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

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

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

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

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

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

Morphometric analysis of the regenerated myelinated axons.

<p>The median values for the number of the myelinated axons (per 10,000 µm²) at the mid-proximal portion of the regenerated nerve stained with toluidine blue showed no significant difference between the groups (<i>P</i> = 0.760; ANOVA, Kruskal-Wallis on Ranks). Parametric analyses of transmission electron microscopy images of the proximal-mid cross-sections of the regenerated femoral nerve show that median (25^th–75^th) values of the myelinated fiber area and myelin thickness statistically differ between the groups (*<i>P</i><0.05; ANOVA, Kruskal-Wallis on Ranks) when compared to the control femoral nerve (^§<i>P</i><0.05; ANOVA, Kruskal-Wallis on Ranks). The quantitative measurement of the axonal area and the g-ratio (axonal area : myelinated fiber area) showed no difference between the untreated graft and control femoral nerve groups. The irradiated and decellularized groups showed significantly lower g-ratio associated with thinner axonal remyelination when compared to untreated grafts (*<i>P</i><0.05, ANOVA, Kruskal-Wallis on Ranks) and uninjured control femoral nerve (^§<i>P</i><0.05; ANOVA, Kruskal-Wallis on Ranks). The measured morphometric parameters represent results from 3–4 mice per group and analysis of 300–400 myelinated fibers.</p

Untreated venous grafts exhibit more effective regeneration.

<p>(<b>A</b>) Eight weeks post-transplantation, Masson's Trichrome staining revealed absence of collagen matrix and adventitia of the grafted vein (blue) and defined nerve regeneration with perineurium surrounding the nerve bundles (pink) in untreated graft. Thickening of the extracellular matrix and disorganized perineurium was obvious in irradiated and decellularized grafted groups. (<b>B</b>) The cross-section of the regenerated nerve exhibited regenerated NF-positive axons (green) encompassed by FluoroMyelin-positive Schwann cells (red) suggesting proper regeneration of the femoral nerve in untreated control grafts compared to irradiated or decellularized grafts. Undamaged host artery shown with arrowheads was used as a reference point (<b>A, B</b>). (<b>C</b>) Toluidine blue staining and (<b>D</b>) transmission electron microscopy at the mid-proximal sections confirm superior regeneration with larger myelinated axons including presence of myelin-producing Schwann cells (arrows) surrounding the regenerated axons. Absence of connective tissue fibrosis was evident in untreated grafts while both irradiated and decellularized grafts showed poor levels of nerve regeneration indicated by small myelinated Schwann cells with high levels of connective tissue fibrosis marked by collagen. “Sc” corresponds to Schwann cells, “M” to myelin sheath, “Ax” to axon, and “Col” to collagen. (<b>E</b>) Four to six weeks after injury, FISH performed for the detection of Y chromosomes, verified the active contribution of male donor-derived blood vessel progenitor cells (Y chromosome-positive nuclei, red) in the regenerated female femoral nerve (host Schwann cell nuclei counterstained with DAPI), which is shown in blue (see arrows, n = 6). A total of 32 rats were transplanted (24 untreated grafts, 4 irradiated grafts, and 4 decellularized grafts). Scale bars represent 100 µm (<b>A–C</b>) or 10 µm (<b>D</b>).</p

Cell migration is inhibited by irradiation.

<p>The normal untreated and irradiated veins were separately cultured on fibronectin-coated culture plates in EGM2 culture media. (<b>A</b>) Many cells migrated out of the untreated vein after 7 days in culture, while very few cells were detected in the culture dishes containing the irradiated vein. (<b>B</b>) Cells were still visible at the edges of the untreated and irradiated veins (arrows) after 14 days of culture, and continuously outgrew from the untreated veins (arrowheads). (<b>C</b>) Untreated venous graft-derived cells showed a diverse morphology; while clustered cells in the irradiated vein did not migrate out of the vein even after 25 days of culture but remained viable as verified by the MTT assay (purple). Data represent at least three independent experiments. Scale bars represent 200 µm (<b>A</b>) or 100 µm (<b>B</b> and <b>C</b>).</p

Cell proliferation is inhibited by irradiation.

<p>The viable cells from normal untreated and irradiated veins were separately cultured on fibronectin-coated culture plates in EGM2 culture media. Plotted are the normalized cell counts at each time point obtained from the analysis of 3 independent experiments per group. The cells from untreated veins showed an increased growth rate in comparison to cells from irradiated veins (*<i>P</i><0.001, Student's <i>t</i>-test).</p