从生成性肌营养不良患者诱导多能干细胞的：尿源性细胞的有效整合，免费重编程
Summary
This protocol entails detailed procedures for isolation of urine derived cells from muscular dystrophy patients; their efficient and rapid reprogramming through Sendai virus transduction.
Abstract
营养不良性心肌病是肌营养不良症的了解甚少的后果。产生从患者肌营养不良诱导多能干细胞（iPS细胞）可以在体外疾病模型系统的宝贵的蜂窝源和可用于药物的筛选研究。患者来源的尿细胞已被用于在成功的重编程为诱导性多能干细胞，以模拟营养不良的心肌1。寻址整合载体系统的安全问题，我们提出了一个协议使用非整合型仙台病毒载体为山的转导因子成从患者肌营养不良采集的尿液细胞。该协议产生完全重新编程2-3周内克隆。所述多能干细胞是由通道-13载体的无。这些营养不良的iPSC可以分化成心肌细胞和使用，也可以研究疾病的机制或用于药物筛选。
Introduction
心肌病死亡的患者Duchenne型和Becker型肌营养不良症（MD）的第二大原因。虽然在X连锁的抗肌萎缩蛋白基因突变发生在1：3,500男胎，很少有人知道分子和细胞事件，导致进行性心肌损害。从肌营养不良患者衍生的人诱导多能干细胞已成为一种新的工具来研究潜在的疾病的机制和用于药物筛选1,2。
皮肤活检或血液样本的预期可能不适劝阻青少年患者和/或其监护人放弃了参与研究的同意。尿液样本是体细胞是易于进行重新编程的方法的非侵入性的来源。我们最近已表明，从肌营养不良的患者采集的尿液细胞可以是培养的，高效地重新编程成使用逆转录病毒转导的山中因素iPSCs的（的Oct3 / 4，SOX2，Klf4和c-Myc的; OSKM）1。的逆转录病毒基因递送的缺点是随机整合的重新编程的基因到宿主染色体中。为了克服这个限制，我们已经使用了非整合型仙台病毒为尿细胞重编程。
这个协议的细节由肌营养不良患者中分离尿细胞然后可以分化成心肌细胞或其它细胞类型用于进一步研究的仙台病毒重新编程。该协议也可以适用于其他患者特定的疾病。
Protocol
注：患者和/或其监护人应知情同意参与机构审查委员会批准的研究。 1.缓冲区和媒体准备洗涤缓冲液：向制备100毫升的洗涤缓冲液，加入1 ml 100×青霉素/链霉素溶液（链霉素100U / ml青霉素+ 100微克/毫升）的99毫升磷酸盐缓冲盐水（PBS）。 尿祖细胞（UPC）介质：要准备UPC中，混合的角质形成细胞无血清（KSF）中等+祖细胞培养基等体积。 KSF培养基：制备角?…
Representative Results
最祖细胞从人尿中分离的阳性尿路上皮的祖和周细胞标记物如CD44，CD73，和CD146（97.37％，97.09％，和97.3％分别; 图1A和1B）。这些细胞还表达其他间充质标记物如α-平滑肌肌动蛋白和波形蛋白（ 图1B）。 RT-PCR分析提供了细胞中，所述培养物的混合群的证据存在是细胞角蛋白-7的弱表达（CK-7）和Uroplakin（UP）-Ia＆-IIIa，尿路上皮谱系的标记物（ 图1C）。</stro…
Discussion
造型采用iPSCs的心血管疾病已成为了解遗传贡献4-6的常用方法。获得细胞样品从病人的一些未预料到的困难，尤其是幼儿，能够避免通过提供一种非侵入性的方法的选择，如尿液收集。在这个年轻的患者群中，通常难以收集的尿量足够的体积，以产生足够的尿细胞进行重新编程。执教年轻患者饮用液体前尿液收集30分钟，提高了尿细胞分离成功。然而，重复尿收集可能需要在该人群中。我?…
Disclosures
The authors have nothing to disclose.
Acknowledgements
Development of this protocol was supported by the Advancing a Healthier Wisconsin and the National Center for Advancing Translational Sciences, National Institutes of Health, through Grant Number 8UL1TR000055. Its contents are solely the responsibility of the authors and do not necessarily represent the official views of the NIH.
Materials
| List of Materials | |||
| Name of Reagent/Material/Equipment | Company | Catalog Number | Comments |
| 4 Oz. Specimen Cup with Lid | STL Medical Supply | M9AMSAS340 | For Urine Sample Collection |
| 15 mL BD-Falcon Tubes | Fisher Scientific | 352097 | |
| 50 mL BD-Falcon Tubes | Fisher Scientific | 352098 | |
| Round Glass Coverslips | Fisher Scientific | 12-545-81 | |
| CytoTune-iPS Sendai Reprogramming Kit | Life Technologies | A1378-001 | |
| PBS, pH 7.4 | Life Technologies | 10010-023 | |
| Pen-Strep w/o Glutamine | Life Technologies | 15140-122 | Warm in 37 °C water bath before use |
| RPMI Medium 1640 | Life Technologies | 11875-093 | Warm in 37 °C water bath before use |
| B-27 Supplement w/Insulin (50x) | Life Technologies | 17504-044 | Warm in 37 °C water bath before use |
| B-27 Supplement w/o Insulin (50x) | Life Technologies | 0050129SA | Warm in 37 °C water bath before use |
| DMEM/F12 (1:1) | Life Technologies | 11330-032 | Warm in 37 °C water bath before use |
| Versene, 1:5000 | Life Technologies | 15040066 | Warm in 37 °C water bath before use |
| bFGF (10 µg) | Life Technologies | 13256-029 | Warm in 37 °C water bath before use |
| Cell Counter Cartridges | Life Technologies | C10228 | |
| Knockout Serum (500 mL Bottle) | Life Technologies | 10828-028 | Warm in 37 °C water bath before use |
| Recovery Cell Culture Freezing Medium | Life Technologies | 12648-010 | |
| Keratinocyte-SFM (1X), Liquid | Life Technologies | 17005-042 | Warm in 37 °C water bath before use |
| DMEM, High Glucose, Glutamax | Life Technologies | 10566-016 | Warm in 37 °C water bath before use |
| Ham's F-12 Nutrient Mix | Life Technologies | 11765-054 | Warm in 37 °C water bath before use |
| EGF Recombinant Human Protein, Liquid Form | Life Technologies | PHG0311L | Warm in 37 °C water bath before use |
| Insulin, Human Recombinant, Zinc Soln | Life Technologies | 12585-014 | Warm in 37 °C water bath before use |
| TeSR-E8 | Stem Cell Technologies | 5940 | Warm in 37 °C water bath before use |
| ROCK Inhibitor (Y-27632) | Selleck | S1049 | Warm in 37 °C water bath before use |
| Matrigel | BD Biosciences | 354277 | hESC Qualified Matrix, LVED Free |
| RNeasy Mini Kit | Qiagen | 74104 | |
| iScript cDNA Synthesis Kit | Bio-Rad | 170-8890 | |
| DreamTaq Green PCR Master Mix (2X) | Thermo-Scientific | K1081 | |
| FBS-Qualified | Sigma Aldrich | F6178 | Warm in 37 °C water bath before use |
| ADENINE BIOREAGENT | Sigma Aldrich | A2786-5G | Warm in 37 °C water bath before use |
| CHOLERA TOXIN FROM VIBRIO CHOLERAE | Sigma Aldrich | C8052-.5MG | Warm in 37 °C water bath before use |
| HYDROCORTISONE | Sigma Aldrich | H0888-1G | Warm in 37 °C water bath before use |
| HOLO-TRANSFERRIN FROM HUMAN | Sigma Aldrich | T0665-50MG | Warm in 37 °C water bath before use |
| 3,3',5-TRIIODO-L-THYRONINE SODIUM SALT | Sigma Aldrich | T6397-100MG | Warm in 37 °C water bath before use |
| DAPI | Santa Cruz Biotechnology | SC-3598 | |
| Fluoromount Aquous mounting medium | Sigma Aldrich | F4680 | |
| 16% Paraformaldehyde | Alfa-Aesar | 43368 | |
| List of Antibodies | |||
| Name of Reagent/Material/Equipment | Company | Catalog Number | Comments |
| Mouse anti CD44 – labelled with FITC | BD Biosciences | 560977 | |
| Mouse anti CD146 – labelled with PE | BD Biosciences | 561013 | |
| Mouse anti CD73 – labelled with PE | BD Biosciences | 561014 | |
| Mouse anti-α-smooth muscle actin | Sigma Aldrich | A2547 | |
| Mouse anti-Vimentin | Abcam | ab8978-100 | |
| Mouse Anti – TRA-1-81 | Life Technologies | 411100 | |
| Rabbit anti Oct 3/4 | Santa Cruz Biotechnology | SC-9081 | |
| Mouse Anti Dystrophin | Leica Biosystems | NCL-DYSB | |
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Cite This Article
Afzal, M. Z., Strande, J. L. Generation of Induced Pluripotent Stem Cells from Muscular Dystrophy Patients: Efficient Integration-free Reprogramming of Urine Derived Cells. J. Vis. Exp. (95), e52032, doi:10.3791/52032 (2015).