In vitro and in vivo differentiation potential of the CD34 iPS cells. (A) Embryoid body-mediated differentiation of CD34 iPS cells. Differentiation of embryoid bodies (EB) consisting of tight clusters of differentiating cells was observed by day 7 and will cavitate, becoming cystic, by day 10. Images were acquired with a standard microscope (Nikon) with a 10× objective. (B) In vitro-differentiated human CD34 iPS cells demonstrate gene expression from all 3 embryonic germ layers. Semiquantitative RT-PCR performed on undifferentiated (U) and embryoid body-differentiated (D) iPS cells shows up-regulated expression of lineage markers from the 3 embryonic germ layers (endoderm, GATA4 and AFP; mesoderm, RUNX1 and GATA2; and ectoderm, NESTIN and N-CAM). β-ACTIN is shown as a positive amplification and loading control. (C) In vitro–differentiated human CD34 iPS cells demonstrate gene expression of hematopoietic lineage markers. Semiquantitative RT-PCR performed on undifferentiated iPS cells and embryoid bodies differentiated in hematopoietic inducing medium shows up-regulated expression of KDR, BMP4, GATA1, and TIE-2. (D) Embryoid bodies derived from CD34 iPS cells yield myeloid colonies in semisolid methylcellulose media: colony-forming unit-granulocyte (CFU-G), colony-forming unit-macrophage (CFU-M), and colony-forming unit-granulocyte macrophage (CFU-GM). Images were acquired with a standard microscope (Nikon) with a 20× objective. (E) Hematoxylin and eosin staining of teratomas derived from immunodeficient mice injected with human CD34 iPS cells shows tissues representing all 3 embryonic germ layers, including respiratory epithelium (endoderm), bone (mesoderm), and immature neural tissue (ectoderm).