Microporation is a valuable transfection method for efficient gene delivery into human umbilical cord blood-derived mesenchymal stem cells.
BACKGROUND
Mesenchymal stem cells (MSCs) are an attractive source of adult stem cells for therapeutic application in clinical study. Genetic modification of MSCs with beneficial genes makes them more effective for therapeutic use. However, it is difficult to transduce genes into MSCs by common transfection methods, especially nonviral methods. In this study, we applied microporation technology as a novel electroporation technique to introduce enhanced green fluorescent protein (EGFP) and brain-derived neurotropfic factor (BDNF) plasmid DNA into human umbilical cord blood-derived MSCs (hUCB-MSCs) with significant efficiency, and investigated the stem cell potentiality of engineered MSCs through their phenotypes, proliferative capacity, ability to differentiate into multiple lineages, and migration ability towards malignant glioma cells.


RESULTS
Using microporation with EGFP as a reporter gene, hUCB-MSCs were transfected with higher efficiency (83%) and only minimal cell damage than when conventional liposome-based reagent (<20%) or established electroporation methods were used (30-40%). More importantly, microporation did not affect the immunophenotype of hUCB-MSCs, their proliferation activity, ability to differentiate into mesodermal and ectodermal lineages, or migration ability towards cancer cells. In addition, the BDNF gene could be successfully transfected into hUCB-MSCs, and BDNF expression remained fairly constant for the first 2 weeks in vitro and in vivo. Moreover, microporation of BDNF gene into hUCB-MSCs promoted their in vitro differentiation into neural cells.


CONCLUSIONS
Taken together, the present data demonstrates the value of microporation as an efficient means of transfection of MSCs without changing their multiple properties. Gene delivery by microporation may enhance the feasibility of transgenic stem cell therapy.
Lim
Jung Yeon
JY
Department of Biomedical Science, College of Medicine, The Catholic University of Korea, Seoul, Korea.
Park
Sun Hwa
SH
Jeong
Chang Hyun
CH
Oh
Ji Hyeon
JH
Kim
Seong Muk
SM
Ryu
Chung Hun
CH
Park
Soon A
SA
Ahn
Jae Geun
JG
Oh
Wonil
W
Jeun
Sin-Soo
SS
Chang
Jong Wook
JW
D000818:Animals; D019208:Brain-Derived Neurotrophic Factor / Q000235:genetics; D002454:Cell Differentiation; D002465:Cell Movement; D049109:Cell Proliferation; D002478:Cells, Cultured; D018274:Electroporation / Q000379:methods*; D005312:Fetal Blood / Q000166:cytology; D017930:Genes, Reporter; D049452:Green Fluorescent Proteins / Q000235:genetics; D006801:Humans; D008297:Male; D059630:Mesenchymal Stem Cells / Q000378:metabolism*; D010957:Plasmids; D051381:Rats; D017207:Rats, Sprague-Dawley; D014162:Transfection / Q000379:methods*
D019208:Brain-Derived Neurotrophic Factor; C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins
Proc Natl Acad Sci U S A. 1996 Oct 15;93(21):11382-8
8876144
Mol Ther. 2009 Feb;17(2):302-8
19066595
Cytotherapy. 2004;6(5):476-86
15512914
Biotechniques. 1990 Jan;8(1):16-20
2182073
Proc Natl Acad Sci U S A. 1999 Apr 13;96(8):4262-7
10200250
J Neurosci. 1995 Aug;15(8):5765-78
7643217
Neurosci Res. 1999 Jan;33(1):71-8
10096474
Science. 1999 Apr 2;284(5411):143-7
10102814
Exp Hematol. 2006 Apr;34(4):433-42
16569590
Neurology. 2002 Aug 27;59(4):514-23
12196642
Neuroreport. 2004 Aug 6;15(11):1731-4
15257137
Exp Neurol. 2005 Feb;191(2):344-60
15649491
J Neurosci Res. 2008 Aug 1;86(10):2168-78
18438930
Biol Blood Marrow Transplant. 2001;7(11):581-8
11760145
Hum Gene Ther. 2002 Jul 20;13(11):1337-47
12162816
Nat Genet. 2000 Jan;24(1):49-52
10615126
Bioelectrochemistry. 2001 Jan;53(1):1-10
11206915
J Natl Cancer Inst. 2004 Nov 3;96(21):1593-603
15523088
Proc Natl Acad Sci U S A. 2004 Oct 12;101(41):14835-40
15466696
Cancer Res. 2005 Apr 15;65(8):3307-18
15833864
Mol Ther. 2003 Mar;7(3):354-65
12668131
Cloning Stem Cells. 2005;7(3):154-66
16176125
Acta Neurochir (Wien). 2005 Sep;147(9):985-92; discussion 992
16010451
J Orthop Res. 1991 Sep;9(5):641-50
1870029
Curr Drug Deliv. 2004 Apr;1(2):153-63
16305381
Biochim Biophys Acta. 2005 Aug 5;1724(3):270-80
15951114
Mol Ther. 2004 Feb;9(2):189-97
14759803
Cancer Res. 2002 Jul 1;62(13):3603-8
12097260
Gene Ther. 2004 Jan;11(2):224-8
14712307
J Gene Med. 1999 Mar-Apr;1(2):121-33
10738576
Annu Rev Biochem. 2003;72:609-42
12676795
Cell Transplant. 2002;11(3):265-74
12075992
Hum Gene Ther. 2001 May 20;12(8):861-70
11387052
Mol Membr Biol. 2003 Jan-Mar;20(1):35-43
12745924
Bone Marrow Transplant. 2007 Oct;40(7):609-19
17603514
Scand J Immunol. 2003 Jan;57(1):11-20
12542793