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Bio-ingénierie

MicroRNA 移植成人骨髓造血干细胞促进细胞工程与磁性靶向结合的协议

Published: June 18, 2018
doi:
10.3791/57474
, , , ,
1Reference and Translation Center for Cardiac Stem Cell Therapy (RTC), Department of Cardiac Surgery,Rostock University Medical Center, 2Department Life, Light and Matter of the Interdisciplinary Faculty,Rostock University

Summary

该协议说明了一个安全和有效的程序, 以修改 CD133+造血干细胞。所提出的非病毒, 磁性 polyplex 公司的方法可以为优化治疗干细胞效应, 并通过磁共振成像监测所管理的细胞产品提供了依据。

Abstract

虽然 CD133+造血干细胞 (SCs) 已被证明在再生医学领域提供了很高的潜力, 他们注射到受伤的组织后保持率低, 以及观察到的大量细胞死亡率导致非常限制治疗效果。为了克服这些限制, 我们寻求在其管理之前为合适的细胞工程学建立一个非病毒基础的协议。microRNA (和平号) 加载的磁性 polyplexes 对人体 CD133+表达 SCs 的修饰涉及吸收效率和安全性, 以及细胞的靶向电位。依靠我们的协议, 我们可以达到高和平号的80–90% 吸收率, 而 CD133+干细胞的性质仍然不受影响。此外, 这些修饰的细胞提供了磁性靶向的选择。我们在这里描述一个安全和高效的程序, 以修改 CD133+ SCs。我们期望这种方法提供一个标准的技术, 以优化治疗干细胞效应, 并通过磁共振成像 (MRI) 监测所管理的细胞产品。

Introduction

CD133是一种异种茎和祖细胞种群, 具有良好的再生医学潜力。他们的造血, 内皮和肌分化电位1,2,3使 CD133+细胞,例如, 通过分化促进血管新生过程通过分泌机制4,5,6,7进入新形成的血管和激活支持血管生成信号。

尽管在30多项经批准的临床试验 (ClinicalTrails.gov) 中表现出很高的潜力, 但其治疗结果仍处于争议性讨论4。事实上, 临床应用的 SCs 是阻碍了低滞留在器官的利益和大规模的初始细胞死亡5,8,9。额外的工程 CD133+ SCs 前移植可以帮助克服这些挑战。

有效细胞治疗的一个先决条件是减少大量的初始细胞死亡, 以提高治疗相关细胞的植入10。目前的研究表明, 在高度灌注的器官, 如大脑和心脏在第一 90–99%, 独立于移植细胞类型或应用路线11,12,13 , 巨大的细胞损失。,14,15,16,17,18,19,20,21。SC 标记使用磁性纳米粒子 (MNPs), 使一个创新的无创战略的目标细胞到感兴趣的地点22,23,24,25,26同时允许使用 MRI27和磁性粒子成像 (MPI) 进行细胞监测。最有效的体内研究应用磁化细胞靶向使用的细胞保留在地方管理以后倾向于细胞教导在静脉注射以后23,24,28.因此, 我们的小组设计了一个交付系统由超顺磁性氧化铁纳米粒子29组成。通过这种技术, CD133+ SCs 和人脐静脉内皮细胞 (血管内皮细胞) 可以有效地被靶向, 如在体外尝试30,31证明。

SC 疗法的另一个障碍是被影响的组织的敌对炎症环境在移植以后, 导致最初的细胞死亡32。除几项预适应研究外, 对治疗相关的大鹏湾的应用进行了33项试验;成功地证明, 抗凋亡的大鹏湾抑制体外凋亡, 增强细胞植入在体内33。这些由20–25核苷酸组成的小分子, 扮演信使 rna 转录后水平调制器 (基因) 的关键角色, 从而影响干细胞的命运和行为34。此外, 对大鹏湾的外源引入避免了不理想的稳定集成到宿主基因组34。

目前试图有效地引入核酸 (NAs) 到初级 SCs 主要是基于重组病毒8,35。尽管高转染效率, 重组病毒操作是一个主要的障碍, 对床边的翻译,例如, 无法控制的基因表达, 致病性, 免疫原学, 和插入诱变35 ,36。因此, 非病毒传递系统, 如聚合物基结构是至关重要的发展。其中, 聚乙烯亚胺 (裴) 代表了一个有效的运载工具, 为大鹏湾提供好处, 如 NA 冷凝, 以防止退化, 细胞摄取, 并通过细胞膜逃逸37,38的胞内释放。此外, 和平号配合物在临床试验中表现出高度的生物相容性39。因此, 我们的交付系统包括一个生物素化分支 25 kDa 裴绑定到一个链亲和素涂层的自然人-核心30,31,40。

在这篇手稿中, 我们提出了一个全面的协议, 描述 (i) 人工隔离 CD133+ sc 从人骨髓 (BM) 捐赠与 sc 产品的详细描述和 (ii) 一个有效和温和的转染策略的基于磁性非病毒聚合物的 CD133 遗传工程输送系统的应用.CD133+ SCs 是孤立和磁性丰富的人胸骨 BM 吸出物使用表面抗体为基础的磁性活化细胞分拣 (mac) 系统。然后利用流式细胞仪对细胞活力和细胞纯度进行分析。随后, 和平号/裴/自然人流动综合体已准备就绪, CD133+ SCs 被转染。18 h 转染后, 分析了其吸收效率和转染对 SC 标记表达和细胞活力的影响。此外, 利用四色标记和结构光照显微镜 (SIM) 对转染复合物的胞内分布进行了评价。

Protocol

胸骨人类细胞分离是从知情的捐赠者那里获得的, 他们书面同意使用他们的样本进行研究, 根据《赫尔辛基宣言》。罗斯托克大学的道德委员会批准了这项研究 (注册号: 2010 23, 延长于 2015年)。 1. 细胞制剂 注: 使用肝素钠 (250 毫升) 防止凝固的 bm 检查。 CD133+ SC 隔离 准备所需的解决方案 将996毫升 1x PBS 与4毫升 EDTA (0…

Representative Results

提出的协议描述了人工隔离和磁性丰富的人 BM 衍生 CD133+ SCs 与随后的病毒独立细胞工程战略, 作为一种非侵入性技术的体外细胞操作和在体内监测工具。 这三步隔离技术允许跨国公司从预消化的胸骨 BM 通过密度梯度离心分离。然后, 使用适当的隔离技术, CD133+细胞分数可以被磁性丰富。这允许丰?…

Discussion

近年来, CD133+ SCs 已成为一个有希望的细胞群体的 SC 为基础的治疗, 证明了几个阶段 I, II 和 III 临床试验43,44,45,46,47,48,49,50,51,52,</sup…

Divulgations

The authors have nothing to disclose.

Acknowledgements

这项工作得到了德国联邦教育和研究部 (FKZ 0312138A 和 FKZ 316159)、国家与欧盟结构基金 (ESF/IVWM-B34-0030/10 和 ESF/IVBM-B35-0010/12) 和 DFG (DA1296/2-1) 的联合。德国心脏基础 (F/01/12), BMBF (VIP + 00240) 和潮湿的基础。此外, 卡尔亨利和下午还得到了罗斯托克大学医疗中心的 FORUN 计划 (889001) 的支持。

Materials

7-AAD BD Biosciences 559925
Acetic Acid with Methylene Blue Stemcell Technologies 7060 3%
anti-CD133/2-PE (clone: 293C3) Miltenyi Biotec GmbH 130-090-853
anti-CD34-FITC (clone: AC136) Miltenyi Biotec GmbH 130-081-001
anti-CD45-APC-H7 (clone: 2D1) BD Biosciences 560178
rhodamine dye; Atto 565 dye conjugated to biotin ATTO-TEC GmbH AD 565-71
BD FACS LSRII flow cytometer BD Biosciences
BD FACSDiva Software 6.1.2 BD Biosciences
BSA Sigma-Aldrich GmbH A7906
CD133 antibody-linked superparamagnetic iron dextran particles; CD133 MicroBead Kit Miltenyi Biotec GmbH 130-097-049
collagenase B Roche Diagnostics GmbH 11088831001
counting chamber Paul Marienfeld GmbH & Co. KG
Cyanine 3 dye labelled precursor miR; Cy3 Dye-Labeled Pre-miR Negative Control #1 Ambion AM17120
Cyanine 5 dye miR labelling kit; Cy5 dye Label IT miRNA Labeling Kit Mirus Bio MIR 9650
DNAse I Roche Diagnostics GmbH 10104159001 (100 U/mL)
ELYRA PS.1 LSM 780 confocal microscope Carl Zeiss Jena GmbH
FcR Blocking Reagent, human Miltenyi Biotec GmbH 130-059-901
bright green protein labeling kit; Oregon Green 488 Protein Labeling Kit Thermo Fisher Scientific O10241
aqueous mounting medium; Fluoroshield Sigma-Aldrich GmbH F6182
density gradient centrifugation tube; Leukosep Centrifuge Tube Greiner Bio-One 89048-932
MACS magnet holder; MACS MultiStand Miltenyi Biotec GmbH 130-042-303
MACS pre-separation filter Miltenyi Biotec GmbH 130-041-407 30 µm
MACS separation column (MS / LS) Miltenyi Biotec GmbH 130-042-201 / 130-042-401
MACS permanent magnet; MACS Separator Miltenyi Biotec GmbH 130-042-302
Millex-HV PVDF Filter Merck SLHV013SL 0.45 μm
mouse IgG 2b-PE Miltenyi Biotec GmbH 130-092-215
amine reactive dye; Near-IR LIVE/DEAD Fixable Dead Cell Stain Kit Thermo Fisher Scientific L10119
human lymphocyte separating medium; Pancoll Pan Biotech GmbH P04-60500 density: 1.077 g/mL
PBS Pan Biotech GmbH P04-53500 without Ca and Mg
PEI Sigma-Aldrich GmbH 408727 branched; 25 kDa
Penicillin/Streptomycin Thermo Fisher Scientific 15140122 100 U/mL, 100 μg/mL
PFA Merck Schuchardt OHG 1040051000
unlabelled precursor miR; Pre-miR miRNA Precursor Negative Control #1 Ambion AM17110
RBC lysis buffer eBioscience 00-4333-57
RNAse decontamination solution; RNaseZap Thermo Fisher Scientific AM9780
human lymphocyte medium; Roswell Park Memorial Institute (RPMI) 1640 medium Pan Biotech GmbH P04-16500
recombinant human cytokine supplement; StemSpan CC100 Stemcell Technologies 2690
serum-free haematopoietic cell expansion medium; StemSpan H3000 Stemcell Technologies 9800
Streptavidin MagneSphere Paramagnetic Particles Promega Corporation Z5481
Trypan Blue solution Sigma-Aldrich GmbH T8154 0.4 %
UltraPure EDTA Thermo Fisher Scientific 15575020 0.5 M; pH 8.0
ZEN2011 software Carl Zeiss Jena GmbH
NanoDrop 1000 Spectrophotometer Thermo Fisher Scientific
Sonorex RK 100 SH sonicating water bath Bandelin electronic Ultrasonic nominal output: 80 W; Ultrasonic frequency: 35 kHz
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Tags

MicroRNA TransferAdult Bone Marrow-derived Hematopoietic Stem CellsCell EngineeringMagnetic TargetingRegenerative MedicineHematopoietic Stem Cell ModificationMesenchymal Stem CellsBone Marrow CollectionDensity Gradient CentrifugationMononuclear Cell IsolationMagnetic Selection

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Hausburg, F., Müller, P., Voronina, N., Steinhoff, G., David, R. Protocol for MicroRNA Transfer into Adult Bone Marrow-derived Hematopoietic Stem Cells to Enable Cell Engineering Combined with Magnetic Targeting. J. Vis. Exp. (136), e57474, doi:10.3791/57474 (2018).
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