Electroporation: A Sustainable and Cell Biology Preserving Cell Labeling Method for Adipogenous Mesenchymal Stem Cells.

Kathrin Von Der Haar,Antonina Lavrentieva,Peter Vogt,Birgit Weyand,Cornelia A Blume,Rebecca Jonczyk,Frank Stahl,André Jochums,Thomas Scheper

doi:10.1089/biores.2019.0001

Kathrin Von Der Haar, Antonina Lavrentieva + Show 7 more

Open Access

https://doi.org/10.1089/biores.2019.0001

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Abstract

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.

Highlights

IntroductionHuman mesenchymal stem cells (hMSCs) that are present in a high quantity, for example, in human adipose tissue (AD-hMSCs)[1,2,3] are frequently used in regenerative medicine,[4] since these cells can be differentiated in various cell types.[1,5] Despite being only multipotent, they demonstrate great therapeutic potential through the release of growth factors, chemokines, or antimicrobial signal molecules.[6,7] A sustainable labeling of hMSCs is relevant to track their way into a Kathrin von der Haar et al 2019; Published by Mary Ann Liebert, Inc
Human mesenchymal stem cells that are present in a high quantity, for example, in human adipose tissue (AD-hMSCs)[1,2,3] are frequently used in regenerative medicine,[4] since these cells can be differentiated in various cell types.[1,5]
Nonsense DNA and egfp DNA were detectable in high amounts after electroporation and further cultivation of 14 days as well as after long-term cultivation

Summary

Introduction

Human mesenchymal stem cells (hMSCs) that are present in a high quantity, for example, in human adipose tissue (AD-hMSCs)[1,2,3] are frequently used in regenerative medicine,[4] since these cells can be differentiated in various cell types.[1,5] Despite being only multipotent, they demonstrate great therapeutic potential through the release of growth factors, chemokines, or antimicrobial signal molecules.[6,7] A sustainable labeling of hMSCs is relevant to track their way into a Kathrin von der Haar et al 2019; Published by Mary Ann Liebert, Inc. EGFP can be detected, for example, by in vivo flow cytometry at available body regions.[12] The efficiency of transfecting primary cells and especially stem cells is usually not as high as in cell lines[13,14,15] and some transfection techniques for AD-hMSCs are questioned to affect cell biology in terms of proliferation or differentiation, affecting the therapeutic use.[16] In general, only stable transfection methods with genomic integration of target DNA are suggested to be sustainable enough for cell therapy, whereas after transient transfection, target DNA diminishes by dilutional effects during cell division.[11,17] On the contrary, viral presence—after stable DNA transfer— may produce immunogenicity, cytopathic effects, cancerogenicity, or severe toxicity in the recipient,[18,19,20,21] and this technique, requires a large number of safety measures as a prerequisite for its performance.[22]

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