Figure 4.

Mesodermal differentiation of chorionic mesenchymal stem cells. (A): Adipogenic differentiation, bright light image (Oil Red staining for fat, red). (A1): Control cells. (A2): Cells grown in adipogenic medium. (B): Osteoblast differentiation, bright light image (red staining with alizarin red for calcium precipitates in cells). (B1): Control cells. (B2): Cells grown in osteogenic medium. (C): Capillary-like structures and capillary network formed by chorionic cells grown on Matrigel in standard growth medium. Phase-contrast image. (D): Chorionic mesenchymal stem cells grown in Methocult medium supporting hematopoietic differentiation, staining for nuclei with DAPI (blue). Two distinct cell populations were present: cells attached to the surface formed a monolayer, and cells not attached to the surface grew as loose spherical bodies (arrows). (E, F): Chorionic mesenchymal stem cells grown on media stimulating endothelial differentiation (Matrigel) for 5 days, followed by the addition of Methocult medium for next 7 days. Immunofluorescent images. Two distinct cell populations were present: cells attached to the Matrigel substrate had an endothelial phenotype, and cells not attached to the surface (bodies) showed phenotype of blood or undifferentiated cells. (E): Confocal microscopy z-stack three-dimensional image. Staining for CD45 (phycoerythrin [PE], red) and for CD31 (fluorescein isothiocyanate [FITC], green); costaining for nuclei with DAPI. Surface cells displayed CD31 (endothelial cell marker) or both CD31 and CD45 leukocyte marker (yellow arrow). Cells inside spherical bodies expressed CD45 but not CD31 (red arrow). (F, H): Split confocal images. Upper left: blue channel (nuclei); upper right: green channel; lower left: red channel; lower right: combined image. (F): Staining for CD117 (PE, red) and for CD31 (FITC, green); costaining for nuclei with DAPI (blue). Surface attached cells displayed CD31 (endothelial cell marker, yellow arrow). Cells inside spherical bodies expressed CD117 but not CD31 (red arrow). (G): Embryoid body-like structures, grown on media stimulating endothelial differentiation (Matrigel) for 5 days. Cells inside the embryoid body expressed OCT-4 (green), whereas cells at the outer layer differentiated into endothelial cells (expression of KDR, red). Confocal microscopy z-stack image: immunostaining for KDR (AF663, red) and for OCT-4 (FITC, green); costaining for nuclei with DAPI. (H): Same as (G): image of embryoid body demonstrating expression of KDR on the outer layer of the embryoid body structure and extensive expression of OCT-4 in cells inside the embryoid body structure. Additional split images are shown in supplemental online Figure 4. Abbreviations: DAPI, 4′,6-diamidino-2-phenylindole; KDR, type III receptor tyrosine kinase, also known as vascular endothelial growth factor receptor 2.