Anabolic Factors and Myokines Improve Differentiation of Human Embryonic Stem Cell Derived Skeletal Muscle Cells

Abstract

Skeletal muscle weakness is linked to many adverse health outcomes. Current research to identify new drugs has often been inconclusive due to lack of adequate cellular models. We previously developed a scalable monolayer system to differentiate human embryonic stem cells (hESCs) into mature skeletal muscle cells (SkMCs) within 26 days without cell sorting or genetic manipulation. Here, building on our previous work, we show that differentiation and fusion of myotubes can be further enhanced using the anabolic factors testosterone (T) and follistatin (F) in combination with a cocktail of myokines (C). Importantly, combined TFC treatment significantly enhanced both the hESC-SkMC fusion index and the expression levels of various skeletal muscle markers, including the motor protein myosin heavy chain (MyHC). Transcriptomic and proteomic analysis revealed oxidative phosphorylation as the most up-regulated pathway, and a significantly higher level of ATP and increased mitochondrial mass were also observed in TFC-treated hESC-SkMCs, suggesting enhanced energy metabolism is coupled with improved muscle differentiation. This cellular model will be a powerful tool for studying in vitro myogenesis and for drug discovery pertaining to further enhancing muscle development or treating muscle diseases.

Keywords: human embryonic stem cell; myokines; myosin heavy chain; myotubes; skeletal muscle.

Figure 1

Skeletal muscle differentiation of hESCs. (A) Differentiation protocol for the derivation of SkMCs from hESCs. HS = horse serum, E = hr-EGF, C = CHIR99021, AI = ALK5 inhibitor, Dex = dexamethasone, AA = ascorbic acid, I = insulin, SB = SB431542, P = hr-PDGF, F = hr-FGFB, H = hr-HGF, O = oncostatin, IG = hr-IGF1, N = necrosulfonamide. (B) hESC-SkMCs expressed high levels of SkMC markers dystrophin, a-actinin, MF20 (MYH all isoforms) and embryonic (MYH3) and perinatal (MYH8) myosin. Scale bar = 100 μm. (C) IPA transcriptomic analysis of upstream regulators between hESCs and hESC-SkMCs and their activation status. (D) IPA transcriptomic analysis of differentially regulated canonical pathways in hESCs and hESC-SkMCs. (E) RNASeq data showing hESC-SkMCs express markers specific to skeletal muscle lineage. Shown are data pooled from 3 independent biological replicates.

Figure 2

Anabolic factors and myokines enhance terminal differentiation of hESC-SkMC. (A) Image analysis pipeline. (B) Example pre- and post-filtered images of untreated and treated hESC-SkMC. Each color in the post-filtered MF20 images represents one segment of MF20 as determined by Harmony, the analysis software. Scale bar = 100 μm. (C–F) Image quantification of hESC-SkMCs in different treatments: MyHC area (C), total nuclei (D), nuclei within MyHC+ fibers (E) and fusion index (F). N = 3 for each condition. Results are the averages of four independent technical replicates over three independent experiments. Statistical analysis was performed using one-way ANOVA with Benjamini–Hochberg FDR correction. ** p < 0.01, *** p < 0.001. (G) TFC enhanced expression of several key myogenesis markers assessed by RT-qPCR. Statistical analysis was performed using a two–tailed t-test. ** p < 0.01, *** p< 0.001, ns: not significant.

Figure 3

Transcriptomic profiling of NTC and TFC-treated hESC-SkMC. (A) Volcano plot of differentially expressed genes between TFC and NTC. (B) RT-qPCR validation of several top differentially expressed genes as identified via RNASeq. Statistical analysis was performed using a two-tailed t-test. * p < 0.05, ** p < 0.01, *** p< 0.001. (C) Heatmap comparison of various myosin and sarcomere genes between hESC, NTC and TFC-treated hESC-SkMC. (D) IPA comparative analysis of differentially regulated pathways in treated hESC-SkMCs compared to NTC. Shown are data pooled from three independent biological replicates.

Figure 4

Proteomic profiles of NTC and TFC-treated hESC-SkMCs. (A) Volcano plot of differentially expressed proteins between TFC and NTC. (B) Heatmap comparison of various myosin and sarcomere proteins among hESC, NTC and TFC-treated hESC-SkMCs. (C) IPA comparative analysis of differentially regulated pathways in treated hESC-SkMCs compared to NTC. (D) Heatmap comparison of various mitochondrial genes at RNA (left panel) and proteins (right panel) levels among hESC, NTC and TFC-treated hESC-SkMC. (E) Heatmap comparison of nuclear (Histone H4) vs. mitochondrial protein ratios in NTC and TFC-treated SkMCs. (F) IPA metabolomic analysis (Log2FC > 1, padj < 0.05) of differentially expressed metabolites between TFC-treated cells and NTC. Shown are data pooled from three independent biological replicates.

Figure 5

TFC treatment enhanced oxidative phosphorylation in hESC-SkMC. (A) Quantitative determination of ATP between NTC and TFC-treated hESC-SkMC. N = 8 for each condition. (B) Mitotracker signal in NTC and TFC-treated hESC-SkMC. Scale bar = 50 μm. (C–F) Quantification of Mitotracker measurement between NTC and TFC for Mitotracker signal intensity (C), Mitochondria area, determined by Mitotracker area (D), Number of nuclei (E) and Normalised mitochondria area per nuclei (F). One representative biological replicate with N = 10 technical replicates is shown for each condition. (G) Representative image of NTC and TFC-treated hESC-SkMC co-stained with human mitochondria antibody and MF20. Scale bar = 50 μm. (H–K) Quantification of mitochondria measurement between NTC and TFC for Mitochondria signal intensity (H), Mitochondria area within MF20+ myotubes (I), Number of myotubes (J) and Normalised mitochondria area per myotube (K). N = 3 independent biological replicates for each condition. Analysis performed with a two-tailed t-test. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001, ns: not significant.