西亚试剂优势供应上万种化学试剂产品，欢迎各位新老客户咨询、选购！
中国
联系我们
联系方式：400-990-3999 / 邮箱：sales@xiyashiji.com
西亚试剂 —— 品质可靠，值得信赖
The direct reprogramming of somatic cells to produce induced pluripotent stem cells (iPSCs) is a prominent recent advance in stem cell biology (Takahashi and Yamanaka, 2006). Generation of iPSCs without genomic integration of extrinsic genes is highly desirable. Initially, human dermal fibroblasts were used to derive human iPSCs (hiPSCs) (Takahashi et al., 2007,Yu et al., 2007). However, recent studies have shown that other human somatic stem cells can be used (Aasen et al., 2008,Eminli et al., 2009,Kim et al., 2009,Ye et al., 2009). It is difficult to obtain human somatic stem cells, but human terminally differentiated circulating T cells (hTDCTCs) are readily available from peripheral blood. Here, we show that a combination of activated T cell cultivation and a temperature-sensitive mutated Sendai virus (SeV) that encodes human OCT3/4, SOX2, KLF4, and c-MYC allows the generation of hiPSCs easily, efficiently, and safely within a 1 month time frame.
Sampling of peripheral blood is one of the least invasive procedures performed routinely in clinics, and surplus peripheral blood samples are often left unused after clinical examinations. Among peripheral blood mononuclear cells (PBMCs), T cells can be readily cultured in vitro by means of a plate-bound anti-CD3 monoclonal antibody and recombinant (r)IL-2 (Desai-Mehta et al., 1996), and we used such an approach to expand hTDCTCs from peripheral blood samples. From 1 ml of whole blood, PBMCs were separated on a Ficoll gradient and then cultured with plate-bound anti-CD3 monoclonal antibody and rIL-2 (Figure 1A). Although PBMC fractions contain lymphocytes and monocytes, T cells are selectively cultured under these conditions. In culture, the number of activated T cells increased gradually but consistently. Five days after blood sampling, the cultured cells were morphologically identical to pure CD3-positive T cells collected by fluorescence-activated cell sorting (FACS) (Figure 1B). We used a whole-PBMC culture method because it is technically simpler than FACS, in which the sorted cells are frequently damaged by laser emission and the process of single-cell sorting.
