![]() (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. Images were acquired with a standard microscope (Nikon) with a 20× objective. Fibroblasts surrounding human iPS colonies serve as internal negative controls for immunohistochemistry staining. 4,6-Diamidino-2-phenylindole (DAPI) staining indicates the total cell content per field (G,J,M). (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). All images were acquired with a standard microscope (Nikon, Tokyo, Japan) with a 10 × objective. (B) Morphology of the CD34 + blood cells. (A) Schematic drawing representing the strategy used in this study for reprogramming human CD34 + cells from the mobilized peripheral blood. Reprogramming of human blood CD34 + cells to pluripotent iPS cells. In total, we picked and expanded 8 independent Gfp − colonies and fully characterized 2 lines, CD34 iPS1 and CD34 iPS2. Given that the retroviral transgenes contain a Gfp marker, transduced cells were initially Gfp +, but as described previously, 4 we observed silencing of Gfp in the successfully reprogrammed colonies ( Figure S2). From 5 × 10 4 CD34 + cells, we routinely observed approximately 5 to 10 hES cell-like colonies (data from 3 independent experiments). We first detected colonies approximately 14 days after transduction most developed into granulated cell clusters that did not have hES cell properties ( Figure 1C), whereas others exhibited distinct flat and compact morphology with clear-cut round edges characteristics of hES cells ( Figure 1D). Human ES (hES) cell medium supplemented with 10 ng/mL basic fibroblast growth factor was added on day 5 ( Figure 1A). Three days after transduction, cells were harvested and plated onto feeder MEF cells ( Figure 1A). 13 Therefore, we performed viral transduction of the CD34 + cells on day 4 of culture, when the majority of the cells were still expressing CD34 and were actively proliferating ( Figure S1). The presence of a high proportion of differentiated or mature cells has a strong suppressive effect on the expansion capacity of hematopoietic progenitors. Analysis by flow cytometry over the course of the 6-day culture period revealed a progressive decrease in the percentage of CD34 +/CD38 − hematopoietic progenitor cells and a simultaneous increase in the percentage of cells with differentiated phenotypes (CD14, CD15 Figure S1B,C). 11 Because the integration and expression of retroviral constructs require mitotic division of the target cells, we first cultured CD34 + cells in vitro with a combination of hSCF, hFlt3L, and IL-3 cytokines, 12 which resulted in proliferation and expansion of CD34 + cells by several orders of magnitude ( Figures 1A,B, S1A). To avoid risks associated with bone marrow harvest, 10 we obtained mobilized CD34 + hematopoietic stem/progenitor cells isolated from peripheral blood. 8 We therefore chose to reprogram human blood progenitor cells. MethodsĪ recent attempt to reprogram mouse B cells reported that terminally differentiated B cells were more refractory to reprogramming than progenitor B cells. The ability to reprogram cells from the human blood will facilitate the development of a reliable method to generate patient-specific stem cells. Reprogramming from human blood cells represents a novel way of establishing iPS cells from donor cells that require little manipulation time in culture. The CD34 +-derived iPS cells are molecularly and functionally indistinguishable from human embryonic stem (ES) cells. In the present study, we show that mobilized human CD34 + peripheral blood cells are amenable to direct reprogramming. Thus, we set out to determine whether iPS cells could be derived from primary, nontransgenic human hematopoietic cells. 8 However, in the study, “secondary” iPS cells were derived from primary B cells of adult spleen, bone marrow, lymph nodes, or embryonic liver of mice engineered to carry doxycycline-inducible Oct4, Sox2, Klf4, and Myc retroviruses in every tissue. The report of reprogramming of murine B cells to pluripotency establishes that somatic cells of the hematopoietic lineage are receptive to reprogramming into iPS cells.
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