Clones of ectopic stem cells in the regeneration of muscle defects in vivo

Abstract

Little is known about whether clones of ectopic, non-muscle stem cells contribute to muscle regeneration. Stem/progenitor cells that are isolated for experimental research or therapeutics are typically heterogeneous. Non-myogenic lineages in a heterogeneous population conceptually may compromise tissue repair. In this study, we discovered that clones of mononucleated stem cells of human tooth pulp fused into multinucleated myotubes that robustly expressed myosin heavy chain in vitro with or without co-culture with mouse skeletal myoblasts (C2C12 cells). Cloned cells were sustainably Oct4+, Nanog+ and Stro1+. The fusion indices of myogenic clones were approximately 16-17 folds greater than their parent, heterogeneous stem cells. Upon infusion into cardio-toxin induced tibialis anterior muscle defects, undifferentiated clonal progenies not only engrafted and colonized host muscle, but also expressed human dystrophin and myosin heavy chain more efficaciously than their parent heterogeneous stem cell populations. Strikingly, clonal progenies yielded ∼9 times more human myosin heavy chain mRNA in regenerating muscles than those infused with their parent, heterogeneous stem cells. The number of human dystrophin positive cells in regenerating muscles infused with clonal progenies was more than ∼3 times greater than muscles infused with heterogeneous stem cells from which clonal progenies were derived. These findings suggest the therapeutic potential of ectopic myogenic clones in muscle regeneration.

Figure 1. Characterization of heterogeneous stem/progenitor cells of the dental pulp.

Immunostaining at passage 2 for Stro1(green) (A), CD146 (green) (B) and CD133 (red) (C) over DAPI-stained nuclei. Scale bars: 100 µm. D: Quantitative expression of CD146, Stro1 and CD133 at passages 2, 5 and 17 (N = 3).

Figure 2. Myogenic clones in comparison with their parent stem/progenitor cells.

A: Cumulative population doubling (PD) of two myogenic clones and their parent stem/progenitor cells showing a lack of statistically significant differences in PD kinetics. B: Expression of CD146, Stro1, CD133, Oct4 and Nanog by the two tested myogenic clones, B6 and C3, as well as their parent stem/progenitor cells. P: cell passage. Strikingly, B6 and C3 were overwhelmingly Oct4+, Nanog+ and Stro1+, and sustained the expression of these markers up to the tested 5 passages. C–H: Representative immunofluorescence of Stro1, Oct4 and Nanog by the two identified myogenic clones (passage 5) merged with DAPI-stain nuclei. Scale bar: 100 µm.

Figure 3. Myogenic potential of clonal progeny of ectopic dental stem cells in chemically defined medium.

A: Phase contrast image of a representative heterogeneous dental stem cell (DSC) population showing typical spindle-shaped cells. Upon exposure to chemically defined medium, DSCs assumed spherical shape but failed to fuse into multinucleated cells (B) or express myosin heavy chain (C). Strikingly, two representative clones (B6 and C3) in chemically defined medium for 4 wks readily fused into tubular structures (D,E) that are positive for myosin heavy chain (MHC) immunoblotting (F,G). Quantitatively, MHC mRNA expression is significantly greater by B6 and C3 cells than their heterogeneous parent cells (DSC) (H). The y axis represents fold change related to heterogeneous DSC. Scale bar: 100 µm.

Figure 4. Myogenic potential of clonal progeny of ectopic dental stem cells upon co-culture with mouse skeletal myoblasts.

Upon co-culture with mouse myoblast cell line (C2C12 cells) for 1 wk, some of the B6 and C3 cells again fused into multinucleated cells (A,B). Human nuclear staining indicates that the presence of human nuclei in some of the fused myotube like, MHC positive structures (A,B). Quantitatively, human nuclear fusion index (C) and human MHC mRNA expression of B6 and C3 (D) are significantly greater than DSC+C2C12 group. In (D), y axis represents fold change related to DSC+C2C12 group. Scale bar, 50 µm.

Figure 5. Engraftment of undifferentiated ectopic stem cell clone in damaged muscle.

The tibialis anterior (TA) muscles in NOD/SCID mice were injured by multi-point cardiotoxin (CTX) injection. 24 hrs following CTX injections, dental stem cells (DSC) and a tested clone (B6) were separately infused in CTX-injured TA muscles with contralateral TA muscles as controls. A: H&E staining shows the presence of centralized nuclei in the representative DSC infused sample. B: The representative B6 infused sample showed abundant centralized nuclei. In contrast, the representative normal TA muscle has peripheral nuclei (C). Immunohistochemistry staining (brown) of human specific nuclei (D) and immunefluorescent staining of human specific dystrophin (green) and human nuclei (red) (E,F) indicates the presence of transplanted human cells in host TA muscle in the representative B6 infused group. We then harvested in vivo muscle samples, isolated RNA for real-time PCR analysis of myogenic differentiation in vivo. Quantitative RT-PCR assay revealed that human MHC gene expression in B6 infusion group after 4 wk injection is ∼8 times greater than DSC infusion group (N = 3, **p<0.01) (G). Quantification of human dystrophin positive cells present in the tibialis anterior (TA) muscle shows that the expression of human dystrophin mRNA was ∼3 times greater following B6 infusion than DSC infusion (N = 3, **p<0.01) (H). In (D) and (F), the arrows indicate the human nuclei. In G, y axis represents fold change relative to heterogeneous DSC. Scale bars: A–C, E, F: 50 µm; D: 20 µm.

Figure 6. Contribution of transplanted myogenic clones and their parent stem/progenitor cells to angiogenesis.

Immunolocalization of human-specific PECAM to a representative blood vessel (A) showing human cell derived endothelium upon infusion of a myogenic clone (B6). Similarly, infusion of heterogeneous stem/progenitor cells (DSC) also yielded human specific PECAM-positive blood vessel (B). Scale: 20 µm. C: Quantitative real-time PCR analysis showed a lack of significant differences in mRNA expression of human-specific PECAM by B6 and DSCs (p>0.05). Y axis represents fold change relative to heterogeneous DSCs.