Osteoblast differentiation potential of BM-hMSCs after culture in an FBS+bFGF-, hPL- or PR-hPL-containing medium.

<p>Differentiation was induced using the specific medium. The calcium deposit was stained using Alizarin Red S (<b>a</b>) and the extracellular matrix using Von Kossa (<b>b</b>). Representative photographs of experiments with hMSCs from n = 3 BM. Black arrows and white stars indicated positively stained areas. Quantification of Alizarin Red S (<b>c</b>) or Von Kossa (<b>d</b>) was expressed as a percentage of positive area. ALP activity measurement was performed using a commercially available kit (<b>e</b>). NS: <i>not significant versus</i> FBS.</p

Proliferation of BM-hMSCs cultured in an FBS-containing medium.

<p>Eight references of FBS were tested at a dose of 10% with 1 ng/mL bFGF. Results are presented as amplification yields for means of triplicates. NS: <i>not significant</i>; ***: <i>p<0</i>.<i>001 versus</i> FBS 3 (one-way ANOVA and Bonferroni posttests).</p

Expression of CD13, CD34, CD40, CD44, CD45, CD73, CD80, CD86, CD90, CD105 and HLA-DR in BM-hMSCs cultured in an FBS+bFGF-, hPL- or PR-hPL-containing medium assessed by flow cytometry.

<p>Results are presented as percentages of positive cells for means of experiments with hMSCs from n = 3 BM.</p

Pathogen reduction through additive-free short-wave UV light irradiation retains the optimal efficacy of human platelet lysate for the expansion of human bone marrow mesenchymal stem cells

<div><p>Background</p><p>We recently developed and characterized a standardized and clinical grade human Platelet Lysate (hPL) that constitutes an advantageous substitute for fetal bovine serum (FBS) for human mesenchymal stem cell (hMSC) expansion required in cell therapy procedures, avoiding xenogenic risks (virological and immunological) and ethical issues. Because of the progressive use of pathogen-reduced (PR) labile blood components, and the requirement of ensuring the viral safety of raw materials for cell therapy products, we evaluated the impact of the novel procedure known as THERAFLEX UV-Platelets for pathogen reduction on hPL quality (growth factors content) and efficacy (as a medium supplement for hMSC expansion). This technology is based on short-wave ultraviolet light (UV-C) that induces non-reversible damages in DNA and RNA of pathogens while preserving protein structures and functions, and has the main advantage of not needing the addition of any photosensitizing additives (that might secondarily interfere with hMSCs).</p><p>Methodology / Principal findings</p><p>We applied the THERAFLEX UV-Platelets procedure on fresh platelet concentrates (PCs) suspended in platelet additive solution and prepared hPL from these treated PCs. We compared the quality and efficacy of PR-hPL with the corresponding non-PR ones. We found no impact on the content of five cytokines tested (EGF, bFGF, PDGF-AB, VEGF and IGF-1) but a significant decrease in TGF-ß1 (-21%, n = 11, <i>p<0</i>.<i>01</i>). We performed large-scale culture of hMSCs from bone marrow (BM) during three passages and showed that hPL or PR-hPL at 8% triggered comparable BM-hMSC proliferation as FBS at 10% plus bFGF. Moreover, after proliferation of hMSCs in an hPL- or PR-hPL-containing medium, their profile of membrane marker expression, their clonogenic potential and immunosuppressive properties were maintained, in comparison with BM-hMSCs cultured under FBS conditions. The potential to differentiate towards the adipogenic and osteogenic lineages of hMSCs cultured in parallel in the three conditions also remained identical.</p><p>Conclusion / Significance</p><p>We demonstrated the feasibility of using UV-C-treated platelets to subsequently obtain pathogen-reduced hPL, while preserving its optimal quality and efficacy for hMSC expansion in cell therapy applications.</p></div

Proliferation of BM-hMSCs cultured for 10 days in an FBS+bFGF-, hPL- or PR-hPL-containing medium.

<p>Six units of PR-hPL and their respective hPL controls were tested at doses ranging from 0% to 15%. FBS (from 2% to 15%) with 1 ng/mL bFGF was used as a control. Proliferation was evaluated using the CellTiter-Glo assay. *: <i>p<0</i>.<i>05</i>; **: <i>p<0</i>.<i>01</i>; *** <i>p<0</i>.<i>001 versus</i> hPL / PR-hPL (two-way ANOVA and Bonferroni posttests).</p

Immunosuppressive properties of BM-hMSCs after culture in an FBS+bFGF-, hPL- or PR-hPL-containing medium.

<p>T-cell proliferation was induced using Con A (<b>a</b>) or MLR assay (<b>b</b>). Results are presented as the percentage of inhibition of T-cell proliferation in experiments performed in quadruplicates. Experiments were performed with MSC:T-cell ratios of 1:20, 1:10, 1:5 and 1:1 (<b>a</b>) and MSC:T-cell:PBMC ratios of 1:5:5 and 1:1:1 (<b>b</b>) NS: <i>not significant</i>, *: <i>p<0</i>.<i>05</i>; **: <i>p<0</i>.<i>01</i> and ***: <i>p<0</i>.<i>001 versus</i> FBS (two-way ANOVA and Bonferroni posttests).</p

Growth factor contents in hPL and PR-hPL measured using commercially available ELISA kits.

<p>Results are presented as concentrations of PDGF-AB, IGF-1, TGF-ß1 (<b>a</b>), and bFGF, VEGF and EGF (<b>b</b>), (individual values and means of dosages in n = 11 units of PR-hPL and their respective hPL controls). **: <i>p<0</i>.<i>01</i> hPL <i>versus</i> PR-hPL (Student’s t-test).</p

Additional file 2: Figure S2. of Identification in GRMD dog muscle of critical miRNAs involved in pathophysiology and effects associated with MuStem cell transplantation

Clinical follow-up. Clinical scores of mock GRMD dogs (---) and MuStem cell-injected dogs (—) are represented as mean ± SD. The clinical score of each GRMD dog was assessed weekly and expressed as a percentage of a theoretical healthy dog score. Limits of the MuStem cell delivery window are indicated (dashed lines). (PDF 32 kb

Representative histogram overlays for expression of CD13 (a), CD44 (b), CD73 (c), CD90 (d), CD105 (e), CD34 (f), CD45 (g), HLA-DR (h), CD40 (i), CD80 (j) and CD86 (k) of BM-hMSCs cultured in an hPL- (red curves <i>versus</i> isotype controls in black) or PR-hPL-containing medium (green curves <i>versus</i> isotype controls in blue).

<p>Representative histogram overlays for expression of CD13 (a), CD44 (b), CD73 (c), CD90 (d), CD105 (e), CD34 (f), CD45 (g), HLA-DR (h), CD40 (i), CD80 (j) and CD86 (k) of BM-hMSCs cultured in an hPL- (red curves <i>versus</i> isotype controls in black) or PR-hPL-containing medium (green curves <i>versus</i> isotype controls in blue).</p

Proliferation of BM-hMSCs cultured in an FBS+bFGF-, hPL- or PR-hPL-containing medium during three consecutive passages.

<p>Results are presented as cumulative population doubling (<b>a</b>) and generation time (<b>b</b>), for means of n = 3 experiments. *: <i>p<0</i>.<i>05 versus</i> hPL / PR-hPL (two-way ANOVA and Bonferroni posttests).</p