Generation of Induced Pluripotent Stem Cells from Human Terminally Differentiated Circulating T Cells
Generation of Induced Pluripotent Stem Cells from Human Terminally Differentiated Circulating T Cells
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DOI：
10.1016/j.stem.2010.06.003
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发表时间：
2010-07-02
期刊：
影响因子：
23.9
通讯作者：
Fukuda, Keiichi
中科院分区：
文献类型：
--
作者：
Seki, Tomohisa;Yuasa, Shinsuke;Fukuda, Keiichi
体细胞直接重编程以产生诱导多能干细胞（iPSCs）是干细胞生物学最近的一个突出进展（Takahashi和Yamanaka， 2006）。没有外部基因基因组整合的iPSCs的产生是非常可取的。最初，人类真皮成纤维细胞被用来获得人类iPSCs (hipsc)（Takahashi等人，2007；Yu等人，2007）。然而，最近的研究表明，其他人类体细胞干细胞也可以被使用（Aasen et al., 2008; Eminli et al., 2009; Kim et al., 2009; Ye et al., 2009）。人类体细胞干细胞很难获得，但人类终分化循环T细胞（hTDCTCs）很容易从外周血中获得。在这里，我们展示了激活T细胞培养和温度敏感突变仙台病毒（SeV）的结合，该病毒编码人类OCT3/4、SOX2、KLF4和c-MYC，可以在1个月的时间内轻松、有效和安全地生成hiPSCs。外周血采样是临床常规操作中侵入性最小的程序之一，多余的外周血样本通常在临床检查后未被使用。在外周血单核细胞（PBMCs）中，T细胞可以很容易地通过板结合抗cd3单克隆抗体和重组(r) IL-2在体外培养（Desai-Mehta et al., 1996），我们使用这种方法从外周血样本中扩增hTDCTCs。从1ml全血中，用Ficoll梯度分离PBMCs，然后用板结合抗cd3单克隆抗体和rIL-2培养（图1a）。虽然PBMC部分含有淋巴细胞和单核细胞，但T细胞是在这些条件下选择性培养的。在培养中，活化T细胞的数量逐渐增加，但持续增加。采血5天后，培养的细胞在形态上与荧光活化细胞分选（FACS）收集的纯cd3阳性T细胞相同（图1b）。我们使用了全pbmc培养方法，因为它在技术上比FACS更简单，在FACS中，分选的细胞经常受到激光发射和单细胞分选过程的破坏。为了避免iPSC生成过程中的转基因整合，我们使用了SeV载体，这是一种负链RNA病毒，不会整合到宿主基因组中，对人类也不会致病（Li et al., 2000）。在这些实验中，我们使用温度敏感的突变SeV载体来减少转基因表达和SeV残留。这种形式的SeV载体产生较弱的转基因表达，并且不能在标准培养温度下增殖（数据未显示）。SeV可以有效地转导到人类T细胞中，并且可以表达外源基因（Okano et al., 2003）。我们首先通过SeV以剂量依赖的方式将绿色荧光蛋白（GFP）导入人T细胞；在使用的病毒剂量下，对感染细胞的毒性最小（图1c）。为了从hTDCTCs中生成iPSCs，我们在培养的第6天使用SeV将编码干细胞特异性转录因子（如OCT3/4， SOX2， KLF4和c-MYC）的多个转基因传递到细胞中。基因导入2天后，将细胞复制到SNL细胞的饲养层上。第9天，将细胞转移到含有4 ng/ml bFGF的人胚胎干细胞（ESC）培养基中。在感染3周内，我们在T细胞衍生物中发现了一个类似人类ESCs （hESCs）的集落。第25天，选择与hesc样菌落更大且形态相似的菌落（图1 D）。在结晶紫染色鉴定的这些初始菌落中，大多数碱性磷酸酶(ALP…
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 1 A). 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 1 B). 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. To avoid transgene integration during iPSC generation, we used an SeV vector, which is a minus-strand RNA virus that is not integrated into the host genome and is not pathogenic for humans (Li et al., 2000). We used a temperature-sensitive mutated SeV vector in these experiments to reduce transgene expression and SeV residue in generated lines. This form of SeV vector generates weaker transgene expression and cannot proliferate at standard culture temperatures (data not shown). SeV can be efficiently transduced into human T cells and can express exogenous genes (Okano et al., 2003). We first introduced green fluorescent protein (GFP) into human T cells by SeV in a dose-dependent manner; toxicity for the infected cells was minimal at the virus dosages used (Figure 1 C). To generate iPSCs from hTDCTCs, we used SeV to deliver multiple transgenes that encoded stem cell-specific transcription factors, such as OCT3/4, SOX2, KLF4, and c-MYC, into cells on day 6 of culture. Two days after gene introduction, the cells were replated onto feeder layers of SNL cells. On day 9, the cells were transferred to human ES cell (ESC) medium that contained 4 ng/ml bFGF. Within 3 weeks of infection, we identified a colony that resembled human ESCs (hESCs) among the T cell derivatives. On day 25, colonies that were larger and morphologically similar to hESC-like colonies were picked (Figure 1 D). Of these initial colonies, which were identified by crystal violet staining, most were positive for alkaline phosphatase (ALP …