Skeletal Muscle Regenerative Potential of Human MuStem Cells following Transplantation into Injured Mice Muscle

Abstract

After intra-arterial delivery in the dystrophic dog, allogeneic muscle-derived stem cells, termed MuStem cells, contribute to long-term stabilization of the clinical status and preservation of the muscle regenerative process. However, it remains unknown whether the human counterpart could be identified, considering recent demonstrations of divergent features between species for several somatic stem cells. Here, we report that MuStem cells reside in human skeletal muscle and display a long-term ability to proliferate, allowing generation of a clinically relevant amount of cells. Cultured human MuStem (hMuStem) cells do not express hematopoietic, endothelial, or myo-endothelial cell markers and reproducibly correspond to a population of early myogenic-committed progenitors with a perivascular/mesenchymal phenotypic signature, revealing a blood vessel wall origin. Importantly, they exhibit both myogenesis in vitro and skeletal muscle regeneration after intramuscular delivery into immunodeficient host mice. Together, our findings provide new insights supporting the notion that hMuStem cells could represent an interesting therapeutic candidate for dystrophic patients.

Keywords: DMD; MuStem; cell therapy; human adult stem cells; regenerative medicine; skeletal muscle.

Figure 1

Morphological Features of hMuStem Cells and Myoblasts (A) 7 days after the end of the isolation protocol, phase contrast microscopy revealed that hMuStem cells formed a colony unit composed of round and thin cells (arrow) as well as short spindle-shaped cells. A part of the cells remained in the supernatant as floating cells, corresponding to small and highly refractile cells positioned above adherent cells (arrowhead in insert). (B) Typical lengthened and large spindle-shaped cells were observed in myoblast-derived primary culture. (C) hMuStem-cell-derived primary culture was characterized by a large majority of thin elongated cells aligned in networks (arrowhead) and a permanent presence of some refractile round cells (arrow). (D) Monolayer of spindle-shaped cells and thick multinucleated cells characterized the myoblast-derived primary cultures. Scale bars, 100 μm.

Figure 2

Cell Lineage-Specific Phenotype of hMuStem Cells (A) Immunolabelings against CD56, CD29, CD82, CD318, and desmin were performed on hMuStem cells (P5) cultivated in growth medium. The upper line shows the profile of a representative cell batch (n1) made exclusively of CD56⁺ cells, whereas the lower line depicts a representative cell batch (n2) containing CD56^+/− cells. Isotype control and specific signal are in white and gray, respectively. For labelings done on culture chamber slides, nuclei were counterstained with 10 μg/mL DAPI (blue). Scale bars, 100 μm. (B) Detection of transcripts specific to SC and myogenic cell markers on the two representative CD56⁺ (n1) and CD56^+/− (n2) cell batches (P5). Myoblasts and RPS18 were used as positive control (T+) and housekeeping gene, respectively. (C) Representative flow cytometry images revealing the expression of perivascular (CD140b and CD146) and mesenchymal (CD201) markers in gated CD56⁺ and CD56⁻ cells. Isotype control and specific signal are in white and gray, respectively. (D) Representative flow cytometry profiles revealing the homogeneous expression of MSC markers CD44, CD73, CD90, and CD105 by hMuStem cells and the lack of expression for the hematopoietic (CD34, CD45, and CD117) and endothelial (CD31, CD144, VEGFR1, and VEGFR2) markers. Control- and specific antibody-stained cells are shown in white and gray profiles, respectively.

Figure 3

Multilineage Potential and Pluripotent Phenotype of Long-Term Cultured hMuStem Cells (A) Cultured hMuStem cells were grown in low-serum medium for 7 days, then fixed and submitted to May-Grümwald Giemsa (MGG) staining and sarcMyHC immunolabeling to reveal multinuclei myotubes. hMuStem cells placed on growth medium were used as negative control. Fusion index was calculated on sarcMyHC⁺ myotubes. Nuclei were counterstained with 10 μg/mL DAPI (blue). Scale bars, 100 μm. (B) Top: after 14 days in adipogenic induction medium, the differentiation of hMuStem cells was assessed by detection of lipid vesicles through oil red O staining. Bottom: at 80% of confluency, hMuStem cells were placed in osteogenic differentiation medium for 21 days. Alizarin red staining revealed the formation of calcium deposits. Scale bars, 100 μm. (C) RT-PCR revealed the presence of adiponectin and LPL RNA on hMuStem cells only after the adipogenic induction. (D) hMuStem cells were cultivated in endothelial medium for 7 days before being plated with a Matrigel coating for a further 3 days. Phase contrast microscopy revealed the formation of capillary-like structures. Scale bars, 300 μm. (E) Expression of classical pluripotent marker mRNA. (F) Immunolabelings against KLF4 and NANOG were performed on hMuStem cells (P5) cultivated in growth medium. Nuclei were counterstained with 10 μg/mL DAPI (blue). Scale bars, 50 μm. MSCs, human umbilical vein endothelial cells (HUVECs) and iPSCs were used as positive controls for (B)–(D) and (E) and (F), respectively.

Figure 4

Contribution of Both Long-Term Cultured CD56⁺ and CD56^+/− hMuStem Cells to Myofiber Regeneration hMuStem cell batches (P5) made exclusively of CD56⁺ cells (left) or containing a CD56⁻ cell fraction (right) were injected into cryodamaged TA muscle of Rag2⁻IL2rβ⁻ mice. (A–L) 3 weeks later, frozen sections of recipient muscle were submitted to HES staining (A and B) and co-labeled with specific Abs against human lamin A/C (red; C–J), murine dystrophin (green; C and D and I–L), murine laminin (light blue, I–L), human spectrin (red; E and F), and human dystrophin (red; G and H). (C–L) All nuclei were counterstained using DAPI (dark blue). Foci of regeneration composed of numerous centronucleated fibers (inset, A and B) and scattered mononucleated cells were observed. A large number of human nuclei were found in muscle tissue (C and D). Also, numerous human spectrin⁺ and dystrophin⁺ fibers were detected. Insets (E–H) show the presence of myofibers characterized by both detection of cytoplasmic donor nuclei and human spectrin or dystrophin expression. In return, hMuStem cells were rarely found in a satellite cell location (arrowhead, I–L). Scale bars, 100 μm (A–D), 150 μm (E–H), and 50 μm (I–L).