Figure 4

Characterization of sheep OSE cells post SSEA-4 based immunomagnetic cell separation. SSEA-4 positive separated cells comprised of spherical stem cells of two distinct sizes and high nucleo-cytoplasmic ratio were observed in (A) Bright field and after (B) H & E staining. (C) Germ cell nests were also observed with few attached epithelial cells. The cells that did not get selected mainly comprised of (D, E) epithelial cells seen under bright field and after H & E staining respectively. The sorted cells expressed both (F, G) nuclear and cytoplasmic OCT-4, (H) cell surface SSEA-4 and (I) CD133, (J, K) STELLA in both stem cells, (L) NANOG, by confocal microscopy. (M) Negative control was employed by omission of primary antibody. (F-M is a composite of four panels representing fluorescent channels, bright field and merged images of both by confocal microscopy). (N) RT-PCR for Oct-4A, Oct-4 and Gapdh in SSEA-4 positive sorted fractions showed bands of expected size (290, 225 and 232 base pairs respectively using PCR conditions and primers described earlier, [12]). Low intensity bands were also detected in the negative fraction possibly because of the germ cell nests which were observed in both the fractions. Scale bar = 10 μm in A, 20 μm in B-E and 5 μm in F-M.