Metabolomics Identifies Metabolic Markers of Maturation in Human Pluripotent Stem Cell-Derived Cardiomyocytes

Abstract

Cardiovascular disease is a leading cause of death worldwide. Human pluripotent stem cell-derived cardiomyocytes (hPSC-CMs) hold immense clinical potential and recent studies have enabled generation of virtually pure hPSC-CMs with high efficiency in chemically defined and xeno-free conditions. Despite these advances, hPSC-CMs exhibit an immature phenotype and are arrhythmogenic in vivo, necessitating development of strategies to mature these cells. hPSC-CMs undergo significant metabolic alterations during differentiation and maturation. A detailed analysis of the metabolic changes accompanying maturation of hPSC-CMs may prove useful in identifying new strategies to expedite hPSC-CM maturation and also may provide biomarkers for testing or validating hPSC-CM maturation. In this study we identified global metabolic changes which take place during long-term culture and maturation of hPSC-CMs derived from three different hPSC lines. We have identified several metabolic pathways, including phospholipid metabolism and pantothenate and Coenzyme A metabolism, which showed significant enrichment upon maturation in addition to fatty acid oxidation and metabolism. We also identified increases in glycerophosphocholine and the glycerophosphocholine:phosphocholine ratio as potential metabolic biomarkers of maturation. These biomarkers were also affected in a similar manner during murine heart development in vivo. These results support that hPSC-CM maturation is associated with extensive metabolic changes in metabolic network utilization and understanding the roles of these metabolic changes has the potential to develop novel approaches to monitor and expedite hPSC-CM maturation.

Keywords: Biomarkers.; Cardiomyocytes; Human Pluripotent Stem Cells; Maturation; Metabolomics.

Figure 1

Long term culture of hPSC-CMs induces morphological and metabolic maturation. (A) H9 hESCs were differentiated to CMs and maintained in RPMI supplemented with B27 for 1 month (1m) or 3 months (3m). Single cells were replated on Matrigel-coated 12 well plates and maintained in the same medium for 3 days prior to immunofluorescent staining for α-actinin, F-actin and cTnT. Representative images are shown. Scale bars, 25 µm. Cell morphological parameters including (B) cell perimeter (µm), (C) cell area (µm²), and (D) cell circularity which was defined as 4*π*Area/(Perimeter)² was calculated using immunostaining images and ImageJ software (N=112 from two independent replicates). (E) Cells were analyzed for Ki-67 and MF20 at indicated time points using flow cytometry and (F) quantification of flow cytometry data using three biological replicates. (G) OCR profiles of hPSC-CMs either pretreated with ETO or DMSO (control) in response to oligomycin, FCCP, and rotenone and antimycin (Rtn/AA) using the Seahorse flux analyzer (*p<0.05 between 3m CM-ETO and 3m CM+ETO). (H) Maximal respiration rate defined as the difference between the OCR after addition of FCCP and OCR after addition of oligomycin to 1m and 3m hPSC-CMs (*p<0.05). Data represent mean +/- S.E.M. from at least three biological replicates.

Figure 2

Long term culture of hPSC-CMs induces ventricular maturation. (A) Representative immunostaining for MLC2v and MLC2a in 1m and 3m hPSC-CMs (Scale bars, 25 µm). (B) Quantification of percentage of cells expressing MLC2v, MLC2a or double positive for MLC2v and MLC2a assessed by examination of immunostaining images. Data were collected from two replicates for 1m and 3m hPSC-CMs, with N>120 in each sample. *p<0.05 comparing 1m and 3m hPSC-CMs.

Figure 3

Metabolomics reveal global time-dependent metabolic changes in hPSC-CMs. Three different parent hPSC lines (H9, ES03, 19-9-7) differentiated to either day 6 (D6) cardiac progenitors or to CMs and maintained in culture for 1 month (1m) and 3 months (3m). NMR analysis was performed on cell lysates and concentration data were auto-scaled prior to analysis. (A) Hierarchical clustering was performed using Pearson distance measure and Ward's linkage. (B) PLS-DA scores plot showing the two components accounting for highest variance on x and y axes. Each data point on the plot represents a unique biological sample and ellipses represent 95% confidence intervals. (C) Variable importance in projection (VIP) scores for top 15 metabolites along the first component which account for culture time-dependent metabolic changes. Colors represent average concentrations from three biological replicates.

Figure 4

Metabolite-metabolite correlation analysis (MMCA) highlights differences in metabolic physiological states in 1m and 3m hPSC-CMs. The metabolic concentrations from (A) 1m and (B) 3m hPSC-CMs were used to evaluate pairwise Pearson correlation coefficients. The correlation table was imported in Cytoscape and Metscape plugin and the cut-off was set to 0.8 (p<0.01). Organic layout was used for plotting the network. The thickness of the edges is proportional to the magnitude of correlation coefficient and the color represents positive (red) or negative (blue) correlation coefficient. The size of the nodes is proportional to the degree of each node.

Figure 5

Pathway enrichment analysis identifies significantly enriched metabolic pathways upon extended hPSC-CM culture. Metabolite concentrations from 1m and 3m hPSC-CMs were compared with day 6 cardiac progenitors and the pathways significantly (FDR<0.1 and p<0.05) enriched in either 1m or 3m hPSC-CMs were included in the analysis. Grey color represents pathways which were not significantly enriched (FDR>0.1 or p>0.05). Q statistic was used to evaluate the level of enrichment.

Figure 6

Potential biomarkers of maturation in hPSC-CMs and validation in mice hearts. A) The normalized and auto-scaled concentrations of top six metabolites which showed significant differences between 1m and 3m hPSC-CMs are shown with p values indicated. The red dotted line highlights the cut-off for distinguishing the two groups and achieving highest discrimination (based on receiver operating characteristic (ROC) curve analysis). B) Heart tissues from mice were isolated at day 20 and 3 months and were used for metabolic profiling. The same six metabolite concentrations in 20 day old mice and 3m old mice heart tissues are shown. Student's t-test was used to calculate the significance.