
Fang, Z. Q. et al. SOX21 ensures rostral forebrain identity by suppression of WNT8B during neural regionalization of human embryonic stem cells. Stem Cell Rep. 13, 1038–1052 (2019).
Doench, J. G. et al. Optimized sgRNA design to maximize activity and minimize off-target effects of CRISPR-Cas9. Nat. Biotechnol. 34, 184–191 (2016).
Bertero, A. et al. Optimized inducible shRNA and CRISPR/Cas9 platforms for in vitro studies of human development using hPSCs. Development 143, 4405–4418 (2016).
Love, M. I., Huber, W. & Anders, S. Moderated estimation of fold change and dispersion for RNA-seq data with DESeq2. Genome Biol. 15, 550 (2014).
Zhou, Y. et al. Metascape provides a biologist-oriented resource for the analysis of systems-level datasets. Nat. Commun. 10, 1523 (2019).
Members, C.-N. & Partners Database resources of the National Genomics Data Center, China National Center for Bioinformation in 2024. Nucleic Acids Res. 52, D18–D32 (2024).
Chen, T. et al. The genome sequence archive family: toward explosive data growth and diverse data types. Genom. Proteom. Bioinform. 19, 578–583 (2021).
Acknowledgements
We are grateful to Dr. Su-Chun Zhang for generously providing the pAAVS1-TRE3G-EGFP plasmid. The working model was generated using BioRender. The study was supported by National Key R&D Program of China (2021YFA1100400 to Y.J., H.L., and L.L.), the Natural Science Foundation of Shanghai (19ZR1428300 to H.L.), and the Innovative Research Team of High-level Local Universities in Shanghai (SHSMU-ZLCX20210201 to Y.J.).
Author information
Authors and Affiliations
Contributions
H.L. and Y.J. conceived and designed the study and wrote the manuscript. X.W., C.W., and C.Z. performed the majority of the experiments and analyzed the results. H.L. assisted in conducting some key experiments and contributed to data analysis. H.J., Y.H., L.Y., M.S., and F.Z. conducted the bioinformatics analysis. C.X. and H.J. carried out the VEGFA165 iOE-related experiments. R.T. performed some western blot experiments. H.Z. and Q.W. helped to convert primed hESCs to naïve hESCs by the 5i/L/FA protocol. J.G. prepared radiation-inactivated MEFs. H.W. and B.L. established the NANOG iKD hESC line. Y.Z. helped with constructs. L.L. contributed to the design of the bioinformatics analysis. All authors reviewed and approved the final manuscript. Y.J. and H.L. supervised the study.
Corresponding authors
Ethics declarations
Competing interests
The authors declare no competing interests.
Peer review
Peer review information
Nature Communications thanks Jacob Hanna and the other anonymous reviewer(s) for their contribution to the peer review of this work. A peer review file is available.
Additional information
Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Source data
Rights and permissions
Open Access This article is licensed under a Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International License, which permits any non-commercial use, sharing, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if you modified the licensed material. You do not have permission under this licence to share adapted material derived from this article or parts of it. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by-nc-nd/4.0/.
About this article
Cite this article
Wu, X., Wen, C., Zhu, C. et al. Endogenous VEGF signaling acts as a guardian of human primed pluripotency. Nat Commun (2026). https://doi.org/10.1038/s41467-026-70526-9
Received:
Accepted:
Published:
DOI: https://doi.org/10.1038/s41467-026-70526-9
