Summary

从人体外周血单个核细胞融合,无诱导多能干细胞的生成方法游离型载体

Published: January 01, 2017
doi:

Summary

This protocol describes a detailed method for efficient generation of integration-free iPSCs from human adult peripheral blood cells. With the use of four oriP/EBNA-based episomal vectors to express the reprogramming factors, KLF4, MYC, BCL-XL, or OCT4 and SOX2, thousands of iPSC colonies can be obtained from 1 mL of peripheral blood.

Abstract

诱导多能干细胞(iPS细胞)持有疾病建模和再生疗法大有希望。我们以前报道的使用游离型载体(EV)来产生从外周血单核细胞(PB跨国公司)自由集成-iPS细胞。所使用的附加型载体是与来自爱泼斯坦-巴尔(EB)病毒oriP和EBNA1元件,其分别允许在哺乳动物细胞中质粒的复制和长期retainment,掺入的DNA的质粒。随着进一步的优化,数以千计的iPSC集落可以在1毫升的外周血中获得。为实现高的重编程效率的两个关键因素是:1)利用一个2A的“自切割”肽连结OCT4和SOX2,从而实现这两个因素的等摩尔的表达; 2)使用两个向量来表达MYC和KLF4个别。在这里,我们描述了一个一步一步的协议,用于生成免费的集成-IP成人外周血标本旺。生成的iPS细胞是整合无残留游离质粒后5代检测不到。虽然重编程效率比得上仙台病毒(SV)矢量,EV质粒是比市售的SV矢量相当更经济。这个经济实惠的EV重新编程系统适用于在再生医学临床应用潜力,并为PB跨国公司的自由集成-间质干细胞,神经干细胞的直接重编程的方法。

Introduction

几个转录因子的强制表达后 OCT4,SOX2,MYC和KLF4),体细胞可以被重新编程诱导多能干细胞(iPS细胞),其保持在再生医学和细胞替代疗法1-3应用大有希望。迄今为止,多种方法已被开发,以提高重编程4-7的成功率。病毒载体诱导重新编程被广泛地用于有效地产生iPSCs的,因为病毒整合导致高电平时,重新编程因子的稳定表达。然而,该载体DNA导入细胞基因组的永久整合可能诱发插入诱变5。此外,重新编程因子失活不足可能扰乱的iPSC分化8。这样,不重编程因子整合使用的iPSC的是必要的,尤其是对在细胞治疗应用中使用。

<p class="jove_content">附加型载体(电动汽车)被广泛应用于无融合-iPS细胞的产生。最常用的EV是含有两种元素,病毒复制(oriP)和EB核抗原1(EBNA1)的原点,从爱泼斯坦-巴尔(EB)病毒9的质粒。的oriP元件促进在哺乳动物细胞中的质粒复制,而EBNA1元件系绳含oriP-质粒DNA的染色体DNA,其允许所述附加体的宿主细胞的分裂过程中的分区。相较于其他自由整合的办法,包括仙台病毒(SV)和RNA转染,电动汽车具备多种优势5,6,10。作为质粒DNA,电动车可以很容易地生产并在内部修改,使它们非常实惠。此外,电动车进行重新编程是较少的劳动密集的过程,因为与电动汽车的单个转染足以的iPSC产生,而几的RNA转染所必需的成功重新编程。

ðermal成纤维细胞已在许多重编程研究中使用。然而,皮肤活检不仅是一种侵入性和痛苦的过程,而且还费时扩大细胞足够的数量进行重新编程。更令人关注的,成年供体的皮肤细胞经常被暴露于长期紫外光辐射,这可能会导致与肿瘤有关,从而限制了从皮肤成纤维细胞11,12衍生的iPSC应用突变。最近,已经报道,人正常皮肤细胞积累的体细胞突变与多种癌症的基因,包括大多数皮肤鳞状细胞癌的主要驱动力,是在强正选择13。

在对比皮肤成纤维细胞,外周血(PB)的细胞是细胞重新编程的一个优选来源,因为1)血细胞可以通过微创方法来容易地得到,2)的外周血细胞是造血干细胞的后代居住在骨髓,从而防止有害的辐射。外周血单核细胞(PB跨国公司)可以在从血沉棕黄层层以下用Ficoll-Hypaque上(1.077克/ mL)的一个简单的梯度离心一小时进行收集。得到的PB跨国公司是由淋巴细胞,单核细胞和少数造血祖细胞(高性能计算机)14。虽然人类T淋巴细胞是在PB中的主要细胞类型中的一种,成熟T细胞中含有的T细胞受体(TCR)基因重排和缺一个完整的基因组中从而限制其应用程序15,16的潜力。然而,通过iPSC的生成T细胞的复兴可能在嵌合抗原受体(CAR)T细胞疗法17-19潜在的应用。相比较而言,高性能计算机有一个完整的基因组,并很容易重新编程的。虽然只有0.01 – 0.1%,外周循环细胞是高性能计算机,这些细胞可被体外扩增在红细胞中有利于erythr的增殖OID祖细胞14。

在我们以前的研究中,我们所用的因子的BCL-XL在除山因子(OCT4,SOX2,MYC和KLF4),这导致在PB中重编程efficency 20 10倍的增加。 BCL-X L,也称为BCL2L1,是细胞死亡的有效抑制剂,通过抑制胱天蛋白酶21,22的激活。但是,BCL-X L也可以在维持多潜能21,22发挥着重要作用。最近,我们已进一步通过分别表达MYC和KLF4与两个向量,这导致在重新编程的效率23的大致100倍增加优化了EV重新编程系统。使用该方法,重编程效率,由通过在转染起始细胞数除以细胞集落数来定义,是0.2 – 来自健康供体的0.5%。如下,我们描述了从PB生成自由整合,iPSCs的详细的实验步骤。

Protocol

人类所有的PB样品均从当地研究伦理委员会批准可用的识别信息,从天津血液中心的匿名捐赠者的成人获得。 1.远藤无质粒制备使用商业质粒纯化Maxi试剂盒根据制造商的协议从大肠杆菌中提取附加型载体。对于最后一步,用不含内毒素的无菌水代替TE缓冲以溶解DNA沉淀。 使用商业紫外/可见光分光光度计测量DNA浓度。的浓度通常为大于1微克/微升,用A260 / …

Representative Results

使用此协议中,我们可以从1×10 5 nucleofected PB跨国公司获得数百菌落( 图1A和1B)。重编程效率为约0.2 – 0.5%和菌落表达多潜能标志物( 图1C和1D)。使用所描述的协议产生的iPSC是积分-自由和具有形成畸胎瘤构成3胚层( 图1E和1F)的能力。 <img…

Discussion

健康人或病人采集血样方便,无创,使之成为基础研究和临床细胞治疗一个有吸引力的细胞来源。在这里,我们描述了高效代从外周血免费集成为iPS细胞的协议。这种可重复和经济实惠的方法应该受益的iPSC领域。

我们已经报道有负责高效PB重新编程30两个关键因素。一个是OCT4的表达等摩尔和Sox2用2A连接器31。为了实现这一目标,在pEV-OCT4-2A-SOX2在该协议中使用?…

Divulgaciones

The authors have nothing to disclose.

Acknowledgements

This work was supported by the Ministry of Science and Technology of China (2015CB964902, 2013CB966902 and 2012CB966601), the National Natural Science Foundation of China (81500148, 81570164 and 81421002), the Loma Linda University School of Medicine GCAT grant (2015), and Telemedicine and Advanced Technology Research Center (W81XWH-08-1-0697).

Materials

Hematopoietic Stem Cell Expansion Medium Sigma S0192 Store at 4 °C
Human stem cell factor (SCF) Peprotech 300-07 Store at -20 or -80°C
Interleukin-3 (IL-3) Peprotech AF-200-03 Store at -20 or -80°C
Eryrthropoietin (EPO) Peprotech 100-64 Store at -20 or -80°C
Insulin growth factor-1 (IGF-1) Peprotech 100-11 Store at -20 or -80°C
Dexamethasone Sigma D4902 Store at -20 or -80°C
1-thioglycerol (MTG) Sigma M6145 Store at -20 or -80°C
DMEM/F12 medium Gibco 112660-012 Store at 4 °C
L-glutamine (100x) Gibco 25030-081 Store at -20 °C
Penicillin/Streptomycin (100x) Gibco 15140-122 Store at -20 °C
Non-essential amino acids solution (100x) Gibco 11140-050 Store at 4 °C
Fibroblast growth factor 2 (FGF2) Peprotech 100-18B Store at -20 or -80°C
ITS (100x) Gibco 41400-045 Store at 4 °C
Ascorbic acid Sigma 49752 Store at -20 °C
DMEM (high glucose) medium Thermo SH30243.01B Store at 4 °C
FBS Hyclone SV30087.01 Store at -40 °C
Ficoll GE Healthcare, SIGMA 17-5442-02 Store at RT
Trehalose  Sigma T9531 Store at 4 °C
DMSO Sigma D2650 Store at RT, protect from light
Endofree Plasmid Maxi Kit(10) Qiagen 12362 Store at RT
IMDM Gibco 21056-023 Store at 4 °C
Human CD34+ Cell Nucleofection Kit Lonza VPA-1003 Store at RT, nucleofection buffer and supplement buffer should be stored at 4 °C
Sodium butyrate Sigma B5887 Store at -20 or -80°C
ROCK inhibitor Y27632 STEMGENT 04-0012-10 Store at -20 °C
Essential 8 basal medium (E8) Gibco A15169-01 Store at 4 °C, the supplement should be stored at -20 or -80°C
Matrigel BD 354277 Store at -20 or -80°C
2x EasyTaq PCR SuperMix(+dye) TransGen Biotech AS111 Store at -20 °C
Cell detachment solution STEMGENT 01-0006 Store at -20 °C, Accutase as a cell detachment solution to obtain a single cell suspension
DAPI Sigma D9542-1MG Store at 4 °C or -20 °C
Anti-nanog-AF488 BD 560791 Store at 4 °C, primary antibody used for Immunofluorescence, dilute 1/100 when use
Anti-OCT4 abcam ab19857 Store at 4 °C, primary antibody used for Immunofluorescence, dilute 1/100 when use
AF488 donkey anti-mouse IgG Invitrogen A21202 Store at 4 °C, secondary antibody used for Immunofluorescence,  dilute 1/500 when use
PE anti-human TRA-1-60-R Antibody Biolegend 330610 Store at 4 °C, antibody used for flow cytometry
eFluor 570-conjugated anti-SSEA4 eBioscience 41-8843 Store at 4 °C, antibody used for flow cytometry
Isotype antibody eBioscience 11-4011 Store at 4 °C, antibody used for flow cytometry
Alkaline Phosphatase Detection Kit SiDanSai 1102-100 Store at 4 °C
Genomic DNA Extraction Kit TIANGEN DP304-02 Store at RT
Trypan Blue solution Sigma T8154 Store at RT
Flow cytometry cell analyzer BD LSRII for flow cytometry analysis
Spinning disk confocal microscope (SDC) PerkinElmer UltraVIEW VOX for confocal imaging
Nucleofection device Lonza Nucleofector 2b for the nucleofection of PB MNC
ImageQuant LAS-4010 GE take photo of AP staining in bulk

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Wen, W., Zhang, J., Chen, W., Arakaki, C., Li, X., Baylink, D., Botimer, G. D., Xu, J., Yuan, W., Cheng, T., Zhang, X. Generation of Integration-free Induced Pluripotent Stem Cells from Human Peripheral Blood Mononuclear Cells Using Episomal Vectors. J. Vis. Exp. (119), e55091, doi:10.3791/55091 (2017).

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