Endothelial Progenitor Cells: Their Potential in the Placental Vasculature and Related Complications

Bright-field images of outgrowth cells from human fetal peripheral blood mononuclear cells (PBMNCs). A: CFU-Hill technique. Round cells in the centrum of the colony, spindle shaped cells in the periphery. The colonies appear to be smaller than in adult and spindle shaped cells migrate away from the centre rapidly. (Scale bar: 50μm) B: Vasa technique: monolayer of round adherent cells. (Scale bar: 100μm) C: Lin-technique: Colony formed by a monolayer of cells with cobble stone appearance. The colonies are larger and appear earlier than those in the adult. (Scale bar: 100μm) Unpublished images of the authors.

Flow cytometry technique for surface marker distribution of ECFCs in peripheral blood. A: Typical Foreward Scatter-Side Scatter histogram of lysed blood; Gate 1 set over mononuclear leucocytes. B: 7AAD-monohistogram. Gate 1 applied. Gate 2 set over 7AAD-negative (viable) cells. Non-viable cells have the tendency to non-specifically bind antibodies. Exclusion of non-vital cells helps reduce the number of false-positive events. C: CD45 monohistogram. Gates 1 and 2 applied. Gate 3 set over the CD45 negative population. D: CD31 monohistogram. Gates 1, 2 and 3 applied. Gate 4 set over the CD31 bright population. E: X-axis represents CD133; Y-axis represents CD34. Gates 1, 2, 3 and 4 are applied. Gate 5 set over CD34⁺ and CD133^- populations, while Gate 6 segregates CD34⁺ and CD133⁺ dual positive cells. Gate 5 highlights CD45^-/CD31^Bright/CD133^-/CD34⁺ events, consistence with an ECFC/CEC identity. Non-vital cells are excluded. Unpublished images of the authors. Flow cytometry technique modified from Duda et al. 2007 [111].

Flow cytometry technique for surface marker distribution of CACs in peripheral blood. A: Typical Foreward Scatter-Side Scatter histogram of lysed blood; Gate 1 set over mononuclear leucocytes. B: 7AAD-monohistogram. Gate 1 applied. Gate 2 set over 7AAD-negative (vital) cells. Non-vital cells may non-specifically bind antibodies. Exclusion of non-vital cells helps reduce false-positive events. C: CD45 monohistogram. Gates 1 and 2 applied. Gate 3 set over CD45 dim population. D: CD31 monohistogram. Gates 1, 2 and 3 applied. Gate 4 set over CD31-positive population. E: X-axis represents CD133; Y-axis represents CD34. Gates 1, 2, 3 and 4 applied. Gate 5 set over CD133⁺ and CD34^Bright population. This population is CD45^Dim/CD31⁺/CD133⁺/CD34^Bright. Non-vital cells are excluded. These features are characteristic to CACs. Unpublished images of the authors. Flow cytometry technique modified from Duda et al. 2007 [111].

A comparison of CAC and ECFC characteristics. ECFCs form tubes on matrix, while CACs fail to do so. Both CACs and ECFCs ingest AC-LDL, and express CD31and CD34. CACs are CD45 and CD133 positive; whilst ECFCs fail to express these antigenes. (Scale bar: 100μm (A-H, K-M); Scale bar: 10μm (I-J)) Unpublished images of the authors.