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.