Mesenchymal stem cells are recruited to striated muscle by NFAT/IL-4-mediated cell fusion

Abstract

Mesenchymal stem cells (MSCs) or mesenchymal adult stem cells (MASCs) that are present in the stroma of several organs have been proposed to contribute to the regeneration of different tissues including liver, blood, heart, and skeletal muscle. Yet, it remains unclear whether MSCs can be programmed to differentiate cell-autonomously into fully functional cells or whether they are recruited by surrounding cells via fusion and thereby acquire specialized cellular functions. Here, we demonstrate that Wnt signaling molecules activate the expression of distinct sets of genes characteristic for cardiac and skeletal muscle cells in MASCs. However, such cells lack morphological criteria characteristic for functional muscle cells and do not show contractile activity. In contrast, MASCs fuse efficiently with native myotubes in an IL-4-dependent manner to form functional hybrid myotubes. Injection of genetically labeled MSCs into wild-type mouse blastocysts revealed a contribution to skeletal but not cardiac muscle development. Disruption of IL-4 and NFATc2/c3 reduced or prevented a contribution of adult stem cells to the development of Il-4 and NFATc2/c3 mutant embryos, further emphasizing the apparent inability of adult stem cells to differentiate fully into striated muscle in a cell-autonomous manner.

Figure 1.

Activation of skeletal and heart-muscle-specific genes in mBM-MASCs. (A,B) RT-PCR analysis of RNA isolated from mBM-MASCs1 or mBM-MASCs2 cocultured for 7 d with Wnt11-expressing cells. (A) Expression of skeletal muscle markers Myf5, MyoD, Myogenin, and MRF-4 in Wnt11-treated mBM-MASCs1 (lane 1), Wnt11-treated mBM-MASCs2 (lane 2), untreated mBM-MASCs1/2 (lane 3), and in skeletal muscle (lane 4). (B) Expression of heart muscle markers Nkx2.5, GATA4, α-MHC, β-MHC ANP, BNP, Hand2, TEF-1, and TM (tropomyosin) in Wnt11-treated mBM-MASCs1 (lanes 1,4), untreated mBM-MASCs1 (lanes 2,5), and in the heart (lanes 3,6). GAPDH expression was used as a loading control in A and B. Treatment with Wnt11 leads to activation of a subset of skeletal or heart-muscle-specific markers. (C) Immunofluorescent staining of the cardiac marker cTnI in FGF-2 and FGF-2/BMP-2 treated mBM-MASCs1 and mBM-MASCs2. cTnI expression was undetectable in untreated mBM-MASCs1 and mBM-MASCs2. Nuclei were visualized using DAPI. The photographs in C were taken with a 100× magnification.

Figure 2.

Recruitment of MASCs into functional skeletal and cardiac muscle cells requires cell fusion. Ad-EGFP (A-I), DiI-labeled MASCs (J-O), C2C12 myogenic cells (A-I), and primary cardiomyocytes (J-O) were plated on opposite sides of polycarbonate filters of different pore sizes as indicated. After 5-6 d of culture, cells were stained with antibodies against myosin heavy chain (MyHC) (B,C,E,F,H,I) and cTnI (J,M,L,O). (D-I,M-O) Labeled MASCs that stained positive both for EGFP or DiI and MyHC or cTnI were found only when filters with a relatively larger pore size were used and are indicated by arrows. The photographs in A-L were taken with a 100× magnification.

Figure 3.

Human mesenchymal stem cells are recruited by mouse myogenic cells to form interspecies hybrid myotubes. (A-D) Human Ad-GFP-labeled MASCs and C2C12 myogenic cells were cocultivated, stained for MyHC expression, and treated with DAPI to reveal the origin of the nuclei. Human nuclei (indicated by arrows in A-D) are larger and paler than their mouse counterparts, which fluorescence more brightly. (A) MyHC staining of a hybrid myotube. The inset in A shows the GFP fluorescence of the same myotube. (B) DAPI staining. (C) Overlay of the MyHC staining (red fluorescence), DAPI staining (blue), and GFP fluorescence (green). (D) Overlay of the MyHC staining (red fluorescence), DAPI staining (blue). (E,F) Partially reprogrammed hybrid myotube resulting from the coculture of Ad-GFP-labeled MASCs and C2C12 myogenic cells. (E) Overlay of GFP-staining (green), DAPI staining (blue), and prolyl 4-hydroxylase (red), an antigen not detected in myogenic cells. The inset in E shows the green channel alone. Note that all Ad-GFP-labeled MASCs in the view field have fused to the myotube. (F) Staining of the hybrid myotube with antibodies against prolyl 4-hydroxylase (cytoplasmic antigen, present in the right half of the hybrid myotube) and Myogenin (nuclear antigen, present in the nuclei of the left half of the hybrid myotube). Note the zonal expression of MASCs and myogenic antigens in the hybrid myotube. The photographs in A-D were taken with a 1000× magnification and those in E and F were taken with a 200× magnification.

Figure 4.

Mesenchymal stem cells fuse in an IL-4-dependent manner with myogenic cells. (A,C) GFP-labeled MASCs and C2C12 myogenic cells were cocultivated in the absence or presence of IL-4 and of antibodies against IL-4 or the IL-4 receptor and stained consecutively for MyHC. (C, panel d) Double-labeled myotubes appear orange-yellow and are indicated by arrows. Bars in A indicate the number of cells that were positive for both GFP and MyHC expression. Error bars in A show the standard deviation. (★) P < 0.05. Note that addition of IL-4 stimulated recruitment of MASCs to a myogenic fate by fusion, while addition of antibodies against IL-4 and its receptor inhibited recruitment. (B) RT-PCR analysis of the expression of the IL-13Rα1 (lanes 1-3) and the IL-4α1 receptors (lane 4-6) in hBM-MASCs (lanes 1,3), human fibroblasts (lanes 2,5), and in negative controls (lanes 3,6). The photographs in C were taken with a 50× magnification.

Figure 5.

Robust engraftment of MASC mBM-MASCs into different host embryos of the mouse. Detection of engraftment of genetically labeled mBM-MASCs into host blastocysts of C57/BL6, NFACTc2^-/-, NFACTc2/c3^-/-, and IL-4^-/- mice by PCR. LacZ transgenic and nonchimeric C57/BL6 mice served as positive and negative controls, respectively. LacZ-specific primers were used to detect the presence of mBM-MASC-derived cells in different organs of host embryos. Primers specific for the Fabpi gene (intestinal fatty acid-binding protein) were used as an internal control.

Figure 6.

The contribution of genetically labeled MASCs to skeletal but not heart development depends on NFAT signaling. LacZ staining of transgenic MyLC1/3-LacZ transgenic (A,E,I) and chimeric (B-E,G-J,L-O) mice at E10.5. C57/BL6 (B,G,L), NFATc2^-/- (C,H,M), NFATc2/c3^-/- (C,I,N), and IL-4^-/- (E,J,O) embryos served as hosts. (A-E) Whole-mount preparations. (F-J) Sections through the neural tube and adjacent somites. (K-O) Sections through the heart. (A) Expression of the MyLC1/3-LacZ transgene in the heart (red arrows) and in somatic myotome (black arrows) is clearly visible in transgenic embryos. (F,J) In C57/BL6 hosts only an expression in somites is present. Virtually no expression of the MyLC1/3-LacZ transgene is seen in NFATc2^-/- (C) and NFATc2/c3^-/- (D) host embryos both in the somites (H,I) and in the heart (M,N), while the contribution to muscle development seems reduced in IL-4^-/- (F,J) hosts, indicating a requirement for NFAT-mediated recruitment of MASCs to the myogenic lineage. The photographs F-O were taken using Nomarski optics with a 100× magnification.

Figure 7.

Genetically labeled MASCs contribute to embryonic skeletal muscle development by formation of hybrid myotubes. Combined LacZ (blue nuclear staining) and MyHC (brown cytoplasmic staining) staining of chimeric wild-type (A) and NFATc2/c3^-/- mutant (C) embryos injected with MASCs derived from transgenic MyLC1/3-LacZ mice and of noninjected transgenic MyLC1/3-LacZ mice (B) at E11.5. Ten-micrometer cryosections through the trunk region are shown. LacZ-labeled nuclei are only found in hybrid myotubes of wild-type hosts that also contain host-derived (not labeled) myogenic nuclei. (C) No activation of the transgenic LacZ marker is detectable in NFATc2/c3^-/- mutant embryos. The photographs were taken using Nomarski optics with a 200× magnification.