Heterogeneity of adult masseter muscle satellite cells with cardiomyocyte differentiation potential

Abstract

Although resident cardiac stem cells have been reported, regeneration of functional cardiomyocytes (CMs) remains a challenge. The present study identifies an alternative progenitor source for CM regeneration without the need for genetic manipulation or invasive heart biopsy procedures. Unlike limb skeletal muscles, masseter muscles (MM) in the mouse head are developed from Nkx2-5 mesodermal progenitors. Adult masseter muscle satellite cells (MMSCs) display heterogeneity in developmental origin and cell phenotypes. The heterogeneous MMSCs that can be characterized by cell sorting based on stem cell antigen-1 (Sca1) show different lineage potential. While cardiogenic potential is preserved in Sca1⁺ MMSCs as shown by expression of cardiac progenitor genes (including Nkx2-5), skeletal myogenic capacity is maintained in Sca1^- MMSCs with Pax7 expression. Sca1⁺ MMSC-derived beating cells express cardiac genes and exhibit CM-like morphology. Electrophysiological properties of MMSC-derived CMs are demonstrated by calcium transients and action potentials. These findings show that MMSCs could serve as a novel cell source for cardiomyocyte replacement.

Keywords: Cardiac differentiation; Cranial masseter muscle; Regenerative medicine; Satellite cells.

Figure 1.. Identification and characterization of MMSCs.

(A): Limb or masseter muscles (MM) were isolated from Nkx2-5^Cre/Rosa^RFP mice. Tissue sections stained with wheat germ agglutinin (WGA-FITC, green) were observed under multiple channels on a confocal microscope. Arrow head in magnification view indicates the location of RFP⁺ MMSCs. (B): MMSCs were isolated and maintained in suspension culture. (C): The expression of RFP was observed by microscope after culture of MMSCs in monolayer. (D): Surface markers and RFP in MMSCs were analyzed by FACS after primary cell isolation. Flow cytometric gates (indicated by solid lines) were set using the appropriate isotype control antibody. (E): RFP and Pax7 expressions in Sca1⁺ MMSCs were analyzed by FACS after sorting. (F): Immunostaining of Pax7 and RFP expression in sorted cells was observed by microscope. Nuclei were stained with DAPI.

Figure 2.. Cardiogenic potential of MMSCs.

(A): Representative morphologies of Sca1⁺ or Sca1⁻ MMSCs treated with the differentiation medium were shown under bright field microscopy. (B): Relative gene expression of cardiogenic or myogenic markers in sorted MMSCs and limb skeletal myoblasts (SkM) was analyzed by qPCR. n=4 per group. Versus SkM, * p<0.05, ^ns p>0.05. (C): Immunostaining of Nkx2-5 and Isl-1 in Sca1⁺ MMSCs after cell sorting. Nuclei were stained with DAPI. (D): The protein level of cardiac progenitor or CM markers was detected by Western blotting in Sca1⁺ MMSCs treated with differentiation (Diff.) medium. Identical results were observed in 3 independent experiments, each with technical triplicates. (E): The expression of RFP and GFP in Sca1⁺ MMSCs from transgenic mice was observed by microscope after differentiation.

Figure 3.. Phenotypes of Sca1⁺ MMSC-derived cells.

(A): Myh6 and cTnT were analyzed by Western blotting in MMSCs treated with various growth factors. Identical results were observed in 3 independent experiments, each with technical triplicates. (B): The relative expression of CM markers in undifferentiated MMSCs (Ctrl) or MMSCs treated with or without growth factors (GF) including bFGF and EGF was analyzed by qPCR. n=4 per group. Versus Ctrl, * p<0.05, ^ns p>0.05. (C): CM markers were analyzed by immunostaining in MMSCs after induced with growth factors. The percentage of Myl2⁺ or Mly7⁺ cells in undifferentiated (Ctrl, n=4) or differentiated (Diff. n=5) MMSCs was showed. (D): Representative transmission electron imaging of undifferentiated (Ctrl) or differentiated MMSCs. Yellow and white arrows show M- and Z-lines respectively.

Figure 4.. Electrophysiological properties of Sca1⁺ MMSC-derived cells.

(A): Measurement of Ca²⁺ transient frequency in MMSC-derived beating cells loaded with Fura-2 fluorescent dye. (B): Ca²⁺ transient of MMSC-derived cells in response to caffeine (5mM). (C): Calcium handling gene expression in differentiated MMSCs, undifferentiated MMSCs, and limb skeletal myoblasts (SkM) was analyzed by qPCR. n=5 for each group; *p<0.05.

Figure 5.. Electrophysiological properties of MMSC-derived cells.

(A and B): Action potentials (APs) of the MMSC-derived beating cells were measured by whole-cell patch-clamp recording. Rebound APs of MMSC-derived beating cells after hyperpolarization. Identical results were observed in 3 independent experiments, each with technical triplicates. (C): Contractility analysis of CM-like cells treated with or without β-adrenergic receptor agonist ISO (1μM). (D): Mechanical response of MMSC-derived myocytes to electrical stimulation. Frequencies of electrical stimulation from 0.0 to 3.0 Hz were measured by using video edge detection.