Generation and characterization of iPSCs from nonmobilized CD34⁺-enriched HSCs isolated from peripheral blood. (A) Flow cytometry dot plot analyses of 1 representative experiment of 8 in which CD34⁺-enriched HSCs were isolated from 20 mL peripheral blood and placed in culture in HSC media for 4 days, but numerical data for all 8 experiments is provided in the legend text below. The resultant cells in the culture were labeled with anti-CD34, anti-CD14, anti-CD3, and anti-CD19 antibodies. The upper 3 panels show the isotype antibody controls for setting the gating used in each analysis indicated in the lower panels. Shown on the horizontal axis of the lower panels is the expression of CD34 by cells in this representative culture. Shown on the vertical axis of the 3 lower panels starting from the left is the expression of CD14, CD3, and CD19, respectively. In this representative culture, approximately 88% of cells are CD34⁺. For each of the experiments from 8 donors, the results were 75%D, 84%D, 91%M, 76%M, 88%M, and 69%M CD34⁺, respectively (where D is Dynal and M is Miltenyi bead isolation). The mean for the group is 80% ± 9% CD34⁺, with less than 1% of the CD34 dim or negative cells expressing any mature lineage markers in any of the 8 experiments. The lack of lineage markers on the remaining cells in the culture indicates that even the CD34⁺ dim and negative cells likely derive from proliferation and early loss of CD34 expression by cells that were CD34⁺ when placed in culture. In this representative experiment, there were no mature monocytes, T lymphocytes, or B lymphocytes detected in the upper left quadrant of the lower panels (CD34 negative/lineage marker strongly positive), and for the entire group of 8 independent experiments, mature monocytes, T lymphocytes, or B lymphocytes represented significantly less than 1% of the population in every case. (B) Microscopy detection of TRA-1-60 and AP pluripotency markers in a representative iPSC line generated by reprogramming of ex vivo cultured nonmobilized CD34⁺ cells derived from peripheral blood. Shown from left to right is a bright-field phase contrast image of a typical iPSC colony and the same colony immune-fluorescent stained for TRA-1-60 or Hoechst nucleus marker as viewed in fluorescence imaging. In the right-most panel is another colony from the same iPSC line stained for AP as viewed in bright-field microscopy. These images show that iPSC colonies exhibit characteristic ESC-like morphology and express pluripotency markers (bar = 500 µm). For bright-field microscopy, TRA-1-60, and Hoescht, we used a Nikon Eclipse Ti microscope (Melville, NY), Nikon Digital Sight Ds-Qi MC camera, NIS Elements BR 3.10 software; ×4 objective/0.13 numerical aperture, and fluorochromes Alexa-555 and Hoechst. For AP, we used a AMG AMS-MV64 LCD viewing microscope/camera (AMG; Bothel, WA) and Micron 2.0.0 software. (C) Fluorescence microscopy imaging detection of germ layer differentiation markers in EBs generated from a representative iPSC line reprogrammed from ex vivo cultured nonmobilized CD34⁺ cells derived from peripheral blood. From the left, respectively, the images show that differentiated cells stain positive (red) for smooth muscle actin (mesoderm), α-fetoprotein (endoderm), or class III β-tubulin (ectoderm). Cell nuclei were stained with Hoechst dye in blue (bar = 100 µm). The same Nikon microscopy/camera/software and fluorochromes described above (B) were used: ×10 objective/0.30 numerical aperture. (D) Bright-field microscopy H&E stain demonstration of the differentiation of all 3 germ layers in teratomas arising in immunodeficient NSG mice injected with a representative iPSC line reprogrammed from ex vivo cultured nonmobilized CD34⁺ cells derived from peripheral blood. From the left, respectively, are sections from the same H&E-stained teratoma containing cartilage (mesoderm), intestinal epithelium (endoderm), and neural tissues (ectoderm). We used a Zeiss Axio Imager.Z1 microscope (Thornwood, NY), a PixeLINK PL-A662 camera with PixeLINK Capture SE software (Ottowa, Ontario), and a ×20 objective/0.75 numerical aperture.