Fig 2: 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 3: Isolation of CD34+ HSPCs from UCB MNCsThe percentage of CD34+ cells was 0.36% in UCB MNCs, as tested in the sample used in this protocol. After immunomagnetic isolation of CD34+ HSPCs from UCB MNCs, the percentage of CD34+ cells was 90.1% in the isolated CD34+ HSPCs.

Fig 4: Characterization of primary human PDL cells. (A) hPDL cells were first characterized by flow cytometry analysis of cell surface markers. The expression pattern for all donors is CD34-, CD45-, CD73+, CD90+ and CD105+ (blue color: isotype control, red color: surface marker) and (B) hPDLs differentiation towards adipocytes, chondrocytes and osteoblasts was performed. hPDL cells were grown in osteogenic, adipogenic and chondrogenic differentiation media for 14 days and then stained with alizarin red, Oil red O and Toluidin blue, respectively. The red color represented mineralized calcium depositions after PDL cells differentiated into osteoblast-like cells. A large number of orange lipid vacuoles were seen in PDL cells after culture in AIM and staining with Oil red O. Staining with Toluidine blue revealed excessive production of extracellular matrix components and proteoglycans during the culture process with CIM (scale: 200 µm).

Fig 5: Effect of gamma-aminobutyric acid type B receptor subunit 2 (GABBR2) knockdown on endothelial progenitor cell (EPC) mobilization in peripheral blood in hindlimb post-ischemia. (A) Representative images of CD34+/CD133+ cells in the peripheral blood of mice analyzed by flow cytometry. WT, normal mice; KD, GABBR2-downregulated mice; WT-H, wild-type (WT) mice with hindlimb ischemia; KD-H, GABBR2 knockdown (KD) mice with hindlimb ischemia. (B) Ratio of CD34+ cells in the peripheral blood of normal and ischemic mice. (C) Ratio of CD133+ cells in the peripheral blood of normal and ischemic mice. (D) Ratio of CD34+ and CD133+ cells in the peripheral blood of normal and ischemic mice. *p < 0.05; ***p < 0.001; ****p < 0.0001.