hDPSCs surface antigens expression and <i>in vitro</i> multilineage differentiation ability.

<p>A: Cytofluorimetric analysis of Stro-1, c-Kit and CD34 expression in hDPSCs cultured in FCS-medium and in HS-medium. Dot plots reporting SSC vs fluorescence are shown. In the histograms, the net fluorescence value was calculated by linearizing the fluorescence value from the logarithmic scale and subtracting the linearized value of the unstained sample to the linearized value of the stained one. Data represent the mean±SD of three different experiments. * indicates values of paired <i>t</i>-test HS vs. FCS (*<i>p<</i>0.05). B: In the first line are shown double immunofluorescence images of hDPSCs/C₂C₁₂ co-culture stained by anti-hMit (green) and anti-myosin (red) Abs. DAPI staining is shown in blue. The second line shows oil red staining of hDPSCs differentiated for two weeks towards adipogenic lineage with HS or FCS supplemented medium. Cells were counterstained with Harris haematoxylin. Images, in third line represent anti-β3-Tubulin immunofluorescence labelling on hDPSCs differentiated in culture neurogenic media supplemented with FCS or HS. Bar 50 µm.</p

Comparative histological analysis (haematoxylin/eosin staining) of the critical size cranial defect reconstruction by hDPSCs/collagen constructs 40 days post-surgery.

<p>Images show transversal sections carried out through the central area of the implants. A–C: cranial defect filled with hDPSCs/collagen pre-differentiated with FCS containing medium; D–F: cranial defect closed with hDPSCs/collagen pre-differentiated with HS containing medium; (dotted line delimitates the areas of bone resection; arrowheads indicate vasa; * indicate areas of active bone deposition). Bar: 100 µm. G: morphometric analysis of new-formed bone areas in controls (C), FCS and HS implants. Values are mean ± SD of the percentage of regenerated bone respect to the whole resected bone area. HS and FCS n = 6; C n = 4; white * inside the column indicate values of ANOVA test of HS and FCS vs. C (**<i>p<</i>0.01, ***<i>p<</i>0.001); black * indicate values of ANOVA test of HS vs. FCS (***<i>p<</i>0.001). H: number of vasa in the scaffold not yet reabsorbed and in new-formed bone areas. Data, normalized to areas of the scaffold not yet reabsorbed and of new-formed bone areas respectively, were presented as mean ± SD (vasa number respect to the total implant area) of each experimental group (controls n = 4; treated n = 6). White * inside the column indicate values of ANOVA test of HS and FCS vs. C (***<i>p<</i>0.001); black * indicate values of ANOVA test of HS vs. FCS (*<i>p<</i>0.05).</p

Confocal images of implants pre-differentiated in both the conditions.

<p>A–D: Double fluorescence signals from DAPI (blue) and anti-hMit Ab (green) superimposed to pseudo-phase contrast images (B, D). Arrowheads indicate cells entrapped in calcified bone matrix clearly stained by anti-human mitochondria antibody. E–H: triple fluorescence signals from DAPI (blue) and anti-hMit (green) and anti-OCN (red) Abs superimposed to pseudo-phase contrast images (F, H). Yellow arrowheads indicate osteocytes labelled by the two Abs; cyan arrowheads indicate OCN deposits in extracelluar bone matrix. I–L: triple fluorescence signals from DAPI (blue) and anti-hMit (green) and anti-von Willebrand factor (red) Abs superimposed to pseudo-phase contrast images (J, L). Arrowheads indicate vasa double stained by the two Abs. Bar: 10 µm.</p

hDPSCs growing in FCS, HS and serum free (SF) media.

<p>A: CFDA vital staining of hDPSCs cultured for 1, 4 and 7 days. Green signal indicates viable cells. Bar: 100 µm. B: Proliferation rate of hDPSCs cultured for a week; Values are mean reported in a Log scale, n = 3; * indicates values of paired <i>t</i>-test HS vs. FCS (**<i>p<</i>0.01, ***<i>p<</i>0.001). C: Cumulative population doubling (CPD) of hDPSCs cultured for a total of 5 passages. At each passage cells cultured in HS-medium show a CPD significantly higher than FCS-medium cultured cells (n = 3; ***<i>p<</i>0.001). D shows β-galactosidase activity staining in confluent culture of hDPSCs grown for 5 passages in FCS-medium or in HS-medium as indicated. Arrowheads indicate cells positive to β-galactosidase activity staining. Bar 10 µm. E: western blot analysis of PARP in hDPSCs cultured in FCS-medium and in HS-medium at passages 1, 3 and 5. HL60, treated with etoposide, were loaded as positive control of the presence of cleaved PARP (cPARP). Actin bands were presented as control of the protein loading. Densitometry of cPARP bands was shown on the bottom of western blot images.</p

Additional file 1: Figure S1. of Stem cells isolated from human dental pulp and amniotic fluid improve skeletal muscle histopathology in mdx/SCID mice

Human DPSCs and AFSCs not undergoing demethylation treatment did not show any expression of muscle-specific markers (myogenin, MyHC, and desmin) after culture in myogenic induction medium. Scale bar = 50 μm. hAFSC, human amniotic fluid stem cell, hDPSC, human dental pulp stem cell, MyHC myosin heavy chain. (TIFF 457 kb

image_1_LonP1 Differently Modulates Mitochondrial Function and Bioenergetics of Primary Versus Metastatic Colon Cancer Cells.PDF

<p>Mitochondrial Lon protease (LonP1) is a multi-function enzyme that regulates mitochondrial functions in several human malignancies, including colorectal cancer (CRC). The mechanism(s) by which LonP1 contributes to colorectal carcinogenesis is not fully understood. We found that silencing LonP1 leads to severe mitochondrial impairment and apoptosis in colon cancer cells. Here, we investigate the role of LonP1 in mitochondrial functions, metabolism, and epithelial–mesenchymal transition (EMT) in colon tumor cells and in metastasis. LonP1 was almost absent in normal mucosa, gradually increased from aberrant crypt foci to adenoma, and was most abundant in CRC. Moreover, LonP1 was preferentially upregulated in colorectal samples with mutated p53 or nuclear β-catenin, and its overexpression led to increased levels of β-catenin and decreased levels of E-cadherin, key proteins in EMT, in vitro. LonP1 upregulation also induced opposite changes in oxidative phosphorylation, glycolysis, and pentose pathway in SW480 primary colon tumor cells when compared to SW620 metastatic colon cancer cells. In conclusion, basal LonP1 expression is essential for normal mitochondrial function, and increased LonP1 levels in SW480 and SW620 cells induce a metabolic shift toward glycolysis, leading to EMT.</p

image_2_LonP1 Differently Modulates Mitochondrial Function and Bioenergetics of Primary Versus Metastatic Colon Cancer Cells.PDF

<p>Mitochondrial Lon protease (LonP1) is a multi-function enzyme that regulates mitochondrial functions in several human malignancies, including colorectal cancer (CRC). The mechanism(s) by which LonP1 contributes to colorectal carcinogenesis is not fully understood. We found that silencing LonP1 leads to severe mitochondrial impairment and apoptosis in colon cancer cells. Here, we investigate the role of LonP1 in mitochondrial functions, metabolism, and epithelial–mesenchymal transition (EMT) in colon tumor cells and in metastasis. LonP1 was almost absent in normal mucosa, gradually increased from aberrant crypt foci to adenoma, and was most abundant in CRC. Moreover, LonP1 was preferentially upregulated in colorectal samples with mutated p53 or nuclear β-catenin, and its overexpression led to increased levels of β-catenin and decreased levels of E-cadherin, key proteins in EMT, in vitro. LonP1 upregulation also induced opposite changes in oxidative phosphorylation, glycolysis, and pentose pathway in SW480 primary colon tumor cells when compared to SW620 metastatic colon cancer cells. In conclusion, basal LonP1 expression is essential for normal mitochondrial function, and increased LonP1 levels in SW480 and SW620 cells induce a metabolic shift toward glycolysis, leading to EMT.</p