Int J Stem Cells.  2018 Nov;11(2):149-156. 10.15283/ijsc18037.

Improved Transfection Efficiency and Metabolic Activity in Human Embryonic Stem Cell Using Non-Enzymatic Method

• Kim CY
• Hwang IK
• Kang C
• Chung EB
• Jung CR
• Oh H
• Jeong YH
• Moon SH
• Kim JS
• Hong KS
• Park JH
• Chung HM

• KMID: 2429547
• DOI: http://doi.org/10.15283/ijsc18037

Abstract

Human embryonic stem cells (hESCs) are pluripotent cells widely used in conventional and regenerative medicine due to their ability to self-renew, proliferate and differentiate. Recently, genetic modification of stem cells using genome editing is the most advanced technique for treating hereditary diseases. Nevertheless, the low transfection efficiency of hESCs using enzymatic methods is still limited in in vitro preclinical research. To overcome these limitations, we have developed transfection methods using non-enzymatic treatments on hESCs. In this study, hESCs were transfected following enzymatic (TrypLE and trypsin) and non-enzymatic treatment ethylenediaminetetraacetic acid (EDTA) to increase transfection efficiency. Flow cytometric analysis using an enhanced green fluorescent protein vector showed a significantly increased transfection efficiency of EDTA method compared to standard enzyme method. In addition, the EDTA approach maintained stable cell viability and recovery rate of hESCs after transfection. Also, metabolic activity by using Extracellular Flux Analyzer revealed that EDTA method maintained as similar levels of cell functionality as normal group comparing with enzymatic groups. These results suggest that transfection using EDTA is a more efficient and safe substitute for transfection than the use of standard enzymatic methods.

Keyword

Human embryonic stem cell; Transfection; EDTA; Metabolic activity

MeSH Terms

Cell Survival
Edetic Acid
Genetic Diseases, Inborn
Genome
Human Embryonic Stem Cells*
Humans*
In Vitro Techniques
Methods*
Regenerative Medicine
Stem Cells
Transfection*
Edetic Acid

Figure

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  Fig. 1 Comparison of electroporation groups of hESCs (H9) using GFP-fluorescence (green) microscope (A). FACS analysis data of lipofection (B) and electroporation (C). Scale bar: 100 μm; Data represent the mean±SD. *p<0.05, **p<0.01.

• 

  Fig. 2 Bright field image indicating cell re-attachment after transfection (A). Lipofection transfected cell viability (B) and electroporation transfected cell viability (C). Data were normalized to normal control as 100%. Scale bar: 50 μm; Data represent the mean±SD. *p<0.05, **p<0.01.

• 

  Fig. 3 Extracellular acidification rate (ECAR) comparing with lipofection groups (Normal, EDTA, TrypLE and trypsin) (A). Data between EDTA and TrypLE groups represent the mean±SD. **p<0.01. Extracellular acidification rate (ECAR) comparing with lipofection groups (EDTA, TrypLE and trypsin) (B). Mean glycolytic ECAR and glycolytic capacity±SD. *p<0.05, **p<0.01.

Reference

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