Figure 1.

Establishment and characterization of human integration-free iPS cell lines and iPSC-derived NCSCs and Schwann cells. (A) Human dermal fibroblasts were reprogrammed with episomal vectors containing Oct4, Sox2, Klf4, and c-Myc genes by using electroporation method. Pluripotent stem cell-like colonies were picked up and expanded to obtain stable iPSC lines. (B) To establish NCSC lines, iPSCs were detached and formed embryoid bodies (EBs) in suspension cultures. EBs were then plated on Matrigel-coating culture plates for up to 2 weeks. Subsequently, cells were dissociated into single cells and cultured as monolayer. To obtain homogeneous NCSC populations, magnetic-activated cell sorting (MACS) were used to select p75 positive cells. Expended p75+ NCSCs were further purified by fluorescence-activated cell sorting (FACS) for HNK-1 positive and SSEA4 negative cells to obtain more homogeneous and stable NCSC line. (C) Established iPSC lines showed typical pluripotent stem cell morphology, positive AP staining, and iPSC markers OCT-4, SSEA4, and TRA-1-60. (D) The iPSC-derived NCSC lines showed positive NCSC markers SOX10, HNK-1, and AP2 and negative iPSC marker SSEA4. (E) In vitro differentiation of iPSC-derived NCSCs into peripheral neural lineages (peripheral neurons, TUJ1; Schwann cells, S100β) and mesenchymal lineages (osteoblasts, Alizarin red; adipocytes, Oil red). Nuclei were stained by Hoechst 33342. Scale bar: 50 μm.