Fig. 1.

Generation and characterization of iPS cells. (a) Control (cell line FHB1) and GSS-Y218N-iPS cell (cell line FH10) stained for AP activity. (b) Bisulphite genomic sequencing of the OCT4 and NANOG promoters showing demethylation in FHB1 and FH10 (Y218N) cell lines. (c) RT-qPCR analyses of the expression levels of retroviral-derived reprogramming factors (transgenic) and endogenous expression levels (endogenous) of the indicated genes in FHB1 (two clones) and Y218N-iPS cells (cell line FH10, 2 clones). (d) Low fluorescence photomicrographs of representative colonies of FHB1 and FH10 (Y218N) stained positive for the pluripotency-associated markers OCT4, NANOG and SOX2 (green), SSEA3, TRA-1-81 and SSEA4 (red). (e) Normal karyotypes of FHB1 and FH10 (Y218N) at passage 20. (f) Immunofluorescence analyses of FHB1 and FH10 (Y218N) iPS cells differentiated in vitro show the potential to generate cell derivatives of all three primary germ cell layers including ectoderm (stained for TUJ1, green), endoderm (stained for α-fetoprotein, green, and FOXA2, red) and mesoderm (stained for smooth muscle actin, SMA, red). (g) Direct sequence of genomic DNA from Control (cell line FHB1) and GSS patient (FH10 (Y218N)) identifying the PRNP ^Y218N mutation. Scale bars in a, d and f = 50 μm