MRI reporter gene (RG) construct and transduction of embryonic stem cells (ESCs) using p2K7 lentiviral vector
Firefly luciferase (fluc) was cloned between the N-terminus of HA antigen and the C-terminus of myc
antigen of pDispaly (Invitrogen, Carlsbad, CA), generating a RG consisting of the following sequence: Igκ-HA-fluc-myc
-PDGFR-TM. The murine Ig κ-chain leader sequence directed the fusion protein to the ribosomal secretory pathway and the platelet derived growth factor receptor transmembrane domain (PDGFR-TM) anchored it to the plasma membrane to express the antigens on the surface of ESCs. Following the cloning of this RG into pENTR™
5′-TOPO vector (Invitrogen, Carlsbad, CA), the promoter and the RG were directionally cloned into the p2K7 lentiviral vector using the Gateway LR plus clonase enzyme (Invitrogen, Carlsbad, CA)(17
). Two different promoters (EF-1α or human ubiquitin C) were compared for their expression of the RG. The ESCs were incubated with viral supernatant for 24 hours followed by blasticidin selection for 3 days, generating RG transduced ESCs (ESC-RGs).
Culture of undifferentiated ESCs
The hESCs (H9, WiCell and the National Cell Bank, Madison, WI) and mESCs (E14 from Weissman Laboratory, Stanford University) were maintained in undifferentiated and pluripotent state by culturing on irradiated mouse embryonic fibroblast cells in the culture medium(18
). Both hESCs and mESCs were cultured at 37.1C° and 5% CO2
in a humidified incubator.
Fluorescence activated cell sorter (FACS) analysis of RG expression
To determine the RG expression of myc and HA antigens on the cell surface, ESC-RGs were labeled with either FITC-conjugated anti-myc antibody (FITC-myc-MAb) or PE-conjugated anti-HA antibody (PE-HA-MAb, Miltenyi Biotech, Auburn, CA), followed by analysis for FITC by FL1 channel (excitation at 488 nm by a 15-mW argon laser and detection above 530 nm) and for PE by FL2 channel (excitation at 488 nm by a 15-mW argon laser and detection above 585 nm) with appropriate compensation using FACS Caliber (Becton Dickinson, San Jose, CA). Using FlowJo v7.2.5 (Tree Star, Ashland, OR), histogram was generated and the percentage of myc or HA expressing cells was calculated by subtracting non-transduced cells from the RG transduced cells.
Labeling of viable ESCs with SPIO-MAb
To assess in vitro
MR viability signal of mouse and human ESC-RGs, the cells were labeled with 20 μL of either SPIO-conjugated anti-myc
-MAb) or SPIO-conjugated anti-HA antibody (SPIO-HA-MAb, Miltenyi Biotech, Auburn, CA). The SPIO-myc
- and SPIO-HA-MAb consist of a superparamagnetic iron oxide core with polysaccharide coating, linked covalently to monoclonal antibodies against myc
and HA antigens. The mean diameter of the SPIO is approximately 50 nm. In order to establish the specificity of the RG-mediated assessment of cell viability, 2 in vitro
negative control groups consisting of non-transduced ESCs and apoptotic ESC-RGs incubated under the same conditions were established. After labeling the cells with SPIO-HA- and SPIO-myc-MAbs, all cells were washed twice with PBS (1mL) and centrifuged at 600 RPM for 5 minutes. Apoptosis was induced by incubating ESC-RGs with 10 μM of doxorubicin (Sigma, St. Louis, MO) for 2 hours prior to labeling by SPIO-MAb(19
In vitro optical bioluminescence imaging (BLI)
D-luciferin was added to the culture media of ESC-RGs at a concentration of 15 mg/L. Non-transduced ESCs were used for negative control. Cells were imaged using IVIS –Spectrum (Caliper, Mountain view, CA) for 30 minutes with 1-minute acquisition intervals. Bioluminescence was quantified in units of average photons per second per centimeter squared per steradian (P·s−1
) using Living Image 2.5 software (Caliper, Mountain view, CA)(20
In vitro molecular MRI
There were three 1×106 of SPIO-HA- and SPIO-myc-MAb labeled cell groups: 1) mouse and human ESC-RGs, 2) mouse and human ESCs (non-transduced), and 3) mouse apoptotic ESC-RGs. The cells were suspended in 200 μL of PBS and then placed in a 330 μl PCR microfuge tube. These microfuge tubes containing the cells were stabilized within a phantom made of 0.7% agar and 1% copper sulfate. The phantom was placed in the iso-center of knee coil and scanned using Signa 3.0 T Excite HD scanner (GE Healthcare System, Milwaukee, WI). A GRE sequence using the following parameters optimized T2*-weighted imaging to maximize the signal from SPIO (TR 100 ms, TE 20 to 60 ms, FA 45°, matrix 128×128, NEX 1, FOV 12, slice thickness 1 mm). The images were analyzed using ImageJ 1.41 software (NIH, Bethesda, MD). Contrast-to-noise ratio (CNR) was calculated as CNR = (SIcell − SIphantom)/SD of the image noise.
In vivo molecular MRI
Animal care and interventions were provided in accordance with the Laboratory Animal Welfare Act. The animal protocols were approved by the administrative panel on laboratory animal care at Stanford University. The dosage of SPIO-MAb, the incubation time delay prior to MRI acquisition, and echo time (TE) were optimized using a mouse hind limb model. The hind limb model was generated by transplanting 2×106 mouse ESC-RGs into the hind limb muscles of severe combined immunodeficiency (SCID) mice (n=7). After acquiring pre-administration GRE images using the following parameters (TE minimum to 15 ms, TR 500 ms, NEX 1, matrix 128×128, slice thickness 1 mm) on Signa 3.0 T Excite HD scanner, three different doses (100 μL, 150 μL and 200 μL of SPIO-MAb) were administered to different mice through the tail vein. Both hind limbs of these mice were scanned repeatedly using the same sequence and parameters. In order to achieve optimal T2*-weighted imaging, TE was increased from minimum to 15 ms. The most favorable image quality, based on minimum dephasing artifact extending to the adjacent anatomical structure, was obtained at TE of 10ms.
For creation of myocardial infarction, SCID mice (8–12 weeks old, n=18, Charles River Laboratories, Wilmington, MA) were anesthetized by 3% isoflurane at 1L/min oxygen and endotracheally intubated with a 20-gauge angiocatheter (Ethicon Endo-Surgery, Cincinnati, OH). Ventilation was maintained by 1% isoflurane at 1L/min oxygen with a Harvard rodent ventilator (Harvard Apparatus, Holliston, MA). Myocardial infarction was created in all the mice by permanent ligation of the distal left anterior descending (LAD) artery, resulting in distal anterior or apical infarct. 0.25×106 of mouse ESC-RGs were transplanted into the proximal border of the peri-infarct area at the mid-ventricular level to minimize confounding signal artifacts, which could arise from the infarcted apex. All mice (n=18) underwent LAD ligation and were imaged before and after intravenous administration of SPIO-MAb at post-transplant days 3, 5, 7, 10 and 14. Two mice were euthanized for histology at each time point. For in vivo MRI, a 1-inch-diameter dedicated small animal receiver coil was built and tuned for 3T imaging.
Based on the data from the hind limb model, 80 μL each of SPIO-myc- and SPIO-HA-MAb was administered into each mouse through the tail vein at each time point. SPIO-MAb was allowed to circulate for 12 to 18 hours to enhance specific binding with myc and HA antigens and to remove unbound antibody from the system. To determine the specificity of our in vivo imaging technique, two negative control groups were established. In the first negative control group (n=4), the mice received 160 μL of SPIO-HIS-MAb (Miltenyi Biotech, Auburn, CA), targeted to unrelated antigen, HIS. In the second negative control group (n=2), the mice received 40 μL of non-targeted SPIO (Feridex I.V., Advanced Magnetics, Cambridge, MA).
For in vivo MRI, the mice were anesthetized with 1% isoflurane at 1 L/min oxygen and placed in the supine position. The ECG gating was optimized by two subcutaneous precordial leads and the respiratory gating by an abdominal respiratory pad. Both cardiac and respiratory gating were monitored and controlled by PC-SAM (SA Instrument, Stoney brook, NY). The body temperature was monitored to maintain a normal physiologic status. The mice were imaged using cardiac and respiratory gated GRE sequence with the following parameters (TE 10 ms, FA 60°, matrix 256×256, NEX 10, FOV 3 and slice thickness 1mm, space 0 mm). Three to four slices were acquired for a short axis view to cover the majority of the left ventricle and single frame image was acquired per imaging plane. In vivo images were analyzed using ImageJ 1.41. CNR was calculated as CNR = (SISPIO-ESC-RG − SIseptum)/SD of the image noise before and after SPIO-MAb administration. The CNR values in were obtained by subtracting pre-contrast CNR in the ROI from post-contrast CNR in the ROI.
Longitudinal assessment of the proliferation of viable mESC-RGs in the infarcted myocardium of SCID mice
In vivo BLI
Mice were anesthetized with 1% isoflurane at 1 L/min oxygen and placed in the supine position within the IVIS imaging chamber. Following intraperitoneal injection of D-luciferin (375 mg/kg body weight), mice were imaged for 30 minutes with 3-minute acquisition intervals using IVIS - spectrum. Images were analyzed in the same manner with the in vitro BLI.
Two mice were euthanized after each time point. Hearts were sectioned along the short axis plane and routinely processed for H&E staining. H&E stained slides were interpreted by a pathologist blinded to the study.
Descriptive statistics include mean and standard deviation of mean. Comparison between two groups was performed using student’s t-test. For comparison between multiple groups, ANOVA with Post Hoc test was utilized. All data analyses were done by SPSS 16.0 (SPSS Inc, Chicago, IL). Significance was assumed when p <0.05.