Endogenous VEGF signaling acts as a guardian of human primed pluripotency

Domain: biology
Published: 2026-03-12
Editorial outcome: major_revision

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 actived in primed hESCs, quiescent in naïve cells, and inactivated upon differentiation. Strikingly, targeted VEGF 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, VEGF inhibition activates the BMP pathway and downregulates 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.

Introduction
During mammalian embryogenesis, transient pluripotent cell populations, such as those in the inner cell mass (ICM) and epiblast, arise within blastocysts. These cells give rise to all somatic and germ cells in mammals and serve as the foundation for embryonic stem cells (ESCs) in vitro1-3. Human ESCs possess the capacity of unlimited self-renewal and the potential of differentiation into all cell types of the body. These unique features make them a powerful tool for modeling early embryonic development and human diseases, also offering the great potential for disease treatment. However, challenges remain in generating hESCs with high quality and functional cells with high efficiency. A thorough understanding of the regulatory mechanisms governing hESC pluripotency and fate decision is thus essential. Currently, pluripotency has been categorized into two main states, naïve and primed. The pluripotent cells in the ICM and pre-implantation epiblasts (pre-EPI) are considered to be naïve, while the cells in the post-implantation epiblasts (post-EPI) are thought to be primed4. As to in vitro cultured pluripotent stem cells (PSCs), mouse ESCs (mESCs) exhibit features of naïve pluripotency, whereas routinely cultured human ESCs (hESCs) display characteristics of primed pluripotency, similar to mouse epiblast stem cells (mEpiSCs)4-6. Pluripotency and unlimited self-renewal, the two hallmarks of hESCs, are governed by both intrinsic factors and extrinsic signaling pathways. Transcriptional factors (OCT4, SOX2, and NANOG) form a core regulatory hub in hESCs, interacting with each other and co-occupying their regulatory regions to create autoregulatory loops that stabilize their expression7, 8. They also jointly regulate a network of other transcription factors and key genes crucial for maintaining hESC identity7, 8. Additionally, they control the expression of differentiation-associated genes, thereby modulating lineage commitment9. How ever, phenotypic outcomes of their absence in hESCs are not entirely consistent across studies, likely due to variations in the cell lines and culture conditions applied in different studies. For example, NANOG deficiency was reported to lead to neuroectodermal differentiation or differentiation towards trophectoderm or primitive endoderm10-14. Currently, our understanding of how NANOG safeguards hESC identity is far from complete.

Extrinsic signaling circuitries triggered by growth factors are also critical for hESC maintenance. A precise combination of growth factors, including bFGF, TGF-β/ACTIVIN/NODAL, and INSULIN/IGF, is essential for activating the core transcriptional circuitry and preventing differentiation in hESCs15-20. Among these factors, bFGF activates the MAPK/ERK1/2 pathway, enhancing the self-renewal-related gene expression21. Although excessive ERK1/2 activation can induce differentiation, bFGF also engages the PI3K pathway, which counteracts ERK overactivation and maintains hESC self-renewal. In contrast to the extensive research on exogenous factors, roles of endogenous molecules in hESC fate decisions remain poorly explored. It was reported that ELABELA, an endogenous peptide hormone, promotes hESC self-renewal by activating the PI3K/AKT pathway22. Further exploration of endogenous signaling pathways important for hESC fate decision is necessary to deepen our understanding of early human embryonic development and optimize the hESC culture system.

The vascular endothelial growth factor (VEGF) signaling pathway is well known for its vital roles in vasculogenesis, angiogenesis, and lymphangiogenesis23. However, its role in early human development during the peri-implantation period has not been documented. Among VEGF ligands (VEGFA, VEGFB, VEGFC, VEGFD, and placental growth factor, PlGF), VEGFA, originally known as VEGF, is the most studied. These VEGF ligands bind to transmembrane receptor tyrosine kinases (VEGFR1/FLT1, VEGFR2/KDR/FLK1, and VEGFR3/FLT4) with varying specificity and distinct functional roles24. VEGF signaling has been implicated in hESC differentiation processes, such as mesendodermal and mesodermal endothelium differentiation25-27. In mESCs, VEGF signaling is quiescent under normal conditions, but prolonged culture, even in LIF-supplemented medium, induces spontaneous VEGF secretion and differentiation towards meso-endoderm lineages28. However, the activity and function of VEGF signaling in hESCs remain unclear.

In this study, we screened a panel of small molecules to identify additional signaling pathways required for identity maintenance of primed hESCs and discovered that endogenous VEGF signaling was indispensable for maintaining primed pluripotency and blocking trophoblast differentiation in primed hESCs. Our results demonstrated that these functions were mediated through activating NANOG expression and concurrent suppressing the BMP signaling pathway. Our findings provide valuable insights into how cell fate decision is regulated through interplay between signaling pathways and transcriptional regulation in human PSCs.

Results
Unique activation of endogenous VEGF signaling in primed hESCs
Receptor tyrosine kinases (RTKs) mediate cellular responses to a wide range of signals. However, their roles in maintaining hESC self-renewal and pluripotency remain poorly characterized. To address this, we screened a panel of small-molecule inhibitors targeting RTKs including those for EGF (epidermal growth factor), VEGF, and PDGF (platelet-derived growth factor). We used the primed SHhES8 hESC line, which was derived and characterized in our lab29, throughout the study, unless otherwise indicated. Remarkably, hESCs treated with VEGFR inhibitor Axitinib (Axi) for 2 days exhibited the morphology of extensive and uniform differentiation, accompanied by a moderate level of cell death (Fig. 1a). In contrast, inhibition of PDGFR failed to induce significant morphological changes. Similarly, inhibition of EGFR, despite triggering marked cell death, did not elicit discernible differentiation phenotype. To verify the specificity of VEGFR inhibition, we treated hESCs with two additional pan-VEGFR inhibitors, Tivozanib (Tiv) and Lenvatinib (Len). Both of them induced a comparable differentiation phenotype, yet caused noticeably less cell death and yielded a more synchronous change in the morphology than Axi (Supplementary Fig. 1a). The efficacy of these inhibitors was determined by western blot analysis of levels of phospho-ERK1/2 (T202/Y204) and phospho-AKT (S473) (Supplementary Fig. 1b-c), common downstream effectors of RTKs. The results showed that all three VEGFR inhibitors markedly reduced the phosphorylation levels of ERK1/2 and AKT. In contrast, EGFR inhibition exerted a modest effect on the phosphorylation of these kinases, while PDGFR inhibition failed to decrease phosphorylation of ERK1/2 or AKT, likely due to low basal PDGF pathway activity in hESCs (Supplementary Fig. 1d). These findings indicate that VEGF signaling might play an important role in maintaining the undifferentiated state of primed hESCs.

Given that our culture medium lacked exogenous VEGF ligands, we next examined the expression and activity of VEGF signaling components in hESCs. Real-time quantitative PCR (RT-qPCR) analyses revealed higher levels of VEGFR1, VEGFR2, VEGFA, and VEGFB in undifferentiated hESCs (Fig. 1b). Similar expression patterns were observed in two additional primed hESC lines, H9 and H1 (Supplementary Fig. 1e, f). Notably, highly expressed VEGF signaling components were markedly down-regulated upon differentiation induced by the E6 medium, in a manner similar to the down-regulation of core pluripotency factors (Fig. 1b). Differentiation was indicated by the down-regulation of pluripotency factors (OCT4, NANOG) and up-regulation of lineage-specific markers (PAX6, T and GATA3) (Fig. 1b). Consistently, our immunofluorescence staining results further showed that VEGF ligands (VEGFA and VEGFB) and receptors (VEGFR1 and VEGFR2) were robustly expressed in primed hESCs, and that their levels decreased shortly after differentiation (Fig. 1c). Moreover, VEGFR1 and VEGFR2 were down-regulated upon differentiation induced by either retinoic acid (RA) treatment (Supplementary Fig. 1g-i) or embryoid body (EPB) formation (Supplementary Fig. 1j-l). These results indicate that primed hESCs produce high levels of VEGF signaling components, and their expression is tightly linked to hESC undifferentiated state.

Since primed and naïve hESCs rely on distinct signaling for pluripotency maintenance, we compared the expression pattern of VEGF signaling components between these two pluripotency states. We converted primed hESCs to the naïve state via the 5i/L/FA conversion protocol30 31(Fig. 1d). Intriguingly, high levels of VEGFR1 and VEGFR2 were uniquely detected in primed hESCs, but not in naïve hESCs, whereas hESCs at both states expressed abundant VEGFA and VEGFB ligands (Fig. 1e). It is known that VEGF signaling activation is initiated by ligand binding to receptors, which then dimerize and phosphorylate intracellular tyrosine residues24, 32. To assess VEGF signaling activity in cells at different states, we examined levels of VEGFR1/2 and their phosphorylation in naïve and primed hESCs as well as E6-induced differentiated cells for hESC lines SHhES8 and H9 cells. Our results showed that both VEGFR1 and VEGFR2 were activated in primed hESCs cultured in the mTeSR1 medium, whereas their activation was absent in naïve hESCs and reduced in differentiated cells (Fig. 1f, g). Collectively, these findings demonstrate that endogenous VEGF signaling is specifically activated in primed hESCs and silenced in naïve hESCs.

Essential roles of VEGF signaling in maintaining self-renewal and preventing trophoblast differentiation
To characterize the function of VEGF signaling in hESCs, we inhibited this pathway using two pan-VEGFR inhibitors, Tiv and Len. Compared with DMSO-treated controls, hESC colonies treated with either inhibitor exhibited loosened morphology within 24 hours (h), followed by flattening of cells at the colony periphery and significant cell death by 48 h. Strikingly, all cells differentiated with a uniform cobblestone-like morphology by day 3 (Fig. 2a). Our western blot analysis showed decreases in levels of phosphorylated VEGFR1 and VEGFR2 (pVEGFR1 and pVEGFR2), validating the efficient inactivation of VEGF signaling (Fig. 2b, c) by the inhibitor. Moreover, RT-qPCR analysis revealed significant down-regulation of pluripotency genes and robust up-regulation of trophoblast markers in inhibitor-treated cells, with no or limited changes in markers of the three germ layers (ectoderm, mesoderm, and endoderm) (Fig. 2d). Similar results were obtained in H1 hESCs, ruling out cell line-specific effects (Supplementary Fig. 2a, b) Moreover, our immunofluorescence staining results illustrated the loss of OCT4 as well as drastic induction of trophoblast markers GATA3 and KRT7 induced by VEGFR inhibition (Supplementary Fig. 2c). Thus, VEGF signaling inhibition disrupts self-renewal in primed hESCs and induces differentiation towards the trophoblast lineage.

To address potential off-target effects of small molecules, we generated a hESC line with doxycycline (DOX)-inducible overexpression (OE) of soluble VEGF receptors sFLT1 and sKDR. These soluble receptors lack intracellular domains and act as decoy receptors for VEGF ligands. Two clones (referred to as SS iOE #7 and #10 thereafter) were selected and used in subsequent experiments. Serving as a negative control, a control hESC line transfected with an empty vector (Flag iOE) was also generated. After 6 days of DOX treatment, robust induction of sFLT1 and sKDR was observed in cells of both selected clones, leading to a significant reduction in levels of pVEGFR1 and pVEGFR2 (Fig. 2e-g). Morphological changes indicative of differentiation were observed in DOX-treated SS iOE cells but not in control cells (Fig. 2h). Moreover, RT-qPCR results showed down-regulation of pluripotency markers and up-regulation of trophoblast markers in DOX-treated SS iOE #7 and SS iOE #10 cells compared with untreated counterparts and Flag iOE control cells (Fig. 2i). Furthermore, immunofluorescence staining detected the loss of OCT4 and induction of GATA3 in DOX-treated SS iOE cells (Supplementary Fig. 2d). Therefore, like small molecule inhibitors, enforced expression of soluble VEGF receptors drives primed hESCs to exit the self-renewal state and initiate differentiation towards the trophoblast lineage.

To further validate these findings, we established inducible VEGFR1 and VEGFR2 double knockout (dKO) hESCs using the CRISPR-Cas9 genome editing strategy with a modified protocol 33. The successful dKO was verified by RT-qPCR and western blot analyses (Supplementary Fig. 3a-c). As expected, dKO cells exhibited the differentiated morphology and induction of trophoblast markers (Supplementary Fig. 3d-f), consistent with phenotypes observed in cells treated with VEGFR inhibitors or cells with soluble VEGFR OE. Together, these results obtained from experiments with three loss of function approaches robustly demonstrate the essential role of the VEGF signaling in the maintenance of self-renewal and highlight its importance in repressing trophoblast lineage specification in primed hESCs.

Given that VEGF signaling inhibition robustly activated the trophoblast program in hESCs, we next sought to determine the functional consequences of activating VEGF signaling during trophoblast differentiation. To this end, we established a hESC line with DOX-inducible OE of VEGFA165 (referred to as VEGFA165 iOE thereafter)25, the predominant VEGF isoform. After DOX-induced VEGFA165 OE in hESCs was validated (Supplementary Fig. 3g), edited cells were cultured in the BAP medium, a well-established condition for inducing trophoblast-like cells from hESCs34, with or without DOX for 1 day (Supplementary Fig. 3h). As anticipated, trophoblast genes were efficiently induced by the BAP medium. However, the induction of trophoblast morphology and marker gene expression was evidently suppressed by VEGFA165 OE (Supplementary Fig. 3i, j). Our results indicate that enforced VEGFA165 expression represses trophoblast induction in primed hESCs.

VEGF signaling inhibits BMP pathway activity to block trophoblast induction
To elucidate how VEGF signaling sustained hESC self-renewal and repressed trophoblast lineage specification, genome-wide transcriptional profiles were analyzed by RNA-sequencing (RNA-seq) on hESCs treated with a VEGFR inhibitor or OE of soluble VEGF receptors at multiple time points : Len or Tiv for 3h, 6h, 12h and 18h; Len or Axi for D1, D2 and D3; SS iOE#10 for D0, D1, D2 and D 6. Among these samples, inhibitor-treated cells at day 3 and DOX-induced SS iOEI cells at day 6 contained the most abundant numbers of differentially expressed genes (DEGs, two-fold change and FDR-adjusted p-value < 0.05) (treatment vs control), with 4125 and 5484 DEGs, respectively. Given that VEGF signaling inhibition activated a trophoblast-like transcriptional program, we analyzed up-regulated DEGs in these samples using the Metascape (http://metascape.org), a web-based tool for enrichment analysis. Terms associated with stem cell differentiation and placenta development were enriched (Fig. 3a, b), in accordance with the phenotypic changes observed following VEGF signaling inactivation in primed hESCs. Notably, enriched terms included SMAD protein signal transduction and BMP signaling pathway known to be closely linked to trophoblast and placenta development. Gene Set Enrichment Analysis (GSEA) further validated the enrichment of the BMP pathway in these up-regulated DEGs (Fig. 3c, d). A heatmap illustrated the progressive induction of BMP pathway members following VEGF signaling inactivation (Supplementary Fig. 4a, b), consistent with the outcomes of our GSEA. To experimentally validate activated BMP signaling after VEGF signaling inactivation, we assessed the kinetics of BMP4 pathway activity by measuring the phosphorylation levels of its downstream effectors SMAD1/5/8 in cells treated with DMSO or inhibitors (Tiv and Len) and in SS iOE cells with or without DOX treatment. Our result showed drastic increases in the levels of phosphorylated SMAD1/5/8 at 4 h of the inhibitor treatment (Fig. 3e, f), and at day 6 of the DOX addition in SS iOE cells, although DOX did not alter levels of phosphorylated SMAD1/5/8 in control cells (Fig. 3g, h).

To determine whether elevated BMP pathway would play a pivotal role in trophoblast differentiation triggered by VEGF signaling inactivation, we employed two BMP type I receptor kinase inhibitors (LDN-214117 and DMH1). Consistently, VEGFR inhibitor treatment (Tiv and Len) or OE of soluble receptors down-regulated pluripotency genes and up-regulated trophoblast markers significantly (Fig. 3i, j), accompanied by cell morphological changes (Supplementary Fig. 4c, d). Notably, BMP inhibitors (LDN-214117 or DMH1) suppressed the up-regulation of trophoblast genes and morphological changes caused by VEGF signaling inactivation. However, BMP inhibitor treatment did not rescue the down-regulation of pluripotency genes (Fig. 3i, j). The inhibitory effects of LDN-214117 or DMH1 on SMAD1/5/8 phosphorylation were validated (Supplementary Fig. 4e-h). These results lead to our conclusion that endogenous VEGF signaling suppresses the BMP pathway to block trophoblast differentiation in primed hESCs.

VEGF signaling sustains NANOG expression and pluripotency
As mentioned above, inhibition of the BMP pathway efficiently blocked VEGF signaling inactivation-induced trophoblast differentiation in hESCs, but could not restore the expression of pluripotency factors. This observation suggested that VEGF signaling might modulate additional factors to preserve hESC identity. To identify such factors, we analyzed down-regulated DEGs in VEGFR inhibitor-treated cells at day 3 and in DOX-treated SS iOE cells at day 6. As expected, terms associated with negative regulation of cell differentiation were enriched in these DEGs (Fig. 4a, b), in line with the observed differentiation phenotype following VEGF signaling depletion in hESCs. Of note, terms related to OCT4, SOX2, NANOG and positive regulation of DNA-binding transcription factor activity were also enriched. Consistently, our heatmap illustrated a decline in the expression of pluripotency-associated genes, with NANOG exhibiting a particularly pronounced and rapid reduction in VEGFR inhibitor-treated cells (Supplementary Fig. 5a, b). Similar results were obtained in SS iOE cells after DOX treatment (Supplementary Fig. 5c). The down-regulation of NANOG and OCT4 in response to VEGF signaling inactivation was further validated at a protein level (Fig. 4c-f and Supplementary Fig. 5d-g).

Based on the observation that NANOG levels decreased most rapidly and dramatically among the down-regulated pluripotency factors, we investigated whether VEGF signaling pathway ensures hESC self-renewal and pluripotency by maintaining NANOG expression. To this end, we generated a DOX-inducible NANOG OE hESC line (referred to as NANOG iOE thereafter). The efficient induction of NANOG OE by DOX was verified (Fig. 4g-i). Functionally, NANOG OE substantially diminished the elevation of trophoblast-specific genes and SMAD1/5/8 phosphorylation levels, as well as morphological alterations caused by the VEGFR inhibitor (Fig. 4g-i and Supplementary Fig. 5h). The decrease in pluripotency gene expression was also partially corrected (Fig. 4g). As a negative control, DOX treatment did not provoke any of these responses in wild-type hESCs (Fig. 4h, i and Supplementary Fig. 5i), verifying that NANOG OE was responsible for the rescue effect in NANOG iOE cells. These results demonstrated the capacity of NANOG to counteract differentiation phenotypes induced by VEGF signaling inactivation. To further characterize the role of NANOG in VEGF signaling-mediated functions, we generated a DOX-inducible NANOG OE hESC line in SS iOE #10 cells (referred to as SSN iOE thereafter). In this cell line, the addition of DOX simultaneously induces OE of NANOG as well as two soluble VEGF receptors. Using both SS iOE and SSN iOE cell lines, we showed that NANOG OE largely rescued down-regulation of pluripotency genes, up-regulation of trophoblast genes and elevations in the level of SMAD1/5/8 phosphorylation, as well as morphological changes triggered by OE of soluble VEGF receptors (Fig. 4j-l and Supplementary Fig. 5j). Collectively, these results support the conclusion that NANOG functions as a key effector downstream of the VEGF signaling pathway to maintain hESCs in an undifferentiated and primed pluripotency state, although we did not find a direct link between VEGF signaling and NANOG transcription in hESCs.

NANOG directly regulates BMP signaling and trophoblast genes
To elucidate how NANOG functions downstream of the VEGF signaling pathway, we performed RNA-seq analyses on cells from the following 3 groups: NANOG iOE, SS iOE and SSN iOE, with indicated treatments (Fig. 5a, b). We analyzed DEGs that were altered by the VEGFR inhibitor or the soluble VEGFR OE and subsequently rescued by concurrent NANOG OE (with fold change > 2 or < 0.5 and FDR-adjusted p-value < 0.05). In NANOG iOE cells treated with the VEGFR inhibitor, we identified 202 such DEGs, including 29 down-regulated genes rescued by NANOG OE (referred to as NANOG_iOE_down_rescued) and 173 up-regulated genes rescued by NANOG OE (referred to as NANOG_iOE_Iup_rescued). Moreover, in SS iOE and SSN iOE cells, we detected 863 DEGs, comprising of 499 down-regulated genes rescued by NANOG OE (referred to as SSN_iOE_down_rescued) and 364 up-regulated genes rescued by NANOG OE (referred to as SSN_iOE_up_rescued) (Supplementary Fig. 6a). Our hierarchical clustering analysis revealed that NANOG OE rescued a subset of pluripotency genes down-regulated by VEGF signaling inactivation (e.g., NODAL, DPPA4, PRDM14, LEFTY1, and OCT4) and trophoblast genes up-regulated by VEGF signaling inactivation (e.g., BMP2, BMP4, BMP5, BMP7, GATA2, GATA3, and KRT7) (Fig. 5a, b). Using Metascape, we conducted enrichment analysis for DEGs down-regulated by VEGF signaling inactivation and rescued by NANOG OE in both VEGF signaling inactivation strategies (inhibitor treatment and soluble receptor OE). Enriched terms included signaling pathways regulating pluripotency of stem cells, transcriptional regulation of pluripotency stem cells, and OCT4, SOX2, NANOG activated genes related to proliferation, in accordance with the central role of NANOG in hESC pluripotency maintenance (Fig. 5c, d). Notably, for DEGs up-regulated by VEGF signaling inactivation and rescued by NANOG OE terms related to the BMP signaling pathway, stem cell differentiation, and embryonic placenta development were enriched (Fig. 5e, f). These analyses supported the notion that NANOG could mediate the function of VEGF signaling by sustaining pluripotency gene expression as well as suppressing the BMP pathway and trophoblast differentiation. Further examination of DEGs of NANOG_iOE_up_rescued and SSN_iOE_up_rescued identified a set of BMP pathway genes (Supplementary Fig. 6b, c). Interestingly, some genes in this set were shown to be up-regulated in inducible NANOG knockout (KO) hESCs (RNA-seq data from our lab35), corroborating a suppressive effect of NANOG on BMP pathway transcripts (Supplementary Fig. 6d). These data strongly demonstrate that NANOG is a key player downstream of VEGF signaling for sustaining pluripotency gene expression as well as repressing the expression of certain BMP signaling components and trophoblast genes in primed hESCs.

To determine whether NANOG could directly bind to the genomic loci of BMP pathway members, we analyzed NANOG chromatin immunoprecipitation sequencing (ChIP-seq) data (data from our lab35). Chromatin-state annotations were generated using ChromHMM based on ENCODE data36, 37. We found that NANOG bound to the promoter or enhancer regions of certain BMP pathway genes (Fig. 5g) and key trophoblast markers, such as GATA3, CDX2, TP63, TFAP2A and TEAD1 (Fig. 5h). The deposition of NANOG on these regions was validated by our ChIP-qPCR analyses (Fig. 5i, j). These data indicate that NANOG safeguards human primed pluripotency through both activating of pluripotency-associated genes and suppressing differentiation-associated genes (e.g. BMP pathway components and trophoblast lineage genes). Nevertheless, the underlying mechanisms through which NANOG fulfills its dual regulatory function in gene expression remain incompletely understood.

In addition, we found that NANOG occupied an enhancer within the gene body of VEGFR1 and a weak enhancer located 7 kb upstream of VEGFR2 (Supplementary Fig. 6e), as verified by our ChIP-qPCR assays (Supplementary Fig. 6f). To test whether NANOG could control the expression of VEGF receptors, we examined their transcript levels in NANOG iKO (Supplementary Fig. 6g) and NANOG iKD (Supplementary Fig. 6h) hESCs. NANOG depletion significantly downregulated VEGFR2, while the VEGFR1 transcript level was not significantly changed, indicating distinct regulatory mechanisms for these two receptors. The reciprocal regulation between NANOG and VEGF signaling highlights mutually reinforcing positive feedback loops between transcription factors and signaling pathways that safeguard hESC self-renewal and pluripotency.

VEGF signaling inhibition activates a trophoblast-like transcriptional program
As the treatment of primed hESCs with VEGFR inhibitors (Tiv and Len) robustly activated trophoblast genes and induced uniform morphological changes (Fig. 6a), we further characterized the properties of these differentiated cells. Initially, we assessed the expression of HLA-ABC, a marker known to be present in hESCs but absent in trophoblast-like cells, mononuclear trophoblast marker GATA3, and trophectoderm (TE) cell surface marker TROP2 (also known as TACSTD2) in hESCs cultured in the self-renewal supporting mTeSR1 medium and treated with VEGFR inhibitors (Tiv or Len) for 3 or 5 days. Our flow cytometric analyses revealed that nearly all differentiated cells expressed GATA3 and TROP2, whereas the majority did not express HLA-ABC. Conversely, undifferentiated hESCs showed the opposite pattern (Fig. 6b, c). These findings suggested that the loss of VEGF signaling activity caused hESCs to exit the pluripotency state and undergo differentiation towards the extra-embryonic trophoblast lineage. We then examined the ELF5 promoter, which is known to be specifically hypomethylated in trophoblast cells38. We found that the hypermethylated state of the ELF5 promoter in untreated hESCs was switched to a hypomethylated state following the inhibitor treatment (Fig. 6d), indicating an epigenetic reprogramming accompanied with the cell fate change. These results indicate that the VEGFR inhibition induces nearly homogeneous trophoblast-like differentiation.

Human placenta comprises three main trophoblast subpopulations: cytotrophoblast (CT), extravillous trophoblast (EVT), and syncytiotrophoblast (ST). CT cells are in vivo counterparts of in vitro human trophoblast stem cells (hTSCs) and can differentiate into the other two differentiated cell types. Hence, we examined the expression of specific markers in Tiv or Len-treated cells and found that the inhibitor treatment robustly activated markers characteristic of CT cells, with mild effects on the expression of EVT and ST markers (Fig. 6e). At the protein level, immunofluorescence staining further illustrated the universal expression of CDX2, ELF5, and TP63, which are indicative of trophectoderm or CT cells, in inhibitor-treated cells (Fig. 6f). Therefore, these inhibitor-treated cells appear to acquire several features of the trophectoderm or CT. To test whether these cells would possess the capacity to differentiate towards EVT and ST trophoblast subtypes, we employed a previously published method39. When being cultured in the medium designed to induce EVT or ST differentiation, the inhibitor-treated cells expressed EVT-specific genes (ITGA1, and PECAM1) and ST-specific genes (hCGA, hCGB, and PSG9) (Fig. 6g). Nevertheless, these cells failed to propagate in self-renewal culture conditions, indicating a lack of intrinsic self-renewal capacity. Finally, given that trophoblast and amnion share certain genes, such as GATA3 and KRT7, we examined the expression of a set of genes, including ACMSD, AQP9, CLDN22, TMEM174, MCCD1, HBA1, and SOX11, which have been reported to be highly and specifically expressed in the amnion40. Based on our RNA-seq data, these genes were not upregulated in VEGF signaling deficiency-induced differentiated cells (Fig. 6h). These findings once again indicate that the endogenous VEGF signaling pathway safeguards the primed pluripotency of hESCs. The loss of VEGF signaling activity results in hESCs to exit from the primed pluripotency state and differentiate into trophoblast-like cells.

Methods
Ethics statement
All experiments were conducted in compliance with the ethical guidelines for the study of human embryonic stem cells issued jointly by the Chinese Ministry of Science and Technology and Ministry of Health in 2003 and adhered to relevant international regulations, including the 2021 ISSCR Guidelines for Stem Cell Research and Clinical Translation.

Reagents and resources
Reagents and resources are detailed in the Supplementary Information file as Supplementary Table 1.

Primed hESC culture and differentiation
The hESC line SHhES8 (Karyotype, 46, XX; derived in our lab)29 was used in all experiments unless otherwise indicated. Primed SHhES8, H1 (Karyotype, 46, XY; WA01, WiCell), and H9 (Karyotype, 46, XX; WA09, WiCell) hESCs were routinely cultured in hESC-qualified Matrigel pre-coated plates in the mTeSR1 medium, and dissociated with Dispase every 4 to 5 days, unless otherwise indicated.

For differentiation in the E6 or RA medium, hESCs were dissociated with Accutase and replated (2 x 105 cells/well) onto Matrigel-coated 6-well plates in the TeSR-E6 or mTeSR1 with 1 µM RA for 2 to 4 days. For spontaneous EB formation, hESCs were dissociated by Dispase for about 15 min at 37oC and transferred to low attachment 60 mm dishes supplemented with the TeSR-E6 for 2 to 4 days.

For trophoblastic induction, hESCs were dissociated with Accutase and replated in the mTeSR1. On the next day, cells were adapted to radiation-inactivated mouse embryonic fibroblast (MEF, homemade)-conditioned medium (CM) containing 4 ng/mL bFGF. Twenty-four hours later, the medium was changed to the BAP medium34 (E6 medium supplemented with 10 ng/mL BMP4, 1 μM ALK4/5/7 inhibitor A83-01 and 0.1 μM FGF2-signaling inhibitor PD173074) for 1 day. A summary of the experimental design is shown in Supplementary Fig. 3h. For EVT and ST differentiation induction, experiments were performed as previously described by Okae et al39.

Naïve hESC culture
Naïve SHhES8 and H9 hESCs were converted from primed SHhES8 and H9 hESCs by the 5i/L/FA medium30 , respectively, and cultured on MEF feeders. Naïve hESCs of both lines were cultured in the 5i/L/A medium, comprising a 1:1 mixture of DMEM/F12 and Neurobasal, 1% N2-supplement, 2% B27-Supplement, 2 mM L-GlutaMAX, 0.1 mM β-mercaptoethanol, 1 x MEM NEAA, 1 x penicillin-streptomycin, 1 μM PD0325901, 1 μM IM-12, 0.5 μM SB590885, 1 μIM WH-4-023, 10 μM Y-27632, 20 ng/mL human LIF and 20 ng/mL Activin A. Cells were passaged every 4 days in a 1:2 to 1:3 split ratio after single-cell dissociation with Accutase.

All hESCs and their derivatives were cultured in a humidified incubator at 37°C with 21% O2, 5% CO2. Routine mycoplasma contamination tests were performed to ensure the absence of mycoplasma contamination.

Constructs
All constructs were validated by DNA sequencing.

pAAVS1-TRE3G-NANOG
For inducible overexpression (OE) of NANOG from the AAVS1 locus, its coding sequence was amplified by PCR from SHhES8 hESC complementary DNA (cDNA) using specific primers containing required restriction enzyme recognition sites (NANOG SalI F: TAAAGTCGACATGAGTGTGGATCCAGCTTG and NANOG MluI R: ATGACGCGTTCACACGTCTTCAGGTTGCATG). The amplicon was subcloned into the pAAVS1-TRE3G-EGFP vector (a kind gift from Dr. Su-chun Zhang) to replace the original EGFP coding sequence, generating the plasmid pAAVS1-TRE3G-NANOG for derivation of NANOG iOE hESCs.

pROSA-Bsd-TRE3G-NANOG
For inducible NANOG OE from the ROSA26 locus, its coding sequence was amplified by PCR from SHhES8 hESC cDNA using specific primers (NANOG F2: ACGATGTTCCAGATTACGCTAGTGTGGATCCAGCTTGTCC and NANOG R2: GGCTAGCCATATGACGCGTCACACGTCTTCAGRTTGCA). The amplicon was subcloned into the pROSA-Bsd-TRE3G-3xFLAG-HA vector (homemade), generating the plasmid pROSA-Bsd-TRE3G-NANOG for derivation of SSN iOE hESCs.

pAAVS1-TRE3G-sFLT1-T2A-sKDR
For inducible OE of sFLT1 and sKDR, their coding sequences were respectively amplified by PCR from SHhES8 hESC cDNA using specific primers (TRE-sFLT1-F: CCTACCCTCGTAAAGTCGACATGGTCAGCTACTGGGACACCG; T2A-sFLT1-R: CACGTCACCGCATGTTAGAAGACTTCCCTCTGCCCTCTCCGCTGCCATGTTTTACATTACTTTGTGTGGTA; T2A-sKDR-F: AGAGGAAGTCTTCTAACATGCGGTGACGTGGAGGAGAATCCCGGCCCTATGCAGAGCAAGGTGCGTGCTGG; SV40-sKDR-R: AGGCTAGCCATATGACGCGTTCACTCTGAGTCTTCTACAAGG). The two fragments were cloned into the pAAVS1-TRE3G-EGFP plasmid to replace the EGFP coding sequence, generating the pAAVS1-TRE3G-sFLT1-T2A-sKDR plasmid for derivation of SS iOE hESCs.

Treatment with inhibitors of growth factor receptors
Human ESCs were dissociated with Accutase and replated (5 x 105 cells/well) in Matrigel-coated 6-well plates in the mTeSR1 supplemented with Y-27632 for 1 day. Subsequently, the medium was refreshed daily with the mTeSR1 supplemented with either 0.1% DMSO or individual inhibitors (2 µM Crenolanib, 5 µM Axitinib, 10 µM Erlotinib, 10 µM Tivozanib or 6 µM Lenvatinib) for the indicated durations.

Electroporation
Human ESCs were cultured in the mTeSR1 supplemented with Y-27632 (10 µM) for 2 hours prior to electroporation. Cells were dissociated into single cells using Accutase for 10 minutes at 37°C. A total of 3 × 106 cells were electroporated with appropriate combinations of plasmids in 100 µL of an electroporation solution (Mirus, MIR 50118) using the Lonza Nucleofector 2b device with program B-016 in 0.2 cm electroporation cuvettes.

Generation of the sFLT1/sKDR inducible overexpression (SS iOE) hESC line
SHhES8 hESCs were electroporated with a pair of single-guide RNA (sgRNA) plasmids (pX335-AAVS1-L and pX335-ACAVS1-R57) and a donor plasmid (pAAVS1-TRE3G-sFLT1-T2A-sKDR), and selected using Puromycin (1 µg/mL) for 2 days. Surviving colonies were individually picked, expanded. The transcript levels were analyzed by real-time quantitative PCR (RT-qPCR) using primers specific for exogenous sFLT1 and sKDR (Ex-sFLT1 and Ex-sKDR) after treatment with doxycycline (DOX) for 2 days. Two clones, designated as SS iOE #7 and #10, respectively, were selected and expanded for further study. A control hESC line was parallelly generated by transfecting with an empty donor plasmid, referred to as Flag iOE. Sequences of the sgRNAs are listed in Supplementary Information file as Supplementary Table 2.

Generation of the VEGFR1/2 inducible double knockout (VEGFR1/2 dKO) hESC line
SHhES8 hESCs were electroporated with a pair of sgRNA plasmids (pX335-AAVS1-L and pX335-AAVS1-R) and a donor plasmid (pAAVS1-PDi-CRISPRn-VEGFR1/2-8xsgRNA), and selected using Blasticidin (5 µg/mL) for 3 days. Surviving colonies were individually picked, expanded, and subjected to genomic DNA PCR analysis after doxycycline (0.5 µg/mL) treatment for 2 days. The primers used for PCR were as follows: FLT1-F (5’-GCTTCCCTTGGATCGGACTT-3’), FLT1-R (5’-GTCCTCTGGGAATGGGCTCT-3’), KDR-F (5’-CGCCGCAGAAAGTCCGTCT-3’), and KDR-R (5’-CTTTACGCAGGACAGTTGGCT-3’). Clones that exhibited inducible double knockout of VEGFR1/2 were selected and expanded as the dKO hESC line for further study. In this single clone line, DOX treatment could lead to the simultaneous deletion of both VEGFR1/2 as well as the soluble receptors sFLT1 and sKDR. A negative control hESC line was generated following the identical electroporation, selection, and colony-picking workflow, but with a non-targeting EGFP-sgRNA58, 59. The related experiments were performed in the MEF-CM containing 4 ng/mL bFGF. Sequences of the sgRNAs are listed in Supplementary Information file as Supplementary Table 2.

Generation of NANOG inducible overexpression (NANOG iOE) hESC line
SHhES8 hESCs were electroporated with a pair of sgRNA plasmids (pX335-AAVS1-L and pX335-AAVS1-R) and a donor plasmid pAAVS1-TRE3G-NANOG. The transfected cells were selected using Puromycin (1 µg/mL) for 2 days. The surviving cells were expanded and validated by analyzing NANOG mRNA and protein levels. Sequences of the sgRNAs are listed in Supplementary Information file as Supplementary Table 2.

Generation of the sFLT1/sKDR and NANOG simultaneously inducible overexpression (SSN iOE) hESC line
SS iOE #10 hESCs were electroporated with a pair of sgRNA plasmids (pX335-ROSA26-L and pX335-ROSA26-R) and a donor plasmid pROSA26-TRE3G-NANOG and selected with Blasticidin (5 µg/mL) for 3 days. The surviving cells were expanded and validated by assessing NANOG mRNA and protein levels. Sequences of the sgRNAs used are listed in Supplementary Information file as Supplementary Table 2.

RNA extraction, cDNA synthesis and RT-qPCR
Total RNA was extracted from cells using the TRIzol reagent (Life Technologies, #15596026) according to the manufacturer's instruction. A total of 1.5 µg of RNA was reverse-transcribed into cDNA using the FastQuant RT Kit (TIANGEN, KR106) following the protocol provided by the manufacturer. RT-qPCR was performed on the ABI ViiA7 Real-Time PCR system using SYBR Premix Ex Taq II (Takara, #RR820L). GAPDH was used as an internal control to normalize expression levels of interested genes. Sequences of the primers used for RT-qPCR are listed in Supplementary Information file as Supplementary Table 3.

Western blot analysis
Cells were rinsed once with phosphate-buffered saline (PBS) and lysed in the RIPA buffer (50 mM Tris-HCl pH7.5, 150 mM NaCl, 0.1% SDS, 1% Nonidet P-40) supplemented with a protease inhibitor cocktail (PIC) and a phosphatase inhibitor cocktail (PPIC). The protein concentration was determined using the Pierce BCA Protein Assay Kit according to the manufacturer's instructions. Proteins were separated by 8% SDS-PAGE and transferred to 0.45 µm nitrocellulose membranes (GE Healthcare, #10600002). Membranes were blocked with 5% BSA in TBST (Tris-HCl buffered saline containing 0.1% Tween- 20; pH 7.4) for 1 hour at room temperature and then incubated with primary antibodies overnight at 4°C. The membranes were incubated with horseradish peroxidase (HRP)-conjugated secondary antibodies for 1 hour at room temperature. Chemiluminescent signals were detected using the SuperSignal West Pico Chemiluminescent Substrate.

To quantify the relative protein levels, the intensity of protein bands was measured using the ImageJ software. Subsequently, the ratio of the intensity between the interested protein band and the loading control band was calculated.

Immunofluorescence staining
The cells seeded on Matrigel-coated coverslips (Fisherbrand, #12-545-82) were fixed with 4% paraformaldehyde for 20 min at room temperature, washed 3 times with PBS, permeabilized with 0.2 % Triton X-100 in PBS for 20 min, and incubated in the blocking buffer (3% BSA in PBS) for 1 hour at room temperature. The cells were then incubated with primary antibodies overnight at 4°C. On the next day, the cells were rinsed three times with PBS and incubated with secondary antibodies (FITC, 1:100; Cy3, 1:200) for 1 hour at room temperature. The nuclei were stained with DAPI (Sigma, #D9542). Images were captured using a Zeiss Cell Observer microscope.

RNA-seq and data analysis
Total RNA was extracted from cells using the TRIzol Reagent according to the manufacturer’s instructions. RNA purification, reverse transcription, library construction and sequencing were performed by Annoroad Gene Technology Co., Ltd. (Beijing, China) or Shanghai Majorbio Bio-pharm Biotechnology Co., Ltd. (Shanghai, China) following the manufacturer’s instructions (Illumina, San Diego, CA). The isolated RNA was enriched by Poly(A) tails and fragmented. A paired-end RNA-seq sequencing library was generated and sequenced using the HiSeq X Ten or NovaSeq Xplus sequencer with a read length of 2 × 150 bp. Levels of transcripts were calculated according to the fragments per kilobase of transcript per million mapped reads (FPKM) or the transcripts per million reads (TPM) method. Differentially expressed genes (DEGs) were identified using DESeq260 with the following criteria: |log FC|≧1 and false discovery rate (FDR)-adjusted p-value < 0.05. Pathway and process enrichment analysis for DEGs was conducted using the Metascape (https://metascape.org/)61. The ontology sources included GO Biological Processes, KEGG Pathway, and Reactome Gene Sets. Terms with a p-value < 0.01, a minimum count of 3, and an enrichment factor > 1.5 were collected and grouped into clusters based on their membership similarities. The raw sequence data reported in this paper have been deposited in the Genome Sequence Archive in National Genomics Data Center, China National Center for Bioinformation / Beijing Institute of Genomics, Chinese Academy of Sciences (GSA-Human: HRA010308) that are publicly accessible at https://ngdc.cncb.ac.cn.ac.cn/gsa-human62, 63.

ChIP-qPCR
Approximately 2 × 107 cells were cross-linked with 1% formaldehyde for 10 minutes and quenched with 0.125 M glycine for 5 minutes at room temperature. Cells were rinsed, scraped, and transferred into Eppendorf tubes containing cold PBS supplemented with PIC and PPIC, and then centrifuged to remove the supernatant. The cell pellet was suspended in 1 mL of 1% SDS FA cell lysis buffer (50 mM HEPES-KOH, pH 7.5, 150 mM NaCl, 1 mM EDTA, 1% Triton X-100, 0.1% sodium deoxycholate, and 1% SDS supplemented with PIC and PPIC). After rotating for 15 minutes at 4°C, the suspension was centrifuged at 15,000 × g for 30 minutes, and the supernatant was discarded. The pellet was resuspended in 1 mL of the 0.1% SDS FA cell lysis buffer. After rotating for 5 minutes at 4°C, the suspension was centrifuged at 15,000 × g for 30 minutes, and the supernatant was discarded. The pellet was resuspended in 1 mL of the 0.1% SDS FA cell lysis buffer and sonicated at 50% amplitude (30 seconds ON, 30 seconds OFF) for 2 minutes using a Sonics (UibraCELL) in ice water, followed by centrifugation at 15,000 × g for 10 minutes at 4°C. For the input sample, 50 μL of the supernatant was collected. For the immunoprecipitation, 900 μL of the supernatant was precleared and then incubated with 2.5 μg anti-NANOG antibody overnight at 4°C. On the next day, the reaction mixture was incubated with 100 µL Protein G Magnetic Beads (Millipore, LSKMAGG10) for 2 hours at 4°C, followed by gentle washing with the 0.1% SDS FA lysis buffer (3 times), high salt buffer (0.1% SDS FA cell lysis buffer with 350 mM NaCl), ChIP wash buffer (10 mM Tris-HCl, pH 8.0, 250 mM LiCl, 0.5% NP40, 0.5% sodium deoxycholate, 1 mM EDTA), and TE buffer (10 mM Tris-HCl, 1 mM EDTA, pH 8.0) sequentially. The chromatin was eluted from the beads with 200 µL of the ChIP elution buffer (50 mM Tris-HCl, 10 mM EDTA, 1% SDS, pH 7.5) for 30 minutes at 37°C. The input and eluted chromatin were decross-linked by adding 8 µL of 5 M NaCl and 1 µL RNase A (10 µg/µL) at 65°C for less than 16 hours, followed by adding 2 µL of 20 mg/mL protease K for 2 hours at 58°C. The resulting DNA was purified using a QIAquick PCR Purification Kit and quantified using a Quant-iT PicoGreen dsDNA Assay Kit. The purified DNA was used for the examination of site-specific enrichments of NANOG. For RT-qPCR, 0.5 ng of the input or enriched DNA was used. Results were presented as the relative fold enrichment to the input. All primers used in ChIP-qPCR assays are listed in Supplementary Information file as Supplementary Table 4.

Flow cytometric analysis
Single-cell suspensions of hESCs were prepared through Accutase digestion. For surface marker detection, cells were incubated with the following antibodies at room temperature for 1 hour: FITC-conjugated HLA-ABC, PE-conjugated TROP2. After incubation, cells were washed three times with PBS. For GATA3 staining, cells were first fixed with 4% paraformaldehyde (PFA) in PBS for 15 minutes at room temperature, then permeabilized with 1.0% Triton X-100 in PBS for 15-20 minutes. Following blocking, cells were incubated with primary antibodies for 1 hour at room temperature, washed three times with PBS, and then incubated with a FITC-conjugated donkey anti-mouse secondary antibody (1:200 dilution) in the dark for 30 minutes. Cells were washed three times with PBS after secondary antibody incubation. Data were analyzed using the FlowJo software (version 10, Tree Star Inc.). The gating strategy is shown in Supplementary Fig. 7.

Promoter methylation characterization
The prediction of CpG islands in the promoter regions of ELF5 was performed using the MethPrimer. Genomic DNA was processed according to the manufacturer's instructions of the EZ DNA Methylation-Gold™ Kit (ZYMO RESEARCH, #D5005). Following bisulfite treatment, unmethylated cytosines (C) in the genome were converted to uracils (U). Nested PCR amplification was then conducted using two pairs of primers specific to promoters of ELF5, sequentially. The amplified products were subsequently inserted into pGEM-T Easy vectors and sequenced to analyze the methylation status of these promoters. To quantify methylation levels of the promoter CpG islands, the sequenced files were uploaded to the Quantification tool for Methylation Analysis (QUMA:http://quma.cdb.riken.jp/). Primers used in the methylation PCR assay are listed in Supplementary Information file as Supplementary Table 5.

Gene set enrichment analysis (GSEA)
The GSEA was performed to identify significant pathway activation patterns using the PID_BMP_PATHWAY gene set from the Pathway Interaction Database. Two separate comparisons were conducted: 1) VEGFR inhibitor (VEGFRi)-treated hESCs versus DMSO-treated hESCs, and 2) SS iOE +DOX hESCs versus SS iOE -DOX hESCs. The analysis was carried out using the GSEA software (version 4.3.2) with default parameters, including 1,000 permutations for significance testing, weighted enrichment statistic calculation, and signal-to-noise ratio as the ranking metric. Gene set permutations were selected as the permutation type to maintain the correlation structure among genes. Statistical significance of enrichments was evaluated by one-sided gene set permutation test with 1,000 iterations to generate the null distribution of enrichment scores (ES). Significant enrichment was defined with an FDR cutoff of 25% as recommended for discovery analyses, with nominal p-value < 0.05 considered supportive of enrichment signals. The normalized enrichment score (NES) was calculated to standardize the ES across gene sets with different sizes and correlation structures.

Statistics and Reproducibility
All experiments were designed with independent biological replicates, and each sample in the RT-qPCR assays included technical replicates. Numbers of biological replicates relevant for individual experiments are stated in figure legends. The unpaired two-tailed Student’s t test was used for statistical tests of differences between two groups. For multiple comparison, The one-way ANOVA followed by Dunnett’s post-hoc test was applied to assess differences relative to the control group. Data are shown as mean ± SEM. p < 0.05 was considered statistically significant (* p < 0.05, ** p < 0.01, and *** p < 0.001). All exact p values are provided in Source Data.

No statistical method was used to predetermine sample size. No data were excluded from the analyses. Cells were randomly allocated into experimental groups. The investigators were not blinded to allocation during experiments and outcome assessment.

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 [https://ngdc.cncb.ac.cn/gsa-human/browse/HRA010308]. Source data are provided with this paper.

Peer review rounds
[{"round": 1, "reviewer_comments": {"reviewer_1": "The authors present an interesting article to investigate the role of VEGF signaling in primed and naive human PSCs. The\nfirst and major part of the paper focuses on primed PSCs and shows that VEGF receptors are expressed in primed but not\nnaive cells, and through small molecule manipulation and gene tools they prove nicely and solidly that VEGF signaling\nperturbation leads to primed psc differentiation towards TSC through compromising Nanog expression and BMP signaling\nthat promotes TSC.\nHowever the last part of the paper trying to argue that inhibition of VEGF signaling in naive human PSCs improves their\nmaintenance and also one can replace human naive PSC media like 5iLAF into media which is composed only of LIF\nVEGFRi and ROCK inhibitor. Unfortunately, i find this part of the work unsubstantiated, partially studied and filled with\ncontradictions and non solid data as i detail below. I think the paper is unpublishable if it keeps this part of the work which is\nunfortunate as the first part in primed PSCs is solid and would have been sufficient to include in this publication.\n1) first all 2i/L cells where first kept in 5iLAF and then moved to 2i/L so the question is can the authors make naive cells in\n2i?L conditions, which must be shown to make such a claim.\n2) characterization of naive pluripotency is partial and non satisfactory. the authors should rely on solid reporters for deltaPe-\noct4-gfp and double fluorescent x chromosome lines to accurately report x chromosome status. this is not done, and non\nconclusive facs surface marker analysis is shown and only for limited passages. this is not the gold standard proof tests that\nhave long been established in the field. to make such a major claim.\n3) global DNA hypomethylation analysis should be shown - nobody in human or mice was able to show global DNA\nhypomethylation without FGF/ERK pathway targeting, so the authors claim they merely can do this with ERK and LIF and\nROCKi.? This is a major controversial claim that better be solidly proved. Methylation of a single promoter ot two is non\nsufficient. Global WGBS is a must.\n4) the fact in figure 7 that in the control naive cells XIST promoter is still highly methylated casts serious doubt on the ability\nof the authors to grow naive cells. Human naive cells should have this region hypomethylated as shown in numerous\nstudies.\n5) RNA-seq and immunostaining should be shown for many naive pluripotency markers (TFCP2LF1, ARGFX, DPPA5,\nKLF4, KLF17, DPPA3 etc...) as well as facs for SUSD2+ and CD24- pure population. also cells analyzed after at least 20\npassages in both feeder and feeder free conditions should be analyzed\nthroughout\n6) the authors seem to use feeder free conditions, and permanent addition of ROCKi at the concentration they use has\nrepeatedly shown in the field that it leads to primed or naive stem cell differentiation in 5-10 passage. The authors should\nprove this is not the case by using exacting knock-in reporter alleles as indicated above. these reporters are available on\nAddgene, and the cell lines are available in the community from many groups.\n7) the authors should show that the VEGFR inhibitor used is not influencing Fgf/MEK/Erk pathway and that this is not\nbecause an off target effect of the small molecule. The authors should use a genetic mean to inactivate VEGFR signaling in\nnaive PSCs as they did for primed cells to make this conclusion.\n8) The above point and claim are heavily shadowed by the authors claim that VEGF receptors are not expressed in naive\nhuman PSCs - so how does inhibition of a receptor that is not expressed in a cell helps maintain keeping that cell and in\nvery minimal condition?. This is bizarre to say the least and should be experimentally addressed aggressively. This is a\nmajor major claim and contradiction.", "reviewer_2": "Review of the Article by Wu et al., “Critical Roles of Endogenous VEGF Signalling for Cell Fate Decision in Human\nEmbryonic Stem Cells”\nIn this article, Wu et al. investigate the role of VEGF signaling in maintaining the pluripotent state in both primed and naïve\nhuman embryonic stem cells (hESCs). The authors demonstrate that the VEGF pathway is active in primed hESCs but\nlargely inactive in naïve hESCs, suggesting a potential role for VEGF in sustaining the primed state.\nInterestingly, VEGF signaling is downregulated during the differentiation of primed cells. Consistent with this, VEGF\npathway inhibition promotes differentiation toward the trophoblast lineage. The authors show that VEGF inhibition leads to\nthe downregulation of pluripotency markers, such as NANOG, and concomitant upregulation of BMP signaling. Importantly,\nNANOG overexpression rescues the differentiation phenotype, supporting its role downstream of VEGF signaling.\nIn the final part of the study, the authors report that VEGF inhibition is sufficient to maintain naïve hESCs in their naïve state,\neven in the absence of ERK pathway inhibition. Notably, culturing naïve hESCs in media containing only VEGF inhibition,\nROCK inhibition, and LIF (referred to by the authors as “2iLIF”) is sufficient to maintain pluripotency.\nThis is a comprehensive and well-executed study that provides novel insights into the role of VEGF signaling in hESC\npluripotency and differentiation. The data are extensive and technically solid. However, the manuscript lacks a direct or\nindirect mechanistic link between VEGF inhibition and Nanog downregulation.\nFurthermore, some methodological aspects of the article need to be improved. Throughout the manuscript, western blots and\nRT-qPCR plots are often presented in separate figures and normalized per figure. Despite the fact that, overall, the effect\ndescribed by the authors is indeed clear, this presentation makes it impossible to evaluate whether there is already a\ndifference in basal levels between the controls and the untreated experimental conditions.\nEven though the effect is mild in comparison to the experimental conditions, the use of DOX seems to have an effect on\ntrophoblast genes. Therefore, it should be avoided where possible, such as in situations where media can be supplemented\nwith recombinant factors and iOE is not strictly necessary.\nThroughout the manuscript, unpaired two-tailed Student’s t tests are being used to compare multiple timepoints to timepoint\n0. However, a correction for multiple testing is needed for such comparisons, but this is never mentioned.\nMajor comments\n1) Figure 1A\nThe authors use PDGFR, VEGFR, and EGFR inhibitors and observe differentiation only with VEGFR inhibition. However,\nthe manuscript does not include controls demonstrating the efficacy of PDGFR and EGFR inhibition. The authors should\ninclude validation of the inhibitor activity, such as downstream pathway readouts or phospho-protein analysis.\nAdditionally, EGFRi does show a pretty drastic difference. From the results shown here, it is hard to judge whether it is a\nproliferation-related effect, but saying that there is no effect at all seems overstated.\n2) Figure 1G\nThe authors claim VEGF signaling is inactive in naïve hESCs based on phospho-VEGFR1 (Y1213) and VEGFR2 (Y951)\nlevels. However, activity is shown in isolation. A side-by-side comparison of VEGF signaling in naïve, primed, and\ndifferentiated hESCs via western blot is necessary to support this conclusion. Furthermore, this findings are only shown in\nH1 and H9 cells, but should also be demonstrated in the primarily used cell line.\n3) Extended figure 1a\nThe figures show the expression levels of the VEGF receptors and ligands and some pluripotency factors in H1 and H9\nhESCs in isolation. However, unlike in figure 1b & 1d, there is no comparison with the differentiated conditions, thus\nmaking the graph quite meaningless as such. It is true that the relative expression of some of the markers are similar to the levels\nshown in figure 1b & 1d, but this graph cannot be interpreted as it is now and comparisons to differentiation conditions\nshould be shown here as well to support the conclusion that is drawn from this experiment.\n4) The DOX-inducible system should be considered carefully in the context of trophoblast differentiation given the fact that,\napparently, DOX itself already has an (minor) effect on the expression of multiple trophoblast-associated genes (extended\nfigure 2c). The reason to use the inducible system for the experiments about the decoy-OE and dKO seems obvious given\nthe apparent role of VEGF signalling in primed pluripotency, but the OE of VEGFA can easily be replaced by simply\nsupplementing the BAP medium with recombinant VEFGA.\nThe authors should provide an experiment simply supplementing the differentiation medium with recombinant VEGFA\n(especially given the DOX-effect on trophoblast genes). And checking for pluripotency and differentiation markers?\n5) Furthermore, some control conditions should be reconsidered, added or included in some of the downstream analysis.\n- Regarding the VEGFR dKO experiments. As negative control for the experiments with the inducible CRISPR-Cas9 dKO\nsystem, normal WT cells are used. However, a more appropriate negative control is the inducible system with scramble\ngRNA (undergoing the same colony picking growth as dKO cells).\n- Extended figure 2g & 2h\nWhile the inducible dKO of the VEGFR’s clearly has the effect that is described by the authors, the normalization of the\nmRNA levels should ideally be done relative to the negative control instead of the -DOX condition of the cells that carry the\nconstruct.\nFurthermore, due to the small effect of DOX on the expression levels of several trophoblast genes (see extended figure 2c), it\nwould be interesting to include the NC±DOX in the RT-qPCR as well.\n6) Page 20 – Line 16-18\n“In addition, BMP4, which induces trophoblast marker expression in hESCs, increases SMAD1 occupancy at the NANOG\npromoter, suggesting that BMP signaling could also regulate NANOG expression.”\nSince no full mechanism is demonstrated in the manuscript, this statement opens the door for potential other mechanisms\nthan the one described here, due to the apparent reciprocal interaction between BMP4 and NANOG. The current model\nsuggests that VEGF directly regulates NANOG expression. However, this link is not convincingly demonstrated. It remains\npossible that VEGF inhibition initiates differentiation through other mechanisms. For instance, VEGF signalling could also\ndirectly affect BMP4 expression, which then in turn affects NANOG. The loss of NANOG would then occur as a secondary\neffect. Given the general role of NANOG in safeguarding pluripotency and suppressing differentiation, the modulations of\nNANOG levels performed in the manuscript could just bypass the BMP4-mediated effect. Especially the rapid rise in pSMAD\nlevels upon VEGFRi and the inability of NANOG OE to completely restore BMP pathway activation could support this\nhypothetical mechanism.\nThe rescue of differentiation by NANOG overexpression alone is not sufficient to prove direct regulation, as NANOG is a potent pluripotency factor and has been widely used in reprogramming protocols.\nThe storyline of the manuscript would gain a lot from an exact and detailed mechanistic link between VEGF signalling and\nNANOG, where it is demonstrated that NANOG is indeed the main and direct regulator.\nThe authors should consider to re-write the text specifying that there is no direct link between VEGF and Nanog and\ntherefore Nanog downregulation might not be the direct mechanism regulating trophoblast differentiation upon VEGF\nremoval.\n7) Regarding the NANOG iOE and SSN iOE experiments\nFurthermore, it would be interesting to see to what extent the NANOG iOE rescues the expression levels of some more\npluripotency genes. In panel 4j, it seems like only the recovery in POU5F1 expression levels are meaningful, since the\nrecovery of DPPA5 is limited and it is a marker for naïve rather than primed pluripotency.\n8) The manuscript proposes that Nanog downregulation leads to BMP activation and subsequent trophoblast differentiation.\nHowever, it remains unclear whether modulation of Nanog and BMP alone is sufficient to drive this process. Can BMP\noveractivation combined with Nanog knockdown induce a trophoblast-like state? These experiments would strengthen the\ncausality between these two factors and the phenotype.\n9) The ultimate proof of naïve pluripotency would be the ability of 2i/L cells to generate human blastoids harboring all three\nblastocyst lineages.\nMinor comments\nPage 6 – Line 4-8\nThe sentence where it is stated that the ligands and receptor levels decrease upon differentiation in E6 can be placed before\nthe results of RA and EB differentiation (page 6 line 1-3), since this again refers to more general differentiation conditions. It\nwould make more sense to first finish the statements of the general differentiation and end the paragraph with a few more\nspecific differentiation examples.\nPage 6 – Line 17-19\n“Collectively, these findings reveal that endogenous VEGF signaling is uniquely activated in primed hESCs and required for\nmaintaining the primed pluripotency state.”\nWhile the treatment with the VEGFRi inhibitor demonstrates morphological changes, the necessity for the primed pluripotent\nstate was not further supported by e.g. RT-qPCR (at this phase of the manuscript). Furthermore, most results here purely\ndemonstrate hallmarks of the primed, naïve and differentiated state, but don’t necessarily demonstrate a strict requirement.\nThis conclusion can therefore be an overstatement at this moment of the manuscript.\nPage 6 – Line 22-23\n“To characterize the function of VEGF signaling in hESCs, we inhibited this pathway using two pan-VEGFR inhibitors,\nTivozanib (Tiv) and Lenvatinib (Len)”\nWhy is the previously mentioned VEFGR inhibitor (Axitinib) not used here?\nPage 9 – Line 14-15\n“… consistent with the outcomes of Gene Ontology (GO), pathway and process enrichment analysis, as well as GSEA\nenrichment analysis.”\nThis are three times the same or at least related wordings. The general term for the analysis where you look for enriched\nprocesses is ‘GSEA’, for which GO and PID (the latter of which is used in panels 3c & 3d) are different databases that can be\nused. Using this sentence is therefore essentially saying three times the same thing and can be replaced by simply saying\n‘consistent with the outcomes of our GSEA’.\nPage 14 – Line 1\n“for 3 or 5 days.”\nThere seems to be no uniformity in the timepoints that are being tested. For many experiments, D1, D2 and D6 are used,\nwhile at other times D3 and D5 are tested without justification. Then again, in the next panel D3 and D8 are evaluated.\nPage 16 – Line 18-19\n“We named this new protocol 2i/L”\nThe name 2i/L is confusing with the 2i/L medium used for the maintenance of naïve mESCs. Furthermore, Linneberg-\nAgerholm et al. (2019) used a similar term (h2iL).\nFigure 7f\nThe only two conditions compared in the analysis are primed and naïve pluripotent cells. This means that the variability in\nthe dataset will be determined by the levels of naïve and primed markers. After demonstrating that VEGFRi disrupts primed\npluripotency but blocks the naïve-primed transition, it is therefore not that surprising that the 2i/L cells (obtained after 5i/LA\nconversion), cluster more closely to the 5i/LA cells than to the primed cells. As long as the primed characteristics are not\nrecovered and some naïve hallmarks are maintained, these cells will always cluster closer to 5i/LA cells. This figure could\ngo to supplementary.\nFigure 7i-k\nThe DNA methylation status of the 5i/LA and 2iL cells are compared to primed and naïve cells. However, since the naïve\ncells used throughout the experiments are 5i/LA-grown, what is the reference condition called ‘naïve’ that is shown here? It\ndoesn’t appear to be 5i/LA since the levels of KLF17 are very different.\nFurthermore, in panel 7i, 5i/LA and 2i/L do show quite a difference in methylation status, unlike what is mentioned in the text.\nMethods – Naïve hESC culture and differentiation\nThe methods section mentions that all hESCs and their derivatives (i.e. the naïve cells) are cultured at 21% O2. Naïve\nhESCs are generally grown in hypoxia (5% O2). For the 5i/LA conversion specifically, this is specified in Fisher et al. (2021).\nIs there a particular reason why normoxia is chosen here?", "reviewer_3": "I co-reviewed this manuscript with one of the reviewers who provided the listed reports. This is part of the Nature Communications initiative to facilitate training in peer review and to provide appropriate recognition for Early Career Researchers who co-review manuscripts.", "reviewer_4": "Wu, Wen, Zhu et al.‘s manuscript “Critical roles of endogenous VEGF signaling for cell fate decision in human embryonic\nstem cells” digs into the well explored space of signal transduction within human pluripotency to evaluate the potential role\nfor VEGF signaling factors to support self-renewal and block differentiation. Beginning with a somewhat simple screen, the\nauthors first test several less explored RTK pathways for their ability to impact “primed” state hESC self-renewal, and find a\nconvincing effect with the VEGFR inhibitor Axitinib. After confirming that this effect conforms with the endogenous\nexpression of VEGF receptors and ligands within primed state cells, the authors then thoroughly validate the role of VEGF\nsignaling in promoting self-renewal within this state using multiple independent small molecule inhibitors, the induction of\nsoluble VEGF receptors (which presumably compete for active ligand), and bulk induction of VEGFR1 and 2 targeting\nsgRNAs. They then utilize combinations of small molecule inhibitors and their soluble receptor induction system to\ndemonstrate how VEGF signaling first supports NANOG expression which, upon destabilization, triggers BMP-supported\nspontaneous differentiation towards a trophoblast stem cell-like state (which they confirm via marker-based analyses such\nas IF, FACs, RT-qPCR alongside limited epigenetic analysis of the ELF5 promoter). Finally, the authors demonstrate that\nVEGF related genes are only induced upon the transition from naïve to primed state pluripotency, and apply this concept to\nreplace the conventional 5i/L/A condition with a limited VEGF-inhibitor-based “2i/L” condition that eliminates the historically\nproblematic MEK1 inhibitor PD0325901 with minimal, if not improved, effects on genomic integrity and self-renewal.\nOverall, I think this is a thorough, mature and compelling work, with multiple validations for each experimental observation.\nThe authors offer an overall coherent framework from the initial observation that VEGF signaling appears to specifically\nimpact primed state pluripotency, through the dissection of the genetic pathway from VEGF to NANOG to BMP-based\ninduction of “trophoblast-like cells,” and to the developmental induction of this pathway as part of the naïve to primed\ntransition. However, there are several areas where I feel that the results or interpretation of the results are overstated,\nsometimes critically so. Moreover, a detailed connection for precisely how VEGF signaling might interact with the\npluripotency network (through which arm of its many downstream effectors) is not explored, but needs to be discussed more\nthoroughly in the discussion. I feel that this is particularly important given the authors claims related to the potential of VEGF\ninhibition to act as a substitute for the more conventional MEK1 inhibitor. A paragraph in the discussion highlighting how\nVEGF may interact with more conventional signaling pathway components would provide important context.\nBelow are the areas that I think are the most problematic in terms of language and interpretation, as well as proposed\nsolutions for making sure this very interesting study is clearly communicated to the field.\nIn particular, the reported differentiation of primed state hESCs into bona fide TSCs is still controversial, as this would\nrepresent a bona fide transdifferentiation event that includes global rewiring of the epigenome. As such, it would not conform\nwith the developmental dynamics of the early epiblast in vivo. Instead, BMP or CDX2 induction of primed state cells seems to\ncreate a complex hybrid, possibly amniotic state that still fits many of the somewhat limited criteria tested by the authors for\na trophoblast-like identity (see in particular PMID: 33831365 as well as PMID: 21816365). My suggestion is that the authors\nconsider the pre-existing literature carefully when developing and explaining their model. To me, they must either use the\nincorporated data and criteria from these previously papers to show more completely that their cell types resemble more\nconventional hTSCs compared to primed state derived amnion-like cells or they must substantially revise this section of the\nmanuscript to highlight these prior works. They must make it clear that VEGF blocked primed state pluripotent cells from\nspontaneous differentiation as driven by BMP4 and discuss how this triggers the induction of a trophoblast-like state that\nmay be more equivalent to early amnion, but remove statements or results intended to validate the equivalence of these\ncells to bona fide TSCs. To me, it is a perfectly interesting and sufficient finding on its own without the added utility of being\nsuitable as a model for trophoblast or placental development.\nThe authors have done a convincing job of showing that VEGF signaling is upstream of NANOG, and that BMP4-based\ndifferentiation is downstream. However, the OE/rescue experiments they have conducted are more similar to an “epistasis”\ntest (establishing the genetic order of events) than they are about direct or functional connections. The observation that\nNANOG binds some BMP-associated effectors is interesting, but derived from previously generated/public data and does\nnot prove that NANOG is directly suppressing these targets. The language (and subheader) related to the section “NANOG\ndirectly suppresses expression of genes associated with the BMP pathway and trophoblast lineage in primed hESCs”\nshould be revised accordingly to better utilize wording related to who is acting upstream to stabilize NANOG expression\n(VEGF) or downstream to support spontaneous differentiation (BMP4). The observation that NANOG appears to bind some\nof these promoters can be noted and the authors can suggest that this might suggest that NANOG plays dual activating and\ninhibitory roles, but anything beyond that is speculation not present in the data.\nThe novel “2i/L” experiments are interesting but overall represent a shallow validation. I think this is fine for a wide-ranging\ninvestigation of VEGF signaling in human pluripotent stem cells, but the authors should consider if it is worth adding\nadditional validation to bolster their claim, including testing additional stem cell lines, evaluating their functional potential\naccording to the conventions used for the publication of these prior conditions, and more carefully evaluating the effects of\nthis condition on long term genetic and epigenetic stability. I fully realize that such an investigation is well outside the scope\nof this study. If they elect to proceed with the validation they have already done, the authors should revise their language to\nhighlight that their 2i/L condition offers intriguing preliminary results for simpler and more stable naïve hPSC culture\nconditions, but that further work would be necessary to establish that these conditions are sufficient to capture the\ncomplexities of self-renewal and differentiation potential long term.\nI think the statement on Page 19, Line 28 reading “The finding that the uterine fluid from women with unexplained infertility\ncontains decreased VEGFA levels further implicates a crucial role of VEGF signaling in early human embryonic\ndevelopment” should be removed. The observations or interpretations of this sort of clinical study are much more likely about\nplacentation rather than the differentiation of the epiblast (which the authors have already stated is an endogenous signaling\nrelationship).\nThe fact that the SHhES8 hESC line is female needs to be stated clearly in the text before presenting data related to Xist\nand MeCP2 methylation in Figure 7.\nThe title could be revised to something more specific, such as “VEGF signaling supports primed-state human embryonic\nstem cell self-renewal” or “Endogenous VEGF signaling blocks spontaneous BMP4-based differentiation in primed state\nhuman embryonic stem cells.”"}, "author_response": "2025/11/30\nThe point-by-point response letter for the manuscript entitled “Critical\nroles of endogenous VEGF signaling for cell fate decision in human\nembryonic stem cells” (manuscript # NCOMMS-25-32707-T)\nDear Reviewers,\nWe appreciate your efforts on this manuscript. Your comments are very\nconstructive. We have made our best to address all your concerns. The\nfollowing is our point-by-point response letter.\nThanks again,\nYing\nReviewer #1 (Remarks to the Author):\nComments: The authors present an interesting article to investigate the role of\nVEGF signaling in primed and naive human PSCs. The first and major part of\nthe paper focuses on primed PSCs and shows that VEGF receptors are\nexpressed in primed but not naive cells, and through small molecule manipulation\nand gene tools they prove nicely and solidly that VEGF signaling\nperturbation leads to primed psc differentiation towards TSC through\ncompromising Nanog expression and BMP signaling that promotes TSC.\nHowever, the last part of the paper trying to argue that inhibition of VEGF\nsignaling in naive human PSCs improves their maintenance and also one can\nreplace human naive PSC media like 5iLAF into media which is composed only\nof LIF VEGFRi and ROCK inhibitor. Unfortunately, i find this part of the work\nunsubstantiated, partially studied and filled with contradictions and non solid\ndata as i detail below. I think the paper is unpublishable if it keeps this part of\nthe work which is unfortunate as the first part in primed PSCs is solid and would\nhave been sufficient to include in this publication.\n1\n1) first all 2i/L cells where first kept in 5iLAF and then moved to 2i/L so the\nquestion is can the authors make naive cells in 2i?L conditions, which must be\nshown to make such a claim.\n2) characterization of naive pluripotency is partial and non satisfactory. the\nauthors should rely on solid reporters for deltaPe-oct4-gfp and double\nfluorescent x chromosome lines to accurately report x chromosome status. this\nis not done, and non conclusive facs surface marker analysis is shown and only\nfor limited passages. this is not the gold standard proof tests that have long\nbeen established in the field. to make such a major claim.\n3) global DNA hypomethylation analysis should be shown - nobody in human\nor mice was able to show global DNA hypomethylation without FGF/ERK\npathway targeting, so the authors claim they merely can do this with ERK and\nLIF and ROCKi.? This is a major controversial claim that better be solidly proved.\nMethylation of a single promoter ot two is non sufficient. Global WGBS is a must.\n4) the fact in figure 7 that in the control naive cells XIST promoter is still highly\nmethylated casts serious doubt on the ability of the authors to grow naive cells.\nHuman naive cells should have this region hypomethylated as shown in\nnumerous studies.\n5) RNA-seq and immunostaining should be shown for many naive pluripotency\nmarkers (TFCP2LF1, ARGFX, DPPA5, KLF4, KLF17, DPPA3 etc...) as well as\nfacs for SUSD2+ and CD24- pure population. also cells analyzed after at least\n20 passages in both feeder and feeder free conditions should be analyzed\nthroughout\n6) the authors seem to use feeder free conditions, and permanent addition of\nROCKi at the concentration they use has repeatedly shown in the field that it\nleads to primed or naive stem cell differentiation in 5-10 passage. The authors\nshould prove this is not the case by using exacting knock-in reporter alleles as\nindicated above. these reporters are available on Addgene, and the cell lines\nare available in the community from many groups.\n7) the authors should show that the VEGFR inhibitor used is not influencing\nsmall molecule. The authors should use a genetic mean to inactivate VEGFR\nsignaling in naive PSCs as they did for primed cells to make this conclusion.\n8) The above point and claim are heavily shadowed by the authors claim that\nVEGF receptors are not expressed in naive human PSCs - so how does\ninhibition of a receptor that is not expressed in a cell helps maintain keeping\nthat cell and in very minimal condition?. This is bizarre to say the least and\nshould be experimentally addressed aggressively. This is a major major claim\nand contradiction.\nResponse: We sincerely appreciate your thorough evaluation of our\nmanuscript and for your positive comment that “the first part in primed PSCs is\nsolid and would have been sufficient to include in this publication”.\nWe fully agree that our current study in naïve hPSCs remains preliminary\nand requires substantially more rigorous validation. Similarly, Reviewer #4 also\nviewed our naïve validation as “shallow”. This reviewer pointed out that the\nextensive experiments required to strengthen that claim are “well outside the\nscope of this study”. Reviewer #4 also suggested us to soften our claims.\nCombined with your strong recommendation to delete the section related data\nobtained from naïve hPSCs, the results related to naïve hPSCs in our original\nFig. 7 and Supplementary Fig. 6 are not included in the revised manuscript,\nalong with the deletion of all related claims about VEGF inhibition in naïve hPSC\nmaintenance. In addition, we have edited the title of this manuscript to\n“Endogenous VEGF signaling: a guardian of human primed pluripotency”. With\nall these changes, the revised manuscript is now exclusively focusing on the\ndiscovery and mechanistic insights in primed hESCs: endogenous VEGF\nsignaling safeguards self-renewal and pluripotency in primed hESCs through\nsustaining NANOG expression and inhibiting BMP signaling activation. This\nconstitutes the key conclusion of this study and is fully supported by our\nexperimental evidence.\nIn addition, we fully acknowledge your listed experiments (conversion and\n3\ncharacterization of naive pluripotency, WGBS, X chromosome-reporter lines,\ngenetic VEGFR ablation, etc.) as essential next steps to rigorously validate the\nrole of VEGF signaling in the regulation of human naïve pluripotency. We are\nnow undertaking a project focusing specifically on naïve state regulation. Your\nsuggestions are invaluable in shaping our ongoing work. We will incorporate\nthese suggestions into an independent follow-up study and report related\nresults separately.\nReviewer #2 (Remarks to the Author):\nComment: Review of the Article by Wu et al., “Critical Roles of Endogenous\nVEGF Signalling for Cell Fate Decision in Human Embryonic Stem Cells”\nIn this article, Wu et al. investigate the role of VEGF signaling in maintaining\nthe pluripotent state in both primed and naïve human embryonic stem cells\n(hESCs). The authors demonstrate that the VEGF pathway is active in primed\nhESCs but largely inactive in naïve hESCs, suggesting a potential role for\nVEGF in sustaining the primed state.\nInterestingly, VEGF signaling is downregulated during the differentiation of\nprimed cells. Consistent with this, VEGF pathway inhibition promotes\ndifferentiation toward the trophoblast lineage. The authors show that VEGF\ninhibition leads to the downregulation of pluripotency markers, such as NANOG,\nand concomitant upregulation of BMP signaling. Importantly, NANOG\noverexpression rescues the differentiation phenotype, supporting its role\ndownstream of VEGF signaling.\nIn the final part of the study, the authors report that VEGF inhibition is sufficient\nto maintain naïve hESCs in their naïve state, even in the absence of ERK\npathway inhibition. Notably, culturing naïve hESCs in media containing only\nVEGF inhibition, ROCK inhibition, and LIF (referred to by the authors as “2iLIF”)\nis sufficient to maintain pluripotency.\nThis is a comprehensive and well-executed study that provides novel insights\n4\ninto the role of VEGF signaling in hESC pluripotency and differentiation. The\ndata are extensive and technically solid. However, the manuscript lacks a direct\nor indirect mechanistic link between VEGF inhibition and Nanog\ndownregulation.\nResponse: We appreciate your positive comments on this study. Moreover, we\nagree with you that the mechanistic line between VEGF inhibition and NANOG\ndownregulation is not clearly elucidated in the current manuscript. We\ndiscussed this issue in revised manuscript (discussion section Page 18, Line\n1\n- Page 19, Line 14).\nComment: Furthermore, some methodological aspects of the article need to\nbe improved. Throughout the manuscript, western blots and RT-qPCR plots are\noften presented in separate figures and normalized per figure. Despite the fact\nthat, overall, the effect described by the authors is indeed clear, this\npresentation makes it impossible to evaluate whether there is already a\ndifference in basal levels between the controls and the untreated experimental\nconditions.\nResponse: Thank you for pointing out this issue. To address this concern, we\nhave reorganized the presentation of our western blot and RT-qPCR data\n(revised Fig. 2g, i; Fig. 3g, h; Fig. 4e, f, i; Supplementary Fig. 3a, b, c, e;\nSupplementary Fig. 5c; and Supplementary Fig. 6h). In the revised figures,\nrelevant western and RT-qPCR plots are presented together in the same figure\npanels, allowing for direct comparison between control and experimental\nconditions. Additionally, we have normalized all data to a common baseline\n(untreated control) across the entire dataset, ensuring that any differences in\nbasal levels are clearly visible.\nComment: Even though the effect is mild in comparison to the experimental\nconditions, the use of DOX seems to have an effect on trophoblast genes.\nTherefore, it should be avoided where possible, such as in situations where\n5\nmedia can be supplemented with recombinant factors and iOE is not strictly\nnecessary.\nResponse: We agree that the DOX-inducible overexpression system should\nbe avoided when recombinant proteins are available. As suggested, we tried to\nreplace the inducible OE system with recombinant VEGFA proteins. However,\nwe unexpectedly found that recombinant VEGFA did not produce the effect of\nDOX-induced overexpression. Our preliminary result suggests that, in our\nexperimental condition, VEGFA acts through an intracrine mechanism in\nhESCs, which explains why recombinant VEGFA could not mimic\noverexpressed VEGFA. For more details, please refer to our response to your\nMajor Comment #4.\nComment: Throughout the manuscript, unpaired two-tailed Student’s t tests are\nbeing used to compare multiple timepoints to timepoint 0. However, a correction\nfor multiple testing is needed for such comparisons, but this is never mentioned.\nResponse: Thank you for pointing this out. In the revised manuscript, for\nmultiple comparisons against the same baseline, we have replaced repeated\nunpaired two-tailed Student’s t tests with one-way ANOVA, followed by the\nDunnett’s post-hoc test. Legends of all involved figures, including revised\nSupplementary Fig. 1g, i, j, l; Fig. 2d; Fig. 3f, h; Fig. 4f; Supplementary Fig. 5b,\nc, e, g; and Supplementary Fig. 6h, and the statistics section of the methods\nhave been revised accordingly.\nMajor comments\nComment 1: Figure 1A\nThe authors use PDGFR, VEGFR, and EGFR inhibitors and observe\ndifferentiation only with VEGFR inhibition. However, the manuscript does not\ninclude controls demonstrating the efficacy of PDGFR and EGFR inhibition. The\nauthors should include validation of the inhibitor activity, such as downstream\npathway readouts or phospho-protein analysis. Additionally, EGFRi does show\n6\na pretty drastic difference. From the results shown here, it is hard to judge\nwhether it is a proliferation-related effect, but saying that there is no effect at all\nseems overstated.\nResponse to comment 1: To address this issue, we have added data from two\nadditional VEGFR inhibitors, Tivozanib (Tiv) and Lenvatinib (Len), and validated\nthe activity of PDGFR, VEGFR, and EGFR inhibitors by assessing their effects\non downstream phospho-ERK1/2 (T202/Y204) and phospho-AKT (S473) levels\nvia western blot (new data are shown in revised Supplementary Fig. 1a-c and\nbelow). Our results show that: 1) all three VEGFR inhibitors caused obvious\nhESC differentiation; 2) the VEGFR inhibitor-treatment led to a significant\nreduction in both phospho-ERK and phospho-AKT levels, validating its potent\ninhibitory effect; 3) the EGFR inhibitor showed a moderate but detectable\ndecrease in downstream signaling, though weaker than VEGFR inhibition; 4)\nthe PDGFR inhibitor didn’t show any decrease in phospho-ERK/AKT levels,\nwhich could be due to the low basal activity of this pathway in primed hESCs.\nTo verify this possibility, we analyzed our RNA-seq data from primed SHhES8\nand H9 hESCs and found that PDGFRs are expressed at low levels in hESCs\n(revised Supplementary Fig. 1d). This suggests that PDGF signaling pathway\nis less active in hESCs compared to VEGF signaling, explaining why we did not\nobserve any effects with its inhibitor.\nRegarding to the reviewer’s observation about EGFR inhibition, we agree\nthat the EGFR inhibitor exhibited a drastic effect (Fig. 1a). In fact, we observed\nobvious cell death upon EGFR inhibition. To make more realistic statement, the\nrelated manuscript text has been revised to the following statement: “In contrast,\ninhibition of PDGFR failed to induce significant morphological changes.\nSimilarly, inhibition of EGFR, despite triggering marked cell death, did not elicit\ndiscernible differentiation phenotype.”\nthe undifferentiated state and becomes inactivated upon differentiation.\n(Related to Fig. 1)\na, Bright-field images of SHhES8 hESCs treated with DMSO (control), VEGFR\ninhibitors (VEGFRi: Tivozanib, 10 μM) and (VEGFRi: Lenvatinib, 6 μM),\nrespectively, for 2 days. Scale bars, 500 μm (top) and 100 μm (bottom).\nb, c, Representative western blot (b) and quantification (c) analysis results for\nrelative protein levels of indicated proteins in SHhES8 hESCs treated with\nDMSO (control), PDGFR inhibitor (PDGFRi: Crenolanib, 2 μM), VEGFR\ninhibitor (VEGFRi: Axitinib, 5 μM; Tivozanib, 10 μM; Lenvatinib, 6 μM), and\nEGFR inhibitor (EGFRi: Erlotinib, 10 μM), respectively, for 10 minutes.\nd, Expression levels of PDGF, EGF and VEGF pathway receptors in hESCs.\nTPM (transcripts per million) values were from our RNA-seq data in two\nindependent hESC lines, SHhES8 and H9.\nThe authors claim VEGF signaling is inactive in naïve hESCs based on\nphospho-VEGFR1 (Y1213) and VEGFR2 (Y951) levels. However, activity is\nshown in isolation. A side-by-side comparison of VEGF signaling in naïve,\nprimed, and differentiated hESCs via western blot is necessary to support this\nconclusion. Furthermore, this findings are only shown in H1 and H9 cells, but\nshould also be demonstrated in the primarily used cell line.\nResponse to comment 2: As suggested, we have provided side-by-side\ncomparisons of VEGF pathway activity across naïve, primed and differentiated\ncells in the primarily used hESC lines (SHhES8 and H9) in revised Fig. 1d, f\nand g) and below. Revised Fig. 1d shows bright-field images of SHhES8 and\nH9 hESCs in the naive (5i/L/A medium), primed (mTeSR1 medium), and\ndifferentiated (E6 medium, 2 days) states; revised Fig. 1f, g shows western blot\nresults of total and active VEGFR1/2 protein levels in SHhES8 and H9 hESCs.\nFor the hESC H1 line, we failed to stably convert them into the naïve state\nunder current conditions. Therefore, we only present experimental results from\nits primed and E6-differentiated states below for reviewer’s information. As the\nfull naïve/primed/differentiated comparison are presented for both SHhES8 and\nH9 cell lines (Fig. 1d, f, g), results from the H1 hESCs are omitted from the\nrevised version, but shown below (Response Letter Fig. 1).\nConsistent results were obtained in all three hESC lines tested. 1) Both\ntotal VEGFR1/2 and phosphorylated VEGFR1 (Y1213)/VEGFR2 (Y951)\nproteins were abundant in primed hESCs; 2) levels of both total and\nphosphorylated VEGFR1/2 markedly decreased upon differentiation; 3) naïve\nhESCs (SHhES8 and H9) exhibited negligible VEGFR expression and kinase\nactivity, validating that VEGF signaling is largely inactive in the naïve state.\nThese new results are now incorporated into the revised manuscript (Fig. 1d, f,\ng).\nhESCs.\nd, Bright-field images of SHhES8 and H9 hESCs cultured in the 5i/L/A medium\n(Naïve), mTeSR1 medium (Primed), and E6 differentiation medium (E6 D2),\nrespectively. Scale bars, 100 μm.\nf, g, Representative western blot (f) and quantification (g) analysis results for\nrelative protein levels of indicated proteins in SHhES8 and H9 hESCs cultured\nin the 5i/L/A medium (Naïve), mTeSR1 medium (Primed), and E6 differentiation\nmedium (E6 D2), respectively.\nResponse Letter Fig. 1: VEGF signaling activity is high in primed H1\n10\nhESCs, but inactivated during differentiation. Representative western blot\nresults (left) and quantification analysis of relative protein levels (right) of\nindicated proteins in H1 hESCs at the primed (mTeSR1) or differentiated (E6\nD2) states.\nComment 3: Extended figure 1a\nThe figures show the expression levels of the VEGF receptors and ligands and\nsome pluripotency factors in H1 and H9 hESCs in isolation. However, unlike in\nfigure 1b & 1d, there is no comparison with the differentiated conditions, thus\nmaking the graph quite meaningless as such. It is true that the relative\nexpression of some of the markers are similar to the levels shown in figure 1b\n& 1d, but this graph cannot be interpreted as it is now and comparisons to\ndifferentiation conditions should be shown here as well to support the\nconclusion that is drawn from this experiment.\nResponse to comment 3: We have now included the expression levels of\nVEGF receptors, ligands, pluripotency, and differentiation markers under\ndifferentiated conditions in both H9 and H1 hESCs (Supplementary Fig. 1e, f).\nIn addition, we also compared the expression of VEGF pathway components in\nthe naïve and primed condition in H9 hESCs (Fig. 1e). These new data enable\ndirect comparison between primed and differentiated states, and reinforce the\ninterpretation that VEGF pathway components are abundant in the\nundifferentiated state of primed hESCs. These new data are shown in revised\nSupplementary Fig. 1e, f and Fig. 1e and below.\nthe undifferentiated state and becomes inactivated upon differentiation.\n(Related to Fig. 1)\ne, f, RT-qPCR analysis results for relative mRNA levels of VEGF signaling\nligands and receptors, pluripotency marker genes, and lineage marker genes\nin H9 (e) and H1 (f) hESCs cultured in the mTeSR1 self-renewal medium for 4\ndays or in the E6 differentiation medium for 2 days.\nFig. 1: VEGF signaling components are highly expressed in primed\nhESCs.\ne, RT-qPCR analysis results for relative mRNA levels of VEGF signaling ligands\n12\nand receptors in SHhES8 (left) and H9 (right) hESCs cultured in the 5i/L/A\nmedium (Naïve), mTeSR1 medium (Primed), respectively.\nComment 4: The DOX-inducible system should be considered carefully in the\ncontext of trophoblast differentiation given the fact that, apparently, DOX itself\nalready has an (minor) effect on the expression of multiple trophoblast-\nassociated genes (extended figure 2c). The reason to use the inducible system\nfor the experiments about the decoy-OE and dKO seems obvious given\nthe apparent role of VEGF signalling in primed pluripotency, but the OE of VEGFA\ncan easily be replaced by simply supplementing the BAP medium with\nrecombinant VEFGA. The authors should provide an experiment simply\nsupplementing the differentiation medium with recombinant VEGFA\n(especially given the DOX-effect on trophoblast genes). And checking for\npluripotency and differentiation markers?\n[Redacted]\n13\n[Redacted]\n14\n[Redacted]\n15\n[Redacted]\n[Redacted\n16\n[R edacted]\nComment 5: Furthermore, some control conditions should be reconsidered,\nadded or included in some of the downstream analysis.\n- Regarding the VEGFR dKO experiments. As negative control for the\nexperiments with the inducible CRISPR-Cas9 dKO system, normal WT cells\nare used. However, a more appropriate negative control is the inducible system\nwith scramble gRNA (undergoing the same colony picking growth as dKO cells).\n- Extended figure 2g & 2h\nWhile the inducible dKO of the VEGFR’s clearly has the effect that is described\nby the authors, the normalization of the mRNA levels should ideally be done\nrelative to the negative control instead of the -DOX condition of the cells that\ncarry the construct.\nFurthermore, due to the small effect of DOX on the expression levels of several\ntrophoblast genes (see extended figure 2c), it would be interesting to include\nthe NC±DOX in the RT-qPCR as well.\nResponse to comment 5: During our revision, we generated a new control\nhESC line (negative control expressing sgRNA targeting EGFP, named NC) that\nundergoes identical colony-picking steps as the dKO line. All RT-qPCR and\nwestern blot data are now normalized to the -DOX condition of the NC cells.\nWe also repeated the immunofluorescence staining assays in dKO cells side-\nby-side with the newly established EGFP-sgRNA expressing NC cells. These\nnew data reproduced our original findings and are now shown in the revised\nSupplementary Fig. 3a-f and below.\noverexpression of VEGFA prevents the activation of trophoblast-\nassociated genes in hESCs. (Related to Fig. 2)\na, RT-qPCR analysis results for relative mRNA levels of VEGFR1 and VEGFR2\nin negative control (NC) hESCs and dKO hESCs cultured without or with DOX\nfor 3 days.\nb, c, Representative western blot (b) and quantification (c) analysis results of\nrelative protein levels of VEGFR1 and VEGFR2 in NC and dKO hESCs cultured\nwithout or with DOX for 3 days. α-Tubulin was used as a loading control.\nd, Bright-field images of NC hESCs and dKO hESCs cultured without or with\ne, RT-qPCR analysis results for relative mRNA levels of indicated marker genes\nin NC hESCs and dKO hESCs cultured without or with DOX for 3 days.\nf, Representative immunofluorescence staining results of GATA3, CDX2, and\nTP63 (green) in NC and dKO hESCs cultured without or with DOX for 3 days.\nThe nuclei were counterstained with DAPI (blue). Scale bars, 50 μm.\nComment 6: Page 20 – Line 16-18\n“In addition, BMP4, which induces trophoblast marker expression in hESCs,\nincreases SMAD1 occupancy at the NANOG promoter, suggesting that BMP\nsignaling could also regulate NANOG expression.”\nSince no full mechanism is demonstrated in the manuscript, this statement\nopens the door for potential other mechanisms than the one described here,\ndue to the apparent reciprocal interaction between BMP4 and NANOG. The\ncurrent model suggests that VEGF directly regulates NANOG expression.\nHowever, this link is not convincingly demonstrated. It remains possible that\nVEGF inhibition initiates differentiation through other mechanisms. For instance,\nVEGF signalling could also directly affect BMP4 expression, which then in turn\naffects NANOG. The loss of NANOG would then occur as a secondary\neffect. Given the general role of NANOG in safeguarding pluripotency and suppressing\ndifferentiation, the modulations of NANOG levels performed in the manuscript\ncould just bypass the BMP4-mediated effect. Especially the rapid rise in\npSMAD levels upon VEGFRi and the inability of NANOG OE to completely\nrestore BMP pathway activation could support this hypothetical mechanism.\nThe rescue of differentiation by NANOG overexpression alone is not sufficient\nto prove direct regulation, as NANOG is a potent pluripotency factor and has\nbeen widely used in reprogramming protocols.\nThe storyline of the manuscript would gain a lot from an exact and detailed\nmechanistic link between VEGF signalling and NANOG, where it is\ndemonstrated that NANOG is indeed the main and direct regulator.\nlink between VEGF and Nanog and therefore Nanog downregulation might not\nbe the direct mechanism regulating trophoblast differentiation upon VEGF\nremoval.\nResponse to comment 6: Thank you for the valuable discussion. In the\nrevised manuscript, we have carefully rephrased the relevant sections to avoid\noverstating a direct link between VEGF and NANOG. We have reorganized the\nsummary and discussion sections, including a detailed discussion on the\nrelationship between VEGF, NANOG, and the BMP pathway. We stated: “we\ndid not find a direct link between VEGF signaling and NANOG transcription in\nhESCs” (Page 12, Line 12-13). For more details, please refer to the discussion\nsection (Page 18, Line 1 - Page 19, Line 28).\nIn the revised discussion paragraphs (Page 19, Line 10 - 28), we stated:\n“We sought to identify specific proteins mediating the regulatory effect of VEGF\nsignaling on NANOG expression using proteomic and phosphoproteomic\napproaches. However, we failed to detect convincing substrate candidates.\nThus, it remains unclear how VEGF signaling regulates NANOG expression in\nhESCs. Although NANOG acts as a major effector downstream of VEGF\nsignaling in hESCs, its OE failed to entirely abrogate VEGF pathway deficiency-\ninduced BMP pathway activation, suggesting that there exist NANOG-\nindependent mechanisms contributing to VEGF signaling-mediated\nsuppression of BMP signaling activation. BMP4, which is known to induce\ntrophoblast marker expression in hESCs, was reported to increase SMAD1\noccupancy at the NANOG promoter, suggesting that BMP signaling could also\nregulate NANOG expression.” “Therefore, suppression of BMP signaling\naccounts for, at least in part, the VEGF signaling-mediated inhibition of\ntrophoblast-like lineage specification in primed hESCs. It remains unclear how\nVEGF signaling crosstalks with the BMP pathway in the context of hESC\npluripotency and lineage specification.”\nThese changes ensure that our manuscript accurately reflects the current\n20\nstate of our research and avoids overstatement of the mechanisms involved.\nComment 7: Regarding the NANOG iOE and SSN iOE experiments.\nFurthermore, it would be interesting to see to what extent the NANOG iOE\nrescues the expression levels of some more pluripotency genes. In panel 4j, it\nseems like only the recovery in POU5F1 expression levels are meaningful,\nsince the recovery of DPPA5 is limited and it is a marker for naïve rather than\nprimed pluripotency.\nResponse to comment 7: In the revised manuscript, we expanded the\nNANOG iOE and SSN iOE rescue panel. In addition to OCT4 and DPPA5, the\nrevised Figure 4j includes primed state markers DNMT3B and ETV4. Both\ngenes were downregulated by soluble VEGFR expression and significantly\nrescued by simultaneous NANOG OE. These data are shown in the revised Fig.\n4j and below.\nFig. 4: NANOG acts as a key effector downstream of the VEGF signaling\npathway.\nj, RT-qPCR analysis results for relative mRNA levels of pluripotency and\ntrophoblast marker genes in SS iOE #10 and SSN iOE hESCs cultured without\nor with DOX for 6 days.\nComment 8: The manuscript proposes that Nanog downregulation leads to\nBMP activation and subsequent trophoblast differentiation. However, it remains\nunclear whether modulation of Nanog and BMP alone is sufficient to drive this\n21\nprocess. Can BMP overactivation combined with Nanog knockdown induce a\ntrophoblast-like state? These experiments would strengthen the causality\nbetween these two factors and the phenotype.\nResponse to comment 8: Thank you for the constructive suggestion.\nFollowing your suggestion, we treated hESCs with BMP4 alone using a\npreviously reported protocol2, NANOG knockdown (KD) alone, or combined\nBMP4 supplementation with NANOG KD in DOX-inducible NANOG iKD hESCs\n(Response Letter Fig. 5). The expression of trophoblast genes was assessed.\nThe NT iKD hESC line was used as a control. The results showed that BMP4\nalone or NANOG KD alone markedly down-regulated pluripotency genes and\nup-regulated trophoblast genes, consistent with previous reports2-6. Notably,\nNANOG KD had a stronger effect on the expression of pluripotency and\ntrophoblast genes relative to BMP4 treatment. The combined effect of BMP4\ntreatment and NANOG KD has not been previously reported. Our results\nindicate that, compared to individual treatments, BMP4 treatment together with\nNANOG KD further and significantly enhanced trophoblast gene expression.\nThese findings suggest that BMP pathway overactivation combined with\nNANOG KD is sufficient to drive a robust trophoblast-like transcriptional\nprogram. Therefore, the NANOG loss and BMP pathway activation could act\nsynergistically to promote hESC differentiation. We consider that these data\nare peripheral to the main context of the manuscript, where NANOG\ndownregulation and BMP activation were examined in the context of VEGF\nsignaling deficiency. Therefore, these data are not included in the revised\nmanuscript. They are shown below for your information.\nand NANOG knockdown is sufficient to drive a robust trophoblast-like\ntranscriptional state.\na, Bright-field images of NT and NANOG iKD hESCs cultured with or without\nDOX and BMP4 for 5 days. Scale bars, 500 μm.\nb, RT-qPCR analysis result for relative mRNA levels of pluripotency and\ntrophoblast genes in NT and NANOG iKD hESCs cultured under the indicated\nconditions for 5 days.\nData in the panel b are represented as mean ± SEM (n = 3 independent\nexperiments). Two-way ANOVA followed by Tukey’s post-hoc test was used. *\np < 0.05, ** p < 0.01, and *** p < 0.001.\nComment 9: The ultimate proof of naïve pluripotency would be the ability of\n2i/L cells to generate human blastoids harboring all three blastocyst lineages.\nexperiments are required to ultimately prove the authentic naïve pluripotency\nof the 2i/L cells. Similarly, Reviewer 1 also suggested a list of experiments\nneeded to provide full proof of naïve pluripotency of our 2i/L cells. Moreover,\nboth Reviewers 1 and 4 also considered that our data for the regulatory role of\nVEGF signaling in naïve pluripotency in our original manuscript (original Figure\n7 and supplemental Figure 6) are preliminary and suggested us to focus on the\nrole of VEGF signaling in primed hESCs. In fact, we are working on the\nconversion of primed hESCs to the naive state using our new protocol based\non the VEGF signaling inhibition, and conducting more experiments to validate\nthe naïve nature of converted hESCs, as suggested by all reviewers. Therefore,\nwe plan to combine our ongoing experiment results with data presented in the\noriginal Figure 7/Supplemental Figure 6 to form another story, focusing on the\nrole of VEGF signaling in the naive pluripotency regulation. As for the current\nstory, we would like to report findings solely in primed hESCs, if reviewers and\neditors could allow us. Thus, the data in the original Figure 7 and supplemental\nFigure 6 are not included in the revised manuscript. We will explain the\nreference cells more clearly in the future.\nMinor comments\nMinor comment 1: Page 6 – Line 4-8\nThe sentence where it is stated that the ligands and receptor levels decrease\nupon differentiation in E6 can be placed before the results of RA and EB\ndifferentiation (page 6 line 1-3), since this again refers to more general\ndifferentiation conditions. It would make more sense to first finish the\nstatements of the general differentiation and end the paragraph with a few more\nspecific differentiation examples.\nResponse to minor comment 1: Thank you for the constructive suggestion,\nand we have re-ordered the paragraph as suggested (Page 6, Line 9-12).\nMinor comment 2: Page 6 – Line 17-19\n24\n“Collectively, these findings reveal that endogenous VEGF signaling is uniquely\nactivated in primed hESCs and required for maintaining the primed pluripotency\nstate.”\nWhile the treatment with the VEGFRi inhibitor demonstrates morphological\nchanges, the necessity for the primed pluripotent state was not further\nsupported by e.g. RT-qPCR (at this phase of the manuscript). Furthermore,\nmost results here purely demonstrate hallmarks of the primed, naïve and\ndifferentiated state, but don’t necessarily demonstrate a strict requirement. This\nconclusion can therefore be an overstatement at this moment of the manuscript.\nResponse to minor comment 2: Thank you for pointing this out. We agree\nthat, at this early point in the manuscript, we only showed morphological\nchanges and marker down-regulation. To avoid overstatement, we have\nchanged the concluding sentence to: “Collectively, these findings reveal that\nendogenous VEGF signaling is uniquely activated in primed hESCs and\nregulated by the differentiation state.” (revised Page 7, Line 2-4). The word\n“required” was used only after the functional experiments were performed.\nMinor comment 3: Page 6 – Line 22-23\n“To characterize the function of VEGF signaling in hESCs, we inhibited this\npathway using two pan-VEGFR inhibitors, Tivozanib (Tiv) and Lenvatinib (Len)”.\nWhy is the previously mentioned VEFGR inhibitor (Axitinib) not used here?\nResponse to minor comment 3: Sorry for missing the explanation for this.\nAxitinib (Axi) was used in our initial phenotype screening (Fig. 1a). To verify the\nfinding, two additional pan-VEGFR inhibitors, Tiv and Len, were used. Both Tiv\nand Len produced the differentiation phenotype comparable to that induced by\nAxi, yet caused noticeably less cell death by day 2 and yielded a more\nsynchronous, slightly slower changes than Axi. We therefore chose Tiv/Len for\nall subsequent functional assays. To avoid unexpected introduction of Tiv and\nLen, the revised manuscript now includes data verifying the phenotype with\nboth inhibitors (revised Supplementary Fig. 1a-c) (revised Page 5, Line 13-20),\n25\nshowing equivalent suppression of VEGF signaling activity and consistent\nmorphological outcomes.\nSupplementary Fig. 1: VEGF signaling in hESCs exhibits high activity in\nthe undifferentiated state and becomes inactivated upon differentiation.\n(Related to Fig. 1)\na, Bright-field images of SHhES8 hESCs treated with DMSO (control), VEGFR\ninhibitors (VEGFRi: Tivozanib, 10 μM) and (VEGFRi: Lenvatinib, 6 μM),\nrespectively, for 2 days. Scale bars, 500 μm (top) and 100 μm (bottom).\nb, c, Representative western blot (b) and quantification (c) analysis results for\nrelative protein levels of indicated proteins in SHhES8 hESCs treated with\nDMSO (control), PDGFR inhibitor (PDGFRi: Crenolanib, 2 μM), VEGFR\ninhibitor (VEGFRi: Axitinib, 5 μM; Tivozanib, 10 μM; Lenvatinib, 6 μM), and\nEGFR inhibitor (EGFRi: Erlotinib, 10 μM), respectively, for 10 minutes.\nMinor comment 4: Page 9 – Line 14-15\n“… consistent with the outcomes of Gene Ontology (GO), pathway and process\nenrichment analysis, as well as GSEA enrichment analysis.”\nThis are three times the same or at least related wordings. The general term for\nthe analysis where you look for enriched processes is ‘GSEA’, for which GO and PID\n(the latter of which is used in panels 3c & 3d) are different databases that can be\nused. Using this sentence is therefore essentially saying three times the same thing and can be replaced by simply saying\n‘consistent with the outcomes of our GSEA’.\nResponse to minor comment 4: Thank you for pointing this out. We have\nchanged the sentence to: “…, consistent with the outcomes of our GSEA.”\n26\n(revised Page 10, Line 4).\nMinor comment 5: Page 14 – Line 1.“for 3 or 5 days.”\nThere seems to be no uniformity in the timepoints that are being tested. For\nmany experiments, D1, D2 and D6 are used, while at other times D3 and D5\nare tested without justification. Then again, in the next panel D3 and D8 are\nevaluated.\nResponse to minor comment 5: In this study, timepoints were selected\naccording to the time length required for different strategies of VEGF signaling\ninactivation to produce phenotypes in cells and the kind of phenotypes\nexamined. For instance, small-molecule inhibitors-mediated VEGF signaling\ninhibition was routinely assessed at day 3, whereas soluble receptor\noverexpression-mediated VEGF signaling inhibition was examined at days 1,\n2\nand 6. For flow-cytometric quantification of HLA-ABC (revised Fig. 6b), we\ncollected cells at day 5 when the surface-antigen loss reached a peak, as for\nthe bisulfite analysis of the ELF5 promoter (revised Fig. 6d), we chose days 3\nand 8 to show progressive and lineage commitment-associated silencing. We\napologize for any confusions this might have caused. In the future investigation,\nwe will try to make timepoints more uniform, and thanks for your comment.\nMinor comment 6: Page 16 – Line 18-19\n“We named this new protocol 2i/L”. The name 2i/L is confusing with the 2i/L\nmedium used for the maintenance of naïve mESCs. Furthermore, Linneberg-\nAgerholm et al. (2019) used a similar term (h2iL).\nResponse to minor comment 6: Thank you for bringing this issue up. As I\nexplained in my response to your major comment 9, we plan to remove the\ndata-related to naïve pluripotency regulation presented in our original\nmanuscript. Thus, the name of this new protocol, at least for now, seems not\nan issue. Nevertheless, we will reconsider the name for this new protocol.\nMinor comment 7: Figure 7f\npluripotent cells. This means that the variability in the dataset will be determined\nby the levels of naïve and primed markers. After demonstrating that VEGFRi\ndisrupts primed pluripotency but blocks the naïve-primed transition, it is\ntherefore not that surprising that the 2i/L cells (obtained after 5i/LA conversion),\ncluster more closely to the 5i/LA cells than to the primed cells. As long as the\nprimed characteristics are not recovered and some naïve hallmarks are\nmaintained, these cells will always cluster closer to 5i/LA cells. This figure could\ngo to supplementary.\nResponse to minor comment 7: For the same reason I mentioned above, the\noriginal Fig. 7f would not be included in our revised manuscript. Nevertheless,\nI understand and thanks for your advice.\nMinor comment 8: Figure 7i-k\nThe DNA methylation status of the 5i/LA and 2iL cells are compared to primed\nand naïve cells. However, since the naïve cells used throughout the\nexperiments are 5i/LA-grown, what is the reference condition called ‘naïve’ that\nis shown here? It doesn’t appear to be 5i/LA since the levels of KLF17 are very\ndifferent.\nResponse to minor comment 8: The reference condition labeled “naïve” was\nreferred to naïve hESCs cultured in the 5i/L/A medium at passage 6 before\nbeing switched to the 2i/L formulation. For the same reason, data in original\nFigure 7i-k would not be included in the revised manuscript. We will explain the\nreference cells more clearly in the future.\nMinor comment 9: Furthermore, in panel 7i, 5i/LA and 2i/L do show quite a\ndifference in methylation status, unlike what is mentioned in the text.\nResponse to minor comment 9: Thank you for pointing this out. While the\nmethylation levels of the MECP2 promoter at both P6+6 and P6+11 as well as\nXIST promoter at P6+6 were comparable between 5i/LA and our 2i/L, the XIST\n28\npromoter methylation level was higher in 2i/L than 5i/LA at P6+11. We will\ndescribe the results more precisely in the future. For the same reason, data in\noriginal Figure 7i would not be included in the revised manuscript.\nMinor comment 10: Methods – Naïve hESC culture and differentiation\nThe methods section mentions that all hESCs and their derivatives (i.e. the\nnaïve cells) are cultured at 21% O2. Naïve hESCs are generally grown in\nhypoxia (5% O2). For the 5i/LA conversion specifically, this is specified in Fisher\net al. (2021). Is there a particular reason why normoxia is chosen here?\nResponse to minor comment 10: Yes, naïve hESCs are generally grown in\nhypoxia (5% O ). However, Fischer et al. previously demonstrated the efficacy\n2\nof converting primed hESCs to the naïve state under normoxic conditions7.\nMore importantly, my lab has successfully converted primed hESCs to the naïve\nstate using the 5i/L/FA method under the normoxia condition with consistent\nresults8. In addition, culturing cells under the normoxia condition avoids the\nneed for specialized hypoxic incubators. Therefore, my lab routinely establishes\nand maintains naïve hESCs under the normoxia condition, including in this\ncurrent study.\nReviewer #4 (Remarks to the Author):\nTo answer the question more clearly, I divided Reviewer #4 comments into 7\nquestions below.\nComment 1: Wu, Wen, Zhu et al.‘s manuscript “Critical roles of endogenous\nVEGF signaling for cell fate decision in human embryonic stem cells” digs into\nthe well explored space of signal transduction within human pluripotency to\nevaluate the potential role for VEGF signaling factors to support self-renewal\nand block differentiation. Beginning with a somewhat simple screen, the authors\nfirst test several less explored RTK pathways for their ability to impact “primed”\nstate hESC self-renewal, and find a convincing effect with the VEGFR inhibitor\nAxitinib. After confirming that this effect conforms with the endogenous\n29\nexpression of VEGF receptors and ligands within primed state cells, the authors\nthen thoroughly validate the role of VEGF signaling in promoting self-renewal\nwithin this state using multiple independent small molecule inhibitors, the\ninduction of soluble VEGF receptors (which presumably compete for active\nligand), and bulk induction of VEGFR1 and 2 targeting sgRNAs. They then\nutilize combinations of small molecule inhibitors and their soluble receptor\ninduction system to demonstrate how VEGF signaling first supports NANOG\nexpression which, upon destabilization, triggers BMP-supported spontaneous\ndifferentiation towards a trophoblast stem cell-like state (which they confirm via\nmarker-based analyses such as IF, FACs, RT-qPCR alongside limited\nepigenetic analysis of the ELF5 promoter). Finally, the authors demonstrate that\nVEGF related genes are only induced upon the transition from naïve to primed\nstate pluripotency, and apply this concept to replace the conventional 5i/L/A\ncondition with a limited VEGF-inhibitor-based “2i/L” condition that eliminates the\nhistorically problematic MEK1 inhibitor PD0325901 with minimal, if not\nimproved, effects on genomic integrity and self-renewal.\nOverall, I think this is a thorough, mature and compelling work, with multiple\nvalidations for each experimental observation. The authors offer an overall\ncoherent framework from the initial observation that VEGF signaling appears to\nspecifically impact primed state pluripotency, through the dissection of the\ngenetic pathway from VEGF to NANOG to BMP-based induction of\n“trophoblast-like cells,” and to the developmental induction of this pathway as\npart of the naïve to primed transition. However, there are several areas where\nI feel that the results or interpretation of the results are overstated, sometimes\ncritically so. Moreover, a detailed connection for precisely how VEGF signaling\nmight interact with the pluripotency network (through which arm of its many\ndownstream effectors) is not explored, but needs to be discussed more\nthoroughly in the discussion. I feel that this is particularly important given the\nauthors claims related to the potential of VEGF inhibition to act as a substitute\nfor the more conventional MEK1 inhibitor. A paragraph in the discussion\n30\nhighlighting how VEGF may interact with more conventional signaling pathway\ncomponents would provide important context.\nResponse to comment 1: We sincerely appreciate your thorough evaluation\nof our manuscript and constructive advice. As suggested, paragraphs\ndiscussing how VEGF signaling might link to the known pluripotency network\nand conventional signaling pathway components are included in our revised\nmanuscript. For specific details, please refer to the discussion section (Page\n17, Line 17-30; Page 18, Line 26 - Page 19, Line 14) and are presented below.\n“The FGF signaling pathway, a well-established RTK signaling essential\nfor primed hESC pluripotency, shares downstream branches such as MEK/ERK\nand PI3K/AKT with the VEGF pathway. Given that both FGFR and MEK\ninhibitors could induce trophoblast marker expression, we propose that the\nVEGF and FGF pathways converge on the MEK/ERK cascade to sustain hESC\npluripotency. Our findings demonstrate that VEGF pathway inactivation\ndisrupted primed hESC self-renewal even in the mTeSR1 medium\nsupplemented with FGF, suggesting that exogenous bFGF supplemented in\nthe medium could not replace VEGF signaling. Moreover, VEGF inhibitors not\nonly triggered trophoblast-like lineage specification but also led to cell death,\nwhich is likely associated with the cell survival-linked PI3K/AKT branch. The\nshared and distinct roles of the FGF and VEGF signaling pathways in sustaining\nhuman primed pluripotency represent an intriguing research focus that merits\nin-depth exploration.”\n“As a core pluripotency transcription factor, the mechanisms by which\nNANOG expression is dynamically modulated have remained a key research\nfocus in the stem cell field since its discovery. Previous studies showed that\nboth TGF-β and bFGF could sustain NANOG expression in hESCs, although\nthe underlying mechanism is not completely elucidated. TGF-β superfamily\nmembers (ACTIVIN A, NODAL and TGF-β) were reported to activate SMAD2/3,\nwhich directly enhanced NANOG expression. The observation that NANOG OE\nbypassed the requirement for both TGF-β and FGF signaling in maintaining\n31\nhESC self-renewal implies that these pathways act via the modulation of\nNANOG expression. In this study, we identify a new signaling pathway that\nplays a critical role in sustaining NANOG expression in primed hESCs. Thus,\nNANOG likely functions as a central hub responding to various exogenous and\nendogenous signaling pathways to safeguard hESC identity. Nevertheless, it\nremains an unresolved mystery how VEGF signaling orchestrates NANOG\nexpression in hESCs. We sought to identify specific proteins mediating the\nregulatory effect of VEGF signaling on NANOG expression using proteomic and\nphosphoproteomic approaches. However, we failed to detect convincing\nsubstrate candidates. Thus, it remains unclear how VEGF signaling regulates\nNANOG expression in hESCs.”\nComment 2: Below are the areas that I think are the most problematic in terms\nof language and interpretation, as well as proposed solutions for making sure\nthis very interesting study is clearly communicated to the field.\nIn particular, the reported differentiation of primed state hESCs into bona fide\nTSCs is still controversial, as this would represent a bona fide\ntransdifferentiation event that includes global rewiring of the epigenome. As\nsuch, it would not conform with the developmental dynamics of the early\nepiblast in vivo. Instead, BMP or CDX2 induction of primed state cells seems to\ncreate a complex hybrid, possibly amniotic state that still fits many of the\nsomewhat limited criteria tested by the authors for a trophoblast-like identity\n(see in particular PMID: 33831365IF: 19.8 Q1 as well as PMID: 21816365IF:\n19.8 Q1 ). My suggestion is that the authors consider the pre-existing literature\ncarefully when developing and explaining their model. To me, they must either\nuse the incorporated data and criteria from these previously papers to show\nmore completely that their cell types resemble more conventional hTSCs\ncompared to primed state derived amnion-like cells or they must substantially\nrevise this section of the manuscript to highlight these prior works. They must\nmake it clear that VEGF blocked primed state pluripotent cells from\n32\nspontaneous differentiation as driven by BMP4 and discuss how this triggers\nthe induction of a trophoblast-like state that may be more equivalent to early\namnion, but remove statements or results intended to validate the equivalence\nof these cells to bona fide TSCs. To me, it is a perfectly interesting and sufficient\nfinding on its own without the added utility of being suitable as a model for\ntrophoblast or placental development.\nResponse to comment 2: Thank you for your valuable suggestions. We\nacknowledge that the issue of whether primed-state hESCs give rise to\ntrophoblast lineages remains debated now. In this study, we found that\ninhibition of VEGF signaling disrupted self-renewal of primed hESCs severely,\nleading to differentiation with the morphology and transcriptome similar to those\nof the trophoblast lineage. The uniformly differentiated cells displayed a\nmorphology reminiscent of trophoblasts and expressed human TSC markers,\nsuch as CDX2, TP63 and ELF5. Moreover, these cells could be induced to\nexpress marker genes of ST and EVT cells. Therefore, we considered these\ncells as TSC-like cells in the original manuscript. After reading your comments,\nwe realize that our characterization is not sufficient to claim that they are hTSC-\nlike cells. In fact, these cells lack a self-renewal capacity when they are cultured\nin the medium supporting hESC self-renewal. We have emphasized this fact in\nthe revised manuscript and do not claim them as TSCs in the revised\nmanuscript. Instead, in the revised manuscript, we consider that VEGF\nsignaling inhibition in primed hESCs could induce trophoblast-like\ndifferentiation, based on the following experimental evidence: i) differentiated\ncells derived from VEGFRi-treated hESCs displayed a uniform morphology\nreminiscent of trophoblasts; ii) these cells exhibited lower levels of HLA-ABC,\nwhich is expressed in amnion; iii) they expressed human TE and CT markers,\nsuch as TROP2, GATA3, CDX2, TP63 and ELF5; iv) they had lower\nmethylation levels in the ELF5 promoter compared to undifferentiated hESCs;\nv) they could be induced to express marker genes of ST and EVT cells. In\naddition, a set of genes, including ACMSD, AQP9, CLDN22, TMEM174,\nexpressed in the amnion 9. We found that these genes were not upregulated in\nVEGF signaling deficiency-induced differentiated cells based on our RNA-seq\ndata (Response Letter Fig. 6). Nevertheless, some VEGF signaling inhibition-\nupregulated genes such as GATA2, GATA3, HAND1, TFAP2A, and KRT7 were\nalso expressed in amnion cells. Moreover, there are differences in cell growth\nenvironments between our in vitro culture condition (mTeSR1) and the true in\nvivo developmental environment. Therefore, we do not rule out the possibility\nthat trophoblast-like cells induced by the inactivation of the VEGF signaling\npathway in primed hESCs may exhibit some characteristics of amnion cells.\nThese statements are included in the discussion of the revised manuscript\n(Page 20, Line 19 – Page 21, Line 8).\nResponse Letter Fig. 6: A set of previously reported amnion marker genes\ndo not upregulate during primed hESC differentiation caused by VEGF\nsignaling deficiency.\na, b, Heatmaps showing Log RPKM and Log TPM of selected amnion-,\n10 10\ntrophoblast-, and pluripotency-marker genes in SS iOE hESCs (a) and WT\nhESCs (b) under the indicated conditions, respectively, based on our RNA-seq\ndata. Amnion genes: ACMSD, AQP9, CLDN22, TMEM174, MCCD1, HBA1,\nand SOX11; trophoblast gene: GATA3; pluripotency gene: NANOG.\nc, A heatmap showing Log TPM of selected genes that highly and specifically\n10\nexpressed in the amnion 9. CT, cytotrophoblast.\nIn addition, we have briefly reviewed related literatures and provided our\nown opinion, as followings: “Following embryo implantation in mammalian\n34\ndevelopment, the trophectoderm (TE), arising from the outer cell layer of the\nblastocyst, gives rise to cytotrophoblasts (CTs), which can generate two\ndifferentiated cell types in human placenta, extravillous trophoblasts (EVTs)\nand syncytiotrophoblasts (STs). Human trophoblast stem cells (hTSCs) can be\nderived from the TE or first-trimester CTs, and can also be converted from\nhESCs. BMP4 has long been used to induce primed hESCs to differentiate into\neither the embryonic mesoderm or the extra-embryonic trophoblast, depending\non the specific experimental conditions. However, in recent years, some\nresearchers argued that differentiated cells derived from primed hESCs\nexhibited features resembling amnion rather than trophoblast cells, unlike naive\nhESCs, which can efficiently differentiate into trophoblasts. Indeed, the BMP\npathway also plays a crucial role in amnion development, which share many\ncommonalities with trophoblasts. Nevertheless, more recent works have\nchallenged the notion of absolute lineage restriction in primed hPSCs. Several\nstudies have showed that primed hESCs can generate trophoblast cells and\nhTSCs. For examples, the study by Wei et al. reported the successful\ngeneration of TSCs from primed hPSCs and demonstrated that BMP4\nsignificantly enhanced this process. Seetharam et al. revealed that primed\nhPSCs differentiated into placental trophoblasts, but not amnion cells, by BMP-\ndirected differentiation protocols.” (Page 19, Line 29 - Page 20, Line 19).\nCollectively, we consider that the lineage specification limitation is not\nabsolute. Given that somatic cells could be induced to become pluripotent cells,\nit is possible that a given cell could be induced to any cell types only if the\ncondition is appropriate and the barrier to restrict its potential is removed. The\nsame applies to the potential of primed hESCs to become trophoblast lineage.\nIt is likely that primed hESCs do not differentiate to trophoblast lineages, but to\namnion cells, under basal conditions. However, they could be induced to the\ntrophoblast lineages when the restriction is removed. I think that this is the case\nwhere the deficiency of VEGF signaling, which is robustly active to prevent\n35\nhESCs from aberrant specification at normal conditions, provides a chance for\nprimed hESCs to enter into the trophoblast program.\nComment 3: The authors have done a convincing job of showing that VEGF\nsignaling is upstream of NANOG, and that BMP4-based differentiation is\ndownstream. However, the OE/rescue experiments they have conducted are\nmore similar to an “epistasis” test (establishing the genetic order of events) than\nthey are about direct or functional connections. The observation that NANOG\nbinds some BMP-associated effectors is interesting, but derived from previously\ngenerated/public data and does not prove that NANOG is directly suppressing\nthese targets. The language (and subheader) related to the section “NANOG\ndirectly suppresses expression of genes associated with the BMP pathway and\ntrophoblast lineage in primed hESCs” should be revised accordingly to better\nutilize wording related to who is acting upstream to stabilize NANOG expression\n(VEGF) or downstream to support spontaneous differentiation (BMP4). The\nobservation that NANOG appears to bind some of these promoters can be\nnoted and the authors can suggest that this might suggest that NANOG plays\ndual activating and inhibitory roles, but anything beyond that is speculation not\npresent in the data.\nResponse to comment 3: Based on the following two results, we concluded\nthat NANOG is a major mediator downstream of VEGF signaling: 1) VEGF\nsignaling inhibition dramatically reduced NANOG expression; 2) NANOG OE\ncould largely rescue phenotypes induced by VEGF signaling deficiency.\nHowever, these results do not indicate direct or indirect link between VEGF\nsignaling and NANOG. Yes, VEGF signaling is upstream of NANOG expression.\nHowever, we are currently not clear how VEGF signaling maintains NANOG\nexpression. It is unlikely that VEGFR controls NANOG expression directly, as it\nis not a transcription factor. We have conducted proteomics and phospho-\nproteomics, hoping to find the factor linking VEGFR and NANOG expression.\nUnfortunately, we failed to find such a factor. On the other hand, our results\n36\nindicate that NANOG could directly regulate expression of certain genes\ninvolved in BMP signaling (e.g. Id1, Id4 and BMP4) and trophoblast\ndifferentiation (e.g. GATA3, CDX2, TP63, and TFAP2A), as NANOG bound to\nthe promoters of these genes and its downregulation led to the upregulation of\ntheir expression.\nIn the revised manuscript we have: 1) changed the sub-heading (Page 12,\nLine 14-15) to “NANOG binds to and regulate genes associated with the BMP\npathway and trophoblast lineage in primed hESCs”; 2) re-written the relevant\nparagraph (Page14, Line 4-9): “These data indicate that NANOG safeguards\nhuman primed pluripotency through both activating of pluripotency-associated\ngenes and suppressing differentiation-associated genes (e.g. BMP pathway\ncomponents and trophoblast lineage genes). Nevertheless, the underlying\nmechanisms through which NANOG fulfills its dual regulatory function in gene\nexpression remain incompletely understood.” 3) discussed the relationship\namong VEGF signaling, NANOG expression and BMP signaling in primed\nhESCs. (Page 18, Line 1 - Page 19, Line 28)\nMinor comment 4: The novel “2i/L” experiments are interesting but overall\nrepresent a shallow validation. I think this is fine for a wide-ranging investigation\nof VEGF signaling in human pluripotent stem cells, but the authors should\nconsider if it is worth adding additional validation to bolster their claim, including\ntesting additional stem cell lines, evaluating their functional potential according\nto the conventions used for the publication of these prior conditions, and more\ncarefully evaluating the effects of this condition on long term genetic and\nepigenetic stability. I fully realize that such an investigation is well outside the\nscope of this study. If they elect to proceed with the validation they have already\ndone, the authors should revise their language to\nhighlight that their 2i/L condition offers intriguing preliminary results for simpler\nand more stable naïve hPSC culture conditions, but that further work would be\nnecessary to establish that these conditions are sufficient to capture the\ncomplexities of self-renewal and differentiation potential long term.\nResponse to comment 4: Thank you very much for your constructive\nsuggestions. We fully agree that our 2i/L validation is only preliminary and that\nthe extensive works required to firm-up those claims lies beyond the scope of\nthe present study. Similar comments were also made by Reviewer#1, who think\nthat “the first part in primed PSCs is solid and would have been sufficient to\ninclude in this publication”. Therefore, we have removed the entire 2i/L dataset\n(original Fig. 7 and Supplementary Fig. 6) and all associated statements from\nthe revised manuscript. Therefore, claims about simplified naïve culture\nconditions will not be included in the revised manuscript. The discussion now\ncontains a single, cautiously worded sentence stating that “Whether VEGF\nsignaling exerts a pluripotency stage-specific role during the naïve-to-primed\ntransition of hESCs, and whether its inhibition can promote primed-to-naïve\nconversion or sustain long-term naïve pluripotency, warrants further\ninvestigation.” (Page 17, Line 11-14). By deleting these naïve pluripotency-\nassociated preliminary results, the paper now focuses solely on its mechanistic\ncore: VEGF signaling safeguards primed hESC self-renewal by sustaining\nNANOG expression and restraining BMP-pathway activation.\nWe also appreciate your suggestions for future experiments (additional\nlines, functional assays, long-term genetic/epigenetic stability, etc.).\nComment 5: I think the statement on Page 19, Line 28 reading “The finding\nthat the uterine fluid from women with unexplained infertility contains decreased\nVEGFA levels further implicates a crucial role of VEGF signaling in early human\nembryonic development” should be removed. The observations or\ninterpretations of this sort of clinical study are much more likely about\nplacentation rather than the differentiation of the epiblast (which the authors\nhave already stated is an endogenous signaling relationship).\nResponse to comment 5: As suggested, we have deleted the sentence and\nassociated reference.\nclearly in the text before presenting data related to Xist and MeCP2 methylation\nin Figure 7.\nResponse to comment 6: Thank you for pointing this out. In line with your\ncomments and the strong recommendation of Reviewer #1, we have removed\nthe entire 2i/L dataset (former Fig. 7 and Supplementary Fig. 6) and all\nassociated statements from the revised manuscript. Consequently, no Xist or\nMeCP2 methylation data are presented in the revised manuscript. Nevertheless,\nthe following statement is included in the Method details of the revised\nmanuscript: “SHhES8 (Karyotype, 46, XX; a female hESC line derived in our\nlab)”.\nComment 7: The title could be revised to something more specific, such as\n“VEGF signaling supports primed-state human embryonic stem cell self-\nrenewal” or “Endogenous VEGF signaling blocks spontaneous BMP4-based\ndifferentiation in primed state human embryonic stem cells.”\nResponse to comment 7: Thank you for the constructive suggestion. We have\nreplaced the original title with the more specific and accurate wording:\n“Endogenous VEGF signaling: a guardian of human primed pluripotency”. I\nhope that you agree with this change.\nReferences:\n1. Gerber, H.P. et al. VEGF regulates haematopoietic stem cell survival by an\ninternal autocrine loop mechanism. Nature 417, 954-958 (2002).\n2. Xu, R.H. et al. BMP4 initiates human embryonic stem cell differentiation to\ntrophoblast. Nat. Biotechnol. 20, 1261-1264 (2002).\n3. Hyslop, L. et al. Downregulation of NANOG induces differentiation of human\nembryonic stem cells to extraembryonic lineages. Stem Cells 23, 1035-1043\n(2005).\n4. Zaehres, H. et al. High-efficiency RNA interference in human embryonic stem\ncells. Stem Cells 23, 299-305 (2005).\n5. Roberts, R.M. et al. The role of BMP4 signaling in trophoblast emergence\nfrom pluripotency. Cell Mol Life Sci 79, 447 (2022).\n6. Horii, M. et al. Human pluripotent stem cells as a model of trophoblast\n39\ndifferentiation in both normal development and disease. Proc Natl Acad Sci\nU S A 113, E3882-3891 (2016).\n7. Fischer, L.A., Khan, S.A. & Theunissen, T.W. Induction of Human Naïve\nPluripotency Using 5i/L/A Medium. Methods Mol Biol 2416, 13-28 (2022).\n8. Shao, M. et al. NANOG governs cell metabolism and redox homeostasis in\nhuman naïve embryonic stem cells. EMBO reports, 1-33-33 (2025).\n9. Seetharam, A.S. et al. The product of BMP-directed differentiation\nprotocols for human primed pluripotent stem cells is placental trophoblast\nand not amnion. Stem Cell Rep 17, 1289-1302 (2022).\nRE：Manuscript # NCOMMS-25-32707A)\nDear Editors and Reviewers,\nWe appreciate your efforts on this manuscript. Your comments are very\nconstructive. In accordance with your suggestions and following the discussion\nwith the editor, we have made final revision on the manuscript. Our point-by-\npoint response letter is as follows:\nReviewer #1 (Remarks to the Author):\nComment: The authors have addressed my comments in the revised version\nof their work (that omits all sections related to human naive pluripotency).\nResponse: Thanks.\nReviewer #2 (Remarks to the Author):\nComment: In the revised version of the manuscript entitled “Critical roles of\nendogenous VEGF signaling for cell fate decision in human embryonic stem\ncells,” the authors have done an excellent job addressing all major and minor\ncomments raised by the reviewers. They have performed a substantial number\nof additional experiments, which convincingly address the concerns and\nsignificantly strengthen the manuscript. All of our questions have been clearly\nand satisfactorily answered, and we would like to congratulate the authors for\ntaking the reviewers’ comments seriously and for the thoroughness of their\nrevisions.\nWe particularly appreciated the authors’ hypothesis, also supported by\npreviously published studies, that VEGFA may act through an intracrine\nmechanism in hESCs. We would like to encourage the authors to consider\nwhether this aspect could be included in the manuscript. That said, we fully\nunderstand if this hypothesis is still too preliminary or if its inclusion might shift\nthe focus of the current study and affect the overall flow of the article.\ninclusion of our preliminary data of the VEGFA intracrine mechanism, we would\nlike to report this finding in a separate paper. After discussing with the Editor,\nthe related result is not included in the final revised manuscript. I appreciate\nyour understanding.\nReviewer #3 (Remarks to the Author):\nComment: I co-reviewed this manuscript with one of the reviewers who\nprovided the listed reports. This is part of the Nature Communications initiative\nto facilitate training in peer review and to provide appropriate recognition for\nEarly Career Researchers who co-review manuscripts.\nResponse: Thanks.\nReviewer #4 (Remarks to the Author):\nComment 1: I find the changes made in Wu, Wen, Zhu et al.‘s revised\nmanuscript, now titled “Endogenous VEGF signaling: a guardian of human\nprimed pluripotency,” to have substantially improved and clarified the work.\nThe decision to remove the description of VEGF’s role in naïve state\npluripotency and save these observations for a second, more mature and\nvalidated study is appropriate and doesn’t change the impact of the\nmanuscript’s other key findings. Efforts to more directly connect VEGF to AKT\nand ERK signaling in hESCs add important context and many of the arguments\nmade by the authors to other comments are sound. I thank them very much for\ntheir thorough effort and hard work.\nResponse 1: Thank you very much for your positive comments.\nComment 2: To me, two data points are provided only to reviewers that I feel\nare important for the reader and should be strongly considered for inclusion in\nthe final manuscript: the ambiguity related to the explicit signaling mechanism\n(Reviewer 2 Comment 4, Response Letter Figures 2, 3 and 4) and the\ntranscription-based validation of trophoblast versus amnion markers (Reviewer\n2\nthese findings be incorporated into the extended data and briefly described in\ntext. I agree with the authors that the interpretation that VEGFA may act through\nan intracrine mechanism is largely speculative at this point, but the results that\nrecombinant VEGFA does not rescue BAP induced trophoblast differentiation,\nthat signal peptide deficient VEGFA O/E does, and that VEGFA is not detected\nin supernatant are all important results, even if they do not conform with most\ncurrent literature. Just because these results add some ambiguity to the model\nand include negative findings does not mean they aren’t important to include,\nand I feel that the reader would be misleaded about the apparent mechanism\nwithout them. However, I’m happy to leave this issue to the editor’s\ndiscretion after considering the comments from the other reviewers.\nResponse 2: I understand that you encouraged us to include our interesting\nbut preliminary data in the manuscript. However, we would like to report these\nfindings in a separate paper. After discussing with the Editor, we decide not to\ninclude these data in the current manuscript.\nComment 3: Regarding the heatmaps in Response Letter 6, I would advise\nincluding these to provide additional validation of the transcriptional responses\nthat are and are not typical of their system. They also provide helpful context\nbut are not as important as the mixed findings related to those related to VEGF\nsensing within their system.\nResponse 3: Thanks for your advice, the heatmap shown in our response letter\nfigure 6 is now included in the final revised manuscript (Fig.6h).\nThank you again for your help and I hope that our final revision is\nsatisfactory for its publication in Nature Communications.\nYours Sincerely,\nYing Jin, M.D., Ph.D.\n3"}, {"round": 2, "reviewer_comments": {"reviewer_1": "The authors have addressed my comments in the revised version of their work (that omits all sections related to human naive pluripotency).", "reviewer_2": "In the revised version of the manuscript entitled “Critical roles of endogenous VEGF signaling for cell fate decision in human embryonic stem cells,” the authors have done an excellent job addressing all major and minor comments raised by the reviewers. They have performed a substantial number of additional experiments, which convincingly address the concerns and significantly strengthen the manuscript. All of our questions have been clearly and satisfactorily answered, and we would like to congratulate the authors for taking the reviewers’ comments seriously and for the thoroughness of their revisions.\nWe particularly appreciated the authors’ hypothesis, also supported by previously published studies, that VEGFA may act through an intracrine mechanism in hESCs. We would like to encourage the authors to consider whether this aspect could be included in the manuscript. That said, we fully understand if this hypothesis is still too preliminary or if its inclusion might shift the focus of the current study and affect the overall flow of the article.", "reviewer_3": "I co-reviewed this manuscript with one of the reviewers who provided the listed reports. This is part of the Nature Communications initiative to facilitate training in peer review and to provide appropriate recognition for Early Career Researchers who co-review manuscripts.", "reviewer_4": "I find the changes made in Wu, Wen, Zhu et al.‘s revised manuscript, now titled “Endogenous VEGF signaling: a guardian of human primed pluripotency,” to have substantially improved and clarified the work. The decision to remove the description of VEGF’s role in naïve state pluripotency and save these observations for a second, more mature and validated study is appropriate and doesn’t change the impact of the manuscript’s other key findings. Efforts to more directly connect VEGF to AKT and ERK signaling in hESCs add important context and many of the arguments made by the authors to other comments are sound. I thank them very much for their thorough effort and hard work.\nTo me, two data points are provided only to reviewers that I feel are important for the reader and should be strongly considered for inclusion in the final manuscript: the ambiguity related to the explicit signaling mechanism (Reviewer 2 Comment 4, Response Letter Figures 2, 3 and 4) and the transcription-based validation of trophoblast versus amnion markers (Reviewer 4 Comment 2, Response Letter Figure 6). In both cases, I would propose that these findings be incorporated into the extended data and briefly described in text. In both cases, I would propose that these findings be incorporated into the extended data and briefly described in text. I agree with the authors that the interpretation that VEGFA may act through an intracrine mechanism is largely speculative at this point, but the results that recombinant VEGFA does not rescue BAP induced trophoblast differentiation, that signal peptide deficient VEGFA O/E does, and that VEGFA is not detected in supernatant are all important results, even if they do not conform with most current literature. Just because these results add some ambiguity to the model and include negative findings does not mean they aren’t important to include, and I feel that the reader would be misleaded about the apparent mechanism without them. However, I’m happy to leave this issue to the editor’s discretion after considering the comments from the other reviewers.\nRegarding the heatmaps in Response Letter 6, I would advise including these to provide additional validation of the transcriptional responses that are and are not typical of their system. They also provide helpful context but are not as important as the mixed findings related to those related to VEGF sensing within their system"}, "author_response": "January 30, 2026\nRE：Manuscript # NCOMMS-25-32707A)\nDear Editors and Reviewers,\nWe appreciate your efforts on this manuscript. Your comments are very\nconstructive. In accordance with your suggestions and following the discussion\nwith the editor, we have made final revision on the manuscript. Our point-by-\npoint response letter is as follows:\nReviewer #1 (Remarks to the Author):\nComment: The authors have addressed my comments in the revised version\nof their work (that omits all sections related to human naive pluripotency).\nResponse: Thanks.\nReviewer #2 (Remarks to the Author):\nComment: In the revised version of the manuscript entitled “Critical roles of\nendogenous VEGF signaling for cell fate decision in human embryonic stem\ncells,” the authors have done an excellent job addressing all major and minor\ncomments raised by the reviewers. They have performed a substantial number\nof additional experiments, which convincingly address the concerns and\nsignificantly strengthen the manuscript. All of our questions have been clearly\nand satisfactorily answered, and we would like to congratulate the authors for\ntaking the reviewers’ comments seriously and for the thoroughness of their\nrevisions.\nWe particularly appreciated the authors’ hypothesis, also supported by\npreviously published studies, that VEGFA may act through an intracrine\nmechanism in hESCs. We would like to encourage the authors to consider\nwhether this aspect could be included in the manuscript. That said, we fully\nunderstand if this hypothesis is still too preliminary or if its inclusion might shift\nthe focus of the current study and affect the overall flow of the article.\ninclusion of our preliminary data of the VEGFA intracrine mechanism, we would\nlike to report this finding in a separate paper. After discussing with the Editor,\nthe related result is not included in the final revised manuscript. I appreciate\nyour understanding.\nReviewer #3 (Remarks to the Author):\nComment: I co-reviewed this manuscript with one of the reviewers who\nprovided the listed reports. This is part of the Nature Communications initiative\nto facilitate training in peer review and to provide appropriate recognition for\nEarly Career Researchers who co-review manuscripts.\nResponse: Thanks.\nReviewer #4 (Remarks to the Author):\nComment 1: I find the changes made in Wu, Wen, Zhu et al.‘s revised\nmanuscript, now titled “Endogenous VEGF signaling: a guardian of human\nprimed pluripotency,” to have substantially improved and clarified the work.\nThe decision to remove the description of VEGF’s role in naïve state\npluripotency and save these observations for a second, more mature and\nvalidated study is appropriate and doesn’t change the impact of the\nmanuscript’s other key findings. Efforts to more directly connect VEGF to AKT\nand ERK signaling in hESCs add important context and many of the arguments\nmade by the authors to other comments are sound. I thank them very much for\ntheir thorough effort and hard work.\nResponse 1: Thank you very much for your positive comments.\nComment 2: To me, two data points are provided only to reviewers that I feel\nare important for the reader and should be strongly considered for inclusion in\nthe final manuscript: the ambiguity related to the explicit signaling mechanism\n(Reviewer 2 Comment 4, Response Letter Figures 2, 3 and 4) and the\ntranscription-based validation of trophoblast versus amnion markers (Reviewer\n2\nthese findings be incorporated into the extended data and briefly described in\ntext. I agree with the authors that the interpretation that VEGFA may act through\nan intracrine mechanism is largely speculative at this point, but the results that\nrecombinant VEGFA does not rescue BAP induced trophoblast differentiation,\nthat signal peptide deficient VEGFA O/E does, and that VEGFA is not detected\nin supernatant are all important results, even if they do not conform with most\ncurrent literature. Just because these results add some ambiguity to the model\nand include negative findings does not mean they aren’t important to include,\nand I feel that the reader would be misleaded about the apparent mechanism\nwithout them. However, I’m happy to leave this issue to the editor’s\ndiscretion after considering the comments from the other reviewers.\nResponse 2: I understand that you encouraged us to include our interesting\nbut preliminary data in the manuscript. However, we would like to report these\nfindings in a separate paper. After discussing with the Editor, we decide not to\ninclude these data in the current manuscript.\nComment 3: Regarding the heatmaps in Response Letter 6, I would advise\nincluding these to provide additional validation of the transcriptional responses\nthat are and are not typical of their system. They also provide helpful context\nbut are not as important as the mixed findings related to those related to VEGF\nsensing within their system.\nResponse 3: Thanks for your advice, the heatmap shown in our response letter\nfigure 6 is now included in the final revised manuscript (Fig.6h).\nThank you again for your help and I hope that our final revision is\nsatisfactory for its publication in Nature Communications.\nYours Sincerely,\nYing Jin, M.D., Ph.D.\n3"}]

Decision letter