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Developmental Biology

从培养儿童原发性鼻腔上皮细胞和重新编程为诱导多能干细胞

Published: March 10, 2016
doi:
10.3791/53814
, , , ,
1Pyrosequencing Core,Cincinnati Children’s Hospital, 2Division of Developmental Biology,Cincinnati Children’s Hospital, 3Division of Pulmonary Medicine,Seattle Children’s Hospital, 4Division of Asthma Research,Cincinnati Children’s Hospital

Summary

This publication demonstrates methods for successful sampling and culture of nasal epithelial mucosa from children, and reprogramming these cells to induced Pluripotent Stem Cells (iPSCs).

Abstract

Nasal epithelial cells (NECs) are the part of the airways that respond to air pollutants and are the first cells infected with respiratory viruses. They are also involved in many airway diseases through their innate immune response and interaction with immune and airway stromal cells. NECs are of particular interest for studies in children due to their accessibility during clinical visits. Human induced pluripotent stem cells (iPSCs) have been generated from multiple cell types and are a powerful tool for modeling human development and disease, as well as for their potential applications in regenerative medicine. This is the first protocol to lay out methods for successful generation of iPSCs from NECs derived from pediatric participants for research purposes. It describes how to obtain nasal epithelial cells from children, how to generate primary NEC cultures from these samples, and how to reprogram primary NECs into well-characterized iPSCs. Nasal mucosa samples are useful in epidemiological studies related to the effects of air pollution in children, and provide an important tool for studying airway disease. Primary nasal cells and iPSCs derived from them can be a tool for providing unlimited material for patient-specific research in diverse areas of airway epithelial biology, including asthma and COPD research.

Introduction

从人体样本（iPS细胞）诱导多能干细胞是干细胞研究的一个快速发展的技术。他们提供少得多的伦理和道德的缺点1,2胚胎干细胞（胚胎干细胞）研究的替代品。尽管它们不是表观遗传学上相同的人类胚胎干细胞3-5，人​​iPS细胞提供建模发育和疾病的表型的独特方式，并且可以从相关的疾病状态5-8的组织中获得。正在不断探索产生人iPS细胞的新方法，以确定最佳的细胞类型，开始时，作为一种方法来制备适用于移植的GMP质量iPSCs的，并且也增加了重编程过程6,9-11的及时性和效率。

呼吸道上皮细胞在过敏性炎症12的发展是至关重要的，而上皮的过敏反应和哮喘气道重塑的主要驱动力通过与与免疫相互作用e和间质细胞。气道上皮起着起源和肺疾病如哮喘的持久性的重要作用。然而，较低的气道上皮细胞是难以得到在临床环境，特别是从健康对照患者和小孩。从几个研究的数据支持的前提下，从鼻腔粘膜上皮细胞可用于下呼吸道上皮细胞13-20的有效和实际的代理，学习到的空气污染物和过敏原的反应时尤其如此。鼻粘膜由90％以上的纤毛呼吸道上皮细胞和取样这些鼻上皮细胞（NEC的）可以在儿童年仅四岁或五个可以容易地进行的，因为它比其他细胞/组织取样技术微创和是与不良事件，如感染20-23的最小的风险相关联。它提供了一个快速，简单的方法来品尝健康和患病的孩子不长，不必要的，往往是痛苦的支气管镜procedu使得需要镇静资源。先前的研究已经发现，与哮喘的严重程度的疾病亚型可以在两个鼻粘膜以及哮喘的儿童采取支气管细胞样品区分开，并且两个组织类型之间的基因表达在非无所不在基因22约90％的相似24。为iPS细胞的来源，NEC的报价比其他经常使用的细胞类型的优势。成纤维细胞通常用于的iPSC产生，但尽管这些细胞可以很容易地从皮肤活检培养，这个过程通常需要局部麻醉，切开和缝合线，并与感染的一些风险。因此，获得来自患者的知情同意对于这种类型的活检可能是困难的25。一个替代的成纤维细胞是外周血单核细胞（PBMC）。然而，可能难以从小儿患者获得足够的血液的iPSC产生。此外，还有下游AP的局限性褶皱成纤维细胞和血细胞来源的iPS细胞，特别是它们的分化能力，某些细胞类型5,26。因此，考虑到相对可及性和副作用跟随他们收集低风险，NEC的代表来自儿童人群的产生的iPSC一个理想的细胞来源。

iPS细胞已经收到了很多关注最近在研究人类的发展，产生新的疾病模型的平台，并作为细胞的个性化​​疗法的潜在来源。可实现这一技术的全部潜力之前，重编程过程的分子基础需要被阐明，但现在这个协议和内概述的程序将阐明集中在呼吸道暴露的调查研究，以及提供一种用于平台研究涉及的iPSCs个性化医疗的效果。

几个实验室的协同工作导致了generatio不仅取样鼻粘膜，而且还培养NEC的，和重编程这些细胞的iPSC 23成功技术的n个。本文提供了最佳的取样，培养和重新编程条件的协议的轮廓。

Protocol

以下协议遵循机构的人力研究伦理委员会的指导方针。 1.采样鼻粘膜注：从科目谁是免费的呼吸道病毒感染的迹象索取样品。 参加者访问前准备一个15毫升的锥形新鲜，加2ml BEGM（支气管上皮细胞生长培养基）加20微升无菌青​​霉素/链球菌/两性霉素B（P / S / F）（0.01％）。 打开细胞刷只是把样品面前，一定要保持无菌刷，不要触摸它的任…

Representative Results

鼻子的初始部分，称为鼻前庭，是由软骨29包围的区域。刷需要顺利晃过此区域鼻子，超越鼻瓣（口internum，或“黑洞”，在鼻孔的背面看到的），并且是从下鼻甲（ 图1）获得的样品。鼻甲的骨性结构会增加鼻部29的表面积，使他们采样的理想地点。该地区还通过血管粘膜组织，它非常类似于下呼吸道的衬里，也活跃在与环境暴露的免疫反应?…

Discussion

鼻粘膜上皮细胞（长者邻舍中心）是研究气道疾病可访问的平台，NEC-iPS细胞提供了一条康庄大道，探讨疾病的发展，治疗及治疗1,31,32。长者邻舍中心可以轻松无压力或潜在有害程序6,23获得。根据我们的经验，鼻粘膜如在本协议中所描述的采样似乎是更少的压力比采血儿童感知。因此，这种方法可以是在儿科患者人群特别有用的和相关性研究的气道疾病。在NEC的的另一个优点是，…

Disclosures

The authors have nothing to disclose.

Acknowledgements

笔者想承认多能干细胞基金和共焦成像的核心在辛辛那提儿童医院。这项工作是由R21AI119236（HJ），R21AI101375（HJ），NIH / NCATS 8UL1TR000077-04（HJ），U19 AI070412（HJ）和2U19AI70235（GKKH）的支持。

Materials

15mL conical Fisher Scientific 14-959-49D Protocol Step 1.1.
BEGM Lonza CC-3170 Protocol Step 1.1.
Penn/Strep/Fungicide Life Technologies 15240-062 Protocol Step 1.1.
Penn/Strep Life Technologies 15140-122 Protocol Step 4.a.
cytosoft cytology brush Fisher Scientific 22-263-357 Protocol Step 1.2.
trypan blue Fisher Scientific MT-25-900-CI Protocol Step 2.1.
hemacytometer Fisher Scientific 02-671-54 Protocol Step 2.1.
PBS Fisher Scientific BP2438-4 Protocol Step 2.2.
Cytology Funnel Clips Fisher Scientific 10-357 Protocol Step 2.2.
cytospin funnel Fisher Scientific 23-640-320 Protocol Step 2.2.
Cytospin 4 Fisher Scientific A78300003 Protocol Step 2.2.
blank slide Fisher Scientific S95933 Protocol Step 2.2.
hema 3 stain kit Fisher Scientific 22-122-911 Protocol Step 2.2.
Bovine Dermal Colagen, type 1 Life Technologies A1064401 Protocol Step 3.2.
T25 flask Fisher Scientific 08-772-45 Protocol Step 3.3.
Trypsin Lonza CC-5012 Protocol Step 5.2.
Trypsin Neutralizing Solution Lonza CC-5002 Protocol Step 5.2.
Fetal Bovine Serum (FBS), heat sterilized at 65°C for 30min Sigma-Aldrich F2442 Protocol Step 5.5.
Dimethyl sulfoxide Hybri-Max™, sterile-filtered, BioReagent, suitable for hybridoma, ≥99.7% -  Sigma-Aldrich D2650 Protocol Step 5.5.
polycistonic lentivirus* e.g. Millipore SCR511 Protocol Step 6.4. 
A commercial source of reprogramming vector is listed. We routinely use the 4-in-1 plasmid reported by Voelkel et al (PMID: 20385817) to generate VSV-G-pseudotyped polycistronic reprogramming lentivirus in-house. This plasmid can be obtained by contacting 
polybrene Santa Cruz Biotechnology sc-134220 Protocol Step 6.4.
Irradiated CF1 MEFs GlobalStem  GSC-6301G Protocol Step 6.4.
hESC media See recipe included in protocol Protocol Step 6.11.
SB431542 Stemgent 04-0010 Protocol Step 6.11.
PD0325901 Stemgent 04-0006 Protocol Step 6.11.
Thiazovivin  Stemgent 04-0017 Protocol Step 6.11.
hESC-qualified Matrigel BD Biosciences 354277 Protocol Step 6.13.
Corning plate, 6 well Fisher Scientific 08-772-1B Protocol Step 6.13.
mTeSR1 StemCell 5850 Protocol Step 6.13.
250mL disposable filter flask (0.22µm) Fisher SCGP-U02-RE 
dispase StemCell 7923 Protocol Step 7.3.
DMEM/F12 Life Technologies 11320-033 Protocol Step 7.3.
cell lifter Fisher Scientific 08-100-240 Protocol Step 7.4.
hESC Media** Protocol Step 6.11.
components should be mixed and then filter sterilized. Media can be kept at 4°C for up to two weeks. When warming media, do not leave at 37°C longer than 15 min
DMEM-F12 50/50 media Invitrogen  11330-032 Final Concentration
KO replacement serum (KO-SR) Invitrogen 10828-028 0.2
200mM L-glutamine Invitrogen 25030-081 1mM
55mM ß-mercaptoethanol Invitrogen 21985-023 0.1mM
100x non-essential amino acids Invitrogen 11140-050 1x
2ug/mL Basic-Fibroblast Growth Factor (b-FGF) Invitrogen 13256-029 4ng/mL
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Tags

Nasal Epithelial CellsInduced Pluripotent Stem CellsIPSCsAerated DiseaseAsthmaEpigenetic VariationAir PollutionNasal SamplingReprogrammingBEGMCytology BrushLive/dead Cell Stain
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Ulm, A., Mayhew, C. N., Debley, J., Khurana Hershey, G. K., Ji, H. Cultivate Primary Nasal Epithelial Cells from Children and Reprogram into Induced Pluripotent Stem Cells. J. Vis. Exp. (109), e53814, doi:10.3791/53814 (2016).
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