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首页> 外文期刊>BioResearch open access. >Electroporation: A Sustainable and Cell Biology Preserving Cell Labeling Method for Adipogenous Mesenchymal Stem Cells
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Electroporation: A Sustainable and Cell Biology Preserving Cell Labeling Method for Adipogenous Mesenchymal Stem Cells

机译：电穿孔：可持续和细胞生物学保存细胞标记方法的脂肪性间充质干细胞。

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Human mesenchymal stem cells derived from adipose tissue (AD-hMSCs) represent a promising source for tissue engineering and are already widely used in cell therapeutic clinical trials. Until today, an efficient and sustainable cell labeling system for cell tracking does not exist. We evaluated transient transfection through electroporation for cell labeling and compared it with lentiviral transduction for AD-hMSCs. In addition, we tested whether nonsense DNA or a reporter gene such as enhanced green fluorescent protein (EGFP) is the more suitable label for AD-hMSCs. Using electroporation, the transfection efficiency reached a maximal level of 44.6?±?1.1% EGFP-positive cells after selective and expansive cultivation of the mixed MSC population, and was 44.5?±?1.4% after gene transfer with Cyanin3-marked nonsense-label DNA, which remained stable during 2 weeks of nonselective cultivation (37.2?±?4.7% positive AD-hMSCs). Electroporation with both nonsense DNA and pEGFP-N1 led to a slight growth retardation of 45.2% and 59.1%, respectively. EGFP-transfected or transduced AD-hMSCs showed a limited adipogenic and osteogenic differentiation capacity, whereas it was almost unaffected in cells electroporated with the nonsense-label DNA. The nonsense DNA was detectable through quantitative real-time polymerase chain reaction for at least 5 weeks/10 passages and in differentiated AD-hMSCs. EGFP-labeled cells were trackable for 24?h in vitro and served as testing cells with new materials for dental implants for 7 days. In contrast, lentivirally transduced AD-hMSCs showed an altered natural immune phenotype of the AD-hMSCs with lowered expression of two cell type defining surface markers (CD44 and CD73) and a relevantly decreased cell growth by 71.8% as assessed by the number of colony-forming units. We suggest electroporation with nonsense DNA as an efficient and long-lasting labeling method for AD-hMSCs with the comparably lowest negative impact on the phenotype or the differentiation capacity of the cells, which may, therefore, be suitable for tissue engineering. In contrast, EGFP transfection by electroporation is efficient but may be more suitable for cell tracking within cell therapies without MSC differentiation procedures. Since current protocols of lentiviral gene transduction include the risk of cell biological alterations, electroporation seems advantageous and sustainable enough for hMSC labeling.
机译：源自脂肪组织（AD-hMSCs）的人间充质干细胞代表了组织工程的有希望的来源，并且已经广泛用于细胞治疗性临床试验中。直到今天，还不存在用于细胞跟踪的有效且可持续的细胞标记系统。我们评估了通过电穿孔进行瞬时转染的细胞标记，并将其与慢病毒转导的AD-hMSCs进行了比较。此外，我们测试了无义DNA或报道基因（例如增强型绿色荧光蛋白（EGFP））是否更适合AD-hMSCs的标记。使用电穿孔，混合培养的MSC群体经过选择性和广泛培养后，转染效率最高达到了44.6±±1.1％EGFP阳性细胞水平，并且用花青素3标记的无义标记进行基因转移后，转染效率达到了44.5±±1.4％在非选择性培养的2周内保持稳定的DNA（37.2？±？4.7％阳性AD-hMSCs）。无意义的DNA和pEGFP-N1的电穿孔分别导致45.2％和59.1％的轻微生长迟缓。 EGFP转染或转导的AD-hMSCs具有有限的成脂和成骨分化能力，而在用无义标记DNA电穿孔的细胞中几乎不受影响。通过定量实时聚合酶链反应至少5周/ 10次以及在分化的AD-hMSC中可检测到无义DNA。 EGFP标记的细胞在体外可追踪24小时，并用作测试材料以及用于牙科植入物的新材料7天。相比之下，慢病毒转导的AD-hMSCs改变了AD-hMSCs的天然免疫表型，降低了两种细胞类型定义的表面标志物（CD44和CD73）的表达，并且通过集落数评估，细胞的生长也相应减少了71.8％。形成单位。我们建议用无义DNA电穿孔作为对AD-hMSCs的有效且持久的标记方法，对细胞表型或分化能力的负面影响相对最低，因此可能适合组织工程。相比之下，通过电穿孔进行EGFP转染是有效的，但可能更适合在没有MSC分化程序的情况下进行细胞疗法中的细胞追踪。由于目前的慢病毒基因转导方案包括细胞生物学改变的风险，因此电穿孔对于hMSC标记而言似乎是足够有利和可持续的。

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