Fig 1.
Experimental design of the study.
See Material and method 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).
Fig 2.
Ex vivo expansion of CB-CD34+ cells with MSCs.
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.
Fig 3.
Clonogenic assays of CB-CD34+ cells.
Results of statistical analysis of methylcellulose-based clonogenic assay performed on CB-CD34+ cells plated after 14 days of co-culture with human MSCs. (A) CFU-GEMM count; (B) BFU-E count; (C) CFU-GM count. Values are reported as mean ± SD. The results derive from three independent experiments. Abbreviations: BFU-E, Burst forming unit-erythroid; CFU, Colony forming unit; E, erythrocyte; GM, granulocyte-monocyte; GEMM, granulocyte-erythrocyte-monocyte-megakaryocyte.
Fig 4.
Gene expression analysis of CB-CD34+ cells after co-culture with MSCs.
(A) Supervised analysis of CB-alone vs. Total SN+AD. CB-CD34+ cells were maintained for 10 days in co-culture with MSCs. 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 CB-alone vs Total SN+AD. Enriched gene sets from hallmark gene sets are plotted. Please refer to S2 Table for the complete GSEA details. Abbreviation: CB-alone: CB-CD34+ cells in single culture; Total SN+AD: CB-CD34+ cells grown in presence of MSCs.
Fig 5.
Real-Time Quantitative Polymerase Chain Reaction PCR (qRT-PCR) analysis of CCNA1 gene.
Expression level of CCNA1 gene was assessed by qRT-PCR. Quantification of mRNA relative quantity was calculated as relative changes in gene expression of the target gene normalised to the GAPDH endogenous control and relative to a calibrator sample. The values obtained were represented as relative quantity of mRNA level variations.
Fig 6.
Gene expression profiles analysis of MSCs after co-culturing with CB-CD34+ cells.
(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 S4 Table for the complete GSEA details.
Fig 7.
Real-Time Quantitative Polymerase Chain Reaction PCR (qRT-PCR) analysis of VEGFA gene.
Expression level of VEGFA in co-cultured MSCs was assessed by qRT-PCR. Quantification of mRNA relative quantity was calculated as relative changes in gene expression of the target gene normalised to the GAPDH endogenous control and relative to a calibrator sample. The values obtained were represented as relative quantity of mRNA level variations as compared to MSCs-alone.