Application of NucleofectorTM technology in the transfection of rat nestin-positive cells derived from bone marrow with pancreatic duodenal homeobox-1 gene☆

Wang Hai-lan1, Jiang Ze-sheng2, Li Ai-hui3, Pan Ming-xin2, Gao Yi2

1Department of Internal Medicine, Longgang Central Hospital, Shenzhen 518116, Guangdong Province, China; 2Department of General Surgery, Zhujiang Hospital, Southern Medical University, Guangzhou 510282, Guangdong Province, China; 3Department of Surgery and Oncology, Donghua Hospital, Dongguan, 523000 Guangdong Province, China

Wang Hai-lan☆, Doctor, Associate chief physician, Department of Internal Medicine, Longgang Central Hospital, Shenzhen 518116, Guangdong Province, China
hailan1970@ yahoo.com.cn

Received: 2008-04-09
Accepted:2008-05-06
(54200804070004/ GW)

Wang HL, Jiang ZS, Li AH, Pan MX, Gao Y.Application of NucleofectorTM technology in the transfection of rat nestin-positive cells derived from bone marrow with pancreatic duodenal homeobox-1 gene.Zhongguo Zuzhi Gongcheng Yanjiu yu Linchuang Kangfu 2008;12(29):
5773-5777(China)
[www.zglckf.com/ zglckf/ ejournal/ upfiles/08-29/ 29k-5773(ps).pdf]

Abstract
BACKGROUND: The direct gene transfer can enhance the regulation of the specific differentiation of bone marrow mesenchymal stem cells (BMSCs), thus, efficient gene transfer techniques are key to the use of BMSCs in tissue engineering.
OBJECTIVE: To investigate the optimal transfection method for rat BMSCs termed Nucleofector TM technology, which provide a new method for high-efficient transfection of rat BMSCs in vitro.
DISIGEN, TIME AND SETTING: Comparative observation, cell genetic engineering experiment was conducted between October and December 2007 at the Central Laboratory of the General Military Neurological Research Institute, Zhujiang Hospital, Southern Medical University, Guangzhou, Guangdong Province, China.
MATERIALS: BMSCs from clean Sprague-Dawley rats of 3-4-week-old, weighing 60-70 g.
METHODS: BMSCs were isolated by density gradient centrifugation, nestin-positive cells were obtained by whole-bone-marrow culture followed by neural stem cell induction. BMSCs and nestin-positive cells were transfected with 1, 5, 10 μg recombinant plasmid pEGFP-C1 using A33 and A31 program of Nucleofector TM technology, transfected cells were cultured in 10% and 20% fetal bovine serum medium, respectively.
MAIN OUTCOME MEASURES: Cells expressed green fluorescence and transfection rate counted under LeicaDMIRE fluorescence microscope; cell viability determined on the basis of trypan blue dye exclusion; the expression of pancreatic duodenal homeobox-1 (PDX-1) gene in the transfected cells analyzed by reverse transcription polymerase chain reaction.
RESULTS: The A33 program of Nucleofector TM technology was more efficient in the transfection of neural stem cells than BMSCs, it was also more efficient than A31.The 24-hour transfection rate and survival rate of transfected cells cultured in the medium with 20% fetal bovine serum were markedly higher than those cultured in the medium with 10% fetal bovine serum (P < 0.05). In reverse transcription polymerase chain reaction detection, mRNA of PDX-1 gene was shown in nestin-positive cells transfected with recombinant plasmid, and not found in those without recombinant plasmid transfection.
CONCLUSION：We obtained higher transfection rate and survival rate when we transfected nestin-positive cells derived from bone marrow with 5 μg DNA using A33 program of Nucleofector TM technology and then cultured the transfected cells in the medium with 20% fetal bovine serum.

INTRODUCTION

Type 1 diabetes is an insulin-dependent, autoimmune disorder characterized by the destruction of insulin-producingβ-cells [1-2]. Hence, a reversal of type 1 diabetes could be afforded by replacement of functionalβ-cells. In recent years, attention has been focused on the possibility of gene or cell therapy of diabetes mellitus using artificially prepared non-β-cell–derivedβ-cells. Some studies have highlighted the potential usefulness of pancreatic duodenal homeobox-1 (PDX-1) gene as a reprogramming factor of non- β-cells towardβ-cell–like cells that can be used in diabetes cell/gene therapy [3-5]. The bone marrow mesenchymal stem cells (BMSCs) are thus attractive candidate cells for tissue engineering applications. Cells derived from bone marrow can be cultured and expanded in vitro and retain their ability to differentiate along these multiple mesenchymal pathways [6-7]. If BMSCs could form newβ-cells, they would become a particularly useful target for therapies that aim at β-cell replacement in diabetic patients, because they are abundantly available in the human bone marrow. In addition, the ability to genetically manipulate these BMSCs, by direct gene transfer, would further enable the selective enhancement of specific differentiation pathways. Thus, gene transfer techniques are key to the use of BMSCs in tissue engineering. In this paper, we described the optimization of a new transfection method for rat BMSCs termed the Nucleofector TMM technology method, developed by amaxa Biosystems [8-10]. This novel transfection method is designed for plasmid-based expression constructs and for primary cells that are difficult to transfect. PDX-1 gene has a central role in regulating both pancreas organogenesis and adultβ-cell function. PDX-1 is involved in regulating the expression of multiple β-cell-specific genes and has a key role in pancreatic morphogenesis in mice and humans [11-13]. We reported here the application of a new transfection technique, the amaxa Nucleofection method, to enhance the transfcetion efficiency of rat nestin-positive cells from BMSCs.

MATERIALS AND METHODS

Design
Comparative observation, cell genetic engineering experiment.

Time and setting
The experiment was conducted between October and

December 2007 at the Central Laboratory of the General Military Neurological Research Institute, Zhujiang Hospital, Southern Medical University, Guangzhou, Guangdong Province, China.

Materials
Clean Sprague-Dawley rats, aged 3-4 weeks and weighing 60-70 g, were provided by the Animal Center of Southern Medical University. Rat eGFP-C1 and rat eGFP-C1-PDX-1 was a gift of Professor An Jing(Department of Histology and Embryology of Southern Medical University). Polymerase chain reaction primer (Invitrogen company, shanghai, China).

Methods
Isolation of BMSCs: Bone marrow was obtained from the long bones of Sprague-Dawley rats. The bones were sterilized by immersion in 75% ethanol, followed by removal of the remaining skin and muscles. Bone marrow was exposed by cutting the ends of the bones and was extruded by inserting a needle and forcing cell culture medium with 10% fetal bovine serum (HyClone, USA) through the bone shaft. Gentle pipetting resulted in the generation of a single-cell suspension. Bone marrow cells were cultured (37 °C, 5% CO2) in 25 cm2 culture flask with low glucose DMEM (Gibco, USA) medium containing 10% fetal bovine serum, 25 mmol/L HEPES. Three days later, total media in the culture were changed including removal of floating cells. One week later, adhere cells gaining 80% confluence were passaged by pipetting with the use of trypsin. Following three to four passages, the cells became morphologically homogeneous, with a slim-spindle appearance.
Nestin-positive cells from BMSCs: BMSCs were obtained by the method described above. There cells were cultured (37°C, 5% CO2) in 25 cm2 culture flask with NSCs culture medium (prepared in Central Laboratory of the General Military Neurological Research Institute, China, Patent No. 02134314.4) containing 10% fetal bovine serum, 48 hours later, total media in the culture were changed including removal of the floating cells. The cells were cultured for 7-10 days.
Plasmid DNA purification: All plasmid DNA samples used in transfection were isolated and purified by QIAprep Spin Miniprep Kit (Qiagen Inc, Valencia, CA). Briefly, bacteria containing the recombinant plasmid were cultured using optimized fermentation procedures, and then plasmid DNA was isolated and purified from the bacteria by column method using Qiagen EndoFree Plasmid Purification Kit (Qiagen Inc, Valencia, CA) and was dissolved in sterile, filtered, low endotoxin (<10 U/mL) water. Quality of the purified plasmid DNA preparations was assessed by enzymatic restriction analysis and spectrophotometry on the basis of A260 to A280.
RNA isolation and reverse transcription polymerase chain reaction analysis: Total RNA was isolated from cells by Trizol method for reverse transcription polymerase chain reaction analysis. Oligonucleotide dT was used as the primer in the reverse transcription reaction, followed by a polymerase chain reaction with the gene-specific primers, and 1 μg of total RNA was used in each reaction. All RNA samples were DNAase I treated in the reverse transcription reaction before the polymerase chain reaction, polymerase chain reaction was also done in the absence of reverse transcription to verify the absence of reaction product. All reaction products were visualized by agarose gel electrophoresis followed by ethidium bromide staining. Primer sequences were as Zulewski et al[14] and Cao et al[15]: Rat nestin:forward, 5’-gcg ggg cgg tgc gtg act ac 3’; reverse, 5’-agg caa ggg gga aga gaa gga tgt 3’[14]; size of product 326 bp. Rat PDX-1: forward, 5’-cgg cca cac agc tct aca agg3’; reverse, 5’-gag gtt acg gca caa tcc tgc; size of product 667 bp[15].
Transfection Method: All reagents were prepared for Nucleofector transfection as described by amaxa Biosystems. Briefly, cells were harvested by trypsinization, and pelleted by centrifugation at 1 100 r/min for 5 minutes, resuspended in growth medium, counted, and then resuspended again at (2-3)×106 in 100 μl of amaxa Nucleofector solution on specific for rat NSCs. DNA, 2 μg or 5 μg, was added and the mixture was transferred into the amaxa electroporation cuvette. Electroporation was performed using either program A33 or A31. Immediately after electroporation, the cells were transferred to six-well plates containing DMEM supplemented with 10% or 20% fetal bovine serum. Cells were cultured and observed at 24 or 48 hours post-transfection. Cell viability was determined on the basis of trypan blue dye exclusion, and the cell expressed green fluorescence protein can be observed by fluorescence microscope.
Cell counting: EGFP-positive fluorescent cells were counted to measure the transfection rate at 24-48 hours after transfection. Cells were counted at 10×magnification using an inverse Olympus fluorescent microscope. Five fields were randomly selected in every well and at least 4-6 wells were counted for each sample. Transfection rate was calculated from relation between EGFP- and DAPI-positive cells respectively.
Statistical analysis: Results are expressed as Mean ± SD of the mean. Values were analyzed with SPSS 11.0 software and compared by Student’s t-test (two-tailed). P < 0.05 is considered statistically significant. The variance analysis (ANOVA), two-tailed t -test or non-parametrical U-test were used to compare experimental groups. The difference between groups was considered as statistically significant if P < 0.05.

RESULTS

Morphological observation
Rat BMSCs derived from bone marrow were adherent to tissue culture plastic and displayed a fibroblast-like morphology. Adherent cells were derived from cultures of unsorted bone marrow cells. The unattached cells were removed following 3 days of culture, spindle-shaped adherent cells cultured for an additional 2-3 weeks, until the spindle-shaped adherent cells reached 70%-80% confluence. The cells were then released from the substrate surface with trypsin-EDTA or pipetting and were replated under the same culture conditions for 3-5 passages (Figure1).

Nestin-positive cells
Inverted optical microscopic observation indicated that the inoculated cells were round and suspended; they were nucleolus cells mixed with a small number of red blood cells. At 48 hours, the adherent cells proliferated and filled the plate bottom one week later. If cells were cultured with the existence of adherent cells, big and round cells with plasma granules were found sprouting at 24 hours with plasma granules at the sprouting end disappeared. After that, sprouting end gradually extended and such cells became increased and clustered, which then formed island cell clones that could be induced to differentiate into cells of multiple shapes with long and slim prominences projecting under certain condition (Figure 2).

Plasmid DNA purification
A260/A280 > 1.8 suggested plasmid DNA was purified.

Result of nestin reverse transcription polymerase chain reaction
Expression of nestin mRNA was detected in the cells from NSC culture medium, but not in BMSCs (Figure 3).

Observation of transfected cells under fluorescence microscope
We transferred pEGFP-C1 into BMSCs or nestin-positive cells by Nucleofector TM technology and observed the cells under fluorescence microscope at 6, 24, 48 hours after transfection. Little eGFP fluorescence can be observed at 6 hours, significantly increased at 24 hours and more at 48 hours after tranfection (Figure 4). Furthermore, we attempted to compare the transfection efficiency in transfected cells. For this purpose, the two kinds of cells were transfected with eGFP by A33 and A31, respectively. The transfected nestin-positive cells gained higher efficiency than transfected BMSCs by A33 or A31 (P < 0.001). The data were shown in Figure 5. Result indicates that higher efficiency could be gained by A33 program than A31 (Figure 5). As a result, all subsequent experiments were carried out with nestin-positive cells.

To gain higher transfection efficiency, we transfected nestin-positive cells with 1 μg, 5 μg, 10 μg eGFP plasmid by A33 program. We found transfection efficiency significantly increased when 5 μg DNA was used than 1 μg DNA was used. But there was no significant difference between 5 μg DNA and 10 μg DNA (Figure 6).

We transfected nestin-positive cells with 5 μg EGFP-PDX-1 plasmid by A33 and then inoculated the cells in 10% or 20% low glucose DMEM. We found that transfection efficiency significantly increased when nestin-positive cells inoculated in low glucose DMEM containing 20% fetal bovine serum (44.9±4.76/38.52±3.72, P < 0.05). We therefore tested whether increasing the medium serum concentration from 10%-20% could overcome the high mortality associated with program A33.This change resulted in a dramatic increase in cell viability from (62.6%±6.41)% to (88%±7.21)% (P < 0.05) and higher transfection efficiency (38.52±3.72/44.9±4.76, P < 0.05). All subsequent transfection experiments were therefore carried out in medium containing 20% fetal bovine serum.

Result of PDX-1 reverse transcription polymerase chain reaction
Expression of PDX-1 mRNA was observed in transfected nestin-positive cells, but expression of that was not observed in untransfected nestin-positive cells (Figure 7).

DISCUSSION

BMSCs were transfected with pEGFP-C1, plasmid construct consisting of green fluorescence protein driven by the constitutively active cytomegalovirus early promoter. In initial studies, conventional transfection methods, such as lipofectamine or calcium phosphate, yielded transfection efficiencies less than 4%[16], even with optimal variations suggested by the manufacturers of these various reagents. Such low efficiencies, therefore, posed a technical obstacle for the genetic manipulation of these cells, and indicated the need to explore novel approaches for the optimization of plasmid transfection efficiency in BMSCs.
We gained two kinds of cells from bone marrow. One of them was bone marrow mesenchymal stem cell, which gained by using low glucose DMEM culture medium. Another was nestin-positive cell, which gained by using NSC culture medium. The amaxa A33 or A31 electroporation program were selected according to the manufactrer’s recommendation (rat NSC-specific reagent solution). Using 2 μg of DNA/106 cells, higher transfection efficiencies were obtained in nestin-positive cells.
By using program A33, 5 μg DNA and the NSCs transfection reagent supplied by the manufacture, with 20% serum supplementation, we achieved high-transfection efficiencies and significant cell viability. However, it is noteworthy that a significant number of eGFP-positive cells remained at 3 weeks after transfection.
The method described here allows efficient gene transfer into nestin-positive cells derived from bone marrow. The implement of the method is simple. Furthermore, we showed that changing the amounts of DNA per transfection could control transfection efficiency. Thus, the electroporation-based gene transfer into neural cells could be an alternative to viral gene transfer, opening new perspectives for gene therapy research.
In agreement with the phenotypes of the knockout animals, recent gain-of-function experiments have revealed the potential of PDX-1 in conferring some β-cell like features on non–β-cells. Others have recently shown that the exogenous expression of PDX-1 in two lines of glucagon-producing cells can induce the expression of β-cell–specific genes, including the insulin gene [17-19]. Also, more recently, Ferber et al [20] showed that PDX-1 could endow some cells in the liver with pancreaticβ-cell characteristics in vivo using recombinant adenovirus-mediated gene delivery. These data have highlighted the potential usefulness of PDX-1 as a reprogramming factor of non–β-cells toward β-cell–like cells that can be used in diabetes cell/gene therapy. Hori [21] showed production of IPCs solely through extracellular factor modulation in the absence of genetic manipulations might promote strategies to derive transplantable islet-replacement tissues from human neural progenitor cells. Based on Hori Y’s[21] conclusion and our experiment, we transferred the PDX-1 gene into nestin-positive cells from rat bone marrow.
In the present study, we optimized the transfection protocol and provide evidence that the transfected cells expressed PDX-1 mRNA by reverse transcription polymerase chain reaction. We confirm that it is possible to use the nestin-positive cells from bone marrow mesenchymal stem cells as seed cells in islet transplantation.

REFERENCES

1 Atkinson MA, Eisenbarth GS.Type 1 diabetes: new perspectives on disease pathogenesis and treatment. Lancet 2001; 358(9277):221–229
2 Haller MJ, Atkinson MA, Schatz D. Type 1 diabetes mellitus: etiology, presentation, and management. Pediatr Clin North Am 2005;52(6): 1553-1578
3 Koizumi M, Nagai K, Kida A, et al. Forced expression of PDX-1 induces insulin production in intestinal epithelia. Surgery 2006; 140 (2): 273-280
4 Koizumi M, Doi R, Fujimoto K,et al. Pancreatic epithelial cells can be converted into insulin-producing cells by GLP-1 in conjunction with virus-mediated gene transfer of pdx-1. Surgery 2005;138 (2): 125- 133
5 Wang AY, Ehrhardt A, Xu H,et al. Adenovirus transduction is required for the correction of diabetes using Pdx-1 or Neurogenin-3 in the liver. Mol Ther 2007;15(2):255-263
6 Herzog EL, Chai L, Krause DS. Plasticity of marrow -derived stem cells. Blood 2003;102(10):3483-3493
7 Tuan RS, Boland G, Tuli R. Adult mesenchymal stem cells and cell-based tissue engineering. Ther 2003;5(1):32-45
8 Hamm A, Krott N, Breibach I et al. Efficient transfection method for primary cells. Tissue Eng 2002;8(2):235-245
9 Konstantin C, Marco T, Maike W,et al. Nucleofection is the most efficient non-viral transfection method for neuronal stem cells derived from ventral mesencephali with no changes in cell composition or dopaminergic fate. Stem Cell 2006;24(12):2776-2791
10 Haleem-Smith H, Derfoul A, Okafor C,et al. Optimization of high-efficiency transfection of adult human mesenchymal stem cells in vitro. Mol Biotechnol 2005;30(1):9-20
11 Jonsson J, Carlsson L, Edlund T, et al. Insulin-promoter-factor 1 is required for pancreas development in mice. Nature 1994;371(6498): 606–609
12 Melloul D. Transcription factors in islet development and physiology: role of PDX-1 in beta-cell function. Ann N Y Acad Sci 2004; 1014:28-37
13 Stoffers DA, Zinkin NT, Stanojevic V,et al. Pancreatic agenesis attributable to a single nucleotide deletion in the human IPF1 gene coding sequence. Nat Genet 1997;15:106-110
14 Zulewski H, Abraham EJ, Gerlach MJ,et al. Multipotential nestin-Positive stem cells isolated from adult pancreatic islets differentiate ex vivo into pancreatic endocrine, exocrine, and hepatic phenotypes. Diabetes 2001;50(3):521-533
15 Cao LZ, Tang DQ,Marko E, et al.High glucose is necessary for complete maturation of pdx1-VP16–Expressing hepatic cells into functional insulin-Producing cells. Diabetes 2004;53:3168-3178
16 Hana HS,Assia D,Chukwuka O,et al.Optimization of eigh-efficiency transfection of adult human mesenchymal stem cell in vitro. Mol Biotechnol 2005;30(1):9-20
17 Serup P, Jensen J, Andersen FG,et al.Induction of insulin and islet amyloid polypeptide production in pancreatic islet glucagonoma cells by insulin promoter factor 1. Proc Natl Acad Sci USA 1996; 93:9015-9020
18 Christine M, Florence DF, Richard MJ,et al. Human bone marrow mesenchymal stem cells can express insulin and key transcription factors of the endocrine pancreas developmental pathway upon genetic and/or microenvironmental manipulation in vitro. Stem Cells 2005;23(4):594-604
19 Sapir T, Shternhall K, Meivar-Levy I,et al. Cell-replacement therapy for diabetes: Generating functional insulin-producing tissue from adult human liver cells.Proc Natl Acad Sci U S A 2005;102 (22):7964-7969
20 Ferber S, Halkin A, Cohen H,et al. Pancreatic and duodenal homeobox gene 1 induces expression of insulin genes in liver and ameliorates streptozotocininduced hyperglycemia. Nat Med 2000;6: 568-572
21 Hori Y, Gu X, Xie X,et al. Differentiation of insulin-producing cells from human neural progenitor cells. PLoS Med 2005;2(4):347-356