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First published online December 7, 2006
Stem Cells Vol. 25 No. 3 March 2007, pp. 790 -797
doi:10.1634/stemcells.2006-0235; www.StemCells.com
© 2007 AlphaMed Press

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TECHNOLOGY DEVELOPMENT

Nucleofection Is a Valuable Transfection Method for Transient and Stable Transgene Expression in Adipose Tissue-Derived Stem Cells

Laure-Emmanuelle Zaragosi, Nathalie Billon, Gérard Ailhaud, Christian Dani

Institut de Recherche, Signalisation, Biologie du Développement et Cancer, UMR6543 Centre National de la Recherche Scientifique, Université Nice Sophia-Antipolis, Centre de Biochimie, Faculté des Sciences, Nice, France

Key Words. Adult stem cells • Adipose tissue • Nucleofection • Transgene expression • Transient • Stable • Secretion

Correspondence: Christian Dani, Ph.D., CNRS UMR6543, Centre de Biochimie, Faculté des Sciences, 06108 Nice Cedex 2, France. Telephone: +33 (0) 4 92 07 64 36; Fax: +33 (0)4 92 04 64 04; e-mail: dani{at}unice.fr

Received April 19, 2006; accepted for publication November 28, 2006.
First published online in STEM CELLS EXPRESS   December 7, 2006.


   ABSTRACT
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 Acknowledgments
 References
 
Adipose tissue-derived stem cells are a powerful tool for in vitro study of adult stem cell biology. So far, they have not been extensively used for gain or loss of function studies since they are resistant to most common transfection methods. Herein, we tested several classic transfection methods on human multipotent adipose tissue-derived stem (hMADS) cells. Our results showed that lipofectants and calcium phosphate were poorly efficient for transgene delivery in hMADS cells. In contrast, nucleofection, an electroporation-based method that is assumed to target plasmid DNA directly to the cell nucleus, led to a significant transient transgene expression in hMADS cells (up to 76% enhanced green fluorescent protein [EGFP]-positive cells were detected). Furthermore, after selection of hMADS cells that were nucleofected with a selectable plasmid coding for EGFP, stable EGFP expressing clones could be propagated in culture and efficiently induced to differentiate into EGFP-positive adipocytes and osteoblasts. Finally, we verified that nucleofected hMADS cells could produce a functional, transgene-encoded, secreted protein. To this aim, hMADS cells were nucleofected with a plasmid coding for leukemia inhibitory factor (LIF). This protein was detected at high concentrations in supernatants from pCAG-LIF transfected hMADS cells. Moreover, supernatants were able to maintain mouse embryonic stem cells' undifferentiated phenotype, indicating that hMADS cells could secrete a functional LIF protein. Taken together, our data demonstrate that nucleofection allows both transient and stable gene expression in adipose tissue-derived stem cells, without impairing their differentiation potential.


   INTRODUCTION
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 Acknowledgments
 References
 
Recent studies on the isolation, characterization, and therapeutic use of adult stem cells have attracted the attention of the scientific and political communities. Adult stem cells have been successfully isolated from different tissues, including hematopoietic [1], neural [2], gastrointestinal [3], hepatic [4], and mesenchymal [5] tissues. Adipose tissue has first been identified as a source of multipotent stem cells by Zuk et al. [6, 7]. Rodriguez et al. isolated human multipotent adipose tissue-derived stem (hMADS) cells from the stroma-vascular fraction of infant fat [8]. After being cultured for more than 100 population doublings, these cells display a normal diploid karyotype. They possess multilineage differentiation potential, as they undergo differentiation into adipocytes, osteoblasts, and chondrocytes in vitro [8]. Moreover, after transplantation into skeletal muscle of dystrophin-deficient (mdx) mice, they are able to fuse into tibialis anterior muscle fibers and to restore long-term expression of dystrophin [9]. Thus, hMADS cells provide a powerful system for studying commitment and differentiation toward various lineages and could be an appropriate tool for gene and cell therapy. For both fundamental and clinical purposes, being able to perform genetic manipulation of hMADS cells is crucial. Unfortunately, as do many primary cells [10, 11], adipose tissue derived-stem cells exhibit resistance to classic nonviral transgene delivery methods. Nonviral classic transfection methods include calcium phosphate precipitation, lipofectants, and electroporation. These methods are generally less efficient than are viral-based techniques, they can not be used with slowly proliferating cells, and they generate high mortality. Viral-based methods can be very efficient, but are difficult to set up because they are time-consuming and require specific safety conditions, especially with human cells [12]. In contrast, nucleofection is a recent electroporation-based technique that has been successfully used to transfect several primary cell types, including mouse T cells [13], neurons [14], and keratinocytes [11], as well as human and mouse stem cells of diverse origins [15–17]. This technique consists of a combination of optimized solutions and electrical parameters that are supposed to target plasmid DNA and small interfering RNA (siRNA) straight to the cell nucleus and trigger rapid expression of transgenes.

Herein, we have tested transfection of hMADS cells with different lipofectant- and electroporation-based methods. We have observed that nucleofection is the only non-viral based technique allowing efficient transgene delivery into hMADS cells. Using a plasmid coding for enhanced green fluorescent protein (EGFP) and a puromycin resistance gene, we could obtain more than 75% EGFP+ hMADS cells 48 hours postnucleofection. Furthermore, after selection, we could successfully establish hMADS cell clones stably expressing EGFP. Nucleofection did not alter the differentiation abilities of these clones, because they could still undergo both adipogenic and osteogenic differentiation without loss of transgene expression. Finally, we demonstrated that hMADS cells that were nucleofected with a plasmid coding for leukemia inhibitory factor (LIF) could secrete a functional LIF protein. Thus, nucleofection is a valuable transfection method to induce transient and stable transgene expression in adipose tissue derived-stem cells without altering the differentiation potential of these cells.


   MATERIALS AND METHODS
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosures
 Acknowledgments
 References
 
Plasmids
The pmaxGFP plasmid (3.49 kb) was provided in the Nucleofector kit (Amaxa Biosciences, Cologne, Germany http://www.amaxa.com). In pmaxGFP, maxGFP expression is driven by a cytomegalovirus (CMV) promoter (for more details, see http://www.amaxa.com). pPyCAGGFPIP (7.29 kb) was a gift from Dr I. Chambers (Institute of Stem Cell Research, Edinburgh, United Kingdom); it contains an EGFP coding sequence under the control of a CAG promoter (a combination of chicken β-actin promoter and cytomegalovirus immediate-early enhancer) and also bears an internal ribosome entry site (IRES)-puromycin cassette. pCAG-LIF was a gift from Pr. Austin Smith (Institute of Stem Cell Research); it is currently used for human LIF production from COS cells.

Isolation and Culture of hMADS Cells
hMADS cells were obtained from the stroma of human adipose tissue as described previously [9]. Briefly, we used the stroma-vascular fraction (SVF) of white adipose tissue from young donors (1 month to 7 years old). Adipose tissue was collected, with the informed consent of the parents, as surgical scraps from surgical specimen of various surgeries, as approved by the Centre Hospitalier Universitaire de Nice (Nice, France) review board. Approximately 200 mg of adipose tissue were dissociated with type A collagenase, and the stroma-vascular fraction was separated from the adipocytes fraction by centrifugation. The crude SVF was plated on uncoated culture dishes. Twelve hours after plating, nonadherent cells were removed by a medium change and adherent cells (termed "CA" by Rodriguez et al. [9]) were maintained in the proliferation medium, which is composed of Dulbecco's modified Eagle's medium (DMEM; low glucose) containing 10% fetal calf serum (FCS), 0.01 M HEPES, and 100 units/ml penicillin and streptomycin. After reaching 80% confluency, adherent cells were dissociated in 0.25% trypsin EDTA and seeded at 4,500 cells per cm2. After 60–80 population doublings, hMADS cells were maintained in proliferation medium supplemented with 2 ng/ml fibroblast growth factor 2 (FGF2) [18]. The hMADS cell populations that are included in this study were isolated from the pubic region fat pad of 5-year-old (hMADS2) and 4-month-old (hMADS3) male donors as well as from the scrotum fat pad of a 18-month-old male donor (hMADS6) [9].

In Vitro Adipogenesis and Osteogenesis
For adipocytes, confluent cells were cultured in DMEM/Ham's F12 medium supplemented with 10 µg/ml transferrin, 0.86 µM insulin, 0.2 nM of triiodothyronine, 1 µM dexamethasone, 100 µM isobutyl-methylxanthine, and 20 nM rosiglitazone. Three days later, the medium was changed (dexamethasone and isobutyl-methylxanthine were omitted). Neutral lipid accumulation was assessed by oil red O staining as described previously [19].

For osteoblasts, confluent cells were cultured in {alpha}-minimal essential medium containing 10% FCS, 50 µg/ml L-ascorbic acid phosphate, 10 mM β-glycerophosphate, and 100 nM dexamethasone. Alizarin red staining was performed as previously described [20].

Isolation of hMADS-Resistant Clones
After puromycin selection in 60-mm culture dishes, clones were allowed to proliferate until they contained at least 200 cells. The process, from puromycin selection to dissociation of clones, took approximately 3 weeks. Clones that were selected for propagation arose from single cells that were separated from other cells by at least one optical field (approximately 2.2 mm). Clones were then isolated using trypsin-wet Whatman paper pieces and seeded in 24-well plates. Approximately every 4 days, they could be passaged and plated successively in 35-mm and 100-mm dishes.

Cell Culture of Embryonic Stem Cells
CGR8 mouse embryonic stem (mES) cells were grown as described previously [21]. For the LIF bioassay, ES cells were seeded at 2,500 cells per cm2 and maintained for 5 days in LIF-free conditions supplemented with hMADS cells conditioned medium, before being assayed for alkaline phosphatase (AP). All cultures were maintained at 37°C in a humidified gassed incubator, 5% CO2 in air. Media were changed every other day.

Alkaline Phosphatase Staining
To assess their undifferentiated phenotype, ES cells were stained for AP according to the manufacturer's instructions (Leukocyte Alkaline Phosphatase Kit, Sigma-Aldrich, Lyon, France, http://www.sigmaaldrich.com).

Transfection with Calcium Phosphate and Lipofectants
Calcium phosphate transfection of hMADS cells was carried out as described previously [22]. Sixty percent confluent cells were transfected in 60-mm dishes with 12 µg of plasmid.

Lipofectamine 2000 (Invitrogen, Cergy Pontoise, France, http://www.invitrogen.com), FuGENE 6 (Roche Applied Science, Mannheim, Germany, http://www.roche-applied-science.com), FuGENE HD (Roche), and Exgen 500 (Fermentas, Burlington, Canada, http://www.fermentas.com) were used as recommended by manufacturers in 24-well plates. At transfection time, cell densities were approximately 90% confluency for Lipofectamine 2000 and FuGENE HD, and they were approximately 60% confluency for FuGENE 6 and Exgen. The various recommended ratios between cDNA amounts and reagents volumes were tested. These ratios are indicated in Table 1. Herein presented are only the results obtained by fluorescence-activated cell sorting (FACS) analysis with these ratios (Table 3). Results are the mean (± SE) of the FACS analysis from three transfection experiments using the same batch of cells.


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  		Table 1. Conditions for transfection with lipofectants

 
Nucleofection Procedure
hMADS cells were grown until 80% confluency, and medium was changed the day before nucleofection. Cells were dissociated in 0.25% trypsin EDTA and numbered. For each nucleofection assay, 6 x 105 cells were resuspended in 100 µl of Nucleofector buffer (Human MSC [mesenchymal stem cells] Kit buffer, reference VPE-1001; Amaxa Biosciences) and nucleofected with 3 µg of plasmid DNA. The U-23 and C-17 protocol were tested on the Nucleofector II device (for more details see http://www.amaxa.com). Unless otherwise mentioned, the U-23 protocol was used for the entire study. Immediately after nucleofection completion, cells were plated onto 60-mm dishes. Culture medium was changed 6 hours after nucleofection to remove dead cells as soon as possible. For the establishment of hMADS cells stably expressing EGFP, puromycin treatment (1 µg/ml) was applied 48 hours after nucleofection. For the collection of supernatants for the enzyme-linked solid-phase immunosorbent assay (ELISA), cells were plated into 35-mm dishes. Six hours after nucleofection, the culture medium was changed (1.5 ml per dish).

FACS Analysis
FACS analyses were performed on living hMADS cells after trypsin dissociation and a single phosphate-buffered saline (PBS) wash. Cells were resuspended in 400 µl of PBS and analyzed on a FACSort (BD Biosciences, Franklin Lakes, NJ, http://www.bdbiosciences.com). Signals were acquired for the forward scatter (FSC), side scatter (SSC), and the FL1-H channels, excited by the argon laser (488 nm) using standard protocols. At least 10,000 events were acquired per sample.

Microscopy
Pictures of living cells were taken on a Zeiss LSM 510 META confocal microscope (Carl Zeiss, Thornwood, NJ, http://www.zeiss.com) using a multichannel scan (green fluorescent protein [GFP] and differential interference contrast [DIC]), excited by the argon laser (488 nm).

Enzyme-Linked Solid Phase Immunosorbent Assay
An ELISA kit (reference BMS242, Bender MedSystems, Vienna, Austria, http://www.bendermedsystems.com) was used to quantify human LIF in hMADS cells conditioned medium. Supernatants were collected after a 48-hour incubation and centrifuged to remove debris (10 minutes at 6,000g). Each supernatant was diluted 1/1,000 and assayed immediately in duplicate wells as described by the manufacturer. hMADS cells from three different donors were used in this assay. For each donor, two supernatants obtained from two independent nucleofection experiments were assayed. Results are the averages (± SE) of the LIF concentrations obtained for these independent nucleofection experiments.

Reverse Transcriptase Polymerase Chain Reaction Analysis
Total RNA was extracted using TRI-Reagent Kit (Euromedex, Souffelweyersheim, France, http://www.euromedex.com) according to the manufacturer's instructions, and reverse transcriptase polymerase chain reaction (RT-PCR) analysis was conducted as described previously [23]. Twenty-five PCR cycles were performed for all transcripts, except peroxisome proliferator-activated receptor-{gamma} (PPAR{gamma}; 30 cycles). All primer sequences are detailed in Table 2. An aliquot of PCR products was analyzed on 2% ethidium bromide-stained agarose.


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  		Table 2. Polymerase chain reaction primers; sequences are given 5' to 3'

 
Materials and Chemicals
Cell culture medium and reagents were purchased from Invitrogen, and fetal calf serum from Dutscher S.A. (Brumath, France, http://www.dutscher.fr). FGF2 was from Peprotech (Rocky Hill, NJ, http://www.peprotech.com). Rosiglitazone (BRL4953) was a gift from Dr. J.F. Dole (GlaxoSmithKline, King of Prussia, PA, http://www.gsk.com). LIF was obtained by collecting supernatants from COS cells transfected with pCAG-LIF.


   RESULTS
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 Acknowledgments
 References
 
hMADS Cells Are Efficiently Transfected with Nucleofection
To overexpress transgenes in hMADS cells, we have tried to transfect them with several classic methods, using the pPyCAGGFPIP plasmid. As summarized in Table 3, we obtained very low percentages of EGFP+ cells with calcium phosphate precipitation, Lipofectamine 2000, FuGENE 6, and FuGENE HD. Exgen 500 was also tested, but proved to be highly toxic for hMADS cells. These results prompted us to test the nucleofection technique. To determine the best-suited electrical conditions, we tested the programs that were recommended by the manufacturer Amaxa Biosciences. We calculated recovery as the ratio between the number of cells that had adhered 24 hours after transfection over the number of total original number of cells exposed to nucleofection. We then determined the percentage of EGFP+ cells, among cells that had survived, using FACS analysis. For that purpose, 6 x 105 hMADS cells were nucleofected with 3 µg of pmaxGFP plasmid (3.49 kb, CMV promoter), using the C-17 and U-23 programs. The U-23 program induced the highest mortality. Recovery was 13.6% ± 6.1%, against 31.2% ± 4.5% for C-17. FACS analysis of 48-hour nucleofected hMADS cells detected 67.2% EGFP+ cells with the U-23 program and 38.0% with the C-17 one (Fig. 1A). Fluorescence microscopy observations confirmed that EGFP expression was more frequent and intense in cells transfected with the U-23 program (Fig. 1B). Despite the low recovery observed with the U-23 program, cells that survived were not impaired in their proliferative capacities (data not shown). Therefore, for the subsequent studies, we used the U-23 program.


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  		Table 3. Transfection efficiencies of human multipotent adipose tissue-derived stem cells transfected with pPyCAGGFPIP using common transfection agents

 


Figure 1
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  		Figure 1. Nucleofection efficiency of the U-23 and C-17 programs on human multipotent adipose tissue-derived stem (hMADS) cells. Passage 19 hMADS3 cells were nucleofected with the pmaxGFP plasmid using the U-23 and C-17 programs and expression of the maxGFP protein was detected 2 days post nucleofection by fluorescence-activated cell sorting (FACS) analysis (A) and fluorescence microscopy (B). In (B), nuclei were stained with 4',6-diamidino-2-phenylindole (DAPI) and are shown in blue.

 
To determine whether nucleofection could allow an efficient transgene expression using a plasmid other than pmaxGFP, we nucleofected hMADS cells with pPyCAGGFPIP (7.29 kb). pPyCAGGFPIP is much larger than pmaxGFP and bears a CAG promoter. EGFP+ cells were detected by fluorescence microscopy as soon as 1 day after nucleofection and were maintained for at least 8 days (Fig. 2A). The day after nucleofection, we could number 54.7% ± 1.1% EGFP+ hMADS cells. The maximal percentage of EGFP+ cells (76.1% ± 1.3%) was obtained 2 days post-transfection. The percentage of EGFP+ cells decreased thereafter, but 8 days after nucleofection, 21.8% ± 2.3% EGFP+ cells were still detected (Fig. 2B, 2C). Similar results were obtained with hMADS cells isolated from three different donors (hMADS2, hMADS3, and hMADS6). Supplemental Figure 1 shows the data obtained for hMADS3, over a 9-day period after nucleofection. Analysis of 48-hour nucleofected hMADS6 cells showed that 70.5% of cells express EGFP. Thus, despite high induced mortality, nucleofection allows efficient expression of a transgene over a 8-day period among the cells that survived.


Figure 2
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  		Figure 2. Evolution of enhanced green fluorescent protein (EGFP) expression in human multipotent adipose tissue-derived stem (hMADS) cells after nucleofection over a 9-day period. Passage 20 hMADS2 cells were nucleofected with the pPyCAGGFPIP construct using the U-23 program. EGFP expression was detected by fluorescence microscopy (A). Bars = 100 µm. The percentage of EGFP+ cells was measured by fluorescence-activated cell sorting (FACS) analysis (B) and subsequently quantified (C). For each time point, three nucleofection experiments were performed. Bars: mean ± SE (n = 3).

 
Nucleofection Allows Stable Transgene Expression in hMADS Cells Without Impairing Their Differentiation Abilities
We next investigated the ability of hMADS cells to stably express a transgene after nucleofection and appropriate selection. Cells were nucleofected with pPyCAGGFPIP and then submitted to puromycin selection 48 hours postnucleofection. After a period of 15 days, the formation of EGFP clones was observed. The number of clones obtained per nucleofection of 6 x 105 cells was between one and two, which was sufficient to establish EGFP+ hMADS cells. Indeed, these clones could be propagated in culture for at least 10 passages before they stopped proliferating. During propagation, EGFP expression was maintained in 97.84% of the cells, as measured by FACS analysis (Fig. 3). Two passages after selection of the clones, EGFP+ hMADS cells were allowed to reach confluency and were shifted to appropriate culture conditions to trigger adipocyte and osteoblast differentiation. EGFP+ hMADS cell clones could differentiate at least as well as nontransfected hMADS cells into both cell types, as we could observe lipid droplet-filled adipocytes and mineralized osteoblasts. No extinction of the transgene expression was observed in fully differentiated cells, as no EGFP-negative cells could be detected by fluorescence microscopy (Fig. 4A). Completion of differentiation was confirmed by oil red O staining (adipocytes) and alizarin staining (osteoblasts), as shown in Figure 4B, as well as by the expression of the adipocyte-specific markers adipocyte fatty acid-binding protein (aFABP) and PPAR{gamma} and the osteoblast-specific markers alkaline phosphatase (ALPL) and core binding factor {alpha} 1 (CBFA1; Fig. 4C). Thus, these results demonstrate that, using nucleofection, hMADS cells clones stably expressing EGFP could be obtained and that these clones retain their differentiation capacities.


Figure 3
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  		Figure 3. Fluorescence-activated cell sorting (FACS) analysis for enhanced green fluorescent protein (EGFP) expression in puromycin selected human multipotent adipose tissue-derived stem (hMADS) cells. Passage 20 hMADS2 cells were nucleofected with the pPyCAGGFPIP plasmid using the U-23 program. Puromycin selection was applied 2 days post-nucleofection and was maintained thereafter. After two passages, the percentage of EGFP+ hMADS cells was checked by FACS analysis.

 


Figure 4
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  		Figure 4. Establishment of EGFP+ hMADS cell clones by nucleofection without impairment of their differentiation abilities. Passage 22 EGFP+ hMADS2 clones were induced to differentiate into adipocytes and osteoblasts. (A): Photographs of EGFP+ adipocytes and osteoblasts (the mineralized matrix is shown in black on the DIC channel). DIC shows the transmitted-light photograph. (B): Oil red O and alizarin staining of a differentiated EGFP+ clone and of control (not transfected) hMADS cells. (C): Reverse transcriptase polymerase chain reaction (RT-PCR) analysis showing expression of aFABP, (PPAR{gamma}, ALPL, and core binding factor {alpha} 1 (CBFA1). Abbreviations: ALPL, alkaline phosphatase; aFABP, fatty acid-binding protein; DIC, differential interference contrast; EGFP, enhanced green fluorescent protein; hMADS, human multipotent adipose tissue-derived stem; PPAR, proliferator-activated receptor.

 
Nucleofection Allows Secretion of Functional Proteins
Finally, we investigated the ability of nucleofected hMADS cells to secrete a functional, transgene-encoded protein. We nucleofected hMADS cells that were isolated from three donors with pCAG-LIF and checked for the levels of secreted LIF with an ELISA. Table 4 shows that pCAG-LIF-nucleofected hMADS cells secrete LIF in the medium, as very high levels are detected (from 397.34 ng/ml for hMADS2 to 586.88 ng/ml for hMADS6). In contrast, supernatants from control cells (e.g., pPyCAGGFPIP-nucleofected cells) displayed amounts of LIF that were barely detectable (0.01–0.02 ng/ml).


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  		Table 4. Concentration of LIF within supernatants of nucleofected hMADS cells from 3 donors

 
LIF has been identified as a key factor for maintenance of mES cell pluripotency, and it is routinely included in mES cell culture media to maintain ES cells in an undifferentiated phenotype [24]. To check that the secreted LIF protein was functional, we tested the hMADS cells' supernatants' ability to maintain mES cells in an undifferentiated state in the absence of exogenous LIF. We used alkaline phosphatase staining and RT-PCR analysis of the pluripotency markers Oct4 and Rex1 to detect undifferentiated ES cells. As shown in Figure 5A, supernatants from pCAG-LIF nucleofected cells allowed the formation of compact, AP+ ES cells colonies, indicative of an undifferentiated phenotype. Conversely, supernatants from control cells did not allow maintenance of this undifferentiated phenotype, as the cells were faintly stained with AP and rapidly spread out from colonies to form a monolayer. In addition, ES cells treated with supernatants from pCAG-LIF nucleofected cells maintained the expression of Oct4 and Rex1 to levels that are comparable to those obtained in cells cultivated with LIF. In contrast, ES cells treated with supernatants from control-nucleofected cells lost the expression of Oct4 and Rex 1 expression, indicative of differentiation (Fig. 5B). In conclusion, nucleo-fected hMADS cells could secrete a transgene-encoded functional LIF protein that is able to maintain ES cells in an undifferentiated state.


Figure 5
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  		Figure 5. Secretion of a functional mouse LIF protein by hMADS cells. Passage 21 hMADS cells from two donors (hMADS2 and hMADS3) were nucleofected with the pCAG-LIF construct, using the U-23 program. Three days after nucleofection, supernatant, were harvested, centrifugated, and added to mouse embryonic stem (mES) cells in the absence of LIF. Five days later, the presence of undifferentiated ES cells colonies was detected by alkaline phosphatase (AP) staining (A) and by the expression of Oct4 and Rex1 mRNAs by reverse transcriptase polymerase chain reaction (RT-PCR) (B). Abbreviations: hMADS, human multipotent adipose tissue-derived stem; LIF, leukemia inhibitory factor; SPN, supernatant.

 

   DISCUSSION
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 Acknowledgments
 References
 
Given their convenient isolation and extensive self-renewal capacities in vitro, adipose tissue-derived stem cells are a promising source of human stem cells for regenerative medicine. Moreover, they can be used to identify molecular pathways involved in stem cell self-renewal and plasticity. For this purpose, gain- and loss-of-function approaches are necessary. Until recently, these experiments have been precluded given the resistance of adipose tissue-derived stem cells to classic transfection methods, including lipofectants. We demonstrate here that nucleofection, an electroporation-based method, is a valuable method to achieve transient transgene expression in hMADS cells. We could detect transgene expression as soon as 8 hours after nucleofection, and we could obtain up to 76.1% EGFP+ cells 2 days postnucleofection. Importantly, we ascertained that nucleofected hMADS cells could secrete a functional, transgene-encoded protein.

In the frame of this study, two plasmids bearing different promoters were used: pmaxGFP, which codes for maxGFP expression under the control of a CMV promoter, and pPyCAGGFPIP, which codes for EGFP under the control of a CAG promoter. Differences in EGFP+ cells percentages were noticed for these two constructs (67.2% for pmaxGFP and 80.8% with pPyCAGGFPIP, with the U-23 program). Several parameters could account for these differences. First, the plasmids used here have very different sizes (3.49 kb for pmaxGFP and 7.29 kb for pPyCAGGFPIP). In addition, maxGFP and EGFP might have different detection thresholds, which could result in variations in the determination of the number of GFP+ cells by FACS analysis. Finally, the promoters driving the expression of GFP in these two constructs are different. Further investigations to identify the best-suited promoter for transgene expression in adipose tissue-derived stem cells should be very useful. In this study, three hMADS cell populations, isolated from three donors, were used. Our data concerning transient EGFP expression (Fig. 2, supplemental Fig. 1) and LIF secretion (Table 4) suggest donor-to-donor variability. This variability is acceptable; thus, nucleofection could be widely used to transfect adipose tissue-derived stem cells.

We used the pPyCAGGFPIP plasmid, which bears a puromycin resistance cassette, to establish hMADS cells stably expressing EGFP. After a 3-day selection period, an almost pure population of EGFP+ cells could be obtained. Fifteen days later, EGFP+ clones had formed and could be propagated in vitro for several passages without loss of the transgene. Moreover, they were successfully induced to differentiate into EGFP+ adipocytes and osteoblasts. These data demonstrate that stable stem cell clones can be generated with nucleofection, and that this process does not alter stem cell proliferation and differentiation abilities.

Despite the encouraging results we obtained regarding stable transgene expression in hMADS cells, improvements need to be made. In fact, the number of clones obtained for 6 x 105 nucleofected cells can be considered low, especially when compared to the high percentage of EGFP+ cells that were detected 2 days postnucleofection. Thus, methods that would increase the yield of hMADS cells stably expressing the transgene are of great interest. Recently, two systems have shown to be highly efficient in establishing stable transgene expression in nucleofected stem cells. Quenneville et al. [17] used the integrase from phage {Phi}C31 to establish human myoblasts with stable transgene expression. In this system, the plasmid coding for the protein of interest contains attB sequences. It is conucleofected with a plasmid coding for the {Phi}C31 integrase. The integrase allows insertion of the sequence of interest in "pseudo-attB" sites of the genome. Conucleofection of both plasmids allowed a 15-fold increase in the number of colonies with stable expression of the transgene when compared to nucleofection of the plasmid of interest alone [17]. Another system, the Sleeping Beauty (SB) transposase/transposon system, has yielded promising results in multipotent adult progenitor cells and human primary T cells [25, 26]. In this system, the SB transposase mediates a cut-and-paste mechanism of transposition, which results in the excision of the DNA sequence of interest and its reinsertion into a TA target dinucleotide of the genome. Preliminary data using the SB transposase/transposon system in hMADS cells indicate that it might be a powerful system to increase stable expression in adipose tissue-derived stem cells (personal observations).

Adipose tissue-derived stem cells are phenotypically very close, although not identical, to bone marrow-derived stem cells (unpublished data). Very recently, Aslan et al., [27] as well as Aluigi et al., [15] have reported that nucleofection could be efficiently used to transfect mesenchymal stem cells from bone marrow (hMSCs). However, these authors did not investigate the ability of nucleofection to generate stable transgene expression. Clearly, if mesenchymal stem cells from bone marrow or adipose tissue are to be used as vehicles for gene therapy, it is essential that they express the gene(s) of interest in a stable manner. Peister et al. [28] managed to obtain stably expressing hMSCs using classic electroporation. Their results, although highly encouraging, can not be directly compared with ours, as they used primary cultures of hMSCs. Indeed, they performed the electroporation on cells that had been isolated from bone marrow 10 days earlier. In our study, we used mesenchymal stem cells that had been cultured for more than 20 passages after isolation from adipose tissue.

The major drawbacks of nucleofection are the cost (the Nucleofector machine and specific kits containing buffer and cuvettes are required) and the induced cell mortality. Indeed, as for regular electroporation methods, poor cell viability has been a major concern of the nucleofection technology [29, 30]. Nevertheless, using similar parameters, Aluigi et al. [15] observed cell recoveries that were almost twice as high as the one we measured with hMADS cells. They assumed that this high recovery yield resulted from a medium change after overnight incubation after nucleofection. As we carried out our experiments in a similar way, we believe that this medium change cannot account for the observed differences. Rather, differences in cell types or in media composition could explain these variations. However, despite a recovery rate of 13.6%, the remaining viable hMADS cells expressed high levels of the transgene and showed no impairment of their proliferative and differentiation abilities. With the intended use of cells for functional studies such as overexpression and promoter transactivation, nucleofection can be considered a valuable method because a high percentage of the remaining cells express the transgene. Thus, a low recovery does not prevent the establishment and use of hMADS cells with transient or stable transgene expression, as long as the appropriate culture conditions are used.

In conclusion, our data clearly demonstrate that nucleofection can be used to generate transient and stable transgene expression in hMADS cells, without altering their stem cell features. Thus, nucleofection is a very promising nonviral transfection method to perform gain- and loss-of-function studies in adipose tissue-derived stem cells, and maybe other cell types that are currently classified as difficult to transfect.


   DISCLOSURES
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
Acknowledgments
 References
 
The authors indicate no potential conflicts of interest.


   ACKNOWLEDGMENTS
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 Acknowledgments
References
 
This work was supported in part by funds from the Centre National de la Recherche Scientifique, the Association pour la Recherche sur le Cancer (contract number 3,721). We thank Dr. Ez-Zoubir Amri for helpful technical advice and discussions, as well as for providing of some reagents. The Stem Cells and Differentiation Group is supported by the Fondation pour la Recherche Médicale. Z.L.E. is supported by a fellowship from Yves-Saint Laurent Beauté. D.C. is an Institut National de la Santé et de la Recherche Médicale (INSERM) established investigator.


   REFERENCES
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 Acknowledgments
 References
 

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