Supplementary Figure 1. Vector validation. (A) PCR analysis. Amplification of the vector-borne Oct4 transgene (tgOct4) and endogenous chromosomal Oct4 (eOct4) in nontransfected control HEK293 cells (0) and HEK293 cells transfected with 2-vector combination of the pEP4 E02S CK2M EN2L and pEP4 E02S ET2K plasmids at passages 1 through 5 in serum containing media as indicated. Transfected populations were serially passaged, counted with a haemocytomer at each passage and a portion of each population at each passage was used for DNA isolation, immunostaining and seeding new cultures with defined cell numbers. (B) Immunostaining of Oct4. The first (HEK293:tf+1) and last passage (HEK293:tf+5) of transfected cells showed 5% and 0.5%, respectively, of the cells were immunopositive for Oct4. These findings suggested that episomes were not efficiently replicated and were rapidly lost during population expansion in DMEM15% media.
Immunofluorescence of rosettes and NSP derivatives.
(A). Rosettes. Low magnification image of H9 and iChM5 derived rosettes immunostained as indicated. (B) iChM5Ap4-derived rosettes and NSPs. Dissociated rosettes from candidate colonies were immunostained as indicated. Rosette immunostained for nestin is indicated by asterisk (*). (A,B) Scale bar, in microns as indicated.
Phase images of NSPB6p12 showing early stage differentiation by withdrawal of mitogens in confluent culture in top image. Middle and bottom images show induced differentiation of NSPB6p12 cells and control hVMNSPs, respectively, at day 7. Representative of presumptive axonal extensions are indicated by arrows. Scale bar, in microns as indicated.
Immunostaining of iChM5Ap15 cells and transfected HEK293 cells.
Left panel shows immunostaining of Sox2 in the iChM5Ap15 cultures that are shown in Fig. 3D in the main text. Grayscale insert at 2X magnification shows Sox2 expression in presumptive forming rosette (asterisk), identified by the radial arrangement of cells. Right panel shows HEK293 cells transfected with 2-vector combination of pEP4 E02S CK2M EN2L and pEP4 E02S ET2K plasmids. Grayscale inset at 2X magnification shows Nanog signal at presumptive centrosomes (arrow) that are in the same focal plane. Centrosomes that are out of the focal plane are not visible here. Differential staining for Oct4 (red), Nanog (green) or Oct4 and Nanog (yellow) expression reflects the presence of Oct4 on both vectors and Nanog on one vector. Scale bar, 50 microns.