TY - JOUR

T1 - Electroporation: A Sustainable and Cell Biology Preserving Cell Labeling Method for Adipogenous Mesenchymal Stem Cells

AU - Von Der Haar, Kathrin

AU - Jonczyk, Rebecca

AU - Lavrentieva, Antonina

AU - Weyand, Birgit

AU - Vogt, Peter

AU - Jochums, André

AU - Stahl, Frank

AU - Scheper, Thomas

AU - Blume, Cornelia A.

N1 - Funding information: This study was carried out as an integral part of the BIOFABRICATION FOR NIFE Initiative, which was financially supported by the ministry of Lower Saxony and the VolkswagenStiftung. NIFE is the Lower Saxony Center for Biomedical Engineering, Implant Research and Development, a joint translational research center of the Hannover Medical School, the Leibniz University Hannover, the University of Veterinary Medicine Hannover, and the Laser Center Hannover. The funding sponsors had no role in the design of the study; in the collection, analyses, or interpretation of data; and in the writing of the article. The publication of this article was funded by the Open Access Fund of the Leibniz University Hannover. We thank Constanza Figureido and Dorothee Eicke from the Institute for Transfusion Medicine, Medical School Hannover, for their advice and knowledge concerning lentiviral transduction and for making it possible to perform our lentiviral transduction experiments at their laboratory. We also thank Eva Grün for her assistance at the beginning of this project, and Martin Pähler who performed the quantitative real-time polymerase chain reaction experiments. We also thank Caroline Paterson as a native speaker for editing the article. This study was carried out as an integral part of the BIOFABRICATION FOR NIFE Initiative. The publication of this article was funded by the Open Access Fund of the Leibniz University Hannover.

PY - 2019/1/8

Y1 - 2019/1/8

N2 - 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.

AB - 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.

KW - AD-hMSCs

KW - cell labeling

KW - electroporation

KW - stem cell therapy

KW - transfection

UR - http://www.scopus.com/inward/record.url?scp=85063613066&partnerID=8YFLogxK

U2 - 10.1089/biores.2019.0001

DO - 10.1089/biores.2019.0001

M3 - Article

C2 - 30944770

AN - SCOPUS:85063613066

VL - 8

SP - 32

EP - 44

JO - BioResearch Open Access

JF - BioResearch Open Access

IS - 1

ER -