Characterization of insert-free iPS clones derived from fresh blood.

This table represents the passage numbers at which further characterization of a subset of the iPSCs generated across different blood donors were examined. All clones positively stained for the pluripotency marker Tra-1-81. The clones were free of detectable IgH and TCR gene rearrangements (IgH/TCR). A subset of these clones were also screened by PCR using multiple primer sets for loss of the transfected, oriP/EBNA1 DNA including those for EBNA1. The numbers indicate the passage where amplified products are no longer detected (Loss of oriP). The expression of endogenous genes was verified by RT-PCR and included DNMT3B, REX1, TERT, UTF1, Oct4, Sox2, Nanog, Lin28, Klf4, and C-myc (Endogenous Expression). Clones were deemed free of integrated DNA by PCR using primers specific for segments of the transfected DNA encoding Oct4, Sox2, Nanog, Lin28, Klf4, and C-myc (Integration Free). Genetic integrity of the clones was confirmed by cytogenetic analysis at one or more passages where indicated. Teratomas were generated by injecting iPSCs at the indicated passage number into immunodeficient mice. Clones were also assessed by flow cytometry using antibodies specific for cell surface expression of both Tra-1-81 and S SEA-4. n/a indicates tests not performed with the indicated clone. All clones satisfied our minimal criteria for an iPSC by exhibiting a classic ES-like morphology and stained positively for Tra-1-81 and AP.