Peripheral blood mononuclear cells were prepared within one hour of blood collection and frozen for storage or immediately processed to be transfected with four Yamanaka factors, Oct, Sox2, Klf4, and c-Myc that have been shown to be sufficient for efficient reprogramming. After confirmation of development of human iPSC by karyotyping (data not shown) [22], we characterized these iPSC with immunostaining of sialylated keratan sulfate antigens Tra-1-81 and Tra-1-60, as we previously reported [22]. Since the CytoTune-iPS reprogramming system uses vectors that are non-integrating into the genome, we further confirmed that there was no trace of Sendai viral protein that could be detected by antibody against SeV protein (data not shown). To demonstrate three germ layer differentiation capacities, we tested differentiation in vitro by embryoid body (EB) formation and confirmed the presence of embryonic epitopes (data not shown) using independent antibodies for ectoderm (β III tubulin), mesoderm (smooth muscle actin), and endoderm (α fetoprotein), as we previously reported [22].
