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Conserved mechanisms of self-renewal and pluripotency in mouse and human ESCs regulated by simulated microgravity using a 3D clinostat – Cell Death Discovery

SMG affects the self-renewal of mESCs

To investigate the impact of SMG on the self-renewal of mESC, mESCs were cultured in a microgravity simulated Rotary Cell Culture System (RCCS, National Space Science Center, China) (Fig. 1A). The expression of core pluripotency regulators such as Oct4, Sox2, Nanog and Tbx3 increased at both the transcript and protein levels (Fig. 1B, C). However, the expression of other pluripotency factors, such as Klf2 and Klf5 remained unchanged under SMG (Fig. 1B). To ascertain that the observed upregulation of core pluripotency genes was not a result of shear stress induced by fluid motion, we conducted an experiment involving horizontal shaking of mESCs. We subsequently examined the expression of pluripotency-related genes in mESCs cultured within flasks placed on a horizontal shaker. Interestingly, the expression levels of Oct4, Sox2, and Tbx3 demonstrated only minimal alterations, while the expression of Nanog was downregulated under these shaking conditions (Fig. S1A). Collectively, these findings provide evidence that SMG governs the self-renewal of ESCs by orchestrating the expression of core pluripotency genes.

Fig. 1: Simulated microgravity affects the self-renewal of mESCs.
figure 1

A Schematics of the experimental design. mESCs were cultured under rotary and standard conditions at 37 °C in 5% CO2. Cells were harvested and used for subsequent analyses. B Transcript levels of pluripotency-associated genes in mESCs cultured for 4 days under 1 g and SMG conditions based on qPCR analysis. (n = 3 independent experiments, **p < 0.01,***p < 0.001, n.s, not significant). C Western blot for OCT4, SOX2, NANOG and TBX3 protein levels in mESCs cultured for 4 days under 1 g and SMG conditions; GAPDH served as a loading control. (n = 3 independent experiments). D Heat map depicts the changes in gene expression in mESCs cultured under 1 g and SMG conditions for 4 days. The color represents the Z score (row-wise) of the log2 FPKM values of the 4474 genes affected. E GO ontology analysis for biological processes associated with genes whose expression changed under SMG condition in (D). F Oxygen consumption rate (OCR) of mESCs cultured under 1 g and SMG conditions for 4 days measured with Mito stress test. (n = 3 independent experiments).

To elucidate the underlying mechanism by which SMG influences ESC self-renewal, we performed RNA sequencing (RNA-seq) to analyze global gene expression changes in ESCs under SMG. We identified 2262 significantly up- and 2212 down-regulated genes in response to SMG compared to the 1 g ground environment (Fig. 1D, Table S3). The dysregulation of genes associated with stem cell maintenance was observed, consistent with the perturbed self-renewal of ESCs under SMG (Fig. 1B, C, E). Moreover, we found enrichment of apoptosis-associated gene ontology (GO) terms among the downregulated genes in mESCs under SMG, suggesting the induction of apoptosis in mESCs (Fig. 1E). Indeed, after 96 h of culture under SMG, mESCs exhibited an increased propensity for apoptosis compared to the 1 g environment (Fig. S1B). KEGG enrichment analysis further revealed significant alterations in metabolic pathways involved in amino acid metabolism (glutathione, arginine, and proline metabolism) and carbohydrate metabolism (glyoxylate and dicarboxylate metabolism, and pyruvate metabolism) under SMG (Fig. S1C). Additionally, the mitochondrial oxygen consumption rate (OCR) of mESCs was lower under SMG, with reduced basal respiration (BR) and ATP production (AP) (Figs. 1F and S1D).

SMG regulates LIF/STAT3 pathway in mESCs

The LIF/STAT3 pathway is essential for the maintenance of mouse ESCs [36, 37]. RNA-seq analysis revealed the upregulation of LIF/STAT3 target genes, including Tbx3, Gjb3, Ly6g6e, Jam2, and Jam3, in mESCs cultured for 4 days under simulated microgravity (SMG) conditions (Fig. 1D). qPCR experiments further validated the increased expression of LIF/STAT3 target genes, such as Lama1, Gjb3, Ly6g6e, Tbx3, Mras, Fabp3, Esrrb, Lrrc34, Capns1, and Ppap2b, in response to SMG (Fig. 2A). Time course experiments demonstrated sustained upregulation of LIF/STAT3 target genes in mESCs cultured for 3 days under SMG conditions (Fig. S2A). We subsequently examined the expression of LIF/STAT3 target genes in mESCs cultured within flasks placed on a horizontal shaker. The expression levels of Tbx3, Gjb3, Lama1, Esrrb, and Ly6g6e demonstrated minimal alterations under the shaking conditions (Fig. S2B), ascertaining the observed upregulation of LIF/STAT3 genes was not a result of shear stress induced by fluid motion. Notably, the increased expression of LIF/STAT3 target genes returned to control levels upon transferring the cells back to normal gravity for 24 and 48 h (Fig. 2B). As expected, withdrawal of LIF under SMG conditions restored the expression of LIF/STAT3 target genes (Fig. S2C). Interestingly, while LIF withdrawal resulted in differentiated ESC morphology under normal gravity, ESCs cultured in non-LIF medium under SMG conditions exhibited morphology similar to ESCs in LIF-containing medium under normal gravity (Fig. 2C). This similarity may be attributed to the higher expression of pluripotency genes Oct4, Sox2, Nanog, and LIF/STAT3 target genes in ESCs cultured in non-LIF medium under SMG (Figs. 2D and S2D). ChIP-qPCR experiments confirmed the enhanced enrichment of phosphorylated STAT3 (p-STAT3) at LIF/STAT3 target genes in ESCs cultured under SMG conditions (Fig. 2E). The activation of Janus kinases (JAKs) occurs through the heterodimerization of LIFR and GP130 [38, 39]. Activated JAKs phosphorylate tyrosine residues on GP130, creating a docking site for STAT3 recruitment and subsequent activation through phosphorylation at tyrosine 705 [40, 41]. Western blot analysis revealed increased levels of both STAT3 and p-STAT3 under SMG (Fig. 2F). The elevated levels of p-STAT3 may be attributed to increased levels of JAK1, phosphorylated JAK1 (p-JAK1), GP130, and phosphorylated GP130 (p-GP130) in ESCs under SMG conditions (Fig. 2G, H). BRG1, the catalytic subunit with ATPase activity of the BAF chromatin remodeling complex, is essential for facilitating STAT3 access to its target loci [42]. However, the protein level and binding of BRG1 at STAT3 target genes remained unchanged under SMG conditions (Fig. S2E, F), indicating that SMG does not regulate the binding of STAT3 to its target genes through BRG1. Consistently, deletion of Arid1a, a subunit of the BAF complex, did not affect the upregulation of LIF/STAT3 target genes under SMG (Fig. S2G). The restoration of LIF/STAT3 target gene expression back to wild-type levels at normal gravity upon Arid1a deletion suggests that SMG does not influence STAT3 binding to its target genes through the BAF complex (Fig. S2G). Therefore, we conclude that SMG enhances the activity of the LIF/STAT3 pathway in a BRG1-independent manner.

Fig. 2: Enhanced activation of the LIF/STAT3 pathway in mESCs under SMG condition.
figure 2

A Differential expression of LIF/STAT3 target genes in mESCs after 4-day culture under SMG and 1 g conditions, assessed by qPCR analysis. The data represents 3 independent experiments (n = 3), with statistical significance indicated as *p < 0.05, **p < 0.01, and ***p < 0.001. B qPCR analysis of transcript levels of LIF/STAT3 target genes in mESCs cultured for 4 days under SMG, normal gravity (1 g), and 4 days of culture under SMG followed by 24 h or 48 h of culture under 1 g conditions. The data represents 3 independent experiments (n = 3), with statistical significance indicated as *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001. C The morphology of mESCs in ES medium with or without LIF supplement, cultured under normal gravity (1 g, Ctrl) and microgravity (SMG) conditions for 3 days. Specifically, mESCs were initially cultured under 1 g and SMG conditions for 24 h, followed by LIF withdrawal for 3 days. The data represents 3 independent experiments (n = 3). Scale bars represent 200 μm. D qPCR analysis of transcript levels of LIF/STAT3 target genes in ES medium with or without LIF, cultured under normal gravity (1 g, Ctrl) and microgravity (SMG) conditions for 3 days. The data represents 3 independent experiments (n = 3), with statistical significance indicated as **p < 0.01, ***p < 0.001, and ****p < 0.0001. E Quantification of p-STAT3 levels at LIF/STAT3 target genes in mESCs cultured under normal gravity (1 g) and microgravity (SMG) conditions, determined by ChIP-qPCR. The data represents 3 independent experiments (n = 3), with statistical significance indicated as *p < 0.05 and **p < 0.01. Western blot analysis of STAT3 and p-STAT3 levels (F), JAK1 and p-JAK1 levels (G), and GP130 and p-GP130 levels (H) in mESCs cultured for 4 days under normal gravity (1 g) and microgravity (SMG) conditions. GAPDH was used as a loading control. The data represents 3 independent experiments (n = 3). I Venn diagram illustrating the overlapping genes that are up-regulated in mESCs cultured under microgravity (SMG) conditions and bound by STAT3 protein. The diagram showcases the shared number of genes between the two conditions. J Gene Ontology (GO) ontology analysis revealing biological processes associated with STAT3 target genes that are upregulated under microgravity (SMG) condition. The analysis identifies the functional categories enriched among the upregulated genes.

In addition to the JAK/STAT3 pathway, the binding of LIF to its receptor initiates signaling through the PI3K (phosphoinositide 3-kinase)/AKT pathway and the SHP2 domain-containing tyrosine phosphatase 2/MAPK pathway [43]. Western blot analysis revealed increased phosphorylation of both AKT and MAPK under SMG conditions, indicating that LIF binding to its receptor may be enhanced during SMG [43]. Western blots revealed the increased phosphorylation of both AKT and MAPK under SMG (Fig. S2H), supporting that the binding of LIF with its receptor may increase under SMG.

We performed a comparison between genes affected by SMG and STAT3 target genes and identified 722 upregulated STAT3 target genes under SMG conditions. These genes are associated with ESC maintenance and differentiation, cytoskeleton organization, and MAPK regulation (Fig. 2I, J). Additionally, 688 STAT3 target genes were down-regulated under SMG (Fig. S2I), and they are functionally related to the apoptotic signaling pathway, ATP metabolic process, pyruvate metabolic process, glycolytic process, and oxidative phosphorylation regulation (Fig. S2J). Given the observed increase in apoptosis and decreased ATP production in the mitochondria of ESCs under SMG (Fig. S1B, D, F), it is likely that SMG influences the maintenance, differentiation, apoptosis, and metabolism of ESCs by modulating the activity of the LIF/STAT3 pathway.

SMG regulates LIF/STAT3 pathway via controlling the expression of heat shock protein genes

GO analysis revealed that genes upregulated under SMG were associated with the regulation of heat shock factor 1 (Hsf1)-mediated heat shock response (Fig. 1E). RNA-seq analysis further demonstrated the dysregulation of HSP genes in mESCs under SMG conditions (Fig. 3A). Intriguingly, the expression of hsp genes in barley and Arabidopsis has also been reported to be altered in the space environment [44, 45]. Considering that HSP90 and HSP110 have been implicated in regulating the activity of STAT3 [34, 35], we hypothesized that SMG might modulate the activity of the LIF/STAT3 pathway by influencing the expression of Hsp genes. qPCR experiments confirmed the increased expression of Hspa1b, Hsp90b1, and Hsph1 in mESCs under SMG (Fig. 3B), which was restored upon transfer to normal gravity for 24 h (Fig. S3A). Notably, Hsph1 exhibited the most significant alteration in expression under SMG conditions (Fig. 3A, Table S3). To investigate the impact of Hsph1 on the LIF/STAT3 pathway, we overexpressed Hsph1 in mESCs and examined its effects. Overexpression of Hsph1 resulted in elevated mRNA levels of Stat3 and its target genes (Fig. 3C). Moreover, protein analysis revealed increased levels of STAT3, p-STAT3, JAK1, p-JAK1, GP130, and p-GP130 in mESCs upon Hsph1 overexpression (Fig. 3D, E), providing support for the notion that Hsph1 overexpression enhances the activity of the LIF/STAT3 pathway. Consistent with the increased expression of pluripotency genes observed under SMG conditions (Fig. 1B, C), Hsph1 overexpression also upregulated the expression of Oct4, Sox2, Nanog, and Tbx3 (Figs. 3C, F, S3B). These findings suggest that SMG may regulate the expression of pluripotency genes by modulating Hsph1 expression.

Fig. 3: Regulation of LIF/STAT3 target genes and pluripotency genes by HSF1/HSP proteins.
figure 3

A Heat map depicting the expression changes of heat shock genes in mESCs cultured for 4 days under normal gravity (1 g) and microgravity (SMG) conditions. The color gradient represents the log2 FPKM (Fragments Per Kilobase Million) values of the genes. B qPCR analysis of transcript levels of Hspa1b, Hsp90b1, and Haph1 in mESCs cultured for 4 days under normal gravity (1 g) and microgravity (SMG) conditions. The data represents 3 independent experiments (n = 3), with statistical significance indicated as *p < 0.05 and ****p < 0.0001. C qPCR analysis of transcript levels of LIF/STAT3 target genes Tbx3, Stat3, Eya1, Mras, Esrrb, Fabp3, Ly6g6e, Lrrc34, Gjb3, Lama1 and Hsp90b1 in mESCs with Hsph1 overexpressed for 3 and 4 days, respectively. The data represents 3 independent experiments (n = 3), with statistical significance indicated as *p < 0.05, **p < 0.01, and ***p < 0.001. D Western blot analysis of STAT3 and p-STAT3 levels in mESCs with Hsph1 overexpressed for 3 and 4 days. GAPDH was used as a loading control. The asterisk (*) indicates GFP fused HSP110. The data represents 3 independent experiments (n = 3). E Western blot analysis of JAK1 and p-JAK1, GP130 and p-GP130 levels in mESCs with Hsph1 overexpressed for 3 and 4 days. GAPDH was used as a loading control. The data represents 3 independent experiments (n = 3). F qPCR analysis of transcript levels of pluripotency genes Oct4, Sox2, and Nanog in mESCs with Hsph1 overexpressed for 4 days. The data represents 3 independent experiments (n = 3), with statistical significance indicated as *p < 0.05 and **p < 0.01. G qPCR analysis of transcript levels of indicated genes in mESCs cultured under normal gravity (1 g) and microgravity (SMG) conditions for 3.5 days. Subsequently, the cells cultured under SMG condition were treated with or without HSP inhibitors for an additional 10 h. The data represents 3 independent experiments (n = 3), with statistical significance indicated as *p < 0.05, **p < 0.01, and ***p < 0.001. H Western blot analysis of HSF1 and p-HSF1 levels in mESCs cultured for 4 days under normal gravity (1 g) and microgravity (SMG) conditions. The data represents 3 independent experiments (n = 3). I qPCR analysis of transcript levels of Hsph1 gene in mESCs with Hsf1 overexpressed for 2 days and 3 days. The data represents 3 independent experiments (n = 3), with statistical significance indicated as *p < 0.05, **p < 0.01, and ***p < 0.001. J qPCR analysis of transcript levels of Hsf1 in mESCs with Hsph1 overexpressed for 3 days. The data represents 3 independent experiments (n = 3), with statistical significance indicated as **p < 0.01. K Western blot analysis of HSF1 levels in mESCs with Hsph1 overexpression for 72 and 96 hours, with GAPDH as a loading control. L qPCR analysis of transcript levels of Hsf1, Hsph1 and Hsp70 in mESCs cultured under normal gravity (1 g) and simulated microgravity (SMG) conditions for 1, 2 and 3 h. (n = 3 independent experiments, *p < 0.05). M qPCR analysis of transcript levels of Hsph1 in WT and Hsf1 KO mESCs cultured for 1 day under both normal gravity (1 g) and SMG conditions. (n = 3 independent experiments, *p < 0.05, **p < 0.01). N qPCR analysis of transcript levels of Oct4, Sox2, Nanog and Tbx3 in mESCs with Hsf1 overexpression for 3 days. (n = 3 independent experiments, *p < 0.05, **p < 0.01). GO analysis revealed the enriched GO terms of genes associated with increased (O) H3K27ac levels and decreased (P) H3K27ac levels in mESCs cultured for 11 h under simulated microgravity (SMG) and normal gravity (1 G) conditions, respectively.

In addition to Hsph1, the upregulation of Hsp90b1 was also observed under SMG conditions (Figs. 3B, S3A). Similar to Hsph1, overexpression of Hsp90b1 resulted in increased expression of Stat3, STAT3 target genes, and pluripotency genes Oct4, Sox2, and Nanog (Fig. S3C, D). Notably, the co-overexpression of Hsph1 and Hsp90b1 further enhanced the expression of STAT3 target genes, including Tbx3, Eya1, and Mras (Fig. S3C), suggesting a collaborative regulation of the LIF/STAT3 pathway by different Hsp genes. The upregulation of other Hsp genes such as Hspa8, Hsp90aa1, and Hspb1 under SMG conditions indicates their potential contribution to the upregulation of pluripotency genes and LIF/STAT3 target genes (Fig. 3A). To further confirm the hypothesis that the upregulation of pluripotency genes and LIF/STAT3 target genes under SMG conditions is mediated by Hsp genes, we employed inhibitors to block the induction of HSP proteins, including HSP inhibitor I (HSP70 and HSP105 inhibitor) and 17-AAG (HSP90 inhibitor). The addition of HSP inhibitors to ESCs cultured under SMG conditions significantly impaired the upregulation of pluripotency genes Oct4, Sox2, Nanog, Stat3, and LIF/STAT3 target genes such as Tbx3, Gjb3, Ly6g6e, Mras, and Eya1 (Fig. 3G). This experimental evidence supports our conclusion that the induction of Hsp genes under SMG conditions leads to the upregulation of pluripotency genes and LIF/STAT3 target genes in ESCs.

HSF1, a key regulator of the heat shock response [46], was hypothesized to be involved in the regulation of hsp genes under SMG conditions. Indeed, we observed an increase in the expression of Hsf1 and its active form p-HSP1 at both the mRNA and protein levels under SMG (Figs. 3H and S3E). Overexpression of Hsf1 resulted in the upregulation of Hsph1 (Fig. 3I), while overexpression of Hsph1 upregulated Hsf1 expression (Fig. 3J, K). Notably, as early as 2 h of culture under SMG, we observed the upregulation of Hsph1, Hsf1, and Hsp70 in mESCs (Fig. 3L). These findings suggest that Hsph1 and Hsf1 may be independently upregulated under SMG, while also co-regulating each other. To further investigate this, we employed CRISPR/Cas9 technology to generate Hsph1 and Hsf1 knockout ESCs, and Western blot analysis confirmed the successful deletion of Hsph1 and Hsf1 genes (Fig. S3F–G). Importantly, the deletion of either Hsph1 or Hsf1 did not affect the upregulation of the other gene under SMG conditions (Figs. 3M and S3H), providing further support for the independent regulation of Hsph1 and Hsf1 under SMG. Overexpression of Hsf1 led to an increased expression of pluripotency genes Oct4, Sox2, Nanog, and Tbx3 (Fig. 3N), as well as Stat3 and LIF/STAT3 target genes (Fig. S3I). Addition of HSP inhibitors in Hsf1 KO ESCs cultured under SMG conditions significantly decreased the expression of pluripotency genes Oct4, Sox2, Nanog, and LIF/STAT3 target genes Tbx3, Gjb3, Ly6g6e, Mras, Lama1, and Eya1 (Fig. S3J). Importantly, compared to WT ESCs, the addition of HSP protein inhibitors to Hsf1 KO ESCs cultured under SMG resulted in a greater decrease in the expression of the upregulated pluripotency and LIF/STAT3 target genes (Figs. 3G and S3J, K), further supporting the independent regulation of Hsf1 and Hsp genes in the context of pluripotency and STAT3 target gene regulation.

Microgravity-induced transcriptional alterations have been found to be regulated at the epigenetic level as an adaptive response to the space microgravity environment [6, 47]. To investigate the role of histone modifications in the regulation of gene expression under SMG conditions, we conducted genome-wide profiling of H3K4me3, H3K27ac, and H3K27me3 in mESCs cultured under SMG for 11 h. Differential binding analysis revealed 1331 regions with increased H3K4me3 levels and 190 regions with decreased H3K4me3 levels (Fig. S3L, left). Similarly, 1307 regions showed increased H3K27ac levels, while 696 regions showed decreased H3K27ac levels (Fig. S3L, middle). No significant changes were observed in H3K27me3 levels under SMG conditions (Fig. S3L, right). To further characterize the affected genomic regions, we employed a hidden Markov model (ChromHMM) to classify the genome into distinct segments based on the histone ChIP-seq data from mESCs [48]. A total of 15 states representing different types of promoters, enhancers, and other genomic regions were identified [48]. Analysis of the differential H3K27ac peaks between 1 g and SMG environments revealed their predominant localization in promoter and enhancer regions (Fig. S3M), suggesting the regulation of gene activity through control of promoters and enhancers under SMG conditions. Gene ontology (GO) analysis revealed that genes associated with increased H3K27ac levels were functionally related to HIF-1 and Wnt signaling pathways, as well as metabolism (Fig. 3O). In contrast, genes with decreased H3K27ac levels were associated with cell proliferation, stress-activated MAPK, and JNK regulation (Fig. 3P). Furthermore, genes associated with increased and decreased H3K4me3 modifications were related to cell fate commitment and microtubule depolymerization, respectively (Fig. S3N–O). Consequently, the GO analysis of genes linked to differential H3K27ac and H3K4me3 peaks was consistent with the dysregulated genes identified from RNA-seq data under SMG conditions (Figs. 3O, P and S3N, O, Fig. 1E), providing further support for the regulation of gene expression under SMG conditions through the control of histone modifications.

The promotion of mesoendoderm differentiation in mESCs under SMG environment was achieved by controlling the expression of Tbx3

To investigate the differentiation potential of mESCs under SMG environment, we conducted embryoid body (EB) differentiation assays. EBs generated under SMG exhibited increased size and density compared to those formed under normal gravity (1 g) conditions (Fig. 4A), indicating a significant impact of SMG on mESC differentiation. Additionally, we examined the expression levels of established lineage markers. The expression of mesoderm markers T and Mixl1, as well as endoderm markers Sox17 and Gata6, was significantly upregulated in EBs induced under SMG (Fig. 4B). Previous studies, including our own, have demonstrated that Tbx3 plays a crucial role in regulating mesoendoderm differentiation of mESCs [48,49,50]. Notably, Tbx3 expression was found to be increased under SMG conditions (Fig. 1B, C). Based on these findings, we hypothesized that the elevated expression of Tbx3 under SMG might contribute to the preferential differentiation of mESCs towards mesoendoderm lineages. To test this hypothesis, we induced the formation of EBs using Tbx3 knockout (KO) mESCs [48]. The expression levels of mesoendoderm marker genes T, Mixl1, Sox17, and Gata6 in Tbx3 KO EBs induced under SMG were comparable to those observed in wild-type (WT) EBs under 1 g gravity control conditions (Fig. 4B). Moreover, the size of Tbx3 KO EBs was smaller than that of WT EBs under SMG (Fig. 4C). These findings support our conclusion that the differentiation of mESCs towards mesoendoderm lineages under SMG is partly regulated by Tbx3 expression.

Fig. 4: SMG promotes mesoendoderm differentiation of mESC by regulating the Wnt signaling pathway and Tbx3 Expression.
figure 4

A Morphology of embryoid bodies (EBs) induced under 1 g and SMG conditions at the indicated time points. Scale bars, 500 μm (4×), 200 μm (10x). B qPCR analysis of transcript levels of mesoderm markers T and Mixl1, and endoderm markers Sox17 and Gata6 in EBs induced under 1 g and SMG conditions on day 3. (n = 3 independent experiments, *p < 0.05, **p < 0.01). C Morphology of 3-day EBs induced from WT and Tbx3 KO mESCs under 1 g and SMG conditions. Scale bars, 500 μm. D qPCR analysis of transcript levels of Wnt3 and Wnt3a in mESCs under 1 g and SMG conditions for 4 days. (n = 3 independent experiments, *p < 0.05, **p < 0.01). E qPCR analysis of Tbx3 expression in WT and mESCs with overexpression of Wnt3 or Wnt3a. (n = 3 independent experiments, **p < 0.01). F qPCR analysis of transcript levels of Tbx3 gene in mESCs under SMG condition supplemented with or without 10 μg/ml of IWP2 for 4 days. (n = 3 independent experiments, *p < 0.05, **p < 0.01). G Western blot for β-CATENIN levels in mESCs cultured for 4 days under 1 g and SMG conditions, with GAPDH as the loading control. (n = 3 independent experiments). H β-CATENIN levels at promoter and intronic enhancer regions of Tbx3 gene in mESCs cultured for 4 days under 1 g and SMG conditions determined by ChIP-qPCR. (n = 3 independent experiments, *p < 0.05, **p < 0.01). I qPCR analysis of transcript levels of LIF/STAT3 target genes Ly6g6e, Gjb3, Mras, Lrrc34, and Lama1 in mESCs cultured under SMG conditions with and without 10 μg/ml of IWP-2 for 4 days (n = 3 independent experiments, *p < 0.05, **p < 0.01, ***p < 0.001). J The heat map displays ChIP-seq signals of STAT3 and β-CATENIN surrounding specific binding sites for STAT3, β-CATENIN, and their shared binding sites. The color gradient represents the log2 RPM (Reads Per Million) values.

A previous study by Lei et al. [27] demonstrated that mesoendoderm differentiation of mESCs can be enhanced through modulation of the Wnt/β-catenin pathway under SMG conditions. In our investigation, we observed a significant upregulation of both Wnt3 and Wnt3a under the SMG environment (Fig. 4D). Moreover, overexpression of Wnt3 or Wnt3a resulted in increased expression of Tbx3 (Fig. 4E). To further elucidate the role of the Wnt pathway in Tbx3 regulation, we employed IWP2, an inhibitor of the Wnt pathway, which successfully restored the upregulated expression of Tbx3 under SMG conditions (Fig. 4F). These findings suggest that the activated Wnt/β-catenin pathway under SMG conditions may exert control over mesoendoderm differentiation through regulation of Tbx3 expression. β-catenin, a key effector of the Wnt signaling pathway [51], exhibited an increased protein level under the SMG environment (Fig. 4G). Importantly, our ChIP-qPCR experiments demonstrated augmented binding of β-catenin protein at the intronic enhancer and promoter regions of the Tbx3 gene under the SMG environment [48] (Fig. 4H). Therefore, our findings further support the conclusion that SMG-induced activation of the Wnt pathway upregulates Tbx3 expression, thereby promoting the mesoendoderm differentiation of mESCs.

Enhanced expression of LIF/STAT3 Target genes and pluripotency factors via activation of the Wnt pathway under SMG conditions

The expression levels of LIF/STAT3 target genes were found to be upregulated under the conditions of SMG (Fig. 2A–C). In addition to Tbx3, we observed that inhibiting the Wnt pathway using IWP2 in mESCs cultured under SMG restored the expression of other LIF/STAT3 target genes, including Ly6g6e, Gjb3, Mras, Lrrc34, and Lama1, to levels comparable to those in mESCs under normal gravity conditions (Fig. 4I). This finding suggests the involvement of the Wnt signaling pathway in the regulation of LIF/STAT3 target genes. Further analysis through STAT3 and β-CATENIN ChIP-seq demonstrated their co-binding at LIF/STAT3 target genes such as Lama1, Lrrc34, Eya1, Mras, Jam2/3, Esrrb, Socs3, and Pim3 (Fig. 4J), providing additional support for the regulatory role of the Wnt signaling pathway in LIF/STAT3 gene expression. Moreover, overexpression of Wnt3 or Wnt3a resulted in increased expression of LIF/STAT3 target genes (Fig. S4A, B). We identified 2100 genomic regions co-occupied by β-catenin and STAT3 (Figs. 4J and S4C), with many of these regions overlapping with LIF/STAT3 target genes (Fig. 4J), further supporting the role of the Wnt signaling pathway in regulating LIF/STAT3 gene expression. GO analysis revealed that β-CATENIN and STAT3 co-bound sites are located proximal to genes associated with stem cell maintenance, embryonic development, and cell fate commitment (Fig. S4D). Consistent with previous findings regarding Nanog regulation by Wnt3a [52], overexpression of either Wnt3a or Wnt3 increased the expression of Oct4, Sox2, and Nanog (Fig. S4E, F). ChIP-seq analysis of β-CATENIN revealed its binding to the Oct4, Sox2, and Nanog genes (Fig. S4G), indicating direct regulation of core pluripotency genes by the Wnt pathway. Overexpression of either Hsph1 or Hsf1 resulted in upregulation of Wnt3 and Wnt3a (Fig. S4G–I), suggesting that the increased expression of Hsf1 and Hsp genes under SMG conditions may lead to enhanced activity of the Wnt pathway (Fig. 4D). Therefore, our findings support the conclusion that SMG-induced expression of Hsf1 and Hsp genes increases Wnt pathway activity, thereby promoting the self-renewal of mESCs under ESC maintaining conditions.

SMG enhanced mesendoderm differentiation of human ESCs (hESCs) by controlling WNT pathway activity

We conducted further investigations into the effects of SMG on human embryonic stem cell (hESC) self-renewal. For this purpose, hESCs were cultured under SMG conditions using the RCCS system (Fig. 5A). qPCR analysis revealed a significant increase in the expression of core pluripotency regulators OCT4, SOX2, NANOG, and TBX3 after 4 days of culture under SMG conditions (Fig. 5B). These increased expression levels returned to control levels after 24 h of culture under 1 g conditions (Fig. S5A).

Fig. 5: SMG affects the self-renewal and differentiation of hESCs.
figure 5

A Schematics of the experimental design. hESCs were cultured under rotary and standard conditions at 37 °C in 5% CO2. Cells were harvested and used for subsequent analyses. B qPCR analysis of transcript levels of pluripotency genes OCT4, SOX2, NANOG and TBX3 in hESCs cultured under 1 g and SMG conditions for 4 days. (n = 3 independent experiments, **p < 0.01, ***p < 0.001). C GO ontology analysis for biological processes associated with the up- and down-regulated genes in hESCs cultured under SMG conditions. D The heat map illustrates the expression changes of the indicated heat shock genes in hESCs cultured under both 1 g and SMG conditions for a duration of 4 days. The color gradient represents the Z score (row-wise) of the log2 FPKM (Fragments Per Kilobase Million) values of the genes. E qPCR analysis of transcript levels of HSPH1 and HSF1 in hESCs cultured under SMG and normal gravity (1 g) conditions for a duration of 4 days, as well as 24 h of culture at 1 g following 3 days of SMG. (n = 3independent experiments, *p < 0.05, **p < 0.01). F qPCR analysis of transcript levels of indicated genes in hESCs cultured under 1 g and SMG conditions for 3 days. Subsequently, hESCs cultured under SMG condition were treated with or without HSP inhibitors for 5 h before RNA extraction for further qPCR analysis. (n = 3 independent experiments, *p < 0.05,**p < 0.01, ***p < 0.001). G qPCR analysis of transcript levels of endoderm markers GATA4, GATA6, and SOX17, mesoderm markers T and MIXL1, and neuronal ectoderm marker PAX6 in day 2 EBs induced under both 1 g and SMG conditions. (n = 3 independent experiments, *p < 0.05,**p < 0.01, ***p < 0.001, ****p < 0.0001). H qPCR analysis of transcript levels of pluripotency genes OCT4, SOX2, NANOG and TBX3 in day 2 EBs induced under 1 g and SMG conditions. (n = 3 independent experiments, **p < 0.01, ***p < 0.001). I qPCR analysis of transcript levels of endoderm marks GATA6 and SOX17, mesoderm markers T in day 2 EBs induced under 1 g and SMG conditions, with and without the presence of 10 μg/ml of IWP2. (n = 3 independent experiments, *p < 0.05,**p < 0.01, ***p < 0.001). J Scatter plot showing the log RPKM values of H3K4me3 (left), H3K27ac (middle) and H3K27me3 (right) in hESCs. Significant differential peaks (|log2FC | >1 and q value < 0.01 by DESeq2) are shown in red (up) and blue (down) colors. K Overlap Percentage of Differential H3K27ac and H3K4me3 Peaks. L Chromatin states defined by ChromHMM using the ChIP-seq data of the histone marks. Heatmap representation of the emission probability of each histone modifications (left) and the enrichment of different genomic segments (right). M Genome browser view of H3K4me3 and H3K27ac at the HSP90AA1 locus in hESCs. N ChIP-qPCR analyses of the HSF1, HSPH1, HSP90AA1, HSPA5, HSPB1, and HAPE1 loci from hESCs cultured under 1 g and SMG conditions for 2 days were carried out with H3K27ac antibody. O Enriched Gene Ontology (GO) terms of genes associated with up-regulated H3K27ac in hESCs cultured under SMG and 1 g conditions.

To investigate the underlying mechanism of SMG-induced effects on hESC self-renewal, we performed RNA-seq to analyze global gene expression changes in hESCs under SMG. We identified 764 significantly upregulated and 1187 downregulated genes under SMG compared to the 1 g condition (Fig. S5B, Table S4). SMG led to dysregulation of genes associated with pathways involved in hESC maintenance, such as TGF-beta and Wnt pathways (Fig. 5C), which is consistent with the altered expression of core pluripotency factors (Fig. 5B). Similar to mESCs (Fig. 1E), many affected genes in hESCs were related to apoptosis and the MAPK pathway (Fig. 5C). The expression of human heat shock genes HSPH1, HSP90AA1, HSPB1, HSPA5, and HSPE1 was upregulated under SMG for 4 days (Figs. 5D and S5B), but returned to control levels under 1 g conditions (Figs. 5E and S5C). Similar to mESCs, hESCs cultured under SMG conditions displayed upregulated expression of HSP gene HSF1, HSPH1, HSP90AA1, HSPA5, and HSPB1, even as early as 1 to 3 h of exposure (Fig. S5D). Overexpression of human HSPH1 resulted in increased expression of SOX2, NANOG, and WNT3 (Fig. S5E), suggesting that SMG may regulate the expression of these genes by controlling HSP gene expression. Consistently, the addition of HSP inhibitors to hESCs cultured under SMG conditions significantly decreased the upregulated expression of pluripotency genes OCT4, SOX2, NANOG, WNT3, and WNT3a (Fig. 5F). These findings demonstrate that, similar to mouse ESCs (Fig. 3G), the induction of HSP genes under SMG conditions upregulates the expression of pluripotency genes and the Wnt pathway in hESCs.

GO analysis revealed that the differentially expressed genes were associated with cell differentiation and embryonic development (Fig. 5C), indicating that the differentiation potential of hESCs may be altered under the SMG environment. To validate this observation, we performed an EB assay and examined the expression of representative marker genes from the three germ layers in day 2 and day 4 EBs induced under SMG. The expression of mesoderm markers T and MIXL1, as well as endoderm markers GATA4, GATA6, and SOX17, was significantly increased in EBs induced under SMG (Figs. 5G and S5E). However, the expression of the neuronal ectoderm marker gene PAX6 was lower in EBs under SMG (Figs. 5G and S5F). Therefore, SMG enhanced the differentiation of hESCs towards mesoendoderm, but impaired differentiation towards neuronal ectoderm.

To investigate the mechanism underlying the enhanced differentiation of hESCs towards mesoendoderm under SMG, we examined the expression levels of pluripotency factors OCT4, SOX2, NANOG, and TBX3 in EBs. qPCR analysis revealed that the expressions of OCT4, NANOG, and TBX3 were upregulated, with TBX3 exhibiting the highest increase, in both day 2 and day 4 EBs induced under the SMG environment (Figs. 5H and S5G). However, the expression of SOX2 decreased (Figs. 5H and S5F), which may explain the observed impairment in neuronal ectoderm differentiation (Figs. 5G and S5E). Furthermore, the expressions of WNT3 and WNT3a were elevated in both day 2 and day 4 EBs induced under SMG (Fig. S5H), suggesting their involvement in the regulation of TBX3 expression. To verify whether the increased activity of the WNT pathway contributed to the biased mesoendoderm differentiation of hESCs under SMG, we added the WNT pathway inhibitor IWP2 during EB induction. The inhibition of the WNT pathway by IWP2 restored the elevated expression of endoderm marker genes GATA6 and SOX17, as well as the mesoderm marker gene T, in EBs induced under SMG conditions (Fig. 5I). Additionally, the upregulated expression of TBX3 in day 2 EBs induced under SMG was restored to its control level in the presence of the WNT inhibitor IWP2 (Fig. S5I). Based on these findings, we conclude that the biased mesoendoderm differentiation of hESCs under SMG may be achieved by increasing the activity of the WNT pathway and subsequently upregulating the expression of TBX3.

HSP genes were found to be upregulated as early as 3 h under SMG conditions (Fig. S5J). To investigate the role of histone modifications in the regulation of gene expression under SMG, we analyzed the genome-wide profiles of H3K4me3, H3K27ac, and H3K27me3 in hESCs cultured under SMG for 5 h. Differential binding analysis revealed that only a small fraction of H3K4me3 peaks were altered, with 95 regions showing increased levels and 16 regions showing decreased levels of H3K4me3 (Fig. 5J, left). In contrast, a larger number of regions showed increased H3K27ac modification, with 454 regions exhibiting increased levels and 63 regions showing decreased levels of H3K27ac (Fig. 5J, Middle; Fig. S5K). Notably, 59% of the 517 differentially acetylated H3K27 regions overlapped with the H3K4me3 regions (Fig. 5K), while the remaining 41% of the H3K27ac differential peaks were located in regions without H3K4me3 (Fig. 5L). No differential H3K27me3 signals were observed (Fig. 5J, Right). ChromHMM analysis revealed that the differentially acetylated H3K27 peaks between the 1 g and SMG environments were primarily located in promoter and enhancer regions (Fig. 5L). Therefore, gene expression regulation under SMG conditions mainly occurs through the control of promoter and enhancer activity. Consistently, H3K4me3 and H3K27ac modifications increased in heat shock genes HSP90AA1 and HSPH1 (Figs. 5M and S5L). ChIP-qPCR analysis confirmed the augmented H3K27ac and H3K4me3 modifications in heat shock genes HSF1, HSPH1, HSP90AA1, HSPA5, HSPB1, and HAPE1 in hESCs cultured under SMG conditions for 2 days (Figs. 5N and S5M). Gene ontology analysis showed that genes associated with the increased H3K27ac and H3K4me3 peaks were functionally related to stress, regulation of HSF1-mediated heat shock response, and the apoptosis signaling pathway (Figs. 5O and S5N), while genes associated with the decreased H3K27ac peaks were functionally associated with glycoprotein metabolic processes, regulation of actin cytoskeleton, and Erk1/2 regulation (Fig. S5O). Therefore, under SMG conditions, there is an initial upregulation of genes related to stress by modulating their promoter and enhancer activity in hESCs, followed by regulation of ESC maintenance and differentiation.

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