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a Stem Cell Institute, Hematology, Oncology and Transplantation Division,% a8 D( b) ]0 s6 W2 w D
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b Department of Genetics, Cell Biology and Development, Department of Medicine, University of Minnesota, Minneapolis, Minnesota, USA
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K; A; F: ^6 j) }Key Words. Embryonic stem cells ? Adult stem cells ? Transfection ? Nucleofection% u* W: v' r* a- V
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Correspondence: Catherine M. Verfaillie, M.D., Stem Cell Institute, Department of Medicine, University of Minnesota, 420 Delaware Street, Minneapolis, Minnesota 55455, USA. Telephone: 612-625-0602; Fax: 612-624-2436; e-mail: verfa001@umn.edu7 `# U; ?( J" I+ x& y3 H
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ABSTRACT3 q4 w, s ]# W8 n
z- ?( B- C1 B" Y- J/ ZMouse embryonic stem (ES) cells have been successfully used to study the developmental milestones from a single cell to a mouse. Generation of knockout mouse models by targeted disruption of essential genes provides useful insights into genes that regulate development, allowing investigators to dissect molecular developmental mechanisms. Gene targeting has also been used to create mouse models for many human genetic diseases, facilitating development of therapeutic strategies . ES cells are also useful as an in vitro model system for the study of cell differentiation. Several transcription factors have been demonstrated to regulate differentiation of stem cells to specific cell types such as heart, pancreas, liver, and neuron . Ectopic overexpression of such factors stimulates ES cells to differentiate to certain cell types .* i/ q5 X2 J5 [4 ^1 p. j/ L
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Besides mouse ES cells, several other types of stem cells have been well characterized. Human embryonic carcinoma (hEC) cells isolated from germ line tumors resemble ES cells and have been extensively used to study embryogenesis . Recently, human embryonic stem (hES) cells have been isolated from human blastocysts . Several types of adult stem cells have been isolated from various sources that display varying differentiation potential . Such a repertoire of stem cells and their ability to be coaxed to differentiate into specific cell types provides new opportunities in regenerative medicine.
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5 P( ~) q! ~2 [* V9 vSeveral methods are available to achieve introduction of foreign DNA that carries a gene of interest for ectopic expression in cells. The most commonly used method for generation of transiently and stably transfected mouse ES cells has been electroporation. Several commercial liposome-based methods have also been used, with Lipofectamine (Life Technologies; Invitrogen, Carlsbad, CA) being the most widely used method . Human ES cells have been transfected by Exgen , a liposome-based method, and by electroporation . Viral vectors such as adenovirus, adeno-associated virus, and lentivirus can also be used to overexpress cDNA in mouse and human ES cells . However, the use of these vectors requires each cDNA to be cloned into specific vectors, thereby hindering rapid screening of potential transcription factors involved in differentiation of ES cells into definitive cell types.- G& U/ u0 @0 b: u' o, p
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Here we describe a nonviral, high-efficiency method of transfecting a variety of mouse and human stem cells using the Amaxa Nucleofector (Amaxa Biosystems, Cologne, Germany). The Amaxa Nucleofector technology is an electroporation-based method in which a combination of a specific nucleofector solution and specific electric parameters achieves delivery of plasmid DNA into the cell nucleus, thereby resulting in enhanced gene expression. This method is effective in transfecting hard-to-transfect cells such as T cells and dendritic cells . Using enhanced green fluorescent protein (eGFP) as the reporter, we demonstrate that nucleofection achieves greater than 60% transient transfection efficiency in mouse ES cells and increases stable transfection of mouse ES cells by 10-fold compared with electroporation. Further, stable transfected cells maintain their ES cell properties. Nucleofection can be used for rapid screening to study the effect of ectopic expression of various transcription factors.
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We further show that nucleofection transfects the human EC cell line, Ntera2, and hES, as well as multipotent adult progenitor cells (MAPCs) and hematopoietic progenitors from fresh mouse bone marrow cells. Based on these studies, we conclude that nucleofection is an efficient method for gene transfer into stem cells from human and mouse.* J f3 ?/ O6 b8 c
( t. T: z, L5 {; j7 YMATERIALS AND METHODS0 O3 s% Q' P! X; f& _( D9 G+ m
8 R& v/ I, {- X) z! z3 c+ l2 J4 ~Nucleofection Achieves High Transient Transfection Efficiency Compared with Electroporation. y3 L* d, X: X, D- @
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The mouse ES cell line R1 (mES-R1) was used to study the potential of nucleofection as a transfection method in these cells. The transfection efficiency by nucleofection was compared to electroporation as both methods achieve transfection in solution and the treatment of cells is similar in both, eliminating differences in transfection efficiencies arising from cell handling. The mES-R1 cells were harvested, and 1 million cells were either electroporated at 330 V 250 μF, nucleoporated using the mES cell nucleofection solution (program A23), or left untreated. Figure 1A shows the brightfield and green fluorescence images of cells electroporated (top two panels) and cells nucleoporated (bottom two panels), 24 hours after transfection. Green fluorescent cells were visible in the nucleoporated sample as early as 8–10 hours post-transfection, and fluorescence intensity increased by 24 hours after transfection. In contrast, the number of green fluorescent cells and their intensity did not significantly increase, even after 48 and 72 hours in the case of electroporation (data not shown). Figure 1B shows the average GFP fluorescence 48 hours post-transfection. The percentage of GFP cells was determined by FACS analysis. While the percentage of GFP cells is 6.41% ( 4.59%; n = 3) in the case of electroporation, nucleofection achieved a transfection efficiency of 63.66% ( 9.36%; n = 8) under the conditions tested.) _- G; i( p$ o* f; _% {! X
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Figure 1. Transfection efficiency of mouse embryonic stem cells (mES-R1) with enhanced green fluorescent protein (eGFP). (A): Approximately 1 million mES-R1 cells grown on gelatin were transfected with the plasmid pEGFP-N1, either by electroporation at 330 V and 250 μF or by nucleofection using the program A23. Cells were visualized for GFP 24 hours later using fluorescence microscopy. (B): Transient transfection efficiency: fluorescence-activated cell sorter (FACS) analysis was carried out 24 hours post-transfection to monitor the percentage of GFP cells with no transfection (No Zap, Avg. n = 8), following electroporation (Ep; Avg. n = 3), or following nucleofection (Np; Avg. n = 8). Standard deviation is represented as error bars. (C): Stable transfection efficiency: Cells transfected with pEGFP-N1 either via electroporation (Ep) or by nucleofection (Np) were treated with 400 μg/ml Geneticin 48 hours post-transfection, and the number of G418-resistant cells were counted following 2 weeks of selection. The average of three independent experiments is represented as a bar graph. Standard deviation is represented as error bars.
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Increased Stable Transfection via Nucleofection
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$ _4 h8 Q L ~ lThe mES-R1 cells transfected with pEGFP-N1 plasmid were subjected to drug selection for 2 weeks in the presence of 400 mg/ml Geneticin (G418) to select for ES cells with stably integrated plasmid. Figure 1C shows the percentage of G418-resistant cells relative to untransfected mES-R1 cells maintained in parallel in the absence of G418. Results are represented as an average of three independent sets of experiments. The percentage of stable transfectants arising from electroporation was 0.59% ( 0.33%; n = 3), while the rate of stable transfection via nucleofection was 12.34% ( 2.43%; n = 3).
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Pooled G418-Resistant Clones
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Nucleoporated G418-resistant mES-R1 clones (over 150 clones on a 10-cm dish) were pooled following 2 weeks of selection and cultured for an additional 2 weeks. The cells were then analyzed for expression of ES cell markers SSEA1, Oct4, and Rex1. Figure 2A shows brightfield and fluorescence images of the pooled stable cells showing that the majority of cells still express GFP. In the control unmanipulated mES-R1 cells, 74% were SSEA1 by FACS analysis; 57% of G418-resistant mES-R1 were GFP cells, and 61% of the GFP cells were SSEA1 (Fig. 2B). QRT-PCR analysis revealed identical levels of Oct4 and Rex1 expression in unmanipulated ES cells and pooled G418-resistant clones (Fig. 2C). These results demonstrate that several key stem cell characteristics are maintained in mES-R1 cells following nucleofection.
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Figure 2. Pooled mouse embryonic stem cells (mES-R1) stable clones expressing enhanced green fluorescent protein (eGFP). (A): Brightfield (top panel) and green fluorescence (bottom panel) of GFP-mES-R1 stable cells following 6 weeks in culture. (B): G418-resistant colonies were subjected to fluorescence-activated cell sorter (FACS) analysis to monitor the percentage of cells positive for both GFP and the stem cell marker SSEA1. The left panel shows SSEA1 staining of untransfected mES-R1 cells, and the right panel shows SSEA1 staining of eGFP-transfected, G418-resistant mES-R1 cells. (C): Polymerase chain reaction (PCR) analysis of stem cell markers Oct4 and Rex1 in G418-resistant cells and untransfected mES cells. Abbreviations: NTC, no template control; R1, untransfected mES-R1 cells; R1-G, eGFP-transfected mES-R1 cells.2 \1 ^8 a* t) g$ @+ `+ C- I* ^" v
; ]/ {$ C2 g: Z" M! c5 m pDifferentiation of an EGFPmES-R1 Clone into Cardiac Cells$ I, B9 G* J) ?8 s
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Cardiac differentiation was evoked by culturing the GFP mES-R1 cells as embryoid bodies using a slight modification of the method described by Maltsev et al. . Following 7 days of differentiation, beating cells were either harvested in RLT buffer for RNA isolation and QRT-PCR analysis for cardiac markers (Gata4, Nkx2.5, and cardiac actinin) or seeded for immunocytochemistry. Figure 3 shows staining patterns of cardiac markers in the mES-R1 cells expressing GFP after differentiation. The top panels show staining of GFP cells with the early cardiac marker Nkx2.5, and the bottom panels are cells stained with the late cardiac-specific marker, cardiac troponin T. As seen in the merged panel of GFP and TRITC–labeled cardiac-specific antibodies, stable GFP expressing mES-R1 cells induced to the cardiac lineage express early or late cardiac markers. Further analysis was carried out by QRT-PCR for expression of the cardiac markers Nkx2.5, Gata4, and cardiac actinin. The results obtained are summarized in Table 2. Relative to levels in undifferentiated cells, expression of Gata4 was increased by 79-fold, while Nkx2.5 and cardiac actinin increased by 8- and 24-fold, respectively.9 d# f' o5 ^3 \; o$ e; Y
* J4 K$ o) z! q2 I2 `# {( WFigure 3. Differentiation of green fluorescent protein (GFP)–positive mouse embryonic stem cells (mES-R1) into cardiomyocytes. Immunostaining of GFP-mES-R1 cell–derived cardiomyocytes for Nkx2.5 and cardiac troponin T after 7 days of dimethylsulfoxide (DMSO) treatment.
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- w; N+ x# p. G! U! XTable 2. Quantitative polymerase chain reaction (QPCR) analysis of Nkx2.5, Gata4, and cardiac actin in differentiated and undifferentiated GFP-mES-R1 cells (see Fig. 3)
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Contribution of EGFP-mES-R1 Cells to Chimeras
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The mES-R1 cells nucleoporated with the pEGFP-N1 construct were selected in G418 for 4 weeks, and the resulting colonies were replated into 24-well plates; each clone was expanded separately. The expanded clones with highest fluorescence intensity as determined by fluorescence microscopy and greater than 95% GFP cells determined by FACS analysis were chosen for blastocyst injection. From 10–12 cells were injected into each blastocyst. The injected blastocysts were implanted into pseudopregnant female mice, and pups born were monitored for chimerism based on coat color. Figure 4 shows the pups obtained from one such litter., Q8 {1 A4 _+ L3 f% _/ ~
" R9 B8 Y5 p8 q2 B5 UFigure 4. Contribution of stable green fluorescent protein (GFP) expressing mouse embryonic stem cells (mES-R1) to chimeras. Chimeras obtained from one representative experiment showed one pup with wild-type coat color (black), two pups with less than 20% chimerism, and one pup with more than 20% chimerism as estimated by coat color.
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QPCR analysis for GFP was performed on genomic DNA isolated from tail clips of chimeric animals. The GFP mES-R1 cells used for blastocyst injection were used as a positive control, and the amount of PCR product obtained from these cells was set as 100%. Based on the amount of GFP PCR product relative to the positive control, animals were classified as 0%, 20% chimeric. We used FACS analysis of cells isolated from various organs to detect GFP cells in animals with more than 20% chimerism. In one chimera analyzed in detail, 2%–3% GFP cells were detected in hematopoietic tissue such as peripheral blood, spleen, and thymus, while nearly 16% GFP cells were detected in kidney. Presence of GFP cells was further confirmed by immunohistochemical staining on frozen sections of kidney using anti-GFP antibodies (Table 3). In animals with no contribution (as assessed by QRT-PCR of genomic DNA) FACS and immunohistochemistry failed to detect any GFP cells.
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# ?$ p* ~; o$ uTable 3. Percentage of chimerism in three representative mice, as determined by genomic DNA analysis of tail clips and FACS analysis of isolated kidney cells and peripheral blood (PB) (see Fig. 4)0 Y/ l2 R7 ?0 Y2 M2 O
, B) a7 M* _, j+ J2 l3 LEctopic Expression of Ngn3 in mES-R1 Cells( l. H8 _( J5 [
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Enhanced transfection efficiencies obtained in mES cells via nucleofection are particularly valuable for screens in which stem cells are induced to differentiate into specific cell types by ectopic expression of regulatory genes such as transcription factors. One such gene that has been well characterized is Ngn3, known to be required early during pancreas development . Overexpression of Ngn3 in mouse ES cells activates downstream genes that specify pancreatic development such as Pax6, Is11, and NeuroD . In those studies, Lipofectamine (Invitrogen), which generally achieves 20%–25% transfection efficiency in mES cells, was used as the method of transfection. We hypothesized that nucleofection of mES cells with Ngn3 would yield greater numbers of transfected cells and result in a more homogenous population of differentiating ES cells. The mES-R1 cells were grown in the presence of RA to induce Gata4 expression. Cells were then trypsinized and nucleoporated with either a GFP or an Ngn3-GFP construct and plated on gelatin-coated dishes. After 72 hours, cells were harvested in RLT buffer, and QRT-PCR was performed to monitor for expression of Pax6, Is11, and NeuroD. Figure 5 shows the image of an agarose gel with the QRT-PCR products of various genes tested. Quantitation of the QRT-PCR results are summarized in Table 4. Cells transfected with GFP plasmid alone show levels of Ngn3 mRNA that are 0.3% of that obtained using fresh mouse pancreas. The relative level of Ngn3 (compared with mouse pancreas) increases to 515% in cells transfected with Ngn3-GFP, a 1,618-fold increase over the control GFP-transfected ES cells. This demonstrates that Ngn3 is introduced into the cells and is being transcribed efficiently. To assess functional NGN3 PROTEIN activity, we assayed for levels of genes known to be activated by Ngn3 during pancreas development, including Pax6, Is11, and NeuroD. Expression levels of these genes were found to increase by 32-fold, 32-fold, and 15-fold, respectively, relative to GFP transfected cells (Table 4).
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Figure 5. Overexpression of Neurogenin3 (Ngn3) in mouse embryonic stem cells (mES-R1). Quantitative reverse transcription poly-merase chain reaction (QRT-PCR) analysis of pancreatic markers in mES-R1 cells transfected with GFP (–) or ngn3-GFP ( ).& z0 |8 g i) i, M/ w4 l% X
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Table 4. Quantitative reverse transcription polymerase chain reaction (QRT-PCR) data showing the expression profiles of pancreatic markers in GFP- and Ngn3-GFP–transfected mES-R1 cells (see Fig. 5)
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" m; ?# v: a- C( NTransfection of hEC and hES Cells
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The plasmid pEGFP-N1 was transfected into the hEC cell line, Ntera2, either by electroporation or by nucleofection. FACS analysis of GFP expression 48 hours post-transfection was used to quantitate transfection efficiency. Ntera2 cells were either electroporated or nucleoporated using two different conditions (Fig. 6A). Electroporation yielded 50% transient transfection efficiency, whereas we achieved nearly 95% efficiency using nucleofection. Average transient transfection efficiencies obtained with Ntera 2 cells (n = 3) are twofold higher via nucleofection than by electroporation (Table 5). Recently, Zwaka and Thompson described electroporation conditions of 320 Vand 200 μF as being optimal for obtaining high-efficiency transfection of hES cells. The transient transfection of human H1 ES cells as clumps via this method was compared with nucleofection methods. Figure 6B gives the results of a representative FACS analysis, showing percentage of GFP cells under various electroporation and nucleofection conditions, 72 hours post-transfection. While electroporation at two different electric parameters yielded 5%–6% transfectants, nucleofection achieved 20%–22% transfection (Table 5).
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Figure 6. Transfection efficiency in human embryonic carcinoma (hEC) and human embryonic stem (hES) cells. Cells were transfected with the plasmid pEGFP-N1 and the presence of green fluorescent protein (GFP) was monitored in transfected cells by fluorescence microscopy and fluorescence-activated cell sorter (FACS) analysis. Data were collected from at least three independent experiments to calculate mean values and standard deviation. (A): Representative FACS analysis of NTERA2 cells. Approximately 106 cells were either subjected to electroporation at the electric parameters of 330 Vand 500 μF or 320 V and 200 μF or were nucleoporated using programs A23 or A27. After 58 hours, cells were trypsinized and subjected to FACS analysis for GFP. (B): 1–2 x 106 human H1 ES cells were either electroporated or nucleoporated as clumps. The parameters used for electroporation were 220 V and 960 μF or 320 V and 300 μF. Cells were nucleoporated using programs A23 or A27. Cells were trypsinized 72 hours post-transfection and subjected to FACS analysis for GFP.( a8 A/ M) @+ M- U8 g
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Table 5. Average percentage of GFP cells as measured by FACS analysis (see Fig. 6)
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Adult Stem Cell and Progenitor Cells: j. r! P* ]) V
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MAPCs are a rare population of cells derived from adult bone marrow . These cells must be maintained at low density to maintain multipotency (200–1,000 cells/cm2). Consequently, various transfection methods that require high-density cell cultures such as calcium phosphate (Clontech), AVET (Bender MedSystems), Effectine (Qiagen), and DMRIE-C (Invitrogen), are largely ineffective in transfecting these cells. However, modest transfection efficiency (. U2 H; x6 b' o. z! M0 J
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Table 6. Transfection efficiency of progenitor cells from adult tissue
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4 T V/ E$ E3 @/ H; ^9 e n4 BTotal mouse bone marrow cells were also efficiently transfected via nucleofection. However, this method resulted in nearly 90% cell death following nucleofection under optimum conditions. Total bone marrow cells depleted for lineage-positive cells (Lin–fraction) were also transfected at similar frequencies as total bone marrow cells (25% versus 18%; Table 6). Colony-forming assays of the transfected bone marrow cells on methylcellulose for 2 weeks yielded green fluorescent CFU-M (macrophage) and CFU-GM (granulocyte/macrophage) colonies, indicating that hematopoietic progenitors are indeed stably transfected via nucleofection (data not shown).$ W& F( ~: |8 j
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DISCUSSION
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1 m! N* p3 F* h4 T& yThe authors thank Dr. Gerald Gradwohl (Institut National de la Sant谷 et de la Recherche M谷dicale , Strasbourg, France) for kindly providing the Ngn3-GFP construct, Dr. Janet Rossant, Mount Sinai Hospital, Toronto, Canada, for the mES cells (line R1), and Drs. Yves Heremans and Lucas Chase for their help with Ngn3 overexpression experiments. The SSEA1 and SSEA4 monoclonal antibodies developed by Davor Solter and Barbara Knowles were obtained from the Developmental Studies Hybridoma Bank developed under the auspices of the National Institute of Child and Human Development (NICHD) and maintained by the University of Iowa, Department of Biological Sciences, Iowa City, IA 52242. This work was funded by grants from the Fanconi Anemia Research Foundation (U.L.), the Leukemia and Lymphoma Society (U.L.), the Government of Navarra (B.P.), the Lillehei Heart Institute (B.P.), the Minnesota Medical Foundation (E.C.), and the National Institutes of Health (HL-72000, D.S.K.; HL-67932, C.M.V.). Beatriz Pelacho was not involved with studies involving human embryonic stem cells.
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