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Wilson PG, Payne T.2014. Genetic reprogramming of human amniotic cells with episomal vectors: Neural rosettes as sentinels in candidate selection for validation assays. PeerJ PrePrints2:e449v1https://doi.org/10.7287/peerj.preprints.449v1
The promise of genetic reprogramming has prompted initiatives to develop banks of induced pluripotent stem cells (iPSCs) from diverse sources. Sentinel assays for pluripotency could maximize available resources for generating iPSCs. Neural rosettes represent a primitive neural tissue that is unique to differentiating PSCs and commonly used to identify derivative neural/stem progenitors. Here, neural rosettes were used as a sentinel assay for pluripotency in selection of candidates to advance to validation assays. Candidate iPSCs were generated from independent populations of amniotic cells with episomal vectors. Phase imaging of living back up cultures showed neural rosettes in 2 of the 5 candidate populations. Rosettes were immunopositive for the Sox1, Sox2, Pax6 and Pax7 transcription factors that govern neural development in the earliest stage of development and for the Isl1/2 and Otx2 transcription factors that are expressed in the dorsal and ventral domains, respectively, of the neural tube in vivo. Dissociation of rosettes produced cultures of differentiation competent neural/stem progenitors that generated immature neurons that were immunopositive for Beta III-tubulin and glia that were immunopositive for GFAP. Subsequent validation assays of selected candidates showed induced expression of endogenous pluripotency genes, epigenetic modification of chromatin and formation of teratomas in immunodeficient mice that contained derivatives of the 3 embryonic germ layers. Validated lines were vector-free and maintained a normal karyotype for more than 60 passages. The credibility of rosette assembly as a sentinel assay for PSCs is supported by coordinate loss of nuclear-localized pluripotency factors Oct4 and Nanog in neural rosettes that emerge spontaneously in cultures of self-renewing validated lines. Taken together, these findings demonstrate value in neural rosettes as sentinels for pluripotency and selection of promising candidates for advance to validation assays.
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.