Table 1.
MDS patient characteristics.
Fig 1.
Characterization of CD34+ cells from leukapheresis of HD.
(A) Percentage of CD34+ cells isolated by immunomagnetic beads and the purity determined by flow cytometry. (B) Flow cytometry characterization of MVs released from MSCs of MDS and HD. The upper images are dot-plots of forward and side scatter of MVs. The gate was defined as elements of smaller size than the 1μm beads. The histograms represent the MVs stained with negative (CD34 and CD45) and positive markers for MVs from HD and MDS-MSC (CD90, CD44, CD73) and for MVs markers (CD81 and CD63). Controls (unstained MVs) are shown in gray; the MVs stained with the different antibodies are shown in black. Images on the left are those of the MVs from MSC-HD, while those on the right images are of the MVs from MSC-MDS. (C) Representative images of transmission electronic microscopy of MVs released by MSC from HD(left) and MDS (right) as revealed by TEM. Scale bar, 200nm. Original magnification: x 8000. (D) MVs characterization by Western Blot assay for the expression of CD63. HD-MVs: microvesicles from healthy donors. MDS-MVs: microvesicles from patients with myelodysplastic syndrome.
Fig 2.
Delta Ct values of 5 microRNAs differentially expressed in MSC-derived MVs between MDS patients and healthy donors (HD).
Bars represent median values of Delta Ct per sample category. Mean and confidence interval per sample category are also drawn. Analysis performed over qPCR microRNA arrays. Asterisks denote differential expression p-values: (**) <0.01, (*) <0.05.
Fig 3.
Incorporation of MVs from MSC-MDS and MSC-HD into CD34+ cells.
(A) Representative images of MVs incorporation by CD34+ cells stained with anti-CD90 Ab (red) and anti-CD45 Ab (green). (B) Representative images of MVs previously labeled with Vybrant-Dil cell-labeling solution (red) that were incorporated into CD34+ cells and stained with anti-CD45 Ab (green). (A-B) Images in the top row are from CD34+ cells that incorporated the MVs released from MSC-HD. Images on the middle row show the incorporation of MVs released from MSC-MDS. In the lower row, images of the CD34+ cells (without incorporation) are shown. Nuclei were counterstained with DAPI (blue). Scale bar, 7.5μm. Revealed by confocal microscopy and acquired in layers (z-Stacks) of 1μm.
Fig 4.
Modification of HPC properties.
(A) Variations in microRNAs expression when CD34+ cells were co-cultured with MDS-MVs or HD-MVs. Ratio was calculated dividing the expression of each microRNA from CD34+ + HD-MVs or CD34+ + MDS-MVs by that of CD34+ cells without MVs. Results were summarized as the median. (B) Expression by RT-PCR of TP53 and MDM2 in CD34+ cells cultured with MDS-MVs from patients (grey) and expression of CD34+ cells without MVs (black). Results were summarized as the mean and standard deviation. (C) Capillary Electrophoresis Immunoassay of MDM2 vs Actin as control. CD34+ cells (without MVs), CD34+ cells with MDS-MVs and with HD-MVs. Each bar of the lower graph represents the value of quantified MDM2 protein expression normalized to actin protein abundance. Each bar represents the quantification of both bands of MDM2 from the pseudo-blots, control CD34+ cells vs CD34+ cells + MDS-MVs or HD-MVs.
Fig 5.
MVs content incorporation results in CD34+ cells behavior modification.
(A): Representative FACS plots of annexinV/7AAD staining on CD34+ cells with and without MVs. Percentage of each subset (dead, live, early and late apoptosis) within the total number of CD34+ cells. (B) Percentage of live CD34+ cells. An increase on the percentage of CD34+ viable cells (annexinV-/7AAD-) was observed when cells were cultured with MDS-MVs compared with the other groups is shown. (C) Clonogenic assays. Results are expressed as the ratio between CFU-GM obtained with CD34+ cells that had been cultured with MVs and CD34+ cells without MVs.