集成的无人类诱导多能干细胞的生成方法头发衍生角质形成细胞
Summary
This manuscript provides a step-by-step procedure for the derivation and maintenance of human keratinocytes from plucked hair and subsequent generation of integration-free human induced pluripotent stem cells (hiPSCs) by episomal vectors.
Abstract
Recent advances in reprogramming allow us to turn somatic cells into human induced pluripotent stem cells (hiPSCs). Disease modeling using patient-specific hiPSCs allows the study of the underlying mechanism for pathogenesis, also providing a platform for the development of in vitro drug screening and gene therapy to improve treatment options. The promising potential of hiPSCs for regenerative medicine is also evident from the increasing number of publications (>7000) on iPSCs in recent years. Various cell types from distinct lineages have been successfully used for hiPSC generation, including skin fibroblasts, hematopoietic cells and epidermal keratinocytes. While skin biopsies and blood collection are routinely performed in many labs as a source of somatic cells for the generation of hiPSCs, the collection and subsequent derivation of hair keratinocytes are less commonly used. Hair-derived keratinocytes represent a non-invasive approach to obtain cell samples from patients. Here we outline a simple non-invasive method for the derivation of keratinocytes from plucked hair. We also provide instructions for maintenance of keratinocytes and subsequent reprogramming to generate integration-free hiPSC using episomal vectors.
Introduction
人类诱导多能干细胞（人iPS细胞）的发现已经彻底改变了再生医学的领域中，提供用于产生患者特异性干细胞1-3的一种可行的方法。人iPS细胞已经成功地从不同的体细胞类型，包括成纤维细胞4,5，造血细胞6,7，从尿8肾小管上皮细胞和角质形成细胞9,10产生的。迄今为止，皮肤成纤维细胞和造血细胞代表了最常用的细胞来源，用于产生病人的具体的iPSC。可以说，这是由于这样的事实，即皮肤活检和采血是常规的医疗程序，并已在许多国家建立大型生物库患者血液或皮肤的样品。
相较于血细胞和皮肤成纤维需要侵入性的提取方法，角质代表一个方便的细胞类型的hiPSC产生。 KeratinocytES是形成皮肤的外表皮屏障，并且还发现，在指甲和头发角蛋白11的富上皮细胞。特别是，角朊细胞可以在毛囊，即覆盖毛干连同内根鞘（IRS）的细胞（12， 图1）的外部的蜂窝层的外根鞘（ORS）中找到。由于毛发收集是一个简单的过程，不需要医务人员的协助下，它提供了一个机会，让患者收集并发送自己的头发样品的实验室，这将极大地方便了hiPSC代病人样本的采集。表皮角化细胞也有较高的重编程效率和更快的重编程动力学相比的成纤维细胞，加入到用角质形成细胞作为起始细胞hiPSC代9,13的优点。此外，人iPS细胞还可以使用毛囊内其他细胞群产生，包括位于毛囊14,15的底部的毛乳头细胞。
IPSC的代发使用来源的细胞以前的报告中经常使用逆转录病毒或慢病毒为基础的重编程方法9,14,15。然而，这些病毒方法的重新编程过程中引进国外转基因不良基因组整合。相比较而言，使用游离型载体的代表一个可行的，非病毒重新编程方法来生成积分-自由iPSCs的4。我们以前曾开发出一种简单，经济有效和非病毒的方法来有效地重新编程角化细胞到使用附加型载体13人iPS细胞。在这里，我们提供的角质细胞衍生的人iPS细胞，包括来自弹拨头发，扩展和维护的角质形成和随后的重新编程，以产生iPS细胞的角质形成细胞的推导的生成的详细协议。
Protocol
Representative Results
Discussion
患者特异性人iPS细胞的产生提供了用于在体外研究发病在患病细胞类型的独特方法，并且还提供了用于药物筛选平台，以确定新的分子，可以拯救疾病表型。使用人iPS细胞这种疾病建模方法已取得了令人鼓舞的结果，适用于各种疾病，包括长QT综合征，亨廷顿氏病，帕金森氏病和肌萎缩性侧索硬化22。若干举措已经展开，建立以病人特异性iPS细胞，其中包括在美国，欧洲，澳大利亚?…
Declarações
The authors have nothing to disclose.
Acknowledgements
The authors wish to thank Harene Ranjithakumaran and Stacey Jackson for technical support. This work was supported in part by grants from the National Health and Medical Research Council (R.C.B. Wong, A. Pébay), the University of Melbourne (R.C.B. Wong), Retina Australia (R.C.B. Wong, S.S.C. Hung, A. Pébay) and the Ophthalmic Research Institute of Australia (R.C.B. Wong, S.S.C. Hung, A. Pébay); Australian Research Council Future Fellowship (A. Pébay, FT140100047), Cranbourne Foundation Fellowship (R.C.B. Wong); intramural funding from the National Institutes for Health (R.C.B. Wong, S.S.C. Hung) and operational infrastructure support from the Victorian Government.
Materials
| Antibiotic Mix: | |||
| 250 ng/ml Antimycotic amphotericin B | Sigma | A2942-20ml | Antibiotic mix is made up in PBS. |
| 1X Penicillin/Streptomycin | Invitrogen | 15140-122 | |
| PBS (-) | Invitrogen | 14190-144 | |
| Knockout Serum Replacement (KSR) medium: | KSR medium is filtered using Stericup (Millipore, #SCGPU05RE) before use. bFGF is added fresh to the media before use. | ||
| 20% knockout serum replacement (KSR) | Invitrogen | 10828-028 | |
| DMEM/F12 with glutamax | Invitrogen | 10565-042 | |
| 1× MEM non-essential amino acid | Invitrogen | 11140-050 | |
| 0.5× Penicillin/Streptomycin | Invitrogen | 15140-122 | |
| 0.1 mM β-mercaptoethanol | Invitrogen | 21985 | |
| bFGF (10 ng/ml, added fresh) | Millipore | GF003 | |
| Keratinocyte medium: | |||
| EpiLife with 60 µM Calcium | Invitrogen | M-EPI-500-CA | |
| 1× Human keratinocyte growth supplement (HKGS) | Invitrogen | S-001-5 | |
| Fetal Bovine Serum (FBS) medium: | FBS medium is filtered using Stericup (Millipore, #SCGPU05RE) before use. | ||
| 10% fetal bovine serum (FBS) | Invitrogen | 26140079 | |
| DMEM | Invitrogen | 11995-073 | |
| 0.5× Penicillin/Streptomycin | Invitrogen | 15140-122 | |
| 2 mM L-glutamine | Invitrogen | 25030 | |
| 0.25% trypsin-EDTA | Invitrogen | 25200-056 | |
| Extracellular Matrix (ECM): | |||
| Matrigel | Corning | 354234 | Aliquot Matrigel stock and store in -80°C following manufacturer’s instructions. Stock concentration of Matrigel varies slightly from batch to batch (~9mg/ml). We recommend to use 200µl matrigel for coating a 12-well plate (~150µg/well). |
| Coating Matrix Kit | Invitrogen | R-011-K | |
| Plasmids: | Note that pCXLE-eGFP is only used for monitoring transfection efficiency and is not required for reprogramming. | ||
| - pCXLE-eGFP | Addgene | 27082 | |
| - pCXLE-hOct3/4-shP53F | Addgene | 27077 | |
| - pCXLE-hSK | Addgene | 27078 | |
| - pCXLE-hUL | Addgene | 27080 | |
| Transfection reagent Fugene HD | Promega | E231B | |
| Gelatin (from porcine skin) | Sigma | G1890 | Make up 0.1% gelatin in distilled water. Autoclave before use. |
| Reduced Serum medium: OPTI-MEM | Invitrogen | 31985062 | |
| Accutase | Sigma | A6964-100ml | |
| Mouse embryonic fibroblast (MEF) feeder | MEF can be inactivated by mitomycin C treatment or irradiation as described previously 16. | ||
| 26G needle | Terumo | NN2613R | |
| 6-well plate (tissue culture treated) | BD Biosciences | 353046 | |
| 12-well plate (tissue culture treated) | BD Biosciences | 353043 | |
| 10 cm dish (tissue culture treated) | BD Biosciences | 353003 | |
| Dispase | Invitrogen | 17105-041 | Use at 10mg/ml |
| Collagenase IV | Invitrogen | 17104-019 | Use at 1mg/ml |
| TRA-160 antibody | Millipore | MAB4360 | Use at 5µg/ml |
| OCT4 antibody | Santa Cruz | SC-5279 | Use at 5µg/ml |
| NANOG antibody | R&D Systems | AF1997 | Use at 10µg/ml |
| MycoAlert Detection kit | Lonza | LT07-418 |
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