Figure 5.

Meso-Endothelial Bipotent Progenitors and Endothelial Progenitor Cells Were Further Characterized Based on Level of CD144 Expression In Vivo

(A) CD31Lo population highly express CD144 marker at primary culture. Scale bars, 200 μm (low magnification) and 50 μm (high magnification).

(B) The CD45⁻CD34⁺ populations were examined for CD144 expression in vivo. CD31Lo and CD31Int cells were divided in two categories based on CD144 expression. Sorted cells were cultured in coated plates with EGM2.

(C) The CD31NegCD144Neg progenitors gave rise to mesenchymal cells. The CD31Lo bipotential progenitors were within the CD144⁺ fraction. Highly proliferative EPCs were isolated from the CD31IntCD144⁺ population and CD31HiCD144⁺ cells formed endothelial clusters with no proliferation potential in subsequent passages. Scale bar, 100 μm.

(D–G) The use of CD144 allowed further enrichment in EPCs and bipotent progenitors.

(H) Upon in vitro culture and in passage 2, cells were stained with CD144 antibody and the CD31Neg population gave rise to CD144⁻ cells, CD31Lo/CD144⁺ population gave rise to a mixture of CD144⁺ and CD144⁻ cells, and CD31Int/CD144⁺ ended up with CD144⁺ cells. Scale bar, 50 μm.

(I and J) Representative images of placental sections stained with CD34 and CD144 antibodies. As expected, all positive cells reside in an intimal position within vascular structures. Scale bar, 100 μm.

(K) Placental arteries were firstly washed with PBS and then perfused with FITC-conjugated Ulex europaeus agglutinin I (UEA-I) and a single-cell suspension prepared. The single-cell suspension was stained with antibodies and sorted. On sorting, almost all CD31Neg cells were UEA-I negative, and CD31Hi cells were entirely UEA-I positive. However, CD31Lo and CD31Int populations had UEA-I-positive and -negative fractions.

(L) Placental sections stained with UEA-I antibody; all (UEA-I)⁺ cells reside in intimal position within vascular structures. Scale bar, 50 μm.

(M) Upon in vitro culture and in passage 2, the CD31Neg population gave rise to (UEA-I)⁻ cells; however, the CD31Lo/(UEA-I)⁻ population gave rise to both (UEA-I)⁺ and (UEA-I)⁻ cells and CD31Int/(UEA-I)⁻ ended up with (UEA-I)⁺ cells. Scale bar, 50 μm.

(N) Placental sections stained with CD34 and osteopontin (OPN) antibodies. Some cells are CD34⁺ OPN⁺ (white arrows). Scale bars, 100 μm (left) and 10 μm (right).

(O) Placental sections stained with CD144 and osteopontin (OPN) antibodies. Arrows indicate CD144⁺ OPN⁺ cells. Some cells are subluminal (red arrows) and some are in luminal (white arrows) position. Scale bars, 100 μm (left) and 10 μm (right).