Stem Cell Tracking by Nanotechnologies

Advances in stem cell research have provided important understanding of the cell biology and offered great promise for developing new strategies for tissue regeneration. The beneficial effects of stem cell therapy depend also by the development of new approachs for the track of stem cells in living subjects over time after transplantation. Recent developments in the use of nanotechnologies have contributed to advance of the high-resolution in vivo imaging methods, including positron emission tomography (PET), single-photon emission tomography (SPECT), magnetic resonance (MR) imaging, and X-Ray computed microtomography (microCT). This review examines the use of nanotechnologies for stem cell tracking

Absence of T and B lymphocytes modulates dystrophic features in dysferlin deficient animal model

Dysferlin mutations cause muscular dystrophy (dysferlinopathy) characterized by adult onset muscle weakness, high serum creatine kinase levels, attenuation of muscle regeneration and a prominent inflammatory infiltrate. In order to verify the role of lymphocytes and immune cells on this disease, we generated the Scid/A/J transgenic mice and compared these animals with the age-matched A/J mice. The absence of T and B lymphocytes in this animal model of dysferlinopathy resulted in an improvement of the muscle regeneration. Scid/A/J mice showed increased specific force in the myosin heavy chain 2A-expressing fibers of the diaphragm and abdominal muscles. Moreover, a partial reduction in complement deposition was observed together with a diminution in pro-inflammatory M1 macrophages. Consistent with this model, T and B lymphocytes seem to have a role in the muscle damaging immune response. The knowledge of the involvement of immune system in the development of dysferlinopathies could represent an important tool for their rescuing. By studying Scid/blAJ mice, we showed that it could be possible to modulate the pathological symptoms of these diseases by interfering with different components of the immune system

Improvement of endurance of DMD animal model using natural polyphenols

Duchenne Muscular Dystrophy (DMD), the most common form of muscular dystrophy, is characterized by muscular wasting caused by dystrophin deficiency that ultimately ends in force reduction and premature death. In addition to primary genetic defect, several mechanisms contribute to DMD pathogenesis. Recently, antioxidant supplementation was shown to be effective in the treatment of multiple diseases including muscular dystrophy. Different mechanisms were hypothesized such as reduced hydroxyl radicals, nuclear factor-\u3baB deactivation and NO protection from inactivation. Following these promising evidences, we investigated the effect of the administration of a mix of dietary natural polyphenols (ProAbe) on dystrophic mdx mice in term of muscular architecture and functionality. We observed a reduction of muscle fibrosis deposition and myofiber necrosis together with an amelioration of vascolarization. More importantly, the recovery of the morphological features of dystrophic muscle leads to an improvement of the endurance of treated dystrophic mice. Our data confirmed that ProAbe-based diet may represent a strategy to co-adjuvate the treatment of DMD

Cytoplasmic vesicle localisation.

<p>Fluorescence microscopy images confirming intra-cellular vesicle localisation. (A) Hcc-1, (B) HepG2, (C) Hep3B, and (D) SNU475 cells. Sunitinib autofluorescence (green), CD29 staining (red) and Hoechst 33342 nuclear staining (blue). Original magnification 40x.</p

Immunophenotyping.

<p>The table shows the mean percentage of cells positive for each marker.</p><p>Immunophenotyping.</p

Chemoresistance assays.

<p>A) When treated with sorafenib, the cell lines with giant PGP-positive lysosomes (Hcc-1, HepG2, PLC/PRF/5 and HuH7) showed higher IC₅₀ values than those with normal lysosomes (Hep3B and SNU475) (p<0.01). B) The HCC cell lines were incubated with different concentrations of sorafenib in order to verify their chemosensitivity (green lines). The cells with larger cytoplasmic vesicles were characterised by a curve with a sort of plateau of viability at sorafenib concentrations of between 5 and 20 µmol. One hour of verapamil pre-treatment used to inhibit ABC proteins before co-incubation with sorafenib and sunitinib increased the chemosensitivity of all of the cell lines (black curves). *p<0.01 green vs. black curves. One hour of sorafenib pre-treatment (red lines) before co-incubation with sorafenib and sunitinib enhanced treatment efficacy in comparison with verapamil pre-treatment in the cell lines carrying giant lysosomes. ^§p<0.05 red vs. black curves.</p

Hypothesised mechanism of the enhanced efficacy of drug pre-treatment before verapamil administration and PGP blockade.

<p>A) HCC cells expressing active PGP can expel a drug (e.g. sunitinib) from the cytoplasm or store it in lysosomes. B) Blocking PGP with verapamil before the co-administration sunitinib and verapamil allows the drugs to enter the cell and diffuse into cytoplasm/nucleus. C) If sunitinib is used for pre-treatment, it is stored in giant lysosomes and, after the co-administration of sunitinib and verapamil and subsequent PGP blockade, the drugs can enter the cytoplasm/nucleus from both extra-cellular space and the lysosomes.</p

Flow cytometry evaluation of MDRPs.

<p>LRP and MRP1 were not detected by means of flow cytometry in any cell line. ABCG2 was expressed on only 9% of the Hcc-1 cells, and was almost absent on the surface of the other cell lines. ABCB1 (PGP) was highly expressed on the plasma membrane of HepG2 (58%), Hcc-1 (18%) and HuH7 cells (13%), but expressed on only about 3% of PLC/PRF/5 cells. Left column negative controls; middle column ABCG2 expression; right column PGP expression.</p

Sunitinib accumulation in cytoplasmic vesicles of HCC cell lines.

<p>Representative pictures of cell lines cultured without (left) or with (right) sunitinib 12 µM. Cytoplasmic vesicles are visible in the Hep3B and SNU475 cells only after sunitinib incubation. Original magnification 20x.</p

Immunofluorescence staining of MDRPs.

<p>Representative pictures of MDRP staining in HCC cell lines. The expression and localisation of MDRPs was different in the various HCC cell lines: ABCG2, LRP and MRP1 were localised on the membrane of the few groups of cells on which they were expressed, whereas PGP was mainly found in the cell cytoplasm, particularly on the membrane of intra-cellular vesicles. Nuclei stained with DAPI (blue). Original magnification 20x.</p