Characteristics of the 50 patients and transplant.

<p>Characteristics of the 50 patients and transplant.</p

Clusters of immunologic variables correlated to aGVHD, cGVHD, and relapse by multivariate analysis.

<p>Clusters of immunologic variables correlated to aGVHD, cGVHD, and relapse by multivariate analysis.</p

Comparison of immunological variables between patients with and without acute GVHD.

<p>Comparison of immunological variables between patients with and without acute GVHD.</p

Univariate analysis of immunological variables associated with relapse.

<p>Univariate analysis of immunological variables associated with relapse.</p

Characteristics of the 50 patients and transplant.

<p>Characteristics of the 50 patients and transplant.</p

Steroid effect on lymphocyte subsets and index of B/T cell neogenesis at day +90.

<p>Steroid effect on lymphocyte subsets and index of B/T cell neogenesis at day +90.</p

Comparison of immunological variables between patients with and without chronic GVHD.

<p>Comparison of immunological variables between patients with and without chronic GVHD.</p

<i>Ex vivo</i> expansion of CB-CD34+ cells with MSCs.

<p>5×10⁵ CB-CD34+ cells were cultured alone or in presence of a layer of MSCs for 10 days; the fold increase in total cell number was calculated from the original CD34+ cells seeded at day 0. The absolute number of CB-CD34+ cells was measured by flow cytometry. Values came from 5 independent replicates. Abbreviation: CB-alone: number of CB-CD34+ cells after 10 days of single culture; SN-fraction: CB-CD34+ cells in the supernatant (SN-fraction) of the co-cultures with MSCs; AD-fraction: CB-CD34+ cells grown directly in contact with MSCs layer; Total SN+AD: total number of CB-CD34+ cells after co-culture with MSCs.</p

Experimental design of the study.

<p>See <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0172430#sec002" target="_blank">Material and method</a> section for a complete description of the figure. Ten days of culture were chosen for 2 reasons: first of all, the majority of research articles dealing with MSCs and HSCs range from 7 to 14 days of co-culturing. Secondly, 10 days of co-culturing allow to see a biological effect of MSCs on HSCs without inducing cellular senescence in stromal cells. Indeed, co-cultures were maintained in serum-free medium supplemented with Stem Cell Factor (SCF), Thrombopoietin (TPO) and Granulocyte Colony Stimulating Factor (G-CSF) (see below for details). When MSCs are cultured in this medium for more than 14 days they undergo changes in cell morphology and they begin losing their phenotypic characteristics (data not shown).</p

Gene expression profiles analysis of MSCs after co-culturing with CB-CD34+ cells.

<p>(A) Supervised analysis of MSCs-alone vs. MSCs after 10 days of co-culture with CB-CD34+ cells. A heat map of the genes differentially expressed in a statistically significant manner after co-culture (corrected p value <0.05) is plotted. (B) Gene Set Enrichment Analysis (GSEA) of MSCs-alone vs. MSCs maintained for 10 days in co-culture with CB-CD34+ cells. Top 10 gene sets from hallmark gene sets, canonical/KEGG pathways and oncogenic signatures are plotted. Please refer to <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0172430#pone.0172430.s013" target="_blank">S4 Table</a> for the complete GSEA details.</p