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Stem Cell Reports
Stem Cell Reports
Stem Cell Reports
2213-6711
Elsevier

S2213-6711(24)00221-2
10.1016/j.stemcr.2024.07.012
Article
Medium acidosis drives cardiac differentiation during mesendoderm cell fate specification from human pluripotent stem cells
Liu Weiwei weiweiliu@um.edu.mo
12∗
Hsieh Hsun-Ting 2
He Ziqing 13
Xiao Xia 1
Song Chengcheng 1
Lee En Xin 1
Dong Ji 34
Lei Chon Lok 5
Wang Jiaxian 6
Chen Guokai guokaichen@um.edu.mo
1789∗∗
1 Centre of Reproduction, Development and Aging, Faculty of Health Sciences, University of Macau, Macau SAR, China
2 Biological Imaging and Stem Cell Core Facility, Faculty of Health Sciences, University of Macau, Macau SAR, China
3 GMU-GIBH Joint School of Life Sciences, The Guangdong-Hong Kong-Macau Joint Laboratory for Cell Fate Regulation and Diseases, Guangzhou National Laboratory, Guangzhou Medical University, Guangzhou, China
4 Bioland Laboratory (Guangzhou Regenerative Medicine and Health Guangdong Laboratory), Guangzhou, China
5 Institute of Translational Medicine, Faculty of Health Sciences, University of Macau, Macau SAR, China
6 HELP Stem Cell Innovations Ltd. Co., Nanjing, Jiangsu, China
7 MoE Frontiers Science Center for Precision Oncology, University of Macau, Macau SAR, China
8 Zhuhai UM Science & Technology Research Institute, Zhuhai, China
∗ Corresponding author weiweiliu@um.edu.mo
∗∗ Corresponding author guokaichen@um.edu.mo
9 Lead contact

22 8 2024
10 9 2024
22 8 2024
19 9 13041319
14 2 2024
27 7 2024
29 7 2024
© 2024 The Authors
2024
https://creativecommons.org/licenses/by-nc-nd/4.0/ This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
Summary

Effective lineage-specific differentiation is essential to fulfilling the great potentials of human pluripotent stem cells (hPSCs). In this report, we investigate how modulation of medium pH and associated metabolic changes influence mesendoderm differentiation from hPSCs. We show that daily medium pH fluctuations are critical for the heterogeneity of cell fates in the absence of exogenous inducers. Acidic environment alone leads to cardiomyocyte generation without other signaling modulators. In contrast, medium alkalinization is inhibitory to cardiac fate even in the presence of classic cardiac inducers. We then demonstrate that acidic environment suppresses glycolysis to facilitate cardiac differentiation, while alkaline condition promotes glycolysis and diverts the differentiation toward other cell types. We further show that glycolysis inhibition or AMPK activation can rescue cardiac differentiation under alkalinization, and glycolysis inhibition alone can drive cardiac cell fate. This study highlights that pH changes remodel metabolic patterns and modulate signaling pathways to control cell fate.

Graphical abstract

Highlights

• Medium acidosis plays an active role in lineage-specific differentiation of hPSCs

• Acidic pH alone is sufficient to induce cardiac differentiation

• Prevention of acidification overrides Wnt inhibition to block cardiac differentiation

• Glycolysis inhibition rescues cardiac induction under alkaline pH

In this study, Chen and colleagues show that medium acidosis plays a key role in hPSC cell fate determination. Acidic pH alone can induce cardiac cell fate, while alkaline pH blocks cardiac differentiation even in the presence of Wnt inhibitor. Glycolysis inhibition rescues cardiac induction under alkaline pH. Through metabolic regulation, cardiac differentiation efficiency and consistency can be greatly enhanced.

Published: August 22, 2024
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pmcIntroduction

Human pluripotent stem cells (hPSCs), including human embryonic stem cells (hESCs) and induced pluripotent stem cells (iPSCs), are important research models and starting materials for cell production in regenerative medicine. In order to achieve optimal maintenance or differentiation, proper signaling and nutritional inputs are provided to the hPSCs in cell culture. Activators and inhibitors of specific signaling pathways are commonly used to drive stem cells toward desired cell fates. On the other hand, accumulating evidence shows that metabolism of various nutrients is also closely linked to pluripotency, reprogramming, and differentiation, which may have an impact on cell fate transitions (Dahan et al., 2019; Folmes et al., 2011; Moussaieff et al., 2015; Teslaa and Teitell, 2015).

Besides the exogenous factors present in cell culture (Liu and Chen, 2021; Liu et al., 2019), hPSCs themselves are also dynamic contributors to their local environment. For example, hPSCs produce autocrine mitogenic factors, such as insulin growth factors (IGFs) that affect cell fate specification in mesoderm lineages (Yang et al., 2019). On the nutritional side, hPSCs not only consume large amount of nutrients such as glucose and glutamine but also remodel their surroundings through the autocrine secretion of metabolites (Kumar et al., 2017; Tatapudy et al., 2017). Numerous endogenous metabolic factors are continuously produced by hPSCs. However, it is unclear whether and how these metabolites play active roles in cell fate determination.

We are interested in the differentiation of hPSCs toward mesendodermal cell fates because many important cell types such as cardiomyocytes and hepatocytes are generated through this lineage. For example, cardiomyocytes from hPSCs through targeted differentiation provide important tools for drug screening, toxicology studies, and cell therapy. Cardiomyocyte induction can be effectively achieved through modulation of WNT (Wingless/Integrated) signaling using growth factors or small chemicals. WNT activation drives hPSCs toward mesendodermal progenitors, and subsequent WNT inhibition leads to cardiomyocyte cell fate (Burridge et al., 2014; Lian et al., 2012). In comparison, if mesendodermal progenitors were allowed to spontaneously differentiate without WNT inhibitor, a variety of cell types will emerge, driven by endogenous signals from the cells. Suppression of endogenous IGF or transforming growth factor β (TGF-β) pathways also leads to cardiomyocyte differentiation (Kattman et al., 2011; Yang et al., 2019). These findings demonstrate the key roles of signal transduction pathways in mesendodermal cell fate specification. However, the roles of metabolic processes or metabolites in this process are less clear and need further investigation.

Glycolysis and oxidative phosphorylation are essential metabolic processes to generate energy in hPSCs. In the pluripotent state, glycolysis is the main energetic metabolism process in hPSCs, and lactic acid is generated as a metabolic byproduct, acidifying the culture media. The balance of energetic metabolism shifts from glycolysis toward oxidative phosphorylation when hPSCs differentiate to specific cell types such as cardiomyocytes (Cho et al., 2006; Chung et al., 2007; Cliff et al., 2017; Shyh-Chang et al., 2013; Tsogtbaatar et al., 2020). As a result, acid secretion is high in the early stages of differentiation, covering the critical period of cell fate determination. We reported previously that acidic environment is inhibitory to mesoderm induction by BMP4, while pH neutralization by NaHCO3 enhances mesoderm induction (Liu et al., 2018). In this study, we investigate the functional role of acids in cell fate determination from mesendoderm progenitors and how environmental pH controls lineage-specific differentiation. Cellular secretion of acids acidifies the culture medium and leads to pH fluctuations between daily medium changes. When the secreted acid is neutralized by excess buffering agents during differentiation, culture medium is no longer acidified, and cardiomyocyte generation is inhibited. This inhibition is strong enough to override the impact of WNT inhibitor IWP2, which is commonly used to induce cardiomyocytes. On the other hand, the addition of exogenous acids drives mesendoderm progenitors toward cardiomyocytes without the need for signaling modulators. Upon further investigation, we report that significant changes in signal transduction and metabolic processes occur as a result of pH modulation and contribute to the cell fate changes.

Results

Medium pH significantly affects cell fate determination in spontaneous differentiation of mesendoderm progenitors

Mesendoderm progenitor cells can differentiate to distinct cell types without exogenous stimuli (Yang et al., 2019), and we used this platform to study the impact of environmental pH on cell fate determination. GSK3 inhibitor CHIR-99021, a WNT activator, was applied on H1 hESCs to induce mesendoderm progenitors. Cells were then allowed to spontaneously differentiate in the absence of exogenous growth factors or chemical inducers (Figure S1A). Culture medium was changed every day. To characterize the impact of lactic acid secretion on the microenvironment, we monitored medium pH during differentiation and observed that medium pH decreased significantly within 24 h, fluctuating between 7.3 and 6.6 (Figures 1A and S1B). The amplitude of pH change was most prominent between day 2 and day 4, which coincides with the critical period of mesendodermal cell fate specification. We also observed that medium pH was acidified within 4 h of medium change and reached 6.8 after just 8 h (Figure S1B). This suggested that hPSCs continuously modified medium pH through lactic acid release, and cells were in acidic condition for over 16 h on a 24-h feeding schedule. We then showed that the pH fluctuation could be modulated by exogenous reagents, HCl and NaHCO3 (Figures S1A and S1C). Exogenous HCl kept medium pH at acidic levels (Figure 1B), while NaHCO3 prevented acidification (Figure 1C).Figure 1 Medium pH significantly affects cell fate determination in spontaneous differentiation of mesendoderm progenitors

(A–C) pH values of cell culture medium before and after daily medium change during the induction and spontaneous differentiation of mesendoderm progenitors. See Figure S1A for procedure details. (A) Control condition, (B) HCl (8 mM) applied daily on day 2–7, (C) NaHCO3 (20 mM) applied daily on day 2–7. “DF” refers to differentiation medium (DF medium) (DMEM/F12, vitamin C, selenium, transferrin, lipid).

(D) Heatmap of gene expression levels on day 10 of differentiation, measured by qPCR (n = 3 differentiations). Expression levels were calculated from 2ˆ(-ΔCt), normalized to GAPDH and scaled by column. See Figure 3D for comparison.

(E) Percentage of cells expressing TNNT2, AFP, and CD44, measured by flow cytometry analysis on day 10 of differentiation (n = 3 differentiations). ∗∗∗, p < 0.001 compared to control (one-way ANOVA with Dunnett’s multiple comparison test). NS, not significant.

(F) Immunostaining showing expression of TNNT2, AFP, and CD44 on day 10 of differentiation under control (Ctrl), HCl, and NaHCO3 treatment. Scale bar, 100 μm.

See also Figure S1.

We investigated how pH fluctuation could affect the emergence of various cell types in 10 days of differentiation, when pH was transiently modulated between day 2 and day 7 (Figures 1A–1C). Under spontaneous differentiation condition, medium pH fluctuated daily (Figure 1A), and various cell type-specific genes were detected (Figure 1D), including markers for cardiomyocytes (ISL1, NKX2-5, TNNT2, MYL7, MYL2, MYH7), epicardium (WT1), mesenchymal cells (CD44), endothelium (PECAM, CDH5), hepatocytes (AFP), and intestinal cells (CDX2). The addition of HCl maintained a constantly acidic pH (Figure 1B) and enhanced cardiac differentiation while suppressing other cell fates (Figure 1D). Meanwhile, NaHCO3 neutralized cellular acid secretion to prevent medium acidification (Figure 1C), which promoted mesenchymal, endothelial, and endodermal gene expression but suppressed cardiac differentiation (Figure 1D). Flow cytometry analysis and immunostaining also demonstrated that acidic condition enhanced cardiac differentiation, while alkaline pH suppressed cardiac cell fate and promoted other cell types (Figures 1E and 1F). At the concentration of HCl (8 mM) and NaHCO3 (20 mM) we were using, the percentage of dead cells was slightly higher under HCl treatment, but the majority (∼80%) of cells survived (Figure S1D). Intracellular pH is lower under HCl treatment, as shown by staining with intracellular pH indicator (Figure S1E). These data suggested that environmental pH plays a critical role in the generation of heterogeneous cell fates from mesendoderm progenitors. Exposure to low pH is necessary for the spontaneous generation of cardiomyocytes, and suppression of acidosis prevented cardiac cell fate. In contrast, a constantly acidic pH led to loss of diversity and promoted differentiation toward cardiomyocytes.

Acidic pH alone is sufficient to drive cardiac differentiation

We then further investigated the aforementioned observation that application of acid drives cardiac differentiation. Considering that the medium acidification is caused by lactic acid from glycolysis, we compared HCl and lactic acid in their ability to modulate cell fate. We showed that lactic acid and HCl decreased medium pH in a dose-dependent manner with similar patterns (Figure S2A). With decreased pH in spontaneous differentiation condition, cardiac gene expression was elevated by both lactic acid and HCl, while epicardial markers WT1 and TBX18 were suppressed (Figures 2A and 2B). The cardiac induction by both acids was confirmed by flow cytometry (Figure S2B). These results indicated that decreased pH was a functional factor modulating cell differentiation. To simplify the study, we used HCl as the main tool to study proton function in this paper. 8 mM HCl was used unless otherwise specified. We also showed that acidic medium induced cardiomyocytes from multiple hPSC lines, including H1 and H9 hESCs as well as NL-1 and NL-4 iPSCs (Figures S2C–S2F).Figure 2 Acidic pH drives cardiac differentiation

(A) Heatmap of cardiac and epicardial gene expression levels on day 11 of mesendoderm spontaneous differentiation showing the concentration-dependent effect of HCl treatment (day 2–7).

(B) Heatmap of cardiac and epicardial gene expression levels on day 11 of differentiation showing the concentration-dependent effect of lactic acid treatment (day 2–7). Gene expression was measured by qPCR (n = 3 differentiations. Expression levels were calculated from 2ˆ(-ΔCt), normalized to GAPDH and scaled by row).

(C) FACS analysis of TNNT2-positive cells on day 12 of cardiac differentiation, showing the impact of HCl treatment at different stages of the differentiation.

(D) Expression of TNNT2 and NKX2-5 following HCl treatment at different stages of the differentiation, measured by qPCR (n = 4 technical replicates and are representative of 3 independent experiments).

(E) Western blot showing the levels of non-phosphorylated (active) β-catenin and total β-catenin under NaHCO3, control, or HCl treatment. Mesendoderm progenitors on day 2 of differentiation were subjected to treatment for 24 h and collected on day 3. β-actin was used as a loading control.

(F) Immunostaining of NKX2-5 and TNNT2 in HCl-induced cardiomyocytes. Cells were passaged into confocal dishes on day 12 of differentiation and fixed for immunostaining the next day. Scale bar, 50 μm.

(G) Heatmap showing row-scaled TPM (transcripts per kilobase million) values of differentially expressed genes (DEGs) under control, HCl, and NaHCO3 treatment on day 0, 3, 5, 7, and 11 of spontaneous differentiation, compared to IWP2-induced cardiac differentiation.

(H) Expression profile of representative genes from each cluster in the heatmap in (G).

See also Figure S2, Videos S1 and S2.

Video S1. Beating cardiomyocytes induced by HCl, related to Figure 2

Mesendoderm progenitors were generated from H1 cells using CHIR-99021 (day 0) and then allowed to spontaneously differentiate under 8 mM HCl (applied day 2–7) in DF medium. Video taken on day 11.

Video S2. Beating cardiomyocytes induced by lactic acid, related to Figure 2

Mesendoderm progenitors were generated from H1 cells using CHIR-99021 (day 0) and then allowed to spontaneously differentiate under 8 mM lactic acid (applied day 2–7) in DF medium. Video taken on day 11.

We further analyzed cell differentiation under low pH. We showed that the timing of acid treatment was critical for the emergence of cardiomyocytes (Figures 2C and 2D). When acid was applied before day 2, most cells died by day 3. If acid was applied after day 5, there was no cardiac induction. Acid treatment between day 2 and day 7 yielded the best cardiac differentiation, so we used this schedule to induce cardiac differentiation in the rest of this study. We showed that acidic medium decreased the level of active (non-phosphorylated) β-catenin and total β-catenin, suggesting WNT pathway was inhibited under acid treatment (Figure 2E). We then showed that WNT activator CHIR-99021 partially suppressed cardiac induction by HCl (Figure S2G). These data suggested that acidic pH induced cardiac differentiation at least partially through WNT inhibition.

Under acid treatment, most cells were positive in NKX2-5 and TNNT2 expression by immunostaining (Figure 2F), which was consistent with flow cytometry analysis (Figure S2C). Expression of epicardium cell marker WT1 was suppressed by acid treatment (Figures 2A and S2H). Upon extended culture, well-organized myofibril structures formed in acid-induced cardiomyocytes as shown by α-actinin immunostaining, similar to the cells induced by WNT inhibitor IWP2 (Figure S2I). Patch clamp analysis and microelectrode array analysis demonstrated typical cardiomyocyte electrophysiological profiles similar to the cells induced by IWP2 (Figures S2J and S2K). These data demonstrated that low pH was an inducer for cardiac cell fate in the absence of other exogenous signaling modulators.

Global gene expression is dynamically regulated by medium pH

In order to understand the impact of medium pH on the differentiation process, a time course experiment was conducted to study global gene expression in spontaneous differentiation when different pH was applied to mesendoderm progenitors from day 2 to day 7. Principal-component analysis (PCA) showed that pH modulation led to significant transcriptome changes after just one day of treatment (day 3), and the difference became more prominent in the following days (Figure S2L). At the end of the time course (day 11), gene expression was enriched in liver, kidney, and heart cell types under spontaneous condition without additional pH modulation (Figure S2M, cluster 2 and 4). Acidic pH promoted global gene expression enriched in cardiac cell fate similar to Wnt inhibitor IWP2 (Figure S2M, cluster 3 and 5), while suppressing gene expression in the liver, smooth muscle, adipocytes, and lung (Figure S2M, cluster 1, 2, and 4). Meanwhile, alkaline pH induced a diversity of mesodermal/endodermal cell types (Figure S2M, cluster 1 and 2) but reduced cardiac gene expression (Figure S2M, cluster 3 and 5). Further examination of cardiomyocyte markers confirmed the cardiac-promoting effect of HCl and the inhibitory effect of NaHCO3 (Figure S2N). These data supported the findings that medium pH played critical roles in cell fate determination in the absence of growth factors.

An overview of the gene expression time course revealed that, during the differentiation process, following the loss of pluripotency in response to CHIR-99021 treatment (Figures 2G and 2H, cluster 1), expression of primitive streak and progenitor cell markers was elevated on day 3–5 (Figures 2G and 2H, cluster 2 and 3), and cell type-specific gene expression elevated between day 5–10 (Figures 2G and 2H, cluster 4, 5, and 6). HCl promoted the expression of cardiomyocyte markers (cluster 5), similar to IWP2. We then further investigated the gene expression on day 3, which was the critical period for cell fate specification (Figure S2O). Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis showed that HCl treatment significantly downregulated gene expression in DNA replication, cell cycle, Wnt signaling, TGF-β signaling, and various metabolic pathways (Figure S2O, cluster 1). HCl also upregulated gene expression in multiple signaling pathways, including calcium signaling pathway and mitogen-activated protein kinase (MAPK) and AMP-activated protein kinase (AMPK) signaling pathways, as well as cellular processes such as focal adhesion (Figure S2O, cluster 3 and 5). In comparison, NaHCO3 treatment significantly suppressed gene expression in cyclic AMP and calcium signaling pathways (Figure S2O, cluster 4 and 5). NaHCO3 treatment significantly elevated gene expression in glycolysis, Wnt and TGF-β signaling, and other metabolic pathways (Figure S2O, cluster 1). Further analysis of Wnt pathway components on day 3 showed that NaHCO3 promoted expression of canonical Wnt pathway genes, and HCl elevated several Wnt target genes and negative regulators of Wnt pathway such as NKD2 and DDIT3 (Figure S2P). Taken together, these data suggest that proton levels in the microenvironment affected important cellular processes and signaling pathways involved in cell fate determination, and acidic pH is capable of driving cell fate toward cardiomyocytes without other chemical inducers.

Alkaline pH interferes with signal transduction in cardiac differentiation

Because acidic and alkaline conditions could modulate mesoderm differentiation, we investigated how they interacted with WNT pathway inhibition, which is widely used for inducing cardiomyocytes (Figure S3A). In the presence of WNT inhibitor IWP2 (applied from day 2 to day 4), the medium was also acidified daily by hPSCs within a few hours, and medium pH decreased to 6.8 in around 8 h (Figure S3B). Exogenous HCl kept the medium at acidic pH, and NaHCO3 elevated medium pH (Figures 3A–3C). The overall trend of daily medium acidosis was similar to the one under spontaneous condition (Figures 1 and S1). These data suggested that WNT inhibition did not affect proton generation from glycolysis in mesendoderm progenitors.Figure 3 Alkaline pH interferes with signal transduction in cardiac differentiation

(A–C) pH values of cell culture medium before and after daily medium change during IWP2-induced cardiac differentiation as shown in Figure S3A. (A) Control condition, (B) HCl (8 mM) applied daily on day 2–7, (C) NaHCO3 (20 mM) applied daily on day 2–7. “DF” refers to DF medium (DMEM/F12, vitamin C, selenium, transferrin, lipid).

(D) Heatmap of gene expression levels on day 10 of differentiation, measured by qPCR (n = 3 differentiations). Expression levels were calculated from 2ˆ(-ΔCt), normalized to GAPDH and scaled by column. See Figure 1D for comparison.

(E) Percentage of cells expressing TNNT2, measured by flow cytometry analysis on day 10 of differentiation (n = 3 differentiations). ∗∗, p < 0.01; ∗∗∗, p < 0.001 compared to IWP2 condition (one-way ANOVA with Dunnett’s multiple comparison test).

(F) Flow cytometry analysis of TNNT2-positive cells on day 10 of differentiation (n = 3 differentiations). Cells were treated with or without IWP2 (day 2–4) and NaHCO3 (applied during different time periods).

(G) Western blot showing the levels of non-phosphorylated (active) β-catenin and total β-catenin under IWP2, IWP2/NaHCO3, or IWP2/HCl treatment. Mesendoderm progenitors on day 2 of differentiation were subjected to treatment for 24 h and collected on day 3. β-actin was used as loading control.

(H) Flow cytometry analysis of TNNT2 expression on day 10, showing the inhibitory effect of CHIR-99021 (applied day 2–4) on IWP2-induced cardiac differentiation and its rescue by HCl (applied day 2–7).

See also Figure S3.

We investigated how pH modulation could affect the emergence of cardiomyocytes under WNT inhibition. WNT inhibitor and acid synergistically induced cardiomyocytes according to real-time PCR (Figure 3D) and flow cytometry analysis (Figure 3E). In contrast, NaHCO3 suppressed cardiac differentiation even in the presence of WNT inhibitor IWP2, and it promoted differentiation to other mesoderm and endoderm cell fates (Figures 3D and 3E). Similar impacts of acidic and basic pH on WNT inhibitor-induced cardiac differentiation were observed with H9 hESCs and NL-1 and NL-4 iPSCs (Figures S4C–S4E). To further evaluate the impact of alkalinization, we applied NaHCO3 for different lengths of time during IWP2-induced cardiac differentiation and found that NaHCO3 treatment during day 2–4 is sufficient to suppress cardiomyocyte generation by IWP2, and the effect is similar to day 2–7 treatment. NaHCO3 treatment during day 5–7 did not have much impact (Figure 3F). These results suggest that medium acidification between day 2 to day 4 is a necessary factor for cardiomyocyte cell fate specification. To better understand the mechanism of the pH impact, we examined WNT signaling 24 h after IWP2 and pH treatment. We showed that HCl further suppressed active β-catenin in the presence of IWP2, while the level of active β-catenin was maintained in the presence of NaHCO3 (Figure 3G). WNT pathway activator CHIR-99021 fully suppressed IWP2-induced cardiac differentiation, but HCl was able to partially rescue cardiac differentiation under CHIR-99021 treatment (Figure 3H). It implied that acidic treatment could induce cardiac fate synergistically with WNT inhibition through another pathway.

In addition to differentiation in DMEM/F12-based media, we also carried out spontaneous and IWP2-induced differentiation in a DF medium prepared using RPMI1640 medium (RPMI1640, vitamin C, selenium, transferrin, lipid) and observed similar impacts of acidic and basic pH on cardiac differentiation (Figure S3F). Similar phenotypes were also reproduced when mesendoderm progenitors were induced with a different protocol using BMP4/Activin A (Figure S3G).

PCA showed that HCl led to a transcriptome trajectory similar to IWP2, but NaHCO3 led cells to another path even in the presence of IWP2 (Figure S3H). Day 11 RNA sequencing (RNA-seq) data demonstrated that IWP2 induced gene expression enriched in cardiac fate (Figure S3I, cluster 2), but NaHCO3 suppressed cardiac gene expression and promoted gene expression enriched in the liver, lung, small intestine, and kidney both in the presence and in the absence of IWP2 (Figure S3I, cluster 1 and 3). These data suggested that elevated pH was able to override signaling modulator IWP2 and suppress cardiac fate induced by WNT inhibition.

pH status influences metabolic changes during differentiation

To understand how alkaline pH overrides WNT inhibition to control cell fate, we then investigated the gene expression profile on day 3 of differentiation under IWP2+NaHCO3 treatment (Figure S3J). Compared to the IWP2 group, addition of NaHCO3 promoted gene expression in the Ras signaling pathway, regulation of actin cytoskeleton, DNA replication, cell cycle, and Wnt signaling pathway (Figure S3J, cluster 2 and 3). In addition to the impact on signal transduction and cellular processes, KEGG pathway enrichment analysis also showed involvement of pH-induced differentially expressed genes (DEGs) in multiple metabolic pathways, including purine and pyrimidine metabolism, carbon metabolism, and amino acid metabolism (Figures S4A and S4B, see also Figures S2O and S3J). Based on these findings, we explored whether environmental pH may control cell fate partially through metabolic changes. We examined the expression levels of glycolysis and tricarboxylic acid (TCA) pathway genes through the differentiation process. On day 3 of differentiation, NaHCO3 treatment significantly elevated the expression of genes in the glycolysis pathway, coinciding with early mesendoderm cell fate commitment (Figure 4A). In contrast, differences in TCA cycle genes appeared later in the differentiation process around day 7–11, and levels are elevated in HCl- and IWP2-treated groups, probably as a result of cardiac cell generation (Figure 4B).Figure 4 pH-induced metabolic changes during differentiation

(A) Heatmap showing the time course of gene expression in the glycolysis pathway based on RNA-seq data (TPM values scaled by row).

(B) Heatmap showing the time course of gene expression in the TCA pathway based on RNA-seq data (TPM values scaled by row).

(C) Impact of pH on metabolite levels, measured by LC-MS. Mesendoderm progenitors were subjected to control (Ctrl), HCl, or NaHCO3 treatment on day 2 of differentiation. Spent medium was collected for LC-MS analysis on day 3 and compared with fresh DF medium to calculate the level changes. Data presented as mean ± SD and are normalized to control, n = 3 differentiations. ∗∗, p < 0.01; ∗∗∗, p < 0.001 compared to control well (one-way ANOVA with Dunnett’s multiple comparison test).

(D) Glycolysis Stress Test showing the impact of pH modulation on glycolysis.

(E) Mito Stress Test showing the impact of pH modulation on mitochondrial respiration. Mesendoderm progenitors were treated with HCl or NaHCO3 on day 2 of differentiation, and the assays were carried out on day 3. Data are normalized to total protein (by Bradford assay) and presented as mean ± SD, n = 4 differentiations. ∗, p < 0.05; ∗∗, p < 0.01; ∗∗∗, p < 0.001 compared to control (one-way ANOVA with Dunnett’s multiple comparison test). NS, not significant.

See also Figure S4.

We then examined the impact of pH modulation on hPSC metabolism by liquid chromatography-mass spectrometry (LC-MS) and demonstrated that metabolite levels were affected by pH on day 3 of differentiation (Figure 4C). Acidic environment suppressed glucose consumption, glutamine consumption, and lactic acid release, while NaHCO3 enhanced these processes. Pyruvate consumption was not significantly changed. In comparison, IWP2 treatment did not significantly change day 3 metabolite levels (Figure S4C). Seahorse extracellular flux analysis showed that acidic environment significantly suppressed glycolysis and glycolytic capacity in differentiating cells (Figure 4D) but did not affect mitochondrial respiration (Figure 4E). On the other hand, alkaline environment led to elevated glycolysis and glycolytic capacity (Figure 4D), as well as elevated basal and spare respiratory capacity and ATP production (Figure 4E). Similar phenotypes were observed in the presence of WNT inhibitor IWP2 (Figures S4D and S4E). These data suggested that glycolysis and oxidative phosphorylation were affected by proton levels in the stem cell microenvironment.

Glycolysis inhibition promotes cardiac cell fate

Considering that the level of glycolysis decreases during stem cell differentiation (Shyh-Chang et al., 2013), we hypothesized that NaHCO3 may interfere with cardiac differentiation by maintaining glycolysis. We examined whether glycolysis inhibition could rescue cardiac differentiation under NaHCO3. Glycolysis inhibitor 2-deoxy-D-glucose (2-DG) was applied on mesendoderm progenitors in the presence of IWP2 and NaHCO3. Glycolysis Stress Test showed that 2-DG suppressed glycolysis and glycolytic capacity that were elevated by NaHCO3 (Figures 5A and S5A). In Mito Stress Test, 2-DG elevated spare respiratory capacity but not basal mitochondrial respiration, proton leak, or ATP production (Figures 5B and S5B). When applied during IWP2-induced cardiac differentiation, 2-DG rescued expression of cardiac markers TNNT2 and NKX2.5 under NaHCO3 (Figure 5C). Flow cytometry confirmed that 2-DG increased TNNT2-positive cells in the presence of NaHCO3 (Figure 5D). Transcriptome analysis showed that 2-DG shifted the differentiation trajectory that was affected by NaHCO3 (Figure S5C). Gene Ontology term enrichment analysis on day 3 samples showed that 2-DG treatment elevated gene expression associated with cardiac muscle tissue development and suppressed glycolytic process which was elevated by NaHCO3 (Figure S5D). Cell type analysis on day 11 showed that 2-DG rescued cardiac differentiation in the presence of IWP2/NaHCO3 and promoted development of cardiac muscle fiber, ventricle, and heart bulk tissue (Figure S5E). These data suggested that high pH suppressed cardiac differentiation by maintaining glycolysis, so the inhibition of glycolysis could rescue cardiac differentiation at high medium pH, confirming that metabolic control is part of the mechanism through which NaHCO3 overrides WNT inhibition in cardiac differentiation.Figure 5 Glycolysis inhibition promotes cardiac cell fate through AMPK signaling

(A) Glycolysis Stress Test showing the impact of 2-DG (4 mM) on glycolysis during IWP2-induced cardiac differentiation under alkaline conditions.

(B) Mito Stress Test showing the impact of 2-DG (4 mM) on mitochondrial respiration during IWP2-induced cardiac differentiation under alkaline conditions. Mesendoderm progenitors were treated with IWP2 with or without NaHCO3 or NaHCO3/2-DG on day 2 of differentiation, and the assays were carried out on day 3. Data are normalized to total protein (by Bradford assay) and presented as mean ± SD, n = 4 differentiations. See Figures S5A and S5B for data analysis results.

(C) qPCR analysis of NKX2-5 and TNNT2 expression on day 10 of cardiac differentiation, normalized to GAPDH. IWP2, 3 μM (day 2–4); NaHCO3, 20 mM (day 2–7); 2-DG, 4 mM (day 2–4). Data presented as mean ± SD of four technical replicates and are representative of three independent experiments. ∗∗∗, p < 0.001 (one-way ANOVA with Dunnett’s multiple comparison test).

(D) Flow cytometry analysis of the percentage of TNNT2+ cells on day 10 of IWP2-induced cardiac differentiation. IWP2, 3 μM; NaHCO3, 20 mM; 2-DG, 4 mM. Data presented as mean ± SEM of four independent experiments. ∗∗, p < 0.01 (one-way ANOVA with Dunnett’s multiple comparison test).

(E) Flow cytometry analysis of TNNT2+ cells on day 10 of mesendoderm spontaneous differentiation, showing the impact of 2-DG treatment without IWP2. Data presented as mean ± SD, n = 3 differentiations. ∗∗∗, p < 0.001 (one-way ANOVA with Dunnett’s multiple comparison test).

(F) Western blot showing the levels of phosphorylated AMPK and total AMPK under control (Ctrl), HCl, or NaHCO3 treatment. Mesendoderm progenitors on day 2 of differentiation were subjected to treatment for 24 h and collected on day 3. β-actin was used as loading control.

(G) Western blot showing the effect of 2-DG treatment on AMPK phosphorylation in cells differentiating under control (Ctrl), 2-DG, IWP2, IWP2/NaHCO3, or IWP2/NaHCO3/2-DG treatment. Mesendoderm progenitors on day 2 of differentiation were subjected to treatment for 24 h and collected on day 3. β-actin was used as loading control.

(H) Flow cytometry analysis of the percentage of TNNT2+ cells on day 10 of IWP2-induced cardiac differentiation in the presence or absence of NaHCO3 and AMPK activator A769662. Data presented as mean ± SEM of three independent experiments. ∗∗, p < 0.01 (one-way ANOVA with Dunnett’s multiple comparison test).

(I) Model diagram illustrating the mechanism of pH impact on cell fate. CHIR, CHIR-99021; 2-DG, 2-deoxy-D-glucose; CM, cardiomyocytes.

See also Figure S5.

We further examined the impact of glycolysis inhibition on cell fate determination in spontaneous differentiation from mesendoderm progenitors (without IWP2). 2-DG alone was sufficient to promote cardiac gene expression, while suppressing gene expression in epicardial, mesenchymal, and endothelial lineages (Figure S5F). The induction of cardiac differentiation by 2-DG was confirmed by flow cytometry analysis and immunostaining (Figures 5E and S5G). These data suggested that glycolysis inhibition promoted cardiac cell fate, and metabolic regulation played an active role in cardiac fate induction.

Glycolysis inhibition induces cardiomyocytes through AMPK activation

In order to understand how metabolic modulation controls cell fate, we examined the impact of 2-DG on gene expression in key signaling pathways involved in cardiac differentiation. RNA-seq data showed that HCl treatment elevated the expression of PRKAA2 which encodes AMPK. NaHCO3 suppressed its expression. Under IWP2 treatment, NaHCO3 treatment suppressed PRKAA2 expression, which was rescued by 2-DG (Figure S5H). Consistently, acidic pH upregulated gene expression in AMPK pathway in spontaneous differentiation (Figure S2O). We hypothesized that pH-associated metabolic changes might alter cell fate through AMPK signaling, which is a master regulator of cellular metabolism and cardiac differentiation (Sarikhani et al., 2020). We then examined the level of AMPK phosphorylation in cells differentiating under different pH. Acidic pH (HCl) promoted AMPK phosphorylation, while basic pH (NaHCO3) had an inhibitory effect (Figure 5F). 2-DG promoted AMPK phosphorylation both by itself and under IWP2/NaHCO3 treatment (Figure 5G). These data suggested that glycolysis inhibition by acidic pH or 2-DG activated AMPK pathway. When AMPK activator A769662 was applied, it rescued cardiac differentiation in the presence of IWP2/NaHCO3 as predicted (Figure 5H). Taken together, these data suggest that pH-induced metabolic changes act through AMPK to control cell fate. A model diagram summarizing the mechanisms of pH impact on cell fate can be found in Figure 5I.

In summary, our results show that medium pH has a significant impact on mesendoderm differentiation through regulation of signaling pathways and metabolism. Modulation of environmental pH and cell metabolism provide promising tools for stem cell manipulation and cell fate determination.

Discussion

The cellular microenvironment has a significant impact on stem cell function. In this report, we demonstrate that environmental pH plays an active role in cell fate determination. Medium acidosis and alkalinization significantly shift cell fate through changes in metabolism and signal transduction, and proper environmental pH is a prerequisite for cell type-specific differentiation driven by classic inducers.

Energetic metabolism in hPSCs and mesendoderm progenitors are characterized by high levels of glycolysis, which produces large amounts of lactic acid as a byproduct in a matter of hours. In reality, cells could be in acidic medium most of the time during differentiation when cells are cultured at high density. Our results show that, during in vitro spontaneous differentiation, the daily pH fluctuation is essential for the emergence of diverse cell types. Disturbances of this fine pH balance can lead to significant changes in differentiation trajectories. Continuously acidic pH environment is sufficient to drive mesendoderm progenitors toward cardiomyocytes without WNT inhibition, while elevated pH promotes other cell types but inhibits cardiac fate. The elevated pH can override WNT inhibition in cardiac induction. These outcomes are realized partially through WNT pathway and glycolysis regulation. It is still to be explored how these processes interact with each other under pH regulation.

Most stem cell manipulations focus on the modulation of signaling pathways, and it is quite surprising that elevated pH could override conventional signal modulation in cardiac induction. In order to examine the role of pH in other differentiation platforms, we characterized the role of medium pH during mesoderm induction (Figure S5I). Under BMP4 treatment, expression of mesoderm genes such as TBXT and MIXL1 was elevated by alkaline pH and was inhibited by acidic pH. At the same time, acidic pH promoted the expression of extraembryonic markers TROP2 and CGB (Figure S5J). Apparently, environmental pH is involved in cell fate determination not only in mesendoderm fates but also in the specification of mesoderm and extraembryonic lineages. These findings support the idea that metabolic feedback from medium acidity plays important roles in various cell fate determination processes.

Fluctuations in pH have significant impacts on growth, reproduction, and development through key cellular processes, including cellular metabolism, protein synthesis, and cell cycle (Ceccarini and Eagle, 1971; Fukamachi et al., 2013; Karagiannis and Young, 2001; Mackenzie et al., 1961; Patel et al., 1973). pH has also been reported to control major signal transduction pathways, such as MAPK (Stathopoulou et al., 2006), TGF-β (Fang et al., 2017), and Wnt pathways (Melnik et al., 2018; Oginuma et al., 2017, 2020; White et al., 2018). Elevated intracellular pH was shown to compromise β-catenin stability by promoting β-TrCP binding in Madin-Darby canine kidney (MDCK) cells (White et al., 2018). Oginuma et al. reported that culturing chicken embryos in low-pH buffer reduced the expression of Wnt target AXIN2 and favored differentiation toward neural cell fate, while a basic culture environment promoted paraxial mesoderm differentiation (Oginuma et al., 2020). In another report, acidification of intracellular pH was reported to induce the expression of DDIT3 and cause Wnt inhibition, leading to decreased stemness in cancer cells (Melnik et al., 2018). In our study, we observed inhibition of Wnt signaling by HCl along with elevated levels of DDIT3. Elevation of pH by NaHCO3, on the other hand, promoted canonical Wnt signaling and elevated the level of AXIN2 (Figure S2P). Our findings show that the acid-base balance is a key factor of the stem cell niche and determines cell fate through WNT and metabolic activities. It is likely that pH could be a pleiotropic modulator for stem cell manipulation.

During in vivo embryogenesis, the early development process from the zygote to the blastocyst occurs in the fallopian tube and the uterus. The pH of the follicular fluid and uterine fluid is alkaline (up to 7.7 in human, 7.8 in monkeys, and 7.9 in rabbits and pigs) and rises higher during ovulation and early pregnancy (Ng et al., 2018; Vishwakarma, 1962), suggesting that embryogenesis takes place in an environment less acidic than in vitro cell culture conditions. Our results show that an alkaline environment permits the development of heterogeneous cell types; the in vivo environment for early embryogenesis, being alkaline in pH, therefore supports the development of a variety of cells. One interesting finding from this study is that cardiac development under alkaline conditions requires not only WNT pathway inhibition but also signals from other pathways, for example AMPK activation, and potentially other necessary signals. This indicates that knowledge on differentiation gained from cell culture studies may need to be re-examined in the context of cell microenvironment, which is under continuous modulations by cells themselves. As in vitro differentiation platforms often involve pH fluctuations and a mostly acidic environment, it is possible that some key factors important for differentiation may have been overlooked in the current research platforms. Further studies examining the interaction between medium pH and cell fate determination may reveal new mechanisms and provide novel tools for future applications.

Our results show that changes in medium pH have a significant impact on cell fate. However, the impact of pH is often overlooked in common practice. pH control provides a new angle to improve stem cell differentiation. For example, the efficiency of cardiac induction with WNT inhibition often varies significantly from batch to batch, subject to plating density, growth rate, cell death, and many other processes in the differentiation period. We observed that, when the differentiation conditions are not optimal, acidic medium pH can often enhance the efficiency of cardiac induction. For example, when cells are plated at sub-optimal densities for cardiac differentiation, cardiomyocytes generated by WNT inhibition can be as low as <5%. Yet, with the modulation of pH by HCl, the efficiency of cardiac induction can be increased by 2- to 10-fold (Figure S5K). Interestingly, current cardiac differentiation protocols often call for high plating density, which may reflect the empiric utilization of acidified medium at high cell densities. Our results potentially explain the mechanism underlying this common practice. Taken together, pH modulation can be a powerful tool to enhance the consistency of stem cell applications.

In summary, our work reveals the significant impact of environmental pH in hESCs and demonstrates effective manipulation of cell fate through pH and metabolic modulations. This study provides a valuable tool to guide stem cell differentiation for research and clinical applications.

Experimental procedures

Resource availability

Lead contact

Requests for resources, reagents, and further information should be directed to and will be fulfilled by the lead contact, Guokai Chen (GuokaiChen@um.edu.mo).

Materials availability

All reagents generated in this study will be made available by the lead contact upon request.

Data and code availability

• RNA-seq data have been deposited at GEO and are publicly available as of the date of publication. The accession number is GEO: GSE211196.

• This paper does not report original code.

Experimental model and subject details

Cell lines

hESC lines H1 and H9 (WiCell Research Institute) and human iPSC lines NL-1 and NL-4 (National Institutes of Health) were used in this study between passages 25–50. The cell lines were routinely tested for mycoplasma status, sterility, genomic integrity, and pluripotency. hESC and iPSC studies were approved by the Institutional Review Board at the University of Macau.

Method details

Maintenance of hPSCs

hESCs and iPSCs were maintained in an E8 medium as previously described (Beers et al., 2012). Briefly, cells were cultured on matrigel-coated 6-well plates in 37°C, 5% CO2 incubators. The medium was changed daily until cells reach 60%–70% confluence. Cells were passaged every 3–4 days using the EDTA method in the presence of 5 μM Y-27632 (for 24 h after re-plating) at a split ratio of 1:6 to 1:12.

Differentiation of hPSCs

For spontaneous differentiation of mesendoderm progenitors, hPSCs at 60%–70% confluence were passaged 1:6 onto matrigel-coated 12-well plates in an E8 medium with 5 μM Y-27632 on day 2. The medium was changed on day 1 with fresh E8. Cells were switched to a DF medium (DMEM/F12, L-ascorbic acid [64 mg/L], sodium selenite [13.6 μg/L], transferrin [10μg/mL], chemically defined lipid concentrate (1×), penicillin/streptomycin) containing 5 μM CHIR-99021 on day 0 followed by a DF medium containing insulin (1 mg/L) on day 1. Starting day 2, the resulting mesendoderm progenitor cells were allowed to spontaneously differentiate in DF medium without chemical inducers. Specific treatments (pH modulation or 2-DG) were applied for designated periods of time starting from day 2. 8 mM HCl and 20 mM NaHCO3 were used for pH modulation unless otherwise specified. The medium was changed daily. Cells were collected for analysis between day 10–12.

For cardiac differentiation under WNT inhibition, the same procedures were used to induce mesendoderm progenitors with CHIR-99021 as described earlier. Starting at day 2, cells were cultured in a DF medium with IWP2 (3 μM, applied day 2, 3, and 4) to drive cardiac differentiation. Other treatments were applied as specified. The medium was changed daily. Cells were collected for analysis between day 10–12.

For BMP4-induced mesoderm commitment, hESCs at 60%–70% confluence were passaged 1:18 onto matrigel-coated 6-well plates in an E8 medium with 5 μM Y-27632 on day 1 and switched to an E8 medium with 20μg/mL BMP4 on day 0. The medium was changed daily. Cells were collected for RNA extraction on day 5.

qPCR

Cells were collected in RNAiso Plus reagent (Takara), and total RNA was extracted following the manufacturer’s instructions. cDNA was generated by reverse transcription using high-capacity cDNA reverse transcription kit (Applied Biosystems) and used for qPCR with SYBR Premix Ex Taq kit (Takara). Gene expression was normalized to the level of GAPDH. Heatmaps were generated using the online software Heatmapper (Babicki et al., 2016).

Western blotting

Cells were collected in 2× Laemmli buffer, and total protein levels were measured using bicinchoninic acid (BCA) protein assay kit (Thermo Fisher). After addition of bromophenol blue and β-mercaptoethanol, protein lysate was boiled at 95°C for 2 min, and 15-20 μg total protein/sample was loaded for SDS-PAGE. After the gel run, proteins were transferred onto polyvinylidene fluoride membrane using Bio-Rad Mini Trans-Blot cell. The membrane was blocked for 1 h in 5% milk, 1×TBST (Tris-Buffered Saline with Tween 20), and incubated with primary antibodies (diluted in 1% BSA, 1xTBST) at 4°C overnight, followed by horseradish peroxidase-conjugated secondary antibodies (diluted in 1% BSA, 1×TBST) for 1 h at room temperature. Chemiluminescent signals were generated using SuperSignal West Pico PLUS chemiluminescent substrate and detected using ChemiDoc MP system.

Flow cytometry

For flow cytometric analysis, cells in culture plates were washed with DPBS/EDTA (0.5 mM), treated with TrypLE select enzyme for 10 min at 37°C, neutralized with DMEM/F12 + 10% BSA, and dissociated by repeated pipetting. Cells were washed with 1xPBS, fixed in 1% paraformaldehyde/1xPBS for 10 min at 37°C, and washed again with 1xPBS. Cells were then permeabilized in 90% methanol/10% 1xPBS on ice for 30 min, washed in fluorescence-activated cell sorting (FACS) buffer (1% BSA, 0.05% Triton X-100, 1xPBS), and incubated with primary antibodies (diluted in FACS buffer) at 4°C overnight. After washing with FACS buffer, cells were incubated with secondary antibodies (diluted in FACS buffer) for 1 h at room temperature in the dark and then washed with 1xPBS and analyzed on a CytoFLEX S flow cytometer.

Immunostaining

Cells were either fixed in-well or passaged into matrigel-coated confocal dishes before fixation. Briefly, cells were rinsed with 1×PBS, fixed in 4% paraformaldehyde/1×PBS for 15 min at room temperature, washed with 1×PBS, and permeabilized in 0.5% Triton X-100/1×PBS for 15 min at room temperature. After washing, cells were incubated with primary antibodies (diluted in 1% BSA/1xPBS) at 4°C overnight, washed, and then incubated with secondary antibodies at room temperature for 1 h in the dark. Cell nuclei were stained with Hoechst for 10 min. Imaging was carried out using EVOS FL auto imaging system (Thermo Fisher Scientific) or Nikon A1R confocal microscope.

RNA-seq and bioinformatic analysis

Cells were collected on day 0, 3, 5, 7, and 11 of differentiation, and total RNA was extracted using RNAiso Plus reagent (Takara). Next-generation sequencing library was prepared using VAHTS Universal V8 RNA-seq library prep kit for Illumina, and sequencing was carried out using a 2 × 150 bp paired-end configuration on an Illumina NovaSeq system. Clean data were aligned to reference genome using the software HISAT2 (v2.0.1).

PCA and differential expression comparison were performed using R package PCAtools and DEseq2. For global expression filtration in PCA, genes with total expression in all groups greater than one were selected. Differential expression calculations were conducted across various groups in day 3, 5, and 11 samples. Genes with | log2 (fold change) | ≥1.5 and padj ≤0.05 were selected as significant genes. Z scores were used to plot heatmaps using the R package pheatmap. Function cutree was exerted to split genes into clusters. DEGs were used for cell type enrichment and KEGG pathway enrichment analyses by Enrichr (Chen et al., 2013; Kuleshov et al., 2016; Xie et al., 2021) using ARCHS4_Tissues, Human Gene Atlas, Tabula Sapiens, and KEGG 2021 Human.

Accession number

The accession number for RNA-seq data reported in this paper is GEO: GSE211196.

LC-MS analysis

Quantification of metabolites was carried out using LC-MS as previously described (Xu et al., 2021). Briefly, H1 hESCs were induced toward mesoderm progenitors as described earlier and subjected to control, HCl, or NaHCO3 treatment on day 2 of differentiation. Spent medium was collected after 24 h and centrifuged at 1,000 × g for 5 min. The supernatant was mixed with nine volumes of medium extraction solution, vortexed, and centrifuged at 18,000 × g at 4°C for 10 min. The supernatant was used for LC-MS analysis. Samples were run on Waters Xevo TQD coupled with Waters ACQUITY UPLC system, and data were analyzed using MassLynx with previously reported settings (Xu et al., 2021). Changes in metabolite levels were calculated by subtracting the level of metabolite in fresh DF medium.

Seahorse extracellular flux analysis

Extracellular flex assays were carried out using Seahorse XFe96 analyzer (Agilent). H1 cells were seeded on matrigel-coated XF96 microplate at 20,000 cells/well and induced toward mesendoderm progenitors as described earlier. Cells were subjected to control, HCl, NaHCO3, 2-DG, and IWP2 treatment or their combinations on day 2 of differentiation. Metabolic flux assays were carried out on day 3 of differentiation.

For Mito Stress Test, cells were switched to XF Cell Mito Stress Test assay medium (Seahorse XF base medium, 10 mM glucose, 1 mM pyruvate, 2 mM glutamine) with the same treatments applied (the concentrations of HCl and NaHCO3 were decreased to 1 mM and 2 mM, respectively, to maintain the pH of the assay medium at similar level as that of the HCl- or NaHCO3-supplemented DF medium) and were incubated in a non-CO2 incubator at 37°C for 1 h before loading onto the analyzer for test. The chemicals used for the assay were oligomycin (2 mM), carbonyl cyanide-4-(trifluoromethoxy) phenylhydrazone (FCCP, 1 mM), and rotenone/antimycin A (0.5 mM) from the Seahorse XF Cell Mito Stress Test kit. After the test, spent medium was removed, cells were lysed in lysis buffer (10 mM Tris pH 7.5, 0.1% Triton X-100) on ice, and Bradford reagent was added. Data were normalized to absorbance at 595nm.

For the Glycolysis Stress Test, cells were switched to XF Glycolysis Stress Test assay medium (Seahorse XF base medium, 2 mM glutamine) with the same treatments applied (the concentrations of HCl and NaHCO3 were decreased to 1 mM and 2 mM, respectively, to maintain the pH of the assay medium at similar level as that of the HCl- or NaHCO3-supplemented DF medium) and were incubated in a non-CO2 incubator at 37°C for 1 h before loading onto the analyzer for test. The chemicals used for the assay were glucose (10 mM), oligomycin (2 mM), and 2-DG (50 mM) from the Seahorse XF Glycolysis Stress Test kit. Cell lysis and data normalization were similar to the aforementioned Mito Stress Test.

Quantification and statistical analysis

Flow cytometry data are presented as mean ± standard deviation (SD) of three independent experiments unless specified. qPCR data are presented as mean ± SD of three technical replicates and are representative of three or more independent experiments unless specified. Statistical significance was determined using unpaired two-tailed Student’s t test for experiments with two treatment groups, and one-way ANOVA with Dunnett’s multiple comparison test for experiments with multiple groups. ∗, p < 0.05; ∗∗, p < 0.01; ∗∗∗, p < 0.001; NS, not statistically significant.

Supplemental information

Document S1. Figures S1–S5 and Table S1

Table S2. Quantitative PCR data used to generate heatmaps, related to Figures 1, 2, 3, and S5

Document S2. Article plus supplemental information

Acknowledgments

This work was supported by the 10.13039/501100004733 University of Macau (file no. MYRG-GRG2023-00137-FHS-UMDF ), by the 10.13039/501100006469 Science and Technology Development Fund , Macau SAR (file no. 0059/2019/A1 , 0123/2019/A3 , 0002/2021/AKP , 0085/2023/AMJ , 0002/2023/RIB1 , 0010/2023/AKP , and 0073/2023/ITP2 ), and by the 10.13039/501100002855 MOST Key R&D grant, Macau SAR (file no. 2022YFA1105000 ). We thank Dr. Faxiang Xu and the Proteomics, Metabolomics and Drug Development Core at the University of Macau for their support on LC-MS analysis.

Author contributions

W.L. and G.C. conceived and designed the project; W.L. and H.-T.H. conducted pH measurement and real-time PCR analysis; W.L., C.S., and E.X.L. conducted stem cell maintenance and differentiation; W.L., C.L.L., and J.W. conducted electrophysiological studies; W.L. characterized the differentiated cells; W.L., Z.H., X.X., and J.D. analyzed sequencing data; W.L. and G.C. wrote the manuscript.

Declaration of interests

W.L. and G.C. filed a patent application based on cardiomyocyte differentiation that is induced by acidic pH.

Supplemental information can be found online at https://doi.org/10.1016/j.stemcr.2024.07.012.
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