Presentation1.pdf

<p>The formation and activity of mammalian tissues entail finely regulated processes, involving the concerted organization and interaction of multiple cell types. In recent years the prospective isolation of distinct progenitor and stem cell populations has become a powerful tool in the hands of developmental biologists and has rendered the investigation of their intrinsic properties possible. In this protocol, we describe how to purify progenitors with different lineage history and degree of differentiation from embryonic and fetal skeletal muscle by fluorescence-activated cell sorting (FACS). The approach takes advantage of a panel of murine strains expressing fluorescent reporter genes specifically in the myogenic progenitors. We provide a detailed description of the dissection procedures and of the enzymatic dissociation required to maximize the yield of mononucleated cells for subsequent FACS-based purification. The procedure takes ~6–7 h to complete and allows for the isolation and the subsequent molecular and phenotypic characterization of developmental myogenic progenitors.</p

CD133+ cells were isolated from the peripheral blood of 70 DMD patients and 20 age-matched control subjects and analyzed by flow-cytometry.

<p>Representative panels show the CD133+CXCR4+CD34+ subpopulation in healthy subjects (mean percentage±SD, 1.58±2.39 of total CD133+ cells) (upper right panel in A) and in DMD patients (3.87±0.63)(lower right panel in B). A subpopulation of CD133+CXCR4+CD34-cells was significantly increased in DMD patients (lower right panel in B) compared with healthy controls (lower right panel in A) (mean percentage±SD, 17.38±1.38 vs. 11.0±1.70 of total CD133+ cells). (C) Histogram showing the percentages of CD133+CXCR4+CD34+ cells of healthy controls compared to DMD patients. (D) Histogram demonstrating the percentages of CD133+CXCR4+CD34- cells in healthy controls compared with to DMD patients. *Significance (<i>P</i> < 0.05).</p

Representation of 2 different disease courses in 19 DMD patients stratified into 2 groups, in which a 24-month follow-up was available according to the results obtained from linear regression analysis between age and levels of the CD133+CXCR4+CD34- cells.

<p>MRC% scores are shown over time in 8 patients with a percentage of the cells localized above the threshold level for the corresponding age (A and C) and in 11 patients with values below the regression line during a total follow-up period of 24 months (B and D). At the end of the observation period, in the first group of DMD patients, the mean MRC% decreased by approximately 14%, and the total clinical score approximately 2.25 points. In the second group, the MRC% decreased by approximately 25%. In a subgroup including 4 DMD patients of the first group and in 5 DMD patients of the second group, we measured CD133+CXCR4+CD34- cells levels. Differences between the baseline values of CD133+CXCR4+CD34- cells and those at 2, 4, 6, 12 and 24 months are shown for individual patients in E and F. Despite the significant increase of CD133+CXCR4+CD34- cells compared to the patients of the second group (mean value±SD: 31.36%±14.67% vs. 11.12%±4.9%; P<0.0001), the 4 patients in group 1 displayed major intra-individual variability between successive measurements.</p

Levels of CD133+CXCD4+CD34- and CD133+CXCX4+CD34+ subpopulations are shown in DMD patients (A) and healthy controls (B) stratified for age.

<p>Levels of CD133+CXCR4+CD34- cells were constantly higher than CD133+CXCX4+CD34+ cells in both DMD patients and controls. DMD patients showed a nadir at the age of 4 years and one at the age of 9, with an overall tendency towards reduction with increasing age. Linear regression analysis of DMD patient data revealed a significant negative correlation (r² = 0.056; P = 0.045) between the level of CD133+CXCR4+CD34- cells and age (C), while no significant correlation (r² = 0.003; P = 0.63) between the level of CD133+CXCR4+CD34+ cells and age was observed (D).</p

Correlation of CD133+CXCX4+CD34- and CD133+CXCX4+CD34+ subpopulations with MRC%, FE% and FVC% in DMD patients.

<p>A positive correlation was observed between the level of CD133+CXCX4+CD34- cells and MRC percentage (r² = 0.065; P = 0.046)(A), FE% (r² = 0.065; P = 0.043) (C), and FVC% (r² = 0.089; P = 0.025) (E). No significant correlation was observed between the level of CD133+CXCX4+CD34+ cells and the above variables (B, D, and F).</p

Identification of new murine dystrophic-fibres associated-miRNAs in muscle biopsies of DMD subjects.

<p>(A) The age and type of muscle of healthy subjects (12) and DMD patients (18) were listed. Moreover the type of muscle from which the biopsies were isolated were also reported. (B–C) MiR-15b, miR-128a, miR-206, miR-17 and miR-27a were quantified by absolute Q-PCR in muscle biopsies of control and dystrophic subjects listed in A. The absolute values (pg) of tested miRNAs were represented in the histogram for healthy (white bars) and dystrophic single fibres (black bars). (B) Quantitative analysis showed that only miR-128a, miR-206 and miR-17 were up-regulated in human dystrophic single fibres independently of the muscle type. (Two-tail parametric t-test; *p value <0, 05; **p value <0, 01; ***p value <0,001).</p

MiRNA profile of single fibres isolated from the mdx mouse.

<p>(A) Clustered heat map showing the expression ratios of miRNAs in TA, DIA and VA of age- and sex-matched c57bl (n = 3) and mdx (n = 3) mice. Expression data were normalized on Universal Reference. A total of 14 miRNAs were found over-expressed in dystrophic samples with distinction among myofibres isolated from different muscle type. (B) Fold change values of the 14 up-regulated miRNAs were reported in the table (ns = no significant).</p

Acute muscle damage activates miRNA machinery in isolated myofibers.

<p>Dystrophic-fibres associated miRNAs were quantified by Q-PCR in single fibres isolated from TA of c57bl mice (n = 10) after CTX-injection. Mice were sacrified at the day 2, 5, 7, 10 after CTX-injury. In the table are represented the fold change values of tested miRNAs in CTX-injected muscle compare to controlateral not injected TA. MiR-206, miR-31, miR-21, miR-335-5p, miR-27a, miR-142-5p and miR-223 were significantly up-regulated afterwards muscle damage respect damaged muscle. Otherwise the other 7 miRNAs were not triggered upon CTX-injection.</p><p>(Two-tail parametric t-test; *p value <0, 05; **p value <0, 01; ***p value <0,001).</p

MyomiR-206 modulates Hmgb3 expression during myogenesis.

<p>(A) Hek cells were co-transfected with mimic-miR-206 at a concentration of 25 nM for 48 h and with pGLO-control or pGLOHmgb3 or pGLOHmgb3Mut. MiR-206 down-regulated the expression of luciferase of 32% (p = 0, 0151) in Hek cells co-transfected with pGLOHmgb3. Instead no down-regulation happened when the binding site of miR-206 was mutated. (B) 3T3 cells were transfected with mimic miR-206 at a concentration of 25 nM for 48 h, evidencing a strong down-regulation of endogenous Hmgb3 mRNAs. (C) Quantification of Hmgb3 mRNA by qRT-PCR in single fibres isolated from CTX-injured TA at day 2, 5, 7 and 10 from the injection evidenced a strong down-regulation of Hmgb3 during muscle regeneration. (D–E) Hmgb3 mRNA (D) and miR-206 (E) were respectively quantified by relative qRT-PCR and by absolute Q-PCR in proliferating C₁C₁₂ myoblasts cell line (MB) versus differentiated C₁C₁₂ myotubes (MT). The quantification analysis highlighted an opposite expression trend for Hmgb3 and miR-206, further validating inhibition of Hmgb3 by miR-206 (AU = arbitrary units). (Two-tail parametric t-test; *p value <0, 05; **p value <0,01; ***p value <0,001).</p

Hmgb3 expression-pattern in single fibres.

<p>(A) Immunoblotting experiments confirmed the presence of Hmgb3 protein in single muscle fibers isolated from the TA and VA of c57bl (n = 3) and mdx (n = 3) mice. (B) Densitometric analysis on WB bands evidenced a decreased expression of Hmgb3 in the TA and VA (p = 0.0133) of mdx mice. (C) Quantification of Hmgb3 mRNA by qRT-PCR in single fibres isolated from the TA and VA of mdx mice (n = 10) and of c57bl (n = 10) mice confirmed a down-regulation of Hmgb3 in dystrophic muscle. (Two-tail parametric t-test; *p value <0, 05; **p value <0, 01; ***p value <0,001).</p