Extended and stable gene expression via nucleofection of MIDGE construct into adult human marrow mesenchymal stromal cells

P. L. Mok, S. K. Cheong, Leong Chooi Fun, Chua Kien Hui, O. Ainoon

Research output: Contribution to journalArticle

12 Citations (Scopus)

Abstract

Human mesenchymal stromal cell (hMSC) is a potential target for cell and gene therapy-based approaches against a variety of different diseases. Whilst cationic lipofection has been widely experimented, the Nucleofector technology is a relatively new non-viral transfection method designed for primary cells and hard-to-transfect cell lines. Herein, we compared the efficiency and viability of nucleofection with cationic lipofection, and used the more efficient transfection method, nucleofection, to deliver a construct of minimalistic, immunologically defined gene expression encoding the erythropoietin (MIDGE-EPO) into hMSC. MIDGE construct is relatively safer than the viral and plasmid expression systems as the detrimental eukaryotic and prokaryotic gene and sequences have been eliminated. Using a plasmid encoding the luciferase gene, we demonstrated a high transfection efficiency using the U-23 (21.79 ± 1.09%) and C-17 (5.62 ± 1.09%) pulsing program in nucleofection. The cell viabilities were (44.93 ± 10.10)% and (21.93 ± 5.72)%, respectively 24 h post-nucleofection. On the other hand, lipofection treatment only yielded less than 0.6% efficiencies despite showing higher viabilities. Nucleofection did not affect hMSC renewability, immunophenotype and differentiation potentials. Subsequently, we nucleofected MIDGE-EPO using the U-23 pulsing program into hMSC. The results showed that, despite a low nucleofection efficiency with this construct, the EPO protein was stably expressed in the nucleofected cells up to 55 days when determined by ELISA or immunocytochemical staining. In conclusion, nucleofection is an efficient non-viral transfection approach for hMSC, which when used in conjunction with a MIDGE construct, could result in extended and stable transgene expression in hMSC.

Original languageEnglish
Pages (from-to)203-216
Number of pages14
JournalCytotechnology
Volume64
Issue number2
DOIs
Publication statusPublished - Mar 2012

Fingerprint

Mesenchymal Stromal Cells
Gene expression
Bone Marrow
Gene Expression
Transfection
Erythropoietin
Plasmids
Genes
Cells
Gene therapy
Luciferases
Cell- and Tissue-Based Therapy
Proteins
Transgenes
Genetic Therapy
Cell Survival
Enzyme-Linked Immunosorbent Assay
Staining and Labeling
Technology
Cell Line

Keywords

  • Bone marrow mesenchymal stromal cells
  • Cationic lipofection
  • Erythropoietin
  • MIDGE
  • Nucleofection

ASJC Scopus subject areas

  • Cell Biology
  • Biotechnology
  • Biomedical Engineering
  • Bioengineering
  • Clinical Biochemistry

Cite this

Extended and stable gene expression via nucleofection of MIDGE construct into adult human marrow mesenchymal stromal cells. / Mok, P. L.; Cheong, S. K.; Chooi Fun, Leong; Kien Hui, Chua; Ainoon, O.

In: Cytotechnology, Vol. 64, No. 2, 03.2012, p. 203-216.

Research output: Contribution to journalArticle

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