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. 2021 Feb 16;22(4):1939.
doi: 10.3390/ijms22041939.

Guide Cells Support Muscle Regeneration and Affect Neuro-Muscular Junction Organization

Flavio L Ronzoni  1   2 , Nefele Giarratana  3   4 , Stefania Crippa  5 , Mattia Quattrocelli  6 , Marco Cassano  7 , Gabriele Ceccarelli  1 , Laura Benedetti  1 , Jens Van Herck  8 , Maria G Cusella De Angelis  1 , Marco Vitale  9 , Daniela Galli  9 , Maurilio Sampaolesi  1   3
Affiliations

Guide Cells Support Muscle Regeneration and Affect Neuro-Muscular Junction Organization

Flavio L Ronzoni et al. Int J Mol Sci. .

Abstract

Muscular regeneration is a complex biological process that occurs during acute injury and chronic degeneration, implicating several cell types. One of the earliest events of muscle regeneration is the inflammatory response, followed by the activation and differentiation of muscle progenitor cells. However, the process of novel neuromuscular junction formation during muscle regeneration is still largely unexplored. Here, we identify by single-cell RNA sequencing and isolate a subset of vessel-associated cells able to improve myogenic differentiation. We termed them 'guide' cells because of their remarkable ability to improve myogenesis without fusing with the newly formed fibers. In vitro, these cells showed a marked mobility and ability to contact the forming myotubes. We found that these cells are characterized by CD44 and CD34 surface markers and the expression of Ng2 and Ncam2. In addition, in a murine model of acute muscle injury and regeneration, injection of guide cells correlated with increased numbers of newly formed neuromuscular junctions. Thus, we propose that guide cells modulate de novo generation of neuromuscular junctions in regenerating myofibers. Further studies are necessary to investigate the origin of those cells and the extent to which they are required for terminal specification of regenerating myofibers.

Keywords: guide cells; mesoangioblasts; muscle injury; neuro-muscular junctions; scRNA-seq.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1
Single-Cell RNA Sequencing reveals the presence of guide cells expressing CD44, Ng2, and Ncam2. (A) Flowchart showing the isolation of single cells from murine hind limbs with Smart-seq2 yielding cellular and phenotypic relationships. (B) UMAP plot and k-means clustering of 256 cells from murine skeletal muscle identifying five clusters, fibroblasts, Schwann cells, guide cells, activated satellite cells (MuSCs), and interstitial stromal cells (ISCs, as reported in [26]). Every point represents one cell. (C) Heatmap of k-means clusters of differentially expressed marker genes. (D) Violin plot with median visualizing marker genes (Cd44, Ng2, Ncam2, and Sox2) for the identified clusters.
Figure 2
Figure 2
Characterization of the CD34LOW and CD34HIGH positive guide cells. (A,B) Confocal images of cardiac (panel A) and skeletal muscle (panel B) mesoangioblasts showing co-expression of CD44 (green), CD34 (far-red), and Ncam2 (red). 5-azacytidine induced differentiation of the cardiac mesoangioblasts. Arrowheads indicate triple-positive cells. Scale bar (40 µm) is the same for panels A and B. (C–E) 5-azacytidine induced differentiation of the cardiac mesoangioblasts. (C) Hoechst nuclei staining, (D) myosin heavy chain (MyHC) immunofluorescence, (E) merge of panels C-D. Scale bar (40 μm in C) is the same for panels D and E. (F) Fluorescence-activated cell sorting (FACS) analysis of the surface markers (CD44 and CD34) in the guide cells. Green peaks indicate the positive cells for CD34 or CD44 with respect to isotype negative control in red. (G) Population doublings of the CD34LOW and CD34HIGH cells; ** p ≤ 0.01. (H–O) Myosin expression analysis in the CD34LOW and CD34HIGH positive cells. (H and L) Hoechst nuclei staining of CD34LOW (H) and CD34HIGH cells (L); (I and M) GFP immunofluorescence of the CD34LOW (I) and CD34HIGH cells (M); (J and N) myosin heavy chain (MyHC) immunofluorescence analysis of the CD34LOW (J) and CD34HIGH cells (N) after 5-azacytidine treatment; (K) merge of H, I, and J panels; (O) merge of L, M, and N panels; scale bar in L (20 μm) is the same for all the panels.
Figure 3
Figure 3
In vitro migration ability of CD34LOW and CD34HIGH positive cells. (A–D) Pictures representative of the movie analysis. Color pathways report the cell movements (each color indicates a different cell) after 24 h. Colored pathways are reported on the same microscopic field both at t = 0 (panels A and C, respectively, for CD34LOW and CD34HIGH cells) and t = 24 h (panels B and D, respectively, for CD34LOW and CD34HIGH cells). (E) Quantitative analysis of the guide cell migration speed (expressed as µm h-1) and of the MuSCs (indicated with a black bar in E) ** p ≤ 0.01; *** p ≤ 0.001. (F) qRT-PCR analysis for extracellular matrix and adhesion molecule genes in cardiac mesoangioblasts (ctrl, black bar), CD34LOW (dark grey bar), and CD34HIGH (light grey bar) guide cells. N-cadherin (Cdh2), Emilin1 (Em1), versican (Vcan), neural cell adhesion molecule (Ncam2), integrin alpha 2 (Itga2), metalloproteinase inhibitor (Timp3). * p < 0.05; ** p ≤ 0.01.
Figure 4
Figure 4
Myogenic differentiation (5 days) of C2C12 cells co-cultured with guide cells. (A–C) Hoechst nuclei staining of C2C12 (A), C2C12 co-cultured with CD34LOW (B), or with CD34HIGH cells (C); (D–F) myosin heavy chain (MyHC) immunofluorescence analysis on C2C12 (D), on C2C12 co-cultured with myosin heavy chain (MyHC) immunofluorescence analysis on C2C12 (D), on C2C12 co-cultured with CD34LOW (E), or with CD34HIGH cells (F); (G–I) GFP immunofluorescence analysis of C2C12 (G), C2C12 co-cultured with CD34LOW (H), or with CD34HIGH (I) guide cells; (J) merge of panels A, D, and G. (K) merge of panels B, E, and H. (L) merge of panels C, F, and I. (M) Fusion index of C2C12, C2C12 co-cultured with CD34LOW or with CD34HIGH cells; *** p < 0.001. Scale bar 30 μm for all panels.
Figure 5
Figure 5
Neuro-muscular junction localization in muscles injected with CD34LOW and CD34HIGH cells. (A,B) Hoechst nuclei staining of longitudinal sections of muscles injected with CD34LOW (A) and CD34HIGH cells (B); (C,D) bungarotoxin staining of the same sections as in A and B; (E) merge of A and C; (F) merge of B and D. Scale bar (30 μm in F) is the same for all the panels. (G–J) Examples of hematoxylin/eosin staining of TA muscles after cardiotoxin (CTX) treatment and injected with PBS (H), with CD34LOW cells (I) CD34HIGH cells (J) or uninjected (G). (K) Morphometric analysis of muscles shown in G–J. In the left histogram the distribution of cross sectional area (CSA) values of un-injected (Ctr, blue bars), PBS-injected (Ctr PBS, grey bars), CD34LOW- cells-injected mice (CD34LOW, orange bars) and CD34HIGH-cells injected mice (CD34HIGH, red bars) was determined on H&E-stained Tibialis Anterior (TA) muscle bundles of cross cryosections from six mice per group. In the right panel, the average of CSA values is represented; N = 600 per group, * p < 0.05; ** p < 0.01; **** p < 0.0001.
Figure 6
Figure 6
Expression of neural markers in guide cells under different culture conditions. (A,D,G,J), Hoechst nuclei staining of CD34LOW (A,G) and CD34HIGH cells (D,J) cultured in neural medium (A,D) or D-MEM 2% Horse Serum (G,J); (B,E,H,K) anti-Tuj1 immunofluorescence of CD34LOW (B,H), and CD34HIGH cells (E,K) cultured in neural medium (B,E) or D-MEM 2% horse serum (H,K). Insets B’, E’, K’ represent sub-regions of panels B, E, K respectively; (C) merge of A and B panels; (F) merge of D and E panels; (I) merge of panels G and H; (L) merge of panels J and K. Scale bar (30 μm), in L, is the same for all the panels. (M) Connexin40 mRNA levels in the original population used as reference (C), after 5-azacytidine treatment (5-aza) and in the guide cells used in the experiments, grown both in proliferating medium and in low serum medium (CD34LOW diff and CD34HIGH diff cells). (N) Relative expression of Sox2 (white bar) and Sox17 (black bar) in cardiac mesoangioblasts (ctrl) or guide cells cultured for 24 hours (24h) in proliferating medium and 5 days in low serum medium (5 d); * p < 0.05, ** p < 0.01; **** p < 0.0001.
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