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CD34+ hematopoietic stem/progenitor cells mobilized into the donor's peripheral blood by pretreatment with granulocyte colony-stimulating factor (G-CSF) can be successfully reprogrammed to pluripotency (Loh et al., 2009). To test whether we can reprogram cells from routine peripheral blood (PB) sources, we obtained CD34+ purified blood samples from a healthy 49-year-old male donor who had undergone simple apheresis without cytokine priming. We also isolated mononuclear cells (PBMCs) from the peripheral blood samples collected by venipuncture of four healthy donors (28- to 49-years-old) via Ficoll density centrifugation.
To induce reprogramming of enriched CD34+ blood cells, we infected with lentiviruses expressing OCT4, SOX2, KLF4, and MYC reprogramming factors (Figure 1A). Colonies with well-defined hESC-like morphology were first observed 21 days after transduction (Figure 1B). For reprogramming of fresh peripheral blood mononuclear cells (PBMCs), we employed two rounds of lentiviral infection (day 0 and day 8) and isolated colonies with distinct flat and compact morphology with clear-cut round edges reminiscent of hESCs after a slightly longer latency of around 35 days (Figure 1C). Interestingly, a previous study with a single round of lentiviral infection of PBMCs failed to observe iPSC colony formation (Haase et al., 2009). In a separate set of experiments, we tested the ability of retroviruses encoding the human reprogramming factors to generate iPSCs from human PBMCs, and despite low infection efficiency, we observed iPSC colonies after 25–35 days (Figure 1D).
