Reprogramming of Notch1-induced acute lymphoblastic leukemia cells into pluripotent stem cells in mice.
Reprogramming of Notch1-induced acute lymphoblastic leukemia cells into pluripotent stem cells in mice.
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Notch1诱导的小鼠急性淋巴细胞白血病细胞重编程为多能干细胞
DOI：
10.1038/bcj.2016.57
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发表时间：
2016-07-08
影响因子：
12.8
通讯作者：
Cheng T
中科院分区：
文献类型：
--
作者：
Zhang H;Cheng H;Wang Y;Zheng Y;Liu Y;Liu K;Xu J;Hao S;Yuan W;Zhao T;Cheng T
体细胞可以利用重编程因子(Oct4、Sox2、Klf4和c-Myc，也称为OSKM)重编程为诱导多能干细胞(IPS)。1细胞重编程和肿瘤发生有许多共同特征。IPS技术在癌症中的应用有助于我们更好地了解癌症发生和发展的机制。因此，确定癌细胞的重新编程潜力将为揭示表观遗传机制和开发癌症的新疗法提供独特的机会。由于癌细胞的重编程效率比一般正常细胞的重编程效率低得多，因此只有少数几种癌细胞被使用iPS技术进行了探索。2到目前为止，在造血系统中，EB病毒(EBV)转化的淋巴母细胞系、人慢性髓系白血病细胞、幼年粒单核细胞白血病细胞和原代小鼠混合系白血病-AF9急性髓系白血病细胞已成功分化为iPS细胞。然而，原发恶性白血病T细胞能否被重新编程为iPS细胞仍然是一个谜。我们首先采用了可以用强力霉素(Dox)诱导OSKM因子的OSKM转基因小鼠，并通过用Notch1绿色荧光蛋白逆转录病毒(图1a)导入OSKM小鼠的谱系阴性(LIN−)骨髓细胞建立了T细胞急性淋巴细胞白血病(T-ALL)小鼠模型。小鼠在2个月内患上白血病(补充图S1a)。垂死的小鼠表现为T-ALL表型(补充图S1B-S1D)。流程图显示了T-ALL单元的重新编程方案(图1b)。分离GFP+白血病细胞，接种于小鼠胚胎成纤维细胞饲养层细胞。在形成小鼠胚胎干细胞(ES)样集落后，提取单个克隆并在饲养层细胞上培养，以产生iPS细胞系(图1c)。总体而言，重新编程效率非常低，仅约为0.005±0.0005%(补充图S1E)。类似于我们之前对急性髓系白血病细胞的研究。4绿色荧光蛋白在已建立的白血病iPS(L IPS)细胞中不表达(图1D)，表明逆转录病毒载体在L iPS细胞中沉默。免疫荧光染色(图1E)和qRT-PCR分析(补充图S2A)证实了多能标记Oct4、Nanog和SSEA-1的表达。基因组聚合酶链式反应显示，所有L-iPS细胞均存在异位Notch1和免疫球蛋白重链重排，证实L-iPS细胞确实来源于T-ALL细胞(补充图S2B和S2C)。此外，L-iPS细胞系以二倍体为主，具有正常的(40，XY)核型(补充图S2D)。为了进一步研究L-iPS细胞的发育潜力，我们对严重联合免疫缺陷小鼠进行了畸胎瘤实验。畸胎瘤显示所有三个胚层(图1f-h)。此外，随机选择L-iPS细胞系进行嵌合评估，两个L-iPS细胞系产生8个出生后嵌合体，嵌合体通过毛色反映(图1I)。值得注意的是，嵌合小鼠在50天内出现复发白血病(补充数字S2ES2H)。因此，我们无法使用嵌合体测试生殖系传播。综上所述，我们成功地建立了T-ALL来源的iPS细胞系，并进一步鉴定了L-iPS细胞的多能性。有趣的是，在重新编程的过程中，我们发现了两个…
Somatic cells can be reprogrammed into induced pluripotent stem (iPS) cells using the reprogramming factors (Oct4, Sox2, Klf4 and c-Myc, also called OSKM). 1 Cellular reprogramming and oncogenesis share many common features. The application of the iPS technology in cancers help us better understand the mechanism underlying the initiation and progression of cancer. Therefore, defining the reprogramming potential of cancer cells would provide unique opportunities to reveal epigenetic mechanisms and develop novel therapeutics for cancer. Because the reprogramming efficiency of cancer cells is paradoxically much lower than that of normal cells in general, only some handful types of cancer cells have been explored using the iPS technology. 2 To date, in the hematopoietic system, Epstein-Barr virus (EBV)-transformed lymphoblastoid cell lines, human chronic myeloid leukemia cells, juvenile myelomonocytic leukemia cells and primary murine mixed lineage leukemia-AF9 acute myeloid leukemia cells have been successfully generated into iPS cells. 3-6 However, whether the primary malignant leukemic T cells can be reprogrammed into the iPS cells is still a mystery. We first employed OSKM transgenic mice in which OSKM factors can be induced by doxycycline (Dox) and established the T-cell acute lymphoblastic leukemia (T-ALL) mouse model by transfecting the Lineage negative (Lin−) bone marrow cells from the OSKM mice with a Notch1-green fluorescent protein (GFP) retrovirus (Figure 1a). The mice developed leukemia within 2 months (Supplementary Figure S1A). The moribund mice exhibited a T-ALL phenotype (Supplementary Figures S1B-S1D). The flowchart shows the reprogramming scheme for the T-ALL cells (Figure 1b). GFP+ leukemia cells were sorted and plated on mouse embryonic fibroblast feeder cells. After the formation of mouse embryonic stem (ES)-like colonies, a single colony was picked up and cultured on feeder cells to produce iPS cell lines (Figure 1c). Overall, the reprogramming efficiency was very low, only approximately 0.005±0.0005%(Supplementary Figure S1E). Similarly to our previous study on acute myeloid leukemia cells. 4 GFP was not expressed in the established leukemia iPS (L-iPS) cells (Figure 1d), indicating that the retroviral vector was silenced in L-iPS cells. The expression of pluripotency markers Oct4, Nanog and SSEA-1 was confirmed by immunofluorescence staining (Figure 1e) and qRT-PCR analyses (Supplementary Figure S2A). Genomic PCR demonstrated the presence of ectopic Notch1 and immunoglobulin heavy chain rearrangement in all tested L-iPS cells, confirming that L-iPS cells were indeed derived from T-ALL cells (Supplementary Figures S2B and S2C). Moreover, the L-iPS cell lines were predominantly diploid with the normal (40, XY) karyotype (Supplementary Figure S2D). To further investigate the developmental potential of the L-iPS cells, we performed the teratoma assay in severe combined immunodeficiency (SCID) mice. The teratomas showed all three germ layers (Figures 1f-h). Furthermore, L-iPS cell lines were randomly selected for chimeric assessment, and two L-iPS cell lines generated eight postnatal chimeras, as chimerism reflected by coat color (Figure 1i). Notably, the chimeric mice developed recurrent leukemia within 50 days (Supplementary Figures S2ES2H). Therefore, we were unable to test germ-line transmission using the chimeras. Taken together, we successfully established T-ALL-derived iPS cell lines even at a very low efficiency and further characterized the pluripotency of the L-iPS cells. Interestingly, during the reprogramming process, we found two …
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Notch抑制允许与癌基因无关的IPS细胞产生。
影响因子：
14.8
作者：
Ichida, Justin K.;Julia, T. C. W.;Williams, Luis A.;Carter, Ava C.;Shi, Yingxiao;Moura, Marcelo T.;Ziller, Michael;Singh, Sean;Amabile, Giovanni;Bock, Christoph;Umezawa, Akihiro;Rubin, Lee L.;Bradner, James E.;Akutsu, Hidenori;Meissner, Alexander;Eggan, Kevin
通讯作者：
Eggan, Kevin
影响因子：
64.5
作者：
Takahashi, Kazutoshi;Yamanaka, Shinya
通讯作者：
Yamanaka, Shinya
影响因子：
20.3
作者：
Choi, Su Mi;Liu, Hua;Jang, Yoon-Young
通讯作者：
Jang, Yoon-Young
影响因子：
21.3
作者：
Soria-Valles, Clara;Osorio, Fernando G.;Lopez-Otin, Carlos
通讯作者：
Lopez-Otin, Carlos
影响因子：
5.2
作者：
Li, Wenlin;Zhou, Hongyan;Abujarour, Ramzey;Zhu, Saiyong;Joo, Jin Young;Lin, Tongxiang;Hao, Ergeng;Schoeler, Hans R.;Hayek, Alberto;Ding, Sheng
通讯作者：
Ding, Sheng