Figure 3
Cellular and molecular properties of MSC cultures established by Protocol 1.
(a) Phase contrast images, on 5 days (P0, D5) and 10 days (P0, D10) after placing the explants. TCV attained spheroid like morphology in culture and the cells are seen migrating out of this spheroid (encircled area) (Supplementary online video S1 and S2). Fetal MSC cultures at passage 5 (P5, D3) are also shown (scale bars, 100 μm). (b) FISH images of migratory cell cultures at day 3 of passage 1 (scale bar, 10 μm). A similar FISH analysis was also done at passage 5 (Supplementary Figure S3). (c) Electropherograms showing the perfectly matching STR profile of the MSC (Passage 1) with the STR profile of the baby (cord blood). The detailed STR profile of the mother, her male baby and the cells isolated from the TCV are given in Supplementary Table S1. A similar STR profile analysis was done for the TCV cultures from a female baby and the results are shown in Supplementary Figure S4 and the corresponding Supplementary Table S2. (d) Immunofluorescence images showing a high CK18 and a low E-cadherin expression (scale bars, 50 μm). Image of the antibody negative control is shown in Supplementary Figure S5. (e) Flow cytometry analysis for MSC specific surface markers (first 6 columns) at passage 5 (mean ± standard deviation, n = 3). Histograms for each marker from a representative experiment are shown in Supplementary Figure S6. A relatively lower expression of MSC marker CD90 (84.8%) is noted here. (f) Tri-lineage differentiation of fetal MSC at passage 5 (scale bar, 100 μm). (g) GTG-banded chromosome analysis of male fetal MSC culture at passage 5 exhibiting a normal 46, XY karyotype. Corresponding metaphase spread is shown in Supplementary Figure S7.
