Culture of Undifferentiated hES Cells
H1 and H9 hES cells were obtained from Wicell (Madison, WI). hES cells were maintained on irradiated mouse embryonic fibroblasts (MEFs) at 5% CO2
in medium containing Knockout-Dulbecco’s modified Eagle medium (KO-DMEM), 20% serum replacement, 1% nonessential amino acids, 1 mM l
-glutamine, 0.1 mM β-mercaptoethanol, and 4 ng/ml fibroblast growth factor-2 (FGF-2; GIBCO BRL). The cells were passaged in a 4-day cycle by incubating with 1 mg/ml collagenase IV at 37°
C for 45 min, maintaining the cells in small clumps. To avoid contamination by MEFs in the transfection (liposome, electroporation, nucleofection) analysis, the hES cells were cultured on Matrigel in MEF-conditioned medium for one passage prior to transfection. Approximately, 2
cells were plated uniformly in six-well plates prior to transfection. Samples of the cells were immunostained with anti-SSEA-4 (Chemicon, Temecula, CA) and anti-Oct-4 (Santa Cruz Biotechnology, Santa Cruz, CA) antibodies. Immunofluorescent labeling was analyzed using a Zeiss LSM 510 Confocal Laser Scanning Microscope equipped with a Coherent Mira 900 tunable Ti:sapphire laser for two-photon excitation (Zeiss, Minneapolis, MN).
Construction of pUb-eGFP-Fluc and pUb-eGFP-Fluc-SV40-neomycin
The pUb-eGFP-Fluc construct used for lentiviral transduction has been described previously [18
]. The pUb-eGFP-Fluc-SV40-neomycin was generated by cleaving pUb-GFP-Fluc at the Bam
H1 restriction enzymes and blunt-end ligating the SV40-neomycin construct. This plasmid was used for stable transfection of hES cells using neomycin as a selection marker.
Plasmid Transfection Methods
(a) Lipofection: lipofection was performed 1 day after plating H1 and H9 hES cells (4–5
/well) onto Matrigel at a confluency of 60–70%. Qiagen lipofection 2000 was used according to the manufacturer’s protocol. Briefly, 5
μg of plasmid DNA (pUb-eGFP-Fluc) was diluted in 250
μl of KO-DMEM followed by addition of 10
μl of liposomal reagent. The mixture was incubated for 15–20 min at room temperature. The mixture was then added to 1,000
μl of pre-warmed conditioned medium and transferred to one well of a six-well plates. (b) Electroporation: 5
μg of plasmid DNA (pUb-eGFP-Fluc) was diluted in 500
μl of phosphate-buffered saline (PBS) and added to 4–5
hES H1 and H9 cells suspended in 500
μl of hES culture medium. Electroporation was performed in a 4-mm gap cuvette using a Gene Pulser (BioRad, München, Germany) with the electric parameters set at 300 V and 220
μF. After pulsing, the cells were incubated in the cuvette at room temperature for 10 min. The cells were transferred to new medium and plated onto Matrigel-coated six-well plates at a density of 2
. Each plate was analyzed for eGFP expression under a fluorescence microscope at 24, 48, and 72 h post-electroporation. (c) Nucleofection: 4-5
hES H1 and H9 cells were centrifuged for 5 min at 800 rpm and 4°C. The culture medium was removed and cells were resuspended in 100
μl of the nucleofector solution (Amaxa Biosystems, Cologne, Germany). DNA (5
μg) was added, cells and DNA were gently mixed, and cells were electroporated using the Nucleofector™ device (Amaxa Biosystems). Transfections were performed with a program (A23) that was selected in a series of pilot transfection experiments with the eGFP reporter gene and proven to result in the highest transfection efficiency. Directly after transfection, hES cells were placed into the culture medium.
Lentiviral Vector Preparation and Transduction
A self-inactivating lentivirus was prepared by transient transfection of 293 T cells as described previously [16
]. Briefly, 12
µg of the HIV-1 packaging plasmid (peGFP-Fluc/pRRE; delta-8.9) containing the eGFP-Fluc reporter gene were cotransfected into 293 T cells with 9
µg of vesicular stomatitis virus G glycoprotein-pseudotyped envelope vector (pVSV-G), and 6.25
µg of REV plasmid (pRSV-REV). Lentivirus supernatant was collected after 72 h and concentrated by sediment centrifugation with SW29 rotor at 50,000×g
for 2 hours. Concentrated virus was titrated on 293 T cells. hES H1 and H9 cells (4–5
) were incubated with virus particles at MOI of 10 and 8
µg/ml polybrene (Sigma). The media was changed after 12 h by fresh medium.
Generation of Stable Cell Line
Stable hES cell lines were generated by fluorescence-activated cell sorting (FACS) in the lentiviral transduction group and by G418 selection (50–100
µg/ml) over 2 weeks in the nucleofection group. Colonies were isolated, propagated, and analyzed for eGFP expression and Fluc activity.
Cell Viability Analysis Cell viability was determined using dissociated cells prior to plating, as well as at 24 and 48 h after transfection. Viability was measured using trypan blue exclusion and expressed as a percentage of the initial number of cells used for each transfection.
Immunohistochemistry The H1 and H9 hES cells treated with the various transfection protocols were plated onto cover slips within six-well plates, fixed in 5% paraformaldehyde for 1 min, and blocked with 1% bovine serum albumin in PBS. They were incubated with the primary antibody in PBS at 37°C for 1 h, followed by several PBS washes, and incubation with a secondary antibody at 37°C for 1 h, with final PBS washes. Slides were mounted with Vectorshield (Vector Laboratories, Inc., Burlingame, CA, USA).
Fluorescence-activated Cell Sorter Analysis
Twenty-four hours after transfection, the cells were washed twice with KO-DMEM and dissociated with Accutase II. After centrifugation, the cells were resuspended at 1
cells/ml in PBS and stored on ice for a maximum of 1 h before analysis. Acquisition was performed on a FACS Calibur system (BD Biosciences, Heidelberg, Germany) and samples were analyzed using FlowJo software (Tree Star Inc., Ashland, OR). Analysis-gating criteria for eGFP-expressing cells were set according to the level of auto-fluorescence of a non-transfected control.
Transplantation of hES Cells into Nude Mice
Harvested control hES and stable hES-DF cells (transduced with lentivirus) were kept on ice for <30 min for optimal viability. Adult immunodeficient nu/nu mice (n
5) were injected subcutaneously with 1
hES-DF cells in 50
µL of PBS. Control animals (n
3) received 1
non-transduced hES cells. All animals had an uneventful recovery and underwent bioluminescence imaging at various time points. Study protocols were approved by the Stanford Animal Research Committee.
Optical Bioluminescence Imaging of hES Cell Transplantation
Bioluminescence imaging was performed with the Xenogen In Vivo Imaging System (IVIS, Alameda, CA). Following intraperitoneal injection of the reporter probe d
-luciferin (375 mg/kg body weight), animals were imaged for 30 min with 1-min acquisition intervals. The same mice were scanned at various time points for a 6-week period according to the specific study design. Bioluminescence was quantified in units of maximum photons per second per centimeter squared per steradian (P s−1
), as described previously [18
Quantitative data were expressed as mean
SD. Means were compared using one-way ANOVA and the Student t
values of less than 0.05 were considered statistically significant.