Summary

和无血清收集无饲养小鼠胚胎干细胞条件培养基的无细胞方法

Published: January 08, 2017
doi:

Summary

这个协议提供了小鼠胚胎干细胞的血清衍生的集合(MESC)-conditioned培养基(MESC-CM)的方法(胎牛血清,FBS) – 和馈线(小鼠胚胎成纤维细胞,MEF中)用于小区-free条件 – 免费的做法。它可以是适用于老化和老化有关的疾病的治疗。

Abstract

The capacity of embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs) to generate various cell types has opened new avenues in the field of regenerative medicine. However, despite their benefits, the tumorigenic potential of ESCs and iPSCs has long been a barrier for clinical applications. Interestingly, it has been shown that ESCs produce several soluble factors that can promote tissue regeneration and delay cellular aging, suggesting that ESCs and iPSCs can also be utilized as a cell-free intervention method. Therefore, the method for harvesting mouse embryonic stem cell (mESC)-conditioned medium (mESC-CM) with minimal contamination of serum components (fetal bovine serum, FBS) and feeder cells (mouse embryonic fibroblasts, MEFs) has been highly demanded. Here, the present study demonstrates an optimized method for the collection of mESC-CM under serum- and feeder-free conditions and for the characterization of mESC-CM using senescence-associated multiple readouts. This protocol will provide a method to collect pure mESC-specific secretory factors without serum and feeder contamination.

Introduction

这个协议的目标是从无血清和无饲养培养条件收集小鼠胚胎干细胞(MESC)-conditioned培养基(MESC-CM)和表征其生物学功能。

在一般情况下,胚胎干细胞(ESC)由于它们的多能性和产能自我更新1-3具有用于再生医学,细胞疗法的巨大潜力。然而,干细胞直接移植有几个限制,如免疫排斥和肿瘤形成4,5。因此,无细胞的方法可提供用于再生医学和老化干预6,7-替代的治疗策略。

衰老被视为蜂窝对口组织器官的老化,其特点是生长停滞,改变细胞的生理和行为的永久状态。老化为流行的疾病,包括癌症,心血管疾病,叔的主要危险因素YPE 2型糖尿病,和神经变性8。一个老龄化的明显特征是组织的再生潜能,这是由干细胞衰老和疲惫9造成的下降。许多显著研究显示药理分子,如雷帕霉素9,白藜芦醇10,和二甲双胍11,和血源性系统性的因素,即GDF11 12中,具有一致延缓衰老和延长寿命的能力。

在本研究中,卓制-CM已经收获无血清(胎牛血清,FBS)和馈线(小鼠胚胎成纤维细胞,MEF中)的层以排除血清因子和由MEF分泌因子的污染。这些条件允许的无血清和无饲养的CM那因此启用的MESC特异性分泌因子的准确识别。

这项拟议的协议效率高,相对成本效益,易于进行操作。该技术提供了见解的MESC衍生的可溶性因子表征可以介导的抗衰老作用,其可用于向干预一个安全的和潜在有利无细胞的治疗方法的开发对于衰老相关疾病和其它再生治疗。

Protocol

注:在无血清和无饲养-CM集合协议的示意图示于图1。 1.材料(MEF中的制备,中,印版和解决方案) 制成500毫升之培养基来培养的MEF。有10%FBS(ESC质量),50单位/ ml青霉素和50mg / ml链霉素补充的Dulbecco氏改良的Eagle氏培养基(DMEM)。 从胚胎以下既定程序协议13 MEF中隔离和维护他们MEF培养基。 制成500毫升之培养基培养的mESCs。的DMEM补充有15%FBS和2mM?…

Representative Results

本来,mESCs保持在与FBS等补品( 图1A和2A)卓制中的MEF饲养。的CM是由在低血清媒体mESCs收集无饲养层,FBS,或其它补充剂( 图1B和2B)。这种文化条件允许我们从给料机,FBS,或其他补充的因素来收集MESC特定条件培养基没有潜在的污染。对照培养基在相同的培养条件下收集,无需mESCs。 <p class="jove_content" fo:keep-together.wit…

Discussion

对于无血清和MESC-CM无饲养成功集合,下面的建议,应考虑。最关键的因素是使用早期通道mESCs为MESC-CM的集合。以前,已经显示,相比于晚期通道mESCs早期传代MESC-CM具有更好的抗衰老作用。 mESCs的通道数目已经报道影响其发育潜力16和多能性17。

而分析MESC分泌蛋白质,从而诱发抗衰老效应的具体因素,需要更多的研究,我们目前可以断定MESC-CM足以在细胞水平降…

Offenlegungen

The authors have nothing to disclose.

Acknowledgements

这项研究是由基础科学研究发展计划(2013R1A1A2060930)和医学研究中心计划(2015R1A5A2009124)通过韩国国家研究基金会(NRF),通过科学,信息和通信技术部提供资金,以及未来规划的支持。这项研究也从病童医院(HK宋)初创工作资助。我们要感谢劳拉·巴维尔和萨拉金JS他们在编辑这个手稿和安德拉斯·纳吉博士提供G4卓制线出色的帮助。

Materials

DMEM Invitrogen #11960-044
FBS Invitrogen #30044333 20%, ES cell quality
Penicillin and streptomycin  Invitrogen #15140 50units/ml penicillin and 50mg/ml strepto
-mycin.
L-glutamine  Invitrogen #25030 2mM
Nonessential amino acids (NEAA)  Invitrogen #11140 100uM
β-mercaptoethanol  Sigma #M3148 100uM
Leukemia inhibitory factor  Millipore #ESG1107 100units/ml
OPTI-MEM Invitrogen #22600
X-gal  Sigma #B4252 1mg/ml
Paraformaldehyde (PFA) Sigma P6148 3.70%
Dimethylformamide (DMF) Sigma #D4551
Potassium ferricyanide  Aldrich #455946 5mM
potassium ferrocyanide  Aldrich #455989 5mM
NaCl  Sigma #S7653 150mM
MgCl Sigma #M2393 2mM
Mytomycin C  Sigma #M4287 10ug/ml
Propidium iodide  Sigma #P4170 50ug/ml
TRIzol Ambion #15596018
M-MLV reverse transcript-tase Promega #M170B
Power SYBR Green PCR master mix  Applied Biosystems #4367659
HDFs, NHDF-Ad-Der-Fibroblast  LONZA #CC-2511
Bottle top filter,  Corning #430513 0.2μm

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Bae, Y., Sung, H., Kim, J. Collection of Serum- and Feeder-free Mouse Embryonic Stem Cell-conditioned Medium for a Cell-free Approach. J. Vis. Exp. (119), e55035, doi:10.3791/55035 (2017).

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