Primary culture of cAM-MSCs and the evaluation of CPDL & stem cell markers.
(A) Harvesting of canine amniotic membrane tissue. (B) Phase contrast images of cAM-MSCs. Cells were cultured with DMEM (10% FBS). Cultured cells exhibited a fibroblast-like morphology and spindle shape similar to that displayed by human mesenchymal stem cells. Scale bar = 50 µm. (C) Measuring CPDL of cAM-MSCs. CPDL was evaluated with the formula described in the Materials and Methods section. The CPDL was measured from passage 3 to 20. Cells grew consistently until passage 20. (D–G) Quantitative RT-PCR assay for evaluation of stem cell markers: OCT4, SOX2, NANOG and KLF4. We performed all these analyses in triplicate and the mean +/− the standard deviation plotted (*; p<0.5, **; p<0.01, ***; p<0.001).