Fig. 3

Xeno-free DiPS cells can be spontaneously differentiated into all three germ layers under in vitro and in vivo conditions. a Three germ layer immunostaining markers of EBs, ectoderm (beta-III tubulin (TUJ1)), mesoderm (smooth muscle actin (SMA)), and endoderm (alpha-fetoprotein (AFP)). b DiPS-derived EBs differentiated to neural rosettes and further morphological changes of adherent neurospheres with cell extensions in xeno-free media. (i) EBs generated from AC_SHED iPS cells in AggreWell™800 plates. (ii) On day 5, EBs were harvested and re-plated on matrix (poly-l-ornithine/laminin)-coated culture plates. (iii) Neural rosette formation occurs from day 9–11. (iv) Neural rosettes formed were selected and re-plated on matrix (poly-l-ornithine/laminin)-coated culture plates on day 12. (v) Attached neural rosette-containing clusters start forming neural progenitor cell outgrowths. c Expression of neural markers Nestin and PAX6 by immunofluorescence for neural progenitor cells differentiated from xeno-free grown DiPS and ES lines. d H&E staining of teratoma derived from AC_SHED iPS cells. The teratoma contains structures of all three germ layers (as shown by the arrows)