Generation of highly pure pluripotent stem cell-derived myogenic progenitor cells and myotubes

Abstract

Driving efficient and pure skeletal muscle cell differentiation from pluripotent stem cells (PSCs) has been challenging. Here, we report an optimized protocol that generates skeletal muscle progenitor cells with high efficiency and purity in a short period of time. Human induced PSCs (hiPSCs) and murine embryonic stem cells (mESCs) were specified into the mesodermal myogenic fate using distinct and species-specific protocols. We used a specific maturation medium to promote the terminal differentiation of both human and mouse myoblast populations, and generated myotubes associated with a large pool of cell-cycle arrested PAX7⁺ cells. We also show that myotube maturation is modulated by dish-coating properties, cell density, and percentage of myogenic progenitor cells. Given the high efficiency in the generation of myogenic progenitors and differentiated myofibers, this protocol provides an attractive strategy for tissue engineering, modeling of muscle dystrophies, and evaluation of new therapeutic approaches in vitro.

Keywords: hiPSC; mESC; muscle progenitors; myogenic differentiation; myotubes.

Figure 1

Characterization of the human skeletal myoblasts generated from hiPSCs (A) Schematic representation of the experimental time line. (B) Immunostaining with PAX7, MYOD, MYOG, and DAPI staining in PCi-AFR1-derived PCi-Myo cells, cultured on Geltrex-coated dishes. Arrows indicate PAX7⁺:MYOG⁻ cells and arrowheads indicate PAX7⁻:MYOG⁺ cells. Scale bar, 60 µM. (C) Quantification of the cell populations in the whole culture dish at the amplification stage (PCi-AFR1-derived PCi-Myo) cultured on Geltrex-coated dishes (n = 3, independent experiments). (D and E) Quantification of the (D) PAX7⁺:MYOD⁻ and PAX7⁺:MYOD⁺ cells within the PAX7⁺ cell population and (E) MYOD⁺:MYOG⁺ and MYOD⁻:MYOG⁺ cells within the MYOG⁺ cell population, at the amplification stage (PCi-AFR1-derived PCi-Myo) on Geltrex-coated dishes (n = 3, independent experiments). Error bars, mean ± SEM. (F) Histogram with the normalized reads per gene of PCi-AFR1-derived PCi-Myo RNA-seq data in individual triplicates. Error bars, mean ± SEM. (G) Quantification of the cell populations in the whole culture dish at the amplification stage (1024/PCi-Cau1/PCi-AFR3-derived myoblasts) cultured on Geltrex-coated dishes (n = 3, independent experiments). (H) Immunostaining with PAX3, PAX7, MYOG, and DAPI staining in 1024/PCi-Cau1/PCi-AFR3-derived myoblasts, cultured on Geltrex-coated dishes. Scale bar, 60 µM. (I) Flow cytometry analysis of PCi-AFR1/1024/PCi-Cau1/PCi-AFR3-derived myoblasts, cultured on Geltrex-coated dishes, for CDH13, erbB3/HER, and HGFR/c-met expression.

Figure 2

Characterization of the human skeletal myotubes and PAX7⁺ cells, generated from hiPSC PCi-AFR1 at day 7 (A, A′, A″) Left: immunostaining with PAX7, MYOD, MYOG, and DAPI staining in PCi-MYO cells. Right: immunostaining with MYOG and MHC and DAPI staining in PCi-AFR1-derived PCi-MYO cells at day 2 (A), day 4 (A′), and day 7 (A″), cultured on Geltrex-coated dishes. Scale bar, 60 µM. (B) Quantification of the cell populations in the whole-cell culture dish at the amplification stage (myoblasts) and during differentiation with Phenocell maturation medium (days 2, 4, and 7), cultured on Geltrex-coated dishes (n = 3, independent experiments). (C and D) Quantification of the (C) PAX7⁺:MYOD⁻ and PAX7⁺:MYOD⁺ cells within the PAX7⁺ cell population and (D) MYOD⁺:MYOG⁺ and MYOD⁻:MYOG⁺ cells within the MYOG⁺ cell population, during PCi-AFR1-derived myoblasts differentiation with Phenocell maturation medium (days 2, 4, and 7) on Geltrex-coated dishes (n = 3, independent experiments). Error bars, mean ± SEM. (E and E′) Immunostaining with PAX7, KI67, and DAPI staining in PCi-AFR1 PCi-MYO cells at the amplification stage (myoblasts) (E) and at day 7 (E′) cultured on Geltrex-coated dishes. Arrows indicate PAX7⁺:KI67⁺ cells and arrowheads indicate PAX7⁺:KI67⁻ cells. Scale bar, 60 µM. (F) Quantification of the PAX7⁺:KI67⁺ and PAX7⁺:KI67⁻ cells within the PAX7⁺ cell population at the amplification stage (myoblasts) and during differentiation with maturation medium (days 2, 4, and 7) (n = 3, independent experiments). Error bars, mean ± SEM. (G) Immunostaining with PAX7, MYOG, MHC, and DAPI staining in PCi-MYO cells at day 7 cultured on Geltrex-coated dishes. Arrows indicate PAX7⁺ cells. Scale bar, 60 µM.

Figure 3

Characterization of human myotubes cultured on Geltrex and laminin coatings (A) Quantification of the number of nuclei in the myotubes at day 7 cultured on Geltrex- or laminin-coated dishes (n = 3, independent experiments). Error bars, mean ± SEM. (B) Immunostaining with MYH3, MYH7, MYH8, and DAPI staining in PCi-Myo cells at day 7. Arrowheads indicate nuclei in the myotubes. Scale bar, 60 µM. (B′) Immunostaining with MYH2, MYH4, MHC, and DAPI staining in PCi-Myo cells at day 7 cultured on laminin-coated dishes. Scale bar, 60 µM. (C) Immunostaining with DYSTROPHIN, MHC, and DAPI staining in PCi-Myo cells at day 7 cultured on laminin-coated dishes. Scale bar, 60 µM. (D) MA plot (M (log ratio) and A (mean average)) of PCi-AFR1-derived myotubes on laminin compared to Geltrex RNA-seq data. Significantly dysregulated genes are highlighted in blue (false discovery rate <0.05). (E) Gene Ontology (GO) analysis for biological processes of the upregulated genes. Selected enriched terms are presented according to the fold enrichment. (F) Heatmap with the normalized reads per gene of PCi-AFR1-derived-myotubes on laminin and Geltrex RNA-seq data in individual triplicates. (G) Immunostaining with MHC, ACTN, and DAPI staining in PCi-Myo cells at day 7 of differentiation. Scale bar, 20 µM.

Figure 4

Characterization of the paraxial mesodermal cells generated from mESCs (A) Schematic representation of the experimental time line to generate paraxial mesodermal cells from mESCs. (B) Immunostaining with KLF4, T, PAX3, and DAPI staining during mESC differentiation into paraxial mesoderm (days 2, 4, and 6). Scale bar, 60 µM. (C and C′) Immunostaining with PAX3, PAX7, and DAPI staining during mESC differentiation into paraxial mesoderm at day 6, preceded (C′) or not (C) by replating at day 4. Scale bar, 60 µM. (D) Quantification of the PAX3⁺:PAX7⁻, PAX3⁺:PAX7⁺, and PAX7⁺:PAX3⁻ populations in the whole culture dish of the paraxial mesodermal differentiation at day 6, preceded or not by replating at day 4. Error bars, mean ± SEM. (E) qRT-PCR analyses of mESCs during paraxial mesodermal differentiation (days 2, 4, and 6) (n = 3, independent experiments). Error bars, mean ± SEM.

Figure 5

Characterization of the murine skeletal muscle cells generated from mESC-derived paraxial mesodermal cells (A) Schematic representation of the experimental time line. (B) Immunostaining with PAX3, PAX7, MYOD, and DAPI staining in mESC-derived paraxial mesodermal cells during differentiation with maturation medium at day 8. Scale bar, 60 µM. (B′ and B″) Left: immunostaining with PAX3, PAX7, MYOD, and DAPI staining in mESC-derived paraxial mesodermal cells during differentiation with maturation medium at day 10 (B′) and day 13 (B″). Right: immunostaining with MYOD, MYOG, MHC, and DAPI staining in mESC-derived paraxial mesodermal cells during differentiation with maturation medium at day 10 (B′) and day 13 (B″). (C) Quantification of the cell populations in the whole-cell culture dish during the differentiation of mESC-derived paraxial mesodermal cells with maturation medium (days 8, 10, and 13). (D) Quantification of the PAX3⁺:PAX7⁻, PAX3⁺:PAX7⁺ cells, and PAX7⁺:PAX3⁻ cells within the PAX3/7⁺ cell population during the differentiation of mESC-derived paraxial mesodermal cells with maturation medium (days 8, 10, and 13) (n = 3, independent experiments). Error bars, mean ± SEM. (E and F) Quantification of the (E) MYOD⁺:PAX3⁻:PAX7⁻, PAX7⁺:MYOD⁺, and PAX3⁺:PAX7⁺:MYOD⁺ cells within the MYOD⁺ cell population, and (F) MYOG⁺:MYOD⁻ and MYOD⁺:MYOG⁺ cells within the MYOG⁺ cell population during the differentiation of mESC-derived paraxial mesodermal cells with maturation medium (days 8, 10, and 13) (n = 3, independent experiments). n.d., not detected. Error bars, mean ± SEM. (G) Immunostaining with PAX3, PAX7, MYOG, and DAPI staining in mESC-derived paraxial mesodermal cells during differentiation with maturation medium (days 8, 10, and 13). Scale bar, 60 µM. (H) Quantification of the cell populations in the whole-cell culture dish at day 13 of the differentiation of mESC-derived paraxial mesodermal cells with maturation medium (n = 3, independent experiments). (I) qRT-PCR analyses in mESC-derived paraxial mesodermal cells during myogenic differentiation (days 6, 8, 10, and 13) (n = 3, independent experiments). Error bars, mean ± SEM.

Figure 6

Characterization of the maturity phenotype of the murine myoblasts and myotubes (A) Schematic representation of the experimental time line. (B and C) qRT-PCR analyses of mESC-derived paraxial mesodermal cells during myogenic differentiation (days 6, 8, 10, and 13) and in fetal myoblasts (n = 3, independent experiments). Error bars, mean ± SEM. (D and D′) Immunostaining with MYH3, MYH8, MYH7, and DAPI staining in mESC-derived paraxial mesodermal cells during differentiation with maturation medium, preceded (D) or not (D′) by the replating step at day 4 (days 13 and 16). Scale bar, 60 µM. (E) Immunostaining with PAX3, PAX7, MHC, and DAPI staining in mESC-derived paraxial mesodermal cells at day 13 of differentiation with maturation medium. Scale bar, 60 µM. (F) Immunostaining with PAX7, MYOG, MHC, and DAPI staining in mESC-derived paraxial mesodermal cells at day 13 of differentiation with maturation medium. Arrows indicate PAX7⁺ cells. Scale bar, 60 µM. (G) Immunostaining with PAX7, KI67, and DAPI staining in mESC-derived paraxial mesodermal cells at day 13 of differentiation with maturation medium. Arrows indicate PAX7⁺:KI67⁺ cells and arrowheads indicate PAX7⁺:KI67⁻ cells. Scale bar, 60 µM. (H) Quantification of the PAX7⁺:KI67⁺ and PAX7⁺:KI67⁻ cells within the PAX7⁺ cell population at day 13 of myogenic differentiation (n = 3, independent experiments). Error bars, mean ± SEM.