Enhanced lipoplex-mediated gene expression in mesenchymal stem cells using reiterated nuclear localization sequence peptides.
BACKGROUND
Mesenchymal stem cells (MSC) are widely regarded as a promising tool for cellular therapy applications, and genetic modification by safe, liposome-based vectors may enhance their therapeutic potential.


METHODS
The present study describes the use of a cationic lipid vector (Lipofectamine 2000) to deliver genes to MSC isolated from a number of species in vitro and determined the characteristics of this vector system in terms of dose, toxicity and the time course of expression. In addition, the optimal use of a nuclear localization sequence (NLS) to enhance gene expression was explored.


RESULTS
Lipofection of human MSC did not adversely affect their ability to differentiate into osteogenic- and adipogenic lineages. Although human and rat MSC were found to take up lipoplexes with relative efficiency, lower levels of gene expression were detected in rabbit MSC, demonstrating a crucial effect of species. Peptides containing reiterated motifs of NLS were found to significantly improve on the level of transgene expression. Optimal gene delivery was observed when a three-fold reiterated NLS sequence was included in the liposome formulation.


CONCLUSIONS
Thus, nonviral gene delivery to MSC is feasible with efficiency being species dependent and can be enhanced by use of a three-fold reiterated NLS.
Hoare
Melissa
M
Regenerative Medicine Institute, National Centre for Biomedical Engineering Science & Department of Medicine, National University of Ireland, Galway, Republic of Ireland.
Greiser
Udo
U
Schu
Sabine
S
Mashayekhi
Kaveh
K
Aydogan
Emrah
E
Murphy
Mary
M
Barry
Frank
F
Ritter
Thomas
T
O'Brien
Timothy
T
D000595:Amino Acid Sequence; D000818:Animals; D015415:Biomarkers; D002454:Cell Differentiation; D002462:Cell Membrane / Q000378:metabolism; D005434:Flow Cytometry; D005786:Gene Expression Regulation; D049452:Green Fluorescent Proteins / Q000378:metabolism; D006801:Humans; D008081:Liposomes; D008156:Luciferases / Q000378:metabolism; D059630:Mesenchymal Stem Cells / Q000166:cytology / Q000378:metabolism*; D008969:Molecular Sequence Data; D019913:Nuclear Localization Signals / Q000737:chemistry / Q000378:metabolism*; D010455:Peptides / Q000737:chemistry / Q000378:metabolism*; D011817:Rabbits; D051381:Rats; D014162:Transfection / Q000379:methods*
D015415:Biomarkers; D008081:Liposomes; D019913:Nuclear Localization Signals; D010455:Peptides; C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins; D008156:Luciferases