Reprogramming of human blood CD34⁺ cells to pluripotent iPS cells. (A) Schematic drawing representing the strategy used in this study for reprogramming human CD34⁺ cells from the mobilized peripheral blood. (B) Morphology of the CD34⁺ blood cells. (C) Image of a non-ES cell-like colony. (D) Image of a hES cell-like colony. All images were acquired with a standard microscope (Nikon, Tokyo, Japan) with a 10 × objective. (E-N) Immunohistochemistry of human blood-derived iPS cell colonies expressing markers for Tra-1-81 (E), NANOG (F), OCT4 (H), Tra-1-60 (I), SSEA3 (K), SSEA4 (L), and alkaline phosphatase (AP) (N). 4,6-Diamidino-2-phenylindole (DAPI) staining indicates the total cell content per field (G,J,M). Fibroblasts surrounding human iPS colonies serve as internal negative controls for immunohistochemistry staining. Images were acquired with a standard microscope (Nikon) with a 20× objective. (O) Quantitative RT-PCR analyses for the expression of ES cell-marker genes OCT4, SOX2, NANOG, KLF4, MYC, REX1, and GDF3 in human CD34 iPS and the parental CD34⁺ cells. Individual PCR reactions were normalized against β-ACTIN and plotted relative to the expression level in the parental CD34⁺ cell. (P) Repression of the exogenously introduced transgenes as shown by quantitative RT-PCR analyses of OCT4, SOX2, MYC, and KLF4 expression. Specific primers were designed to probe for either the coding regions (Total) to measure the expression of both the endogenous gene and the transgene, 3′ untranslated region (Endo), which measure the expression of the endogenous gene only, or primers specific (Transgene) to the region of the viral transgenes. β-ACTIN is shown at the bottom as a loading control for each sample. (Q) Bisulfite genomic sequencing of the OCT4 and NANOG promoters reveals demethylation in the iPS cell lines. Each horizontal row of circles represents an individual sequencing reaction for a given amplicon. Open and filled circles represent unmethylated and methylated CpGs dinucleotides, respectively. The cell lines (CD34⁺ and its derivatives CD34 iPS1 and CD34 iPS2) are indicated to the left of each cluster. The values above each column indicate the CpG position analyzed relative to the downstream transcriptional start site.

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).