Generation of craniofacial myogenic progenitor cells from human induced pluripotent stem cells for skeletal muscle tissue regeneration

Abstract

Craniofacial skeletal muscle is composed of approximately 60 muscles, which have critical functions including food uptake, eye movements and facial expressions. Although craniofacial muscles have significantly different embryonic origin, most current skeletal muscle differentiation protocols using human induced pluripotent stem cells (iPSCs) are based on somite-derived limb and trunk muscle developmental pathways. Since the lack of a protocol for craniofacial muscles is a significant gap in the iPSC-derived muscle field, we have developed an optimized protocol to generate craniofacial myogenic precursor cells (cMPCs) from human iPSCs by mimicking key signaling pathways during craniofacial embryonic myogenesis. At each different stage, human iPSC-derived cMPCs mirror the transcription factor expression profiles seen in their counterparts during embryo development. After the bi-potential cranial pharyngeal mesoderm is established, cells are committed to cranial skeletal muscle lineages with inhibition of cardiac lineages and are purified by flow cytometry. Furthermore, identities of iPSC-derived cMPCs are verified with human primary myoblasts from craniofacial muscles using RNA sequencing. These data suggest that our new method could provide not only in vitro research tools to study muscle specificity of muscular dystrophy but also abundant and reliable cellular resources for tissue engineering to support craniofacial reconstruction surgery.

Keywords: Craniofacial myogenesis; Craniofacial myogenic precursor cells; Direct differentiation; Human induced pluripotent stem cells; Muscle tissue engineering.

Fig. 1.. Induction of cranial pharyngeal mesoderm (CPM) from human iPSCs using BMP activation and Notch inhibition within 6 days.

(A) Protocol day 0 to day 6 for mesoderm induction. (B) Relative mRNA expression levels of cranial mesoderm marker genes at day 3 and day 6. Mean ± SEM; n = 3 for each group. Data were analyzed by 1-way ANOVA. Asterisks indicate statistical significance (*P < 0.05, **P < 0.01, and ***P < 0.001). (C) Morphological changes during CPM induction from human iPSCs. Scale bars = 330 μm. (D). MESP1⁺ and PDGFRα⁺ protein expressed colonies at day 3 and day 6, respectively. White dotted boxes indicate a higher magnification. Scale bars = 330 μm.

Fig. 2.. Specification and enrichment of craniofacial myogenic progenitor cells (cMPCs) using dual inhibition of BMP and Rho kinase (ROCK) signaling and sorting strategy.

(A) Protocol day 6 to day 12 for myogenic progenitor cell (MPC) specification. (B) Treatment of BMP inhibitor and ROCK inhibitors. LDN (LDN193189, a BMP inhibitor) suppresses cardiac muscle marker genes (GATA4 and TBX5 for first heart field and ISL1 for second heart field) and enhances a craniofacial muscle marker gene (TBX1) at day 8. Y indicates Y-27632, a Rho kinase inhibitor. Data represent the mean ± SEM; n = 3 for each group. Data were analyzed by 1-way ANOVA. Asterisks indicate statistical significance (*P < 0.05, **P < 0.01, and ***P < 0.001). (C) BMP signaling determines lineage fate from bipotent cardiac/cranial pharyngeal mesoderm (PM). (D) Sorting of MPC at day 12. Representative flow cytometry plots show gating strategy HNK1⁻ERBB3⁺NGFR⁺ for sorting of MYF5⁺ MPCs.

Fig. 3. Characterization of HNK1⁻ERBB3⁺NGFR⁺ sorted craniofacial myogenic progenitor cells (cMPCs).

. (A) Protocol day 12 to day 35 for MPC differentiation. (B) Relative mRNA expression of craniofacial muscle specific marker genes in sorted craniofacial MPCs. Mean ± SEM; n = 3 for each group. Data were analyzed by t-tests. Asterisks indicate statistical significance (*p < 0.05 and **P < 0.01). (C) Immunostaining of craniofacial muscle differentiation marker proteins (embryonic MyHC and TBX1) in sorted craniofacial MPCs derived from normal human iPSCs (GM25256 and GM23476) and an iPSC line from a Duchenne muscular dystrophy (DMD) patient (GM25313). Scale bars = 70 μm.

Fig. 4.. Inhibition of TGF-β signaling with IGF treatment for enhancing the maturation of craniofacial myogenic progenitor cells (cMPCs) in vitro.

(A) Protocol after day 35 for MPC maturation. (B) Immunostaining of myosin heavy chain (MYH3 and MYH1) and human dystrophin (H-Dystrophin) with or without treatment of IGF-1 and TGF-β inhibitors (SB431542) in matured iPSC-cMPCs (HNK1⁻ERBB3⁺NGFR⁺ cells) for 18 days. Green fluorescence indicates proteins by immunostaining and blue fluorescence is DAPI staining. Scale bars = 130 μm. (C) Pseudo-color images represent striation (white arrow) in matured iPSC-cMPCs (HNK1⁻ERBB3⁺NGFR⁺ cells). White pseudo-color indicates late fetal MyHC (MYH1) and green pseudo-color indicates DAPI staining. Scale bars = 130 μm. (D) Quantified percentage of nuclei present in MyHC-positive myotubes with the indicated number of nuclei after 18 days of maturation in HNK1⁻ERBB3⁺NGFR⁺ sorted craniofacial MPCs with or without IGF-1 and SB431542 treatment. Data represent the mean ± SEM; n = 3 for each groups. Data were analyzed by 2-way ANOVA. Asterisks indicate statistical significance (**P < 0.01 and ***P < 0.001). (E) Immunostaining of myosin heavy chain (MYH1) in matured craniofacial MPCs (HNK1⁻ERBB3⁺NGFR⁺ cells) derived from normal human iPSCs (GM23476 and GM23279) and an iPSC line from Duchenne muscular dystrophy (DMD) patient (GM25313). Bottom panel showed represent striations (white arrow) in matured iPSC-cMPCs. Scale bars = 130 μm.

Fig. 5.. RNA-seq analyses of iPSC-derived myogenic progenitor cells (MPCs) compared to primary myoblasts.

(A) Pearson correlation analysis between transcriptome of myoblasts from extraocular (EO), zygomaticus (Zygo), masseter (Mas), circropharyngeus (CP) and tibialis anterior (TA) muscles. Numbers in the box indicates the correlation number, ρ between samples. (B) PCA plot of variant genes in iPSC-derived MPCs and primary myoblast groups. (C) Venn diagram showing the number of commonly or differentially expressed genes in somite and PM method-derived MPCs from human iPSCs. (D) Volcano plot showing differentially expressed genes (DEGs, cut off > 2 fold) in the iPSCs-derived MPCs using somite or PM method. X axis represents log₂ transformed fold change and Y axis represents negative log₁₀ false discovery rate. Red points indicate the upregulated craniofacial muscle development related genes (PITX2, TBX1, ILS1) and a blue point represent the downregulated limb muscle development related gene (PAX3) in PM method-derived MPCs. (E) Venn diagram showing the number of exclusively expressed genes in somite-derived primary myoblast (TA) compared to PM-derived primary craniofacial muscles (EO, Zygo, Mas, and CP myoblasts). (F) Venn diagram showing the number of commonly up-regulated genes in primary craniofacial myoblasts (EO, Zygo, Mas, and CP myoblasts) compared to limb myoblasts (TA). (G) Total Venn diagram indicating the overlap of DEGs across four comparisons. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

Fig. 6.. Scheme of differentiation and enrichment of craniofacial skeletal myotubes from human iPSCs.

Our PM method mimicked the critical signaling pathways to induce cranial/cardiac pharyngeal mesoderm (CPM) from iPSCs by using small molecules. After the bi-potential CPM was established, cells were committed to cranial skeletal muscle lineages with inhibition of cardiac lineages. We purified HNK1⁻ERBB3⁺NGFR⁺ cMPCs using flow cytometry and confirmed that sorted cells expressed myogenic factor 5 (MYF5), a key marker for early skeletal myogenic precursors. To facilitate differentiation into mature myotubes, cells were treated with transforming growth factor-β (TGF-β) inhibitor and IGF.