Intracellular lactate was increased during hESCs spontaneous differentiation

HESCs are maintained in mTESR1 media and they can spontaneously differentiate into three germ layer cells in vitro in DMEM/F12 media [35] (Fig. 1A). Therefore, we firstly investigate the function of lactate in this spontaneous differentiation model. After 48 h of differentiation, the intracellular lactate levels in those cells were measured by colorimetric assays and the results showed that the level of lactate was increased approximately two-fold compared to that in the undifferentiated cells (Fig. 1B). In comparison, both the intercellular and extracellular lactate levels were not changed in hESCs culture when those cells were maintained as stem cells (Figure S1A). qRT-PCR analysis on the differentiated cells confirmed that the expression of pluripotency markers OCT4 (also known as POU5F1) and NANOG were significantly decreased after 48 h (Figure S1B), while the expressions of neuroectoderm markers PAX6 and SOX1 (Figure S1C) and mesoderm markers TBXT and HAND1 (Figure S1D) were significantly increased. No significant change was observed in endoderm markers SOX17 during differentiation (Figure S1E).

Fig. 1

Exogenous lactate promotes hESCs differentiation towards neuroectoderm in the spontaneous differentiation model. (A) Illustration for the hESCs spontaneous differentiation procedure. (B) Relative intracellular lactate levels of Day 0 cells and Day 2 cells in the spontaneous differentiation model (n = 3). (C) 25 mM Sodium lactate or 10 µM LDHA inhibitors were added at Day 0 as illustrated. (D) Relative intracellular lactate levels of Day 2 cells with either sodium lactate or LDHA inhibitors treatments (n = 3). (E-H) qRT-PCR analysis of OCT4, NANOG, PAX6, SOX1, TBXT, HAND1, and SOX17 mRNA expression in Day 2 cells with sodium lactate or Gska treatment (n = 4)

As the intercellular lactate level was increased during hESCs spontaneous differentiation, we used sodium lactate or LDHA inhibitors (Gska or Fx11) to change the intracellular lactate during hESCs spontaneous differentiation (Fig. 1C). Again, the intracellular lactate level was examined after 48 h and the results confirmed that sodium lactate treatment significantly elevated the intracellular lactate level, with an increase of approximately eight-fold; whereas Gska or FX11 treatment significantly suppressed the intracellular lactate level to about 15% of that in the control cells (Fig. 1D). We further analyzed the expression of multiple pluripotency markers and lineage-specific genes under these conditions. Exogenous lactate significantly upregulated the expression of neuroectoderm markers PAX6 and SOX1 with a nearly three-fold increase (Fig. 1F) and downregulated the expression of mesoderm marker HAND1 by 84% (Fig. 1G). No significant changes were observed in the expression of TBXT and SOX17 (Figure S1G&H). At the same time, exogenous lactate partially attenuated the loss of pluripotency markers OCT4 and NANOG during spontaneous differentiation as they were higher in lactated treated cells and untreated cells (Fig. 1E). On the other hand, Gska treatment had no significant effect on most markers except a slight decrease in NANOG expression (Fig. 1E-H).

Collectively, these results showed that exogenous lactate could boost the intracellular lactate level and facilitate the spontaneous differentiation of hESCs towards the neuroectoderm.

Exogenous lactate promoted neuroectoderm differentiation in the induced differentiation model

We observed that exogenous lactate could facilitate the spontaneous differentiation of hESCs towards the neuroectoderm. However, there are mixed cell populations in this spontaneous model. HESCs can be specifically induced to neuroectoderm cells by SMAD inhibition [14, 36] (Fig. 2A). Again, we first confirmed that the induced differentiation was successful in our hands. The decrease of pluripotent marker OCT4 and the increase of neuroectoderm marker PAX6 in this induced model were confirmed by qRT-PCR, western blotting, and immunofluorescent experiments (Figure S2A-C). We also checked the intracellular lactate level at Day 5, at which most cells have differentiated into neuroectoderm cells. The results showed that the intercellular lactate at Day 5 was approximately four-fold of that in Day 0 undifferentiated cells (Fig. 2B). This result was in consistent with what we had observed in the spontaneous differentiation model. We examined the expression of PDHA1, LDHA, and LDHB in this induced differentiation model and the results showed that the expression of LDHA was significantly increased at Day 5, while the levels of PDHA1 and LDHB were significantly decreased at Day 5 (Fig. 2C). Such expression pattern suggested that more pyruvate might be catalyzed to lactate in those neuroectoderm cells.

Fig. 2

Exogenous lactate promotes neuroectoderm differentiation in the induced differentiation model. (A) Illustration for the induced neuroectoderm differentiation model from hESCs. (B) Relative intracellular lactate levels of Day 0 cells and Day 5 cells in the induced differentiation model (n = 3). (C) qRT-PCR analysis of LDHA, LDHB, and PDHA1 mRNA expression in Day 0, Day 3, and Day 5 cells during neuroectoderm differentiation (n = 4). (D) 25 mM Sodium lactate or 10 µM LDHA inhibitors were added at Day 0 as illustrated. (E) Relative intracellular lactate levels of Day 5 cells with sodium lactate or Gska treatment (n = 3). (F-G) qRT-PCR analysis of OCT4, NANOG, PAX6, and SOX1 mRNA expression in Day 5 cells treated with sodium lactate or Gska (n > 5)

Again, we used sodium lactate and Gska to change the intercellular lactate level in this model (Fig. 2D). The intracellular lactate level was significantly upregulated about nine-fold by sodium lactate and reduced to nearly 15% upon Gska treatment (Fig. 2E). Before we examined the effect of intercellular lactate on differentiation, we tested whether changes on lactate levels affect cell viability, proliferation, and apoptosis. Neither sodium lactate nor LDHA inhibitors (Gska or Fx11) had any significant influence on the viability of cells in this model (Figure S3A). No obvious effect on cell numbers was observed under different treatments (Figure S3B). Immunofluorescent experiments with antibodies against cleaved caspase 3 (apoptosis marker) or Ki67 (proliferation marker) on Day 5 cells also revealed no significant changes upon those treatments (Figure S3C-D). Therefore, neither cell proliferation nor apoptosis was affected by those treatments.

Then, we examined the effect of sodium lactate or Gska on neuroectoderm differentiation in this induced differentiation model. The expression of neuroectoderm markers PAX6 and SOX1 were both significantly increased by sodium lactate treatment in Day 5 cells (Fig. 2G); while the expression of pluripotency markers OCT4 and NAONG were decreased to about 50% (Fig. 2F). We did not observe any significant changes upon Gska treatment compared to the control cells (Fig. 2F&G). Immunostaining experiments for OCT4 and PAX6 on Day 5 cells also confirmed these results (Figure S4A&B). As the expression of OCT4 was no longer detectable in Day 5 cells, we tested OCT4 expression in Day 3 cells, and found that sodium lactate treatment indeed downregulated the ratio of OCT4 positive cells, while Gska treatment had no significant effects (data not shown). Together, our results suggested that exogenous lactate further promotes neuroectoderm differentiation without affecting cell viability, proliferation or apoptosis.

Intracellular lactate did not affect BMP or WNT/β-catenin signaling

Differentiation of hESCs to neuroectoderm requires inhibition of BMP signaling and low WNT/β-catenin signaling [36, 37]. It has been reported that lactate can act as a signaling molecule to activate WNT/β-catenin signaling [38, 39], we wonder whether the intracellular lactate level could affect WNT/β-catenin and BMP signaling during neuroectoderm differentiation in our models. Therefore, we examined the levels of phosphorated-SMAD1/5 and active β-catenin by western blot to analyze the activity of BMP and WNT/β-catenin signaling. Neither sodium lactate nor Gska treatment had any effect on the levels of phosphorated-SMAD1/5 or active β-catenin in Day 5 cells (Figure S5A&B) or Day 2 cells (Figure S5C&D) of the induced differentiation model. Moreover, we also analyzed the activity of BMP and WNT/β-catenin signaling on the spontaneous differentiation model. Again, neither sodium lactate nor Gska treatment had any effect on the expression of phosphorylated SMAD1/5 and active β-catenin compared to the control cells (Figure S5E&F). Collectively, our results suggested that changes on the intracellular lactate level do not affect the BMP or WNT/β-catenin signaling during neuroectoderm differentiation.

Histone lactylation was increased during differentiation

It has been reported that lactate is the precursor for lysine lactylation [24, 40], and especially exposed lysine of histone can be lactylatized [24]. Therefore, it is possible that the intracellular lactate might affect the level of protein lactylation, including histone lactylation. We first analyzed the Pan-lysine lactylation levels during induced neuroectoderm differentiation. Besides a band at approximately 70 kDa was decreased in differentiated cells compared to undifferentiated hESCs, there were no significant changes in overall Pan-lysine lactylation during this process (Fig. 3A). On the other hand, the Pan-histone lysine lactylation level (Pan HKla) was significantly increased in neuroectoderm compared to that in the hESCs (Fig. 3B&C). It has been reported that H3K18 and H4K12 are important sites for histone lactylation [25,26,27], therefore we further analyzed the lacylation levels of H3K18 (H3K18la) and H4K12 (H4K12la) by western blot. The results showed that the level of H3K18la were significantly increased in neuroectoderm compared to that in hESCs; while no significant change was detected in the levels of H4K12la (Fig. 3B&C). We also examined the levels of histone lactylation in induced differentiation model upon sodium lactate or Gska treatment. At Day 5, the level of Pan HKla was not significantly affected by sodium lactate treatment; while the levels of H3K18la and H4K12la were upregulated about 1.7 and 1.2 folds, respectively (Fig. 3D&E). Gska treatment can approximately decrease the Pan HKla level to 56%, H3K18la level to 69%, and H4K12la level to 64%, respectively (Fig. 3D&E). These results indicated that changes on intracellular lactate level could affect histone lactylation, especially H3K18la levels during neuroectoderm differentiation.

Fig. 3

Intracellular lactate modulates histone lactylation during neuroectoderm differentiation. (A) Western blotting analysis of Pan lysine lactylation in Day 0, Day 3, and Day 5 cells from the induced neuroectoderm differentiation model (representative graphs of four independent experiments). (B) Representative graphs of Pan HKla, H3K18la, and H4K12la in Day 0, Day 3, and Day 5 cells during the induced neuroectoderm differentiation by western blotting analysis. Quantification of their protein levels were shown in (C) (n = 4). (D) Representative graphs of Pan HKla, H3K18la and H4K12la in Day 5 cells with sodium lactate or Gska treatments in the induced differentiation model by western blotting analysis. Quantification of their protein levels were shown in (E) (n = 3)

Genome-wide analysis of H3K18la during neuroectoderm differentiation showed it was enriched at promoter regions and engaged in gene transcription

Since exogenous lactate could promote histone lactylation, especially H3K18la level during neuroectoderm differentiation, we performed CUT&Tag experiment on cells from induced differentiation model by using H3K18la antibodies to analyze genome-wide chromatin locations of H3K18la. H3K18la peaks were found in over 15,000 genes in both Day 0 and Day 5 cells (Figure S6A), suggesting that the H3K18la modifications were ubiquitous in genome during neuroectoderm differentiation. Further analysis revealed a predominantly enrichment of H3K18la peaks at transcriptional start sites (TSSs), with over 40% of the peaks located within the 3 kb promoter regions (Fig. 4A and S6B). Analysis with deeptools [41] also showed that the enrichment peaks at TSS in Day 5 cells was higher than that in Day 0 cells (Fig. 4B). To elucidate the potential epigenetic impacts of H3K18la in this process, we further analyzed the CUT&Tag data and identified 2,172 genes exhibiting significant changes in H3K18la modification levels between Day 0 and Day 5 cells (Pvalue < 0.05, abs (log2FoldChange) > 0.8). Gene ontology (GO) analysis showed that those genes were enriched in neural development related pathways, including axon development, axonogenesis, and modulation of chemical synaptic transmission (Fig. 4C).

Fig. 4

Genome-wide analysis of H3K18la during neuroectoderm differentiation. (A) The density heatmap of H3K18la binding peaks in Day 0 and Day 5 cells during neurectoderm differentiation which was visualized by the deeptools tool and ordered by signal intensity. (B) H3K18la peaks distribution of Day 0 and Day 5 cells; KS-test & T-test < 2.2e-16. (C) GO analysis (biological process) of 2,172 genes with the significantly different H3K18la binding peaks between Day 0 and Day5 cells. (D) Overlap of our RNAseq-DEGs and H3K18la genes were analyzed and showed in Scatterplot (left) and bar plot (right). 275 genes were divided into 4 groups (H3K18la-up & activated; H3K18la-up & repressed; H3K18la-down & activated; H3K18la-down & repressed) by their H3K18la levels (log2[Day 5/ Day 3]) and transcriptional levels (log2[Day 5/ Day 0]). (E) Similar analysis was performed on published RNA-seq datasets. (F) Intersection analysis for “H3K18la-up & activated” genes between two datasets: 93 genes from (D) and 46 genes from (E). (G) The heatmap showed the qRT-PCR analysis of the expression of PLK3, PAX6, TMEM169, PCDH9, RUSC2, and C1QTNF6 in Day 0, Day 3, and Day 5 cells from the induced differentiation model (n = 4). (H) qRT-PCR analysis of mRNA expressions of PAX6, TMEM169, RUSC2, C1QTNF6 and PCDH9 in Day 5 cells treated with sodium lactate or Gska from the induced differentiation model (n = 4)

To gain insights into the transcriptional landscape during hESCs differentiation towards neuroectoderm, we also conducted RNA sequencing (RNA-seq) analysis of Day 0 and Day 5 cells from induced differentiation model. 3,874 genes were identified as differentially expressed genes (DEGs) under the threshold of P value < 0.05 and a fold-change cutoff of absolute value of abs (log2FoldChange) > 1 (Figure S6C). In order to identify genes potentially regulated by H3K18la modification in neuroectoderm differentiation, we performed an intersection analysis of H3K18la genes (2172 genes) and RNAseq-DEGs (3,874 genes). There are 275 genes exhibiting both robust changes in transcript abundance and H3K18la modification (Figure S6F). Based on their expression patterns and H3K18la levels, these 275 genes were further classified into four groups: H3K18la-up & activated (93 genes), H3K18la-up & repressed (49 genes), H3K18la-down & activated (34 genes), and H3K18la-down & repressed (99 genes) (Fig. 4D). We also downloaded two published transcriptome datasets of hESCs induced differentiation (GSE161531 [33] and GSE137129 [34]) and performed similar analysis. 2,739 DEGs from intersection of GSE161531 and GSE137129 were identified after applying a significance threshold of P value < 0.05 and a fold-change cutoff of absolute value of abs (log2FoldChange) > 1 (Figure S6D&E). After intersection analysis with 2172 H3K18la genes, there are 183 genes exhibiting both robust changes in transcript abundance and H3K18la modification (Figure S6G). They also can be further divided into four groups: H3K18la-up & activated (46 genes), H3K18la-up & repressed (37 genes), H3K18la-down & activated (33 genes), and H3K18la-down & repressed (67 genes) (Fig. 4E). Since we observed that the level of H3K18la was elevated during differentiation, and H3K18la was previously reported as an activator to gene transcription [24, 25], we focused on the “H3K18la-up & activated” genes. After intersection analysis of the 93 H3K18la-up & activated genes from our RNA-seq data and the 46 H3K18la-up & activated genes from public RNA-seq data, there are 19 genes shared by both groups (Fig. 4F). As there was enriched H3K18la signals at the promoter regions, we looked at the H2K18la sites on those 19 genes. Within these 19 genes, there are 6 genes have H3K18la at the promoter regions, which are PAX6, PLK3, TMEM169, RUSC2, PCDH9 and C1QTNF6 (Fig. 4F).

Next, we verified the mRNA expression pattern of those 6 genes during neuroectoderm differentiation by qRT-PCR. Except PLK3, the other 5 genes were confirmed to be upregulated in induced neuroectoderm differentiation (Fig. 4G). As sodium lactate treatment can promote the level of H3K18la, we further tested the expression levels of these 5 genes after sodium lactate treatment. The results showed that the transcription levels of 4 genes, PAX6, TMEM169, RUSC2, and C1QTNF6, could be significantly upregulated by sodium lactate (Fig. 4H).

Moreover, an open chromatin conformation is essential for gene transcriptional process [42]. Therefore, we analyzed chromatin accessibility from published ATAC-seq data (GSE174727), which was induced neuroectoderm differentiation from human induced pluripotent stem cells (iPSCs) [33]. We found that H3K18la modifications of all these 4 genes (PAX6, TMEM169, RUSC2, and C1QTNF6) are located within accessible chromatin regions of their respective promoters in neural stem cells (NSCs) or neural progenitor cells (NPCs), but not in iPSCs (Fig. 5). These findings suggested that the accessibility of chromatin at these gene promoters may facilitate H3K18la-mediated transcriptional activation during neuroectoderm differentiation.

Fig. 5

H3K18la modifications were located within accessible chromatin regions. Genome browser tracks of H3K18la CUT&Tag signal (H3K18la antibody) loci at the promotors of 4 genes (PAX6, TMEM169, RUSC2, and C1QTNF6) were presented here. Day 0 and Day 5 cells are from the induced differentiation model. Open chromatin changes between iPSCs, in vitro-differentiated NPCs, and commercially available NSCs were defined by reported ATAC-seq; ATAC. Yellow box highlights regions where H3K18la overlaps open chromatin on those gene promoters. Scale bar, 1 kb

H3K18la positively regulates PAX6 transcription

PAX6 exhibited a particular higher elevation the mRNA level during induced differentiation (Fig. 4G) and upon lactate treatment compared to other genes (Fig. 4H). Therefore, we focused on PAX6 in the following study. PAX6 a is a key transcriptional factor to induce neurogenesis [43, 44]. The loss of PAX6 could also impair human neuroectoderm differentiation in vitro [45]. We further explored how intracellular lactate promoting PAX6 transcription. To confirm the H3K18la level at PAX6 promoter, we also performed a quantitative chromatin immunoprecipitation (qChIP) analysis on PAX6 promoter by using H3K18la antibody. The results showed that there is H3K18la enrichment at PAX6 promoter and it was significantly elevated during neuroectoderm differentiation (Fig. 6A). Moreover, sodium lactate treatment could significantly enhance the H3K18la level at PAX6 promoter, while Gska treatment would slightly attenuate the H3K18la level at PAX6 promoter in Day 5 cells (Fig. 6B). Collectively, these results demonstrated that lactate promoted H3K18la modification at PAX6 promoter during neuroectoderm differentiation.

Fig. 6

Exogeneous lactate upregulates the H3K18 lactyaltion level and transcription of PAX6. (A) qChIP analysis of the PAX6 promoter was performed using antibodies against H3K18la and control IgG in Day 0 and Day 5 cells from the induced neuroectoderm differentiation model (n = 4). (B) qChIP analysis of the PAX6 promoter was performed using antibodies against H3K18la and control IgG in Day 5 cells with sodium lactate or Gska treatments in the induced neuroectoderm differentiation model (n = 4). (C) Illustration of shPAX6 and sodium lactate treatment in the induced differentiation model. shPAX6 was introduced to human neuroectoderm differentiation in vitro at Day 1 with or without sodium lactate; and cells were harvested at Day 3. (D) qRT-PCR analysis of PAX6, OCT4, SOX2, SOX1, OTX2, and NES mRNA expression in Day 3 cells treated with shPAX6 or scramble shRNA (shScr) with or without sodium lactate

Since H3K18la modifications on the PAX6 promoter can be upregulated by lactate, we hypothesized that lactate treatment could rescue the level of PAX6 mRNA after shRNA treatment. To test this hypothesis, we introduced shPAX6 at day 1 of the induced differentiation model to degrade PAX6 mRNA; then cells were divided into two groups either with or without lactate treatment and all cells were harvested at Day 3 (Fig. 6C). The levels of PAX6 mRNA were quantified by qRT-PCR and the results showed that the expression of PAX6 was significantly suppressed by shPAX6, and it could be rescued by sodium lactate treatment to the level similar to control cells (Fig. 6D). We also examined the level of other markers, including pluripotency marker OCT4, neuroectoderm markers SOX1, SOX2, OTX2, and NES. The transcriptional level of SOX1 was significantly downregulated by shPAX6, while no obvious difference was observed on the expression levels of OCT4, SOX2, OTX2 and NES upon shPAX6 treatment (Fig. 6D). Sodium lactate treatment not only rescued the expression of PAX6 but also that of SOX1 (Fig. 6D), suggesting that PAX6 is upstream of SOX1. These results demonstrated that exogenous lactate could promote PAX6 transcription during neuroectoderm differentiation.