PMC

Teratoma assay. (A) Haematoxylin/eosin-stained MM40.2-teratoma derived sections. (B) immunostaining with antibodies for ectoderm (βIII-tubulin, green), mesoderm (α-actinin, red) and endoderm(α-feto protein, red) markers.

(A) Schematic representation of the vectors used in this study, not drawn to scale. (B) GFP expression in HUES-2, HUES-10 and HT1080 cells 24 h after transduction with pHGNeo4 (left) and pα40 (right) amplicons.

HAC analysis. (A) FISH with a 17α DNA probe (green), and a vector probe (red). The chromosomes are counterstained in DAPI, blue. The HAC are identified by yellow arrows. The insets show DAPI staining only (HAC, red arrows), in black and white. (B) Fibre FISH on HAC clones, with 17α DNA (green) and vector DNA (red) probes. (C) ImmunoFISH with anti-CENP C antibody (green), and 17α HAC probe (red).

Pluripotency analysis. (A) RT–PCR with primers specific for pluripotency genes at time points 0, 30, 60 and 90 days in culture (0, 30, 60, 90) in the presence (on) or absence (off) of selection. (RT-) control PCR performed in the absence of the reverse transcriptase, using GAPDH primers, (-) no template control. (B) Immunostaining with pluripotency marker antibodies (red). The cells are counterstained with DAPI, blue. (C) RT–PCR analysis of germinal layer transcripts on cDNA from in vitro differentiated MM40.2 and SK40.19 cells, with primers specific for pluripotency (Oct4, Sox2, Nanog), endoderm (αFP, HFN3α, α1AT), mesoderm (GATA-2), epidermis (CK-14, CK-5, HSK) and neuronal markers (Pax6). U: undifferentiated cells, D: differentiated cells. (RT-): control PCR performed in the absence of the reverse transcriptase, using GAPDH primers. (-) no template control. (D) Neuronal differentiated cells stained with anti-βIII tubulin antibody (red).