von der Haar Kathrin, Jonczyk Rebecca, Lavrentieva Antonina, Weyand Birgit, Vogt Peter, Jochums André, Stahl Frank, Scheper Thomas, Blume Cornelia A
Institute of Technical Chemistry, Leibniz University Hannover, Hannover, Germany.
Department of Plastic Hand and Reconstructive Surgery, Hannover Medical School Hannover, Hannover, Germany.
Biores Open Access. 2019 Mar 29;8(1):32-44. doi: 10.1089/biores.2019.0001. eCollection 2019.
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 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.
源自脂肪组织的人间充质干细胞（AD-hMSCs）是组织工程的一个有前景的细胞来源，并且已广泛应用于细胞治疗临床试验。直到如今，还不存在一种用于细胞追踪的高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用于细胞标记，并将其与AD-hMSCs的慢病毒转导进行比较。此外，我们测试了无义DNA或诸如增强型绿色荧光蛋白（EGFP）等报告基因是否是更适合AD-hMSCs的标记。使用电穿孔，在对混合的间充质干细胞群体进行选择性和扩增培养后，转染效率达到了EGFP阳性细胞的最高水平，即44.6±1.1%，在用花青素3标记的无义标记DNA进行基因转移后为44.5±1.4%，在非选择性培养的2周内保持稳定（37.2±4.7%的阳性AD-hMSCs）。用无义DNA和pEGFP-N1进行电穿孔分别导致细胞生长略有迟缓，分别为45.2%和59.1%。EGFP转染或转导的AD-hMSCs显示出有限的成脂和成骨分化能力，但在用无义标记DNA电穿孔的细胞中几乎未受影响。通过定量实时聚合酶链反应至少在5周/10代以及在分化的AD-hMSCs中可检测到无义DNA。EGFP标记的细胞可追踪24小时，并作为牙科植入新材料的测试细胞达7天。相比之下，慢病毒转导的AD-hMSCs显示出AD-hMSCs的天然免疫表型发生改变，两种细胞类型定义表面标志物（CD44和CD73）的表达降低，并且通过集落形成单位数量评估，细胞生长显著降低了71.8%。我们建议用无义DNA进行电穿孔作为一种高效且持久的AD-hMSCs标记方法，对细胞的表型或分化能力具有相对最低的负面影响，因此可能适用于组织工程。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间充质干细胞分化程序的细胞治疗中进行细胞追踪。由于当前慢病毒基因转导方案存在细胞生物学改变的风险，电穿孔对于人间充质干细胞标记似乎足够有利且可持续。
