开发，扩展，<em>在体内</em>从人类胚胎干细胞（hESCs）监测人类NK细胞和诱导多能干细胞（iPS细胞）
Summary
本协议描述的发展，扩张和来自人类胚胎干细胞和iPS细胞的NK细胞在体内成像。
Abstract
我们提出一个方法推导自然杀伤（NK）细胞未分化的人类胚胎干细胞和iPS细胞使用馈线的方法。此方法产生高水平的NK细胞培养4周后，可以进行进一步的人工抗原呈递细胞的2 – 日志扩展。在本系统中开发的人类胚胎干细胞和iPS细胞来源的NK细胞有成熟的表型和功能。基因修改的NK细胞的大量生产适用于两个基本机理以及抗肿瘤的研究。萤火虫荧光素酶在人类胚胎干细胞衍生的NK细胞可表达一种非侵入性的方法，遵循NK细胞植入，分布和功能。我们还描述了一种双重成像计划，允许独立的监控两种不同的细胞群，以更清楚地描述他们在体内的相互作用。推导，膨胀， 在体内成像和双此方法提供了可靠的制造NK细胞的方法，其EVALU这是必要的，以改善当前NK细胞过继疗法通报BULLETIN。
Introduction
是未分化的人类胚胎干细胞（hESCs）和诱导多能干细胞（iPS细胞），多能干细胞能无限的自我更新和多谱系分化。人胚胎干细胞已被成功地分化为成熟和功能的子集的每一个胚层，包括细胞的造血系统1-3。自然杀伤（NK）细胞是先天免疫系统的淋巴细胞，可以是来自于人类胚胎干细胞形成胚状体（胚体）4,5或共培养基质细胞系1,2,6-8。 NK细胞具有抗病毒和抗肿瘤能力，有可能可以有效对抗广泛的恶性肿瘤，因为它们不执行需要事先抗原刺激的效应子功能。因此，NK细胞的人类胚胎干细胞衍生的是一个有吸引力的免疫细胞来源。此外，推导出人类胚胎干细胞的NK细胞基因提供了一个适合的系统来研究正常发展<EM>的体外。
因为它们提供了基因听话的系统，人类胚胎干细胞来源的NK细胞可以通过实验修饰以表达荧光和生物发光记者提供一个最佳的模型来研究在体外和体内的 NK细胞的效应子功能。人类胚胎干细胞衍生的NK细胞具有活性，对一系列目标，包括艾滋病毒，白血病（K562）和其他类型的癌症7,8。然而，能够有效地获得足够的NK细胞能够治疗患者仍然是一个重要的障碍的临床翻译是，在较小的程度，广泛的临床前体内研究的NK细胞的发育和抗癌功能的限制。在这里，我们使用一个自旋EB的方法来获得4,5,10从人类胚胎干细胞的造血祖细胞。之后11天的胚体的自旋转移到NK细胞培养28天，带或不带供料器。 NK细胞培养基中，4周后，NK细胞被转修饰以表达膜结合的白细胞介素21（IL-21），作为人工抗原呈递细胞（AAPCS）与K562细胞共培养sferred。适应扩张外周血NK细胞，使用这些人工装甲运兵车11,12的协议，我们能够扩大NK细胞日志，同时保留一个成熟的表型和细胞毒性的能力。
这个过程的发展和扩张提供了足够的人类胚胎干细胞衍生的NK细胞在体内表征广泛。对于体内研究，我们能够以非侵入性监测长期植入，注入萤火虫荧光素酶的动力学表达（涨落+），人类胚胎干细胞来源的NK细胞，利用生物发光成像。此外，我们都能够按照使用双，生物发光或荧光成像计划的与肿瘤细胞的NK细胞间的相互作用。我们先前的研究组利用生物发光成像的抗肿瘤模型遵循肿瘤进展和CLEarance涨落+ K562细胞体内 7。现在，我们的人类胚胎干细胞表达萤火虫荧光素酶13,14工程，我们可以遵循的分布和贩运NK细胞对K562肿瘤细胞表达最近特征的荧光蛋白，turboFP650 15。我们选择了这个双报告系统，以同时跟随两个淋巴细胞的增殖（ 图1）的。大多数双成像机型已经双荧光素酶系统，但这些系统可以在技术上具有挑战性，由于腔肠素的交付要求，表达最海肾和Gaussia的荧光素酶记者16-18所需的基板。荧光报告在体外的许多细胞系和构造允许简单的监控，但由于组织和皮毛的自体荧光和许多共同的发射光谱之间的重叠部分在体内成像了有限的成功mmonly使用荧光记者，包括红色荧光蛋白，绿色荧光蛋白，和TdTomato 15,19。这种担忧鼓励发展的远红荧光蛋白，它可以更好的组织渗透率和较高的特定信号比背景15,19。 TurboFP650，在本系统中所示的荧光蛋白，远红移，克服了许多与成像的荧光蛋白在活的动物所涉及的问题。
这种发展和扩大是来自于人类胚胎干细胞的NK细胞的方法，使我们能够进一步表征人类胚胎干细胞来源的NK细胞， 在体外和体内 ，这是必要的，以便更好地了解NK细胞功能和临床上重要的，以改善电流NK细胞过继疗法。它也适合于iPS细胞衍生的NK细胞的推导和扩展。双荧光和生物发光成像方案广泛适用于系统以外的抗肿瘤模型中，我们已经在这里显示。
Protocol
Representative Results
Discussion
人类胚胎干细胞是一个理想的平台，研究不同的细胞类型，并保持卓越的潜力为临床翻译。我们使用一个定义，自旋EB的方法，对造血祖细胞分化的人类胚胎干细胞/ iPS细胞。的自旋EB的方法已经取得了一致的推导造血祖细胞和NK细胞的分化，变化仍然存在跨细胞系的分化效率，并可能需要进行修改，以生成其他的造血细胞谱系。虽然比较结果可以得到从其他EB或基质培养方法的推导，这个系统是无…
Disclosures
The authors have nothing to disclose.
Acknowledgements
笔者想感谢梅林达·Hexum为我们的实验室内旋EB协议开始。我们想感谢实验室的其他成员，包括劳拉E. Bendzick，迈克尔Lepley，和他们这项工作的技术援助的振亚镍。作者还要感谢布拉德·泰勒在他的专家技术咨询卡尺生命科学。
Materials
| Name of Reagent | Company | Catalog Number | Comments |
| Materials | |||
| Media | |||
| BPEL media for Spin EB generation | |||
| Iscove’s Modified Dulbecco’s Medium (IMDM) | Fisher Scientific | SH3022801 | |
| F-12 Nutrient Mixture w/ Glutamax I | Invitrogen | 31765035 | |
| Bovine Serum Albumin (BSA) | Sigma-Aldrich | A3311 | Commercially available BSA can be cytotoxic to ES cells. Deionizing the solution can reduce the potential for cytotoxicity. |
| Poly(vinyl alcohol) | Sigma-Aldrich | P8136 | |
| Linoleic Acid | Sigma-Aldrich | L1012 | |
| Linolenic Acid | Sigma-Aldrich | L2376 | |
| Synthechol 500X solution | Sigma-Aldrich | S5442 | |
| a-monothioglycerol (a-MTG) | Sigma-Aldrich | S5442 | |
| Protein-free hybridoma mix II | Invitrogen | 12040077 | |
| Ascorbic Acid 2-phosphate | Sigma-Aldrich | A8960 | |
| Glutamax I | Invitrogen | 35050061 | |
| Insulin, Transferrin, Selenium 100X solution (ITS) | Invitrogen | 41400-045 | |
| Pen-Strep | Invitrogen | 15140122 | |
| NK Differentiation Media | |||
| Dubecco’s Modified Eagle Medium (DMEM) | Invitrogen | 11965-118 | |
| F-12 Media | Invitrogen | CX30315 | |
| 15% Human AB serum | Valley Biomed | HP1022HI | |
| 5 ng/ml Sodium Selenite | Sigma-Aldrich | S5261 | |
| 50 uM ethanolamine | Sigma-Aldrich | E9508 | |
| 20 ng/ml ascorbic acid | Sigma-Aldrich | A8960 | |
| 25 uM 2-mercaptoethanol (BME) | Gibco | 21985 | |
| 2 mM L-glutamine | Gibco | CX30310 | |
| 1% Pen-Strep | Invitrogen | 15140122 | |
| Cytokines | |||
| (all cytokines used fresh from frozen aliquots) | |||
| SCF | PeproTech, Inc. | 300-07 | 40 ng/ml in BPEL media; 20 ng/ml in NK medium). As noted by the Elefanty protocol, and as we discovered with a bad lot of BMP4, there can be lot differences with cytokines in regards to hematopoietic differentiation.Especially if buying cytokines in bulk, obtain a sample of the lot to test prior to purchase. Compare head-to-head with old lot. |
| rhBMP-4 | R &D Systems | 314-BP | 20 ng/ml in BPEL media |
| rhVEGF | R&D Systems | 293-VE | 20 ng/ml in BPEL media |
| IL-2 | PeproTech, Inc. | 200-02 | 1 x 105 U/ml given to mice after NK cell injection; 50 U/ml used in NK cell expansion protocol |
| IL-15 | PeproTech, Inc. | 200-15 | 10 ng/ml given to mice after NK cell injection (first 7 days only) |
| IL-3 | PeproTech, Inc. | 200-03 | 5 ng/ml in NK media |
| IL-7 | PeproTech, Inc. | 200-07 | 20 ng/ml in NK media |
| Flt-3-Ligand | PeproTech, Inc. | 300-19 | 10 ng/ml in NK media |
| In vivo Imaging | |||
| D-Luciferin Sodium Salt | Gold BioTechnology | Lucna-500 | |
| TurboFP650 plasmid | Evrogen, Moscow, Russia | FP731 | Subcloned into a Sleeping Beauty transposon based plasmid driven by the mCAGs promoter. Cells were then sorted on their expression of turboFP650 by FACS. Cells and plasmids can be obtained from our lab. |
| Equipment | |||
| IVIS Spectrum Imaging System | Caliper Life Sciences | ||
| Cells | |||
| Membrane bound IL-21 expressing artificial antigen presenting cells | MD Anderson, Houston, TX | contact: Dean A. Lee. http://www.jove.com/video/2540/expansion-purification-functional-assessment-human-peripheral-blood | |
| Firefly luciferase expressing hESCs | University of Minnesota, Minneapolis, MN | contact: Dan S. Kaufman . H9 cells modified with a Sleeping Beauty transposon based method (references 13 and 14). Expression of firefly luciferase is driven by the mCAGGS promoter. Following the firefly luciferase gene is an IRES element at the 5′ end of a GFP:zeocin fusion construct. | |
| TurboFP650 expressing K562 cells | University of Minnesota, Minneapolis, MN | contact: Dan S. Kaufman. Description under plasmid comments section |
Materials Table.
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