Enrichment of Motor Neuron-like Cell Precursors Derived from Mouse Embryonic Stem Cells by Density Gradient Centrifugation / 用密度梯度离心法富集源于小鼠胚胎干细胞的运动神经元样细胞前体

摘要/Abstract

摘要：目的：我们的目标是从源于胚胎体(EBs)的小鼠胚胎干细胞(mESCs)中分离运动神经元样细胞前体(MNLCPs),以用于发展针对运动神经元疾病的药物筛选试验和移植疗法。天然Shh蛋白（或Shh通路激动剂）和维甲酸诱导的胚胎体中, MNLCPs和未分化细胞的含量是不确定的。如果不把未分化的细胞从细胞培养中充分去除, 其可能会干涉药物筛选试验或在移植后增生。我们开发了一种以密度梯度离心法为基础的富集MNLCPs的方法。方法：我们用Wichterle等人2008年改进的方法, 将mESCs (HBG3:eGFP: HB9) 扩大和分化。通过化学和酶学的无研磨处理, 含有绿色荧光蛋白的MNLCPs和未分化细胞的胚胎体被小心轻轻地离解成单细胞。利用OptiprepTM 8％-20％逐步梯度离心技术将 MNLCPs回收。拥有绿色荧光蛋白的MNLCPs的含量由流式细胞仪检测。结果：我们的结果表明，在胚胎体形成前，mESCs在明胶包被的培养板上生长，其分化为MNLCPs的能力减少。比较mESCs在明胶，明胶与PMEFs, 及PMEFs包被的培养板上的生长发现，mESCs在PMEFs包被的培养板上产生含绿色荧光蛋白的MNLCPs的得率为54.1％±11.0％（ ±s；n=12），在明胶包被的培养板上的得率为2.8％±1.1％（ ±s；n=9）。用密度梯度离心法获得的含绿色荧光蛋白的MNLCPs的平均含量为87.7％±5.5%（ ±s；n=3）。结论：我们的数据表明，不使用细胞分选器, 无研磨解离和密度梯度离心法也能用于富集具有高存活率的MNLCPs。有必要对MNLCPs在体外、体内和表型上进行进一步的生理学意义（如神经轴突的生长及形成神经肌肉接头的能力）上的鉴定。

关键词:胚胎干细胞,密度梯度,运动神经元

Abstract:Objective:Our objective was to isolate motor neuron-like cell precursors (MNLCPs) derived from mouse embryonic stem cells (mESCs) within embryoid bodies (EBs) for the development of drug screening assays and transplantation therapies in motor neuron disorders. EBs, induced with native Shh protein (or agonists of Shh pathway) and retinoic acid, contain variable proportions of MNLCPs and undifferentiated cells. Undifferentiated cells may interfere in drug screening assays or proliferate following transplantation if not sufficiently removed from the culture. We have developed a method based on density gradient centrifugation to enrich MNLCPs.Methods:mESCs (HBG3:eGFP: HB9) were expanded and differentiated using a modified protocol by Wichterleet al.(2008). EBs containing GFP (+) MNLCPs and undifferentiated cells were gently dissociated into single cells by chemical and enzymatic treatments without trituration. MNLCPs were recovered after centrifugation of dissociated cells on Optiprep^TM8%~20% step-gradient. The amount of GFP (+) MNLCPs was determined by flow cytometry.Results:Our results show that mESCs grown on gelatinized plates prior to EB formation, decreases the ability of mESCs to differentiate into MNLCPs. mESCs were grown on gelatin, gelatin with PMEFs, and PMEFs alone and found that growing mESCs on PMEF yielded GFP (+) MNLCPs at 54.1% ± 11.0% ( ±s；n=12) compared with gelatin 2.8% ±1.1% ( ±s；n=9). We obtained enriched fractions containing on average 87.7% ± 5.5%( ±s；n=3) GFP(+) MNLCPs by density gradient centrifugation.Conclusion:Our data support that dissociation without trituration and density gradient centrifugation can be used to significantly enrich MNLCPs retaining high viability without the use of a cell sorter. Further characterization of MNLCPs is necessary to design an appropriate assay such as physiological relevancein vitroandin vivoand phenotype such as neurite outgrowth, and the ability to form neuromuscular junctions.

Key words:embryonic stem cells,density gradient,motor neuron

Stephanie Richardson Milazi Ph.D., Benjamin Rix Brooks M.D., Jean-Luc Mougeot Ph.D.. Enrichment of Motor Neuron-like Cell Precursors Derived from Mouse Embryonic Stem Cells by Density Gradient Centrifugation / 用密度梯度离心法富集源于小鼠胚胎干细胞的运动神经元样细胞前体[J]. 神经药理学报, 2011, 1(1): 16-26.

Stephanie Richardson Milazi Ph.D., Benjamin Rix Brooks M.D., Jean-Luc Mougeot Ph.D. . Enrichment of Motor Neuron-like Cell Precursors Derived from Mouse Embryonic Stem Cells by Density Gradient Centrifugation[J]. Acta Neuropharmacologica, 2011, 1(1): 16-26.

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