Specificity of the Cre-Lox system in differentiated myotubes <i>in vitro</i>.

<p>Schematic representation of the Cre/Lox-β-galactosidase system used in the current study (<b>A</b>). (β-gal/LacZ)+ cells were not observed with homogenous fusion of MDC-Lox cells (<b>B</b>). In co-cultured MDC-Cre and MDC-Lox cells, (β-gal/LacZ)+ cells can be either nascent myotubes with 2 nuclei (<b>C</b>, nuclei marked by arrows; 2 days after myogenic differentiation), or mature myotubes with multiple nuclei (<b>D</b>, nuclei marked by arrows; 4 days after myogenic differentiation). No (β-gal/LacZ)+ mononuclear cells were observed. Statistical quantification of (β-gal/LacZ)+ myotubes is shown (<b>E</b>). It is proposed that the presence of any mononuclear cells positive for β-gal expression in the culture have to be released from (β-gal/LacZ)+ myotubes, that is, after some type of stimulation, myotubes formed through the fusion of MDC-Cre and MDC-Lox cells, dedifferentiate and release the mononuclear (β-gal/LacZ)+ cells (<b>F</b>).</p

Cre/Lox-β-galactosidase cell transplantation and muscle injury resulted in β-gal/LacZ positive mononuclear cells.

<p>Three weeks after cell transplantation into MDX/SCID mice, many (β-gal/LacZ)+/dystrophin+ myofibers were observed (<b>A</b>, <b>B</b>). β-gal/LacZ staining and immunofluorescent Pax7/dystrophin staining are shown here; arrows indicate (β-gal/LacZ)+/dystrophin+ myofibers which are the result of the fusion of Cre-Lox myocytes, and arrowheads indicate (β-gal/LacZ)−/dystrophin− host myofiber (<b>A</b>, <b>B</b>). Three weeks after transplantation and four days after muscle injury in SCID mice, some (β-gal/LacZ)+ mononuclear cells were observed in the injured muscle (<b>E–G</b>), but not in the control non-injured muscle (<b>C</b>, <b>D</b>). Results of β-gal/LacZ staining and HE staining are shown here; arrows indicate (β-gal/LacZ)+ myofibers, and arrowheads indicate (β-gal/LacZ)+ mononuclear cells (<b>C–G</b>). Some of the (β-gal/LacZ)+ mononuclear cells were shown to be Pax7+ (<b>H–K</b>). Images H–J are of the same location, and result of β-gal/LacZ staining and immunofluorescent Pax7/dystrophin staining are shown here (<b>H–J</b>); arrows indicate β-gal+/Pax7+ mononuclear cells, and arrowheads indicate β-gal+ myofibers (<b>H–J</b>). Fluorescent co-staining of β-gal and Pax7 are also shown (K); arrowheads indicate β-gal+/Pax7+ mononuclear cells, and arrows indicate Pax7+ cells. Statistical quantification of the results is also shown (<b>L</b>).</p

β-gal/LacZ positive mononuclear cells from injured muscle contain different cell populations (i.e., myoblasts, satellite cells, and MDSCs).

<p>The result of flow cytometry analysis, β-gal/LacZ staining and immune-staining to MyoD or Pax7 showed that, the isolated PP2 (myoblasts) (<b>A–C</b>) and PP5 (satellite cells) (<b>D–F</b>) cell populations both contain (β-gal/LacZ)+ cells, which can be MyoD+ or Pax7+. The result of β-gal/LacZ staining and immune-staining to Sca-1 also showed that, the PP6 cells (MDSCs) also contain (β-gal/LacZ)+ cells, which were shown to be Sca-1+ (<b>G</b>, <b>H</b>). Flow cytometry analysis comparing PP6 cells from non-injured control muscles and the PP6 cells from injured muscle demonstrated a greater number of (β-gal/LacZ)+ cells which also expressed Sca-1 and CD34 in the injured muscle (I–J), indicating that adult stem cells can be dedifferentiated from “terminally” differentiated muscle fibers in injured skeletal muscle of mice. Statistical quantification of the flow cytometry results is shown (<b>K</b>).</p

β-gal/LacZ positive cells can proliferate and contribute to myotube formation <i>in vitro</i>, and blood vessel formation <i>in vivo</i>.

<p>BrdU incorporation assay showed that (β-gal/LacZ)+ cells can proliferate (<b>A–C</b>). A myogenic differentiation assay, which deprives the cultures of serum, showed that (β-gal/LacZ)+ cells, in both cell populations without purification [ around 6% of cells were (β-gal/LacZ)+] (<b>D</b>) and after purification with Fluorescence-activated cell sorting (<b>E</b>), can participate in myotube formation (<b>F</b>). <i>In vivo</i>, ten days after laceration-injury of GM muscles that were transplanted with Cre-cells and Lox-cells for 3 weeks, some (β-gal/LacZ)+ signal was also found to co-localize with CD31+ signal in the blood vasculature (<b>G–L</b>). Images G–I or J–L are of the same location in tissue, and result of β-gal/LacZ staining and immunofluorescent CD31/Utrophin staining are shown here (<b>G–I</b>); arrowheads indicate β-gal+/CD31+ cells, and arrows indicate β-gal+ myofibers (<b>G–I</b>). Fluorescent co-staining of β-gal and Pax7 are also shown (<b>J–L</b>); arrowheads indicate β-gal+/Pax7+ cells, and arrows indicate CD31+ cells (<b>J–L</b>).</p

Transplanted MDSPCs foster repair of critical-size sciatic nerve defects.

<p>(<b>A</b>) A 4- to 5 mm segment of the sciatic nerve was removed from the hind limb of each mouse, (<b>B</b>) resulting in a 6.5 to 7 mm defect. (<b>C</b>) Following transplantation of MDSPCs into the defect, complete regeneration from proximal to distal end was observed <i>(n</i>  =  28). Blood vessel networks (arrowheads) were also present around all regenerated nerves. (<b>D</b>) Many <i>nLacZ</i>-positive cells (blue) were observed between weeks 5 and 9 following injury. “<i>p</i>” corresponds to the proximal stump and “<i>d</i>” to the distal stump. (<b>E</b>) The regenerated nerve exhibited both NF (green) and CNPase (red) immunoreactivity. (<b>F</b>) CNPase (red) staining of the regenerated sciatic nerve revealed nodes of Ranvier-like structures (white circles). (<b>G</b>) Cross-sections of regenerated nerve showed <i>nLacZ</i>-positive cells (blue) and exhibited NF-positive axons (green, inset) encompassed by FluoroMyelin-positive cells (red, inset). (<b>H, I, J</b>) Colocalization of β-gal (red) with (<b>H, I</b>) GFAP (green) or (<b>J</b>) CNPase (green), and DAPI (blue) suggests possible differentiation of the MDSPCs into Schwann cells (double-positive cells denoted by arrows). (<b>K-M</b>) Electron microscopy of semi-thin cross-sections of (<b>K</b>) non-operated (uninjured) control, and (<b>L, M</b>) MDSPC-regenerated peripheral nerve 10 weeks after implantation, show a high number of myelin-producing Schwann cells. Arrows indicate Schwann cells surrounding the myelinated axon. “Sc” corresponds to Schwann cells, “M” to myelin sheath, and “Ax” to axons. (<b>N</b>) Graphical quantification of the g-ratio (axonal area: myelinated fiber area) represents the median values of both uninjured and MDSPC-regenerated nerves (<i>P</i><0.001, Mann-Whitney Rank Sum Test). Sciatic nerve regeneration studies represent three independent experiments. The morphometric parameters represent results from 5 mice (2 controls and 3 treated) and analysis of 1000 fibers. Scale bars represent 100 µm (<b>D, E, and G</b>) or 10 µm (<b>F, H-M</b>).</p

Transplanted MDSPCs assist in functional recovery of critically-sized sciatic nerve defects.

<p>(<b>A</b>) A depiction of representative paw prints from control- and MDSPC-implanted mice at 6 and 10 weeks post-implantation. (<b>B-D</b>) Quantification of paw print analyses, indicating that MDSPC transplantation increases the ability of the mice to walk normally, displayed a (<b>B</b>) decrease in toe spread factor, (<b>C</b>) decrease in print length factor, and (<b>D</b>) an increase in SFI (sciatic functional index) compared to PBS-treated mice. Thirty-five paw prints were analyzed per group for each time point. Error bars indicate s.e.m. (*<i>P</i><0.05 and **<i>P</i><0.001, Mann-Whitney Rank Sum Test).</p

TDCs can regenerate tumors in vivo.

<p>(<b>A, B</b>) Hematoxylin and eosin staining of a tumor generated from TDCs implanted into a sciatic nerve defect in mice (<i>n</i>  =  16) demonstrates the destruction of the bone, as seen by the smooth and pink stained portion (arrows). (<b>C, D</b>) In vitro myogenic analysis demonstrates that the TDCs possess expression of (<b>C</b>) the myogenic marker desmin (red, 12%) and have the ability to form myotubes in vitro, as seen by (<b>D</b>) f-MyHC staining (red). (<b>E, F</b>) In vivo myogenic analysis demonstrates that although (<b>E</b>) parental MDSPCs (blue) showed myofiber regeneration (muscles stained with eosin) after injection into the gastrocnemius muscles of <i>mdx</i> mice and were detected up to 17 weeks without sign of tumor formation (<i>n</i>  =  10), the (<b>F</b>) TDCs (blue) implanted into the muscle formed tumor 100% of the time (<i>n</i>  =  10), and as early as 4 weeks post-implantation. (<b>G</b>) A schematic of the experimental design and results displayed above. Negative control sections were similarly processed without primary antibodies. Scale bars represent 250 µm (<b>A</b>) or 100 µm (<b>B-F</b>).</p

Between weeks 11 and 13, approximately 70% of the mice (<i>n</i> = 28) implanted with MDSPCs formed large neoplastic growths.

<p>(<b>A, B</b>) Representative image of hematoxylin and eosin staining of tumors that formed in mice implanted with MDSPCs. (<b>C-L</b>) The resulting tumors were classified as malignant peripheral nerve sheath tumors with rhabdomyoblastic differentiation (Triton tumors) by showing positivity for (<b>C, D</b>) smooth muscle actin (brown), (<b>E, F</b>) desmin (brown), (<b>G, H</b>) NF (brown), and pockets of (I, J) S100 (brown), (<b>K, L</b>) as well as cells positive for both NF (brown) and S100 (purple). Positive cells (brown, <b>C-J</b>) or double-positive cells (K and L) are indicated by black arrows. Images B, D, F, H, J, and L show a higher magnification image of the image they were preceded by. (<b>M, N</b>) The neoplasias were also positive for FluoroMyelin (green), showing areas of unorganized myelin deposition. (<b>O</b>) Image depicting NF (green) and FluoroMyelin (red) double-stained sections from a regenerated nerve (white arrow) in the area of tumorigenesis. Negative control sections were similarly processed without primary antibodies. Scale bars represent 100 µm.</p

Differentiation of MDSPCs to a neurogenic lineage prior to implantation decreases their ability to respond to environmental cues and stops transformation.

<p>(<b>A-C</b>) Two weeks post-implantation MDSPC-derived neurospheres showed a reduction in muscle regeneration when compared to the parental MDSPCs. (<b>A</b>) The <i>nLacZ</i> donor-derived MDSPC-derived neurospheres (blue) could be detected in eosin stained regenerated myofibers and (<b>B</b>) showed fewer regenerated dystrophin-positive myofibers (red) compared to undifferentiated MDSPCs. (C) Graphical representation of the regeneration index (number of dystrophin positive fibers/100,000 injected cells) of parental MDSPCs and MDSPC-derived neurospheres (NS). NS-injected mice yield a lower regeneration index compared to the undifferentiated MDSPCs. Error bars indicate ± s.d. (**<i>P</i><0.001, Mann-Whitney Rank Sum Test). (<b>D-F</b>) When dissociated MDSPC-derived neurospheres were implanted into a sciatic nerve defect, tumor formation was abrogated. Furthermore, 80% of mice (<i>n</i>  =  5) formed large fibrotic masses, as seen by (<b>D</b>) eosin staining with <i>nLacZ</i> donor MDSPC-derived neurospheres shown in blue, in addition to (<b>E</b>) intense collagen deposits identified with Masson’s Trichrome staining (blue). (<b>F</b>) Graphical representation of the Trichrome-positive area of parental MDSPCs and NS. Parental MDSPCs show minimal signs of fibrosis evident by a lower percentage of collagen-positive area. Error bars indicate ± s.d. (**<i>P</i><0.001, Mann-Whitney Rank Sum Test). (<b>G</b>) A schematic of the experimental design and results. Scale bars represent 10 µm.</p

TDCs maintain their neurogenic and myogenic differentiation potential in vitro.

<p>(<b>A</b>) TDCs grew as neurosphere-like structures in the absence of neurogenic medium, and (<b>B</b>) were <i>nLacZ</i>-positive (blue). (<b>C-H</b>) Immunofluorescent analysis of these spontaneously-occurring neurosphere-like structures demonstrated that they were positive for (<b>C</b>) β-tubulin III, (<b>D</b>) CNPase, (<b>E</b>) GFAP, (<b>F</b>) nestin, and (<b>G</b>) NF, but were negative for (<b>H</b>) Neu-N. For all immunofluorescence, the antibodies used are visualized in red, with the nuclear stain DAPI seen in blue. (<b>I-L</b>) Cell cycle analysis of the parental MDSPCs and TDCs was performed by FACS. Shown is the (<b>I, J</b>) DNA content and (<b>K, L</b>) graphical representation of the results, indicating the apoptotic, G₀/G₁, S, and G₂/M fractions. (<b>M, N</b>) Karyotypic analysis of MDSPCs and TDCs. (<b>M</b>) Depicted is a representative karyotype from the MDSPC population (40, XX). (<b>N</b>) Karyotype of one of the TDCs that has 68 chromosomes, is hypertriploid, and expresses several unidentifiable marker chromosomes, the largest of which appears to be dicentric. Scale bars represent 100 µm.</p