Mesenchymal stem cells in human placental chorionic villi reside in a vascular Niche

Western analysis was used to show that CD49a, 3G5 and STRO-1 were expressed in both PMSC protein extracts (PMSC) and in term placental tissue homogenates (tissue). (A) The CD49a antibody detected bands at 210kDa, 80kDa and 70kDa band in tissue and PMSC protein extracts. (B) The 3G5 antibody, detected a predominant immunoreactive band at 15kDa, with minor bands at 10kDa and 30kDa for both term placental tissue homogenate and PMSC protein extracts. (C) The STRO-1 antibody detected a prominent 27kDa band for both term placental tissue homogenate and PMSC protein extracts. Minor bands at 18kDa and 50kDa were also observed. The control blots with NIRS (A) for CD49a/VLA-1 (A) and IA6.12 (B, C) for STRO-1 and 3G5 showed no bands.

Immunocytochemical staining of isolated PMSCs. Cells were negative for X63 (a), vWF (b) and NIRS, FDO66Q, CD34, and CD117 (data not shown). Cells were positive for 3G5 (c), CD146 (d), CD49a/VLA-1 (e), α-smooth muscle actin (α-SMA) (f), STRO-1 (g) and CD106 (h) (magnification 200×). Cultured cells were clonogenic as shown by colony forming unit assay (Fig. 1i) (magnification 100×).

Differentiation assays for cells in the mesenchymal lineage. Control undifferentiated PMSCs (a, c, e) were grown in α-MEM medium alone. Panels (b), (d), (f) show cells grown for the same time as the controls but in α-MEM medium containing differentiation factors. Staining was with Alcian Blue for chondrocytes (a, b), Oil red O for adipocytes (c, d) and Alizarin Red for osteocytes. Magnification for panels (a) and (b) was 100× and for panels (c)–(f) was 200×. The arrows in panel (d) show lipid droplets. Scale bars represent 50μm.

Immunohistochemical localization on paraffin sections of cell surface markers in first trimester (12 week) human placenta. X63 negative control (a) and FDO66Q, a control antibody staining the syncytiotrophoblast layer (b). STRO-1 (c), CD146 (d), CD49a/VLA-1 (e) and 3G5 antibody staining of cells around the vessels (f). 3G5 antibody staining in scattered cells around the vessels (arrows). CD34 (g) and vWF (h) show antibody staining of endothelial cells lining the villous vessels. Panels (a)–(f) show staining with DAB and a haematoxylin counterstain. Panels (g) and (h) show AEC staining and a methyl green counterstain. Images are 400× magnification and the scale bar represents 50μm. v, Villous vessels; and tr, syncytiotrophoblast cells. Staining of cells is shown by an arrow.

Immunohistochemical localization of cell surface markers in term human placenta. X63 negative control (a) and FDO66Q, a control antibody staining the syncytiotrophoblast layer (b). STRO-1(c), CD146 (d), CD49a/VLA-1 (e) and 3G5 (f) antibody staining in the vascular region of the villous vessels. CD34 (g) and vWF (h) control antibodies staining the endothelial cells of the villous vasculature. Antibodies were used on either frozen (a, b, c, d & f) or paraffin (e, g & h) sections depending on antibody specificity. Panel a and f-h show staining with AEC and a methyl green counterstain. Panels (b)–(e) show staining with DAB and a haematoxylin counterstain. Images are 400× magnification and the scale bar represents 50μm. v, Villous vessels; and tr, syncytiotrophoblast cells. Staining of cells is shown by an arrow.

Dual-label immunofluorescence staining with STRO-1/vWF (a–f) on term frozen placental sections; vWF antibody staining with Cy3 (red) (a), STRO-1 staining with fluorescein detection (green) (b), combined images of (a), (b) are shown in (c) with a DAPI nuclear counterstain stain. Staining from another placenta with vessels of various sizes is shown in panels (d)–(f). vWF antibody staining with Cy3 (d), STRO-1 staining with fluorescein (e), combined images of (d), (e) are shown in (f) with a DAPI nuclear counterstain stain vWF antibody staining with Cy3 detection (f). NIRS and X63 controls showed low or no background staining (data not shown). Images are 400× magnification and the scale bar is 50μm. Arrowheads in (c) and (f) show representative staining of STRO-1 in cells within the vascular region.