Table 1.
Antibodies used for characterizing CMSCs and DMSCs by flow cytometry.
Fig 1.
CMSC phenotypic characterization.
A. (i) Bright field microscopy image of CMSCs at P0. Magnification is 100X and scalebar is 100 μm. (ii) CMSCs from placentae of male newborns were analyzed using interphase FISH on MSC nuclei. CMSCs showed one chromosome X (Spectrum Green) and one chromosome Y (Spectrum Orange) signals. Cell nuclei were stained blue with DAPI. Magnification is 630X. B. Primary CMSCs cell surface markers expression. Histograms of representative primary CMSC at P3 depicting the expression of CD90, CD146, CD166, CD44, CD73, CD105, CD45, HLA-DR, and CD19. The red histogram shows the MSC marker antibody staining while the white histogram shows the corresponding isotype control antibody staining. PE: phycoerythrin dye, APC: allophycocyanin dye, APC-Cy7: allophycocyanin-Cy7 dye. C. Representative photomicrographs showing CMSCs differentiation into mesenchymal lineages. (i) Osteogenic differentiation, Alizarin Red staining in cells after 5weeks growth in osteogenic induction medium. Arrows show calcium depositions. (ii) Adipogenic differentiation, Oil Red O staining in cells after 14 days growth in adipogenic induction medium. Arrows show fat droplets. (iii) Chondrogenic differentiation, Safranin O staining for proteoglycans depositions in cells after 21 days growth in chondrogenic induction medium. Inset shows control uninduced CMSCs. Scalebar is 100 μm.
Fig 2.
DMSC phenotypic characterization.
A. (i) Bright field microscopy image of DMSCs at P0. Magnification is 100X and scalebar is 100 μm. (ii) DMSCs from placentae of male newborns were analyzed using interphase FISH on MSC nuclei. DMSCs showed two X chromosomes (Spectrum Green) signals. Cell nuclei were stained blue with DAPI. Magnification is 630X. B. Primary DMSCs cell surface markers expression. Histograms of representative primary DMSC at P3 depicting the expression of CD90, CD146, CD166, CD44, CD73, CD105, CD45, HLA-DR, and CD19. The red histogram shows the MSC marker antibody staining while the white histogram shows the corresponding isotype control antibody staining. PE: phycoerythrin dye, APC: allophycocyanin dye, APC-Cy7: allophycocyanin-Cy7 dye. C. Representative photomicrographs showing DMSCs differentiation into mesenchymal lineages. (i) Osteogenic differentiation, Alizarin Red staining in cells after 5weeks growth in osteogenic induction medium. Arrows show calcium depositions. (ii) Adipogenic differentiation, Oil Red O staining in cells after 14 days growth in adipogenic induction medium. Arrows show fat droplets. (iii) Chondrogenic differentiation, Safranin O staining for proteoglycans depositions in cells after 21 days growth in chondrogenic induction medium. Inset shows control uninduced DMSCs. Scalebar is 100 μm.
Fig 3.
Histology of CMSCs and DMSCs transplants.
Cross sections are representative of CMSCs transplants (A-B) and DMSCs transplants (E-F) after 8 weeks stained with Haematoxylin and Eosin (H&E). In the transplant, the HA/TCP carrier surfaces (dashed lines) are lined with new bone formation (b), areas of immature bone (ob) together with the surrounding fibrous and hematopoietic tissue (a) and blood vessel (bv). Representative BrdU staining for localization of implanted CMSCs (C-D) and DMSCs (G-H). BrdU-stained implanted cells were found lining the mineralized matrix (black arrows) and surrounding fibrous tissue. Brown nuclear staining is indicative of DAB reactivity. There was no immunoreactivity present in sections stained with isotype-matched antibodies. HA/TCP: hydroxyapatite/tricalcium phosphate particles. Magnification is 100X and scalebar is 500 μm.
Fig 4.
Immunoreactivity of osteogenesis markers after in vivo transplantation of primary CMSCs and DMSCs into immunocompromised mice.
(A-B) BGN: biglycan expression. (C-D) BSP: bone sialoprotein expression. (E-F) OPN: osteopontin expression. (G-H) OCN: osteocalcin expression. (I-J) α-SMA: alpha-smooth muscle actin as negative control. Inset shows representative control sections stained with isotype-matched antibodies. Colour detection was performed using DAB reaction. Magnification is 200X and scalebar is 300 μm. Black arrow show bone-forming surfaces and red arrows show blood vessels.