Transplantation of Mouse Embryonic Stem Cells into the …

Animal and surgical procedures

Mongolian gerbils of both genders aged 36months with healthy external ears were used for this study. The animals were born and raised in low-noise environment [median sound level was 40dB sound pressure level (SPL)] at the Animal Research Facility of the Medical University of South Carolina. All aspects of the animal research were conducted in accordance with the guidelines of the local Institutional Animal Care and Use Committee.

Animals were anesthetized with pentobarbital sodium (50mg/kg) and given atropine (0.2mg/kg) to reduce respiratory secretions. Body temperature was maintained between 37C and 38C using a heating pad. Sterile procedures were used to open the bulla and place about 2040l of a 1mM ouabain (Sigma, O-3125) solution in normal saline in the RW niche. After 30min, the ouabain solution was removed by wicking with a small piece of filter paper. The surface of the bulla was fully closed with dental cement, and the incisions were closed with sutures. Postsurgical discomfort was treated with buprenorphine if necessary. The animals allowed to recover for 13days after ouabain exposure before transplantation were designated as the early post-injury (EPI) transplant group, whereas animals allowed to recover for 7days or longer were designated as the late post-injury (LPI) transplant group.

Approaches used for ESC transplantation and the state of differentiation of ESCs after RA neural induction. A Surgical approach to expose the round window (RW) niche of the gerbil cochlea for ESC transplantation. The basilar membrane comprises the translucent zone and is visible through the RW membrane. The bony osseous spiral lamina, the wall of Rosenthals canal (RC), and the central modiolus are located in the opaque zone. Scale bar=500m. B Schematic diagram illustrating three delivery routes of ESCs into: (1) Rosenthals canal, (2) perilymph of scala tympani (ST), (3) and endolymph of scala media (SM). The photograph was modified from a radial paraffin section of the basal turn from a normal young gerbil. Sa stapedial arteria, Sp.L spiral ligament, SV scala vestibuli. Scale bar=100m. C The majority of the cultured ESCs stained for nestin (green). Nuclei were countstained with bis-benzimide (blue). Scale bar=20m.

Surviving ESCs 34weeks after transplant

To suppress rejection of mouse ESCs, gerbils were given daily injection of cyclosporine A (15mg kg1 day1 s.c., Sandoz Pharmaceutical Corp., NJ) starting 1day before surgery and terminating the day before sacrifice. The same dose was given to all animals in this study.

Mouse ESCs were prepared for transplantation from (1) wild-type D3 cells, (2) D3 cells engineered to express enhanced green fluorescent protein (EGFP), or (3) D3 cells modified to over-express bcl2 (D.I. Gottlieb, Washington University, St. Louis, MO). The D3 cell line was isolated from day4 129/Sv blastocysts (Doetschman et al. 1985). The details for EGFP expression and bcl2 gene modification have been described previously (Adams et al. 2003; Wei et al. 2005). The ESC lines were maintained at low passage (<20) with normal karyotype. Cells were passaged, and neural differentiation was induced using the 4/4+ RA induction protocol (Bain et al. 1995; Bain and Gottlieb 1998). Briefly, undifferentiated cells were maintained in T25 flasks in ES cell growth media (ESGM) consisting of Dulbeccos modified Eagle media (with l-glutamine, without pyruvate, Gibco 11965-043) supplemented with 10% fetal bovine serum, 10% new born calf serum, 8.5g/ml guanosine, 8g/ml adenosine, 7.3g/ml cytidine, 7.3g/ml uridine, 2.4g/ml thymidine, 1,000U/ml of leukemia inhibitory factor (LIF, Gibco 13275-011), and 104M -mercaptoethanol.

For neural lineage induction, cells were harvested from the growth flasks by trypsinization with 0.25% trypsin and ethylenediaminetetraacetic acid (EDTA) in Hankss salt solution (Gibco, 15050) for 10min. One quarter of the cells from a T25 flask were seeded into a standard 100-mm bacterial Petri dish in ESC induction medium (ESIM). The ESIM is similar to ESGM, but without LIF and -mercaptoethanol. After 2days, the cell aggregates and the media were removed from the dish, and cells were allowed to settle for 10min in a 15-ml centrifuge tube. The medium was then aspirated and replaced with fresh ESIM. Cells were then returned to the culture dish for an additional 2days. The culture medium was replaced with ESIM containing 5107M RA (all-trans RA, Sigma R-2625), and cells were cultured for an additional 4days before harvesting for transplant. The state of ESC differentiation after RA 4/4+ neural induction was in agreement with the results of previous studies (Meyer et al. 2005; Wei et al. 2005). Although there was heterogeneity among the cell types generated by this protocol, a great majority (76.23.5%) of the cultured ESCs were positive for neuronal precursor and/or immature neuron markers such as nestin (Fig.1C).

The methods for recording the compound action potential (CAP) response, distortion product otoacoustic emissions (DPOAEs), and endocochlear potential (EP) were similar to those previously described (Lang et al. 2005, 2006). Physiological data were obtained from ESC-implanted ears and a group of untreated ears. The animals were anesthetized as described above and fitted to a head holder in a sound- and vibration-isolated booth. The pinna and surrounding tissue were removed and bulla opened widely. The CAP electrode was placed on the bony rim of the RW niche and an acoustic assembly, including a probe-tube microphone (B&K 4134, Bruel and Kjaer, Norcross, GA) and driver (Beyer DT-48, Beyerdynamic, Farmingdale, NY), was sealed to the bony ear canal with closed-cell foam. Tone pips were calculated in the frequency domain using Tucker Davis Technologies (Alachua, FL) equipment and software. CAP thresholds were obtained visually with an oscilloscope online at half-octave frequencies from 0.5 to 20kHz with tone pips of 1.8-ms total duration with cos2 rise/fall times of 0.55ms. DPOAEs were measured with an Ariel board (Ariel, Canbury, NJ) and CUBeDISP software (Etymotic Research, ELK grove Village, IL). DPOAEs were obtained with an opened bulla after removing the pinna and underlying tissue. The intensity levels of both primaries were fixed at 50dB SPL. Primary tones were swept from f2=4 to 20kHz with f1/f2 ratio of 1.2 and a resolution of 10 points per octave.

EPs were measured with a micropipette filled with 0.2M KCl yielding an impedance of approximately 2030M. The output of the micropipette was tied to an electrometer (World Precision Instruments FD 223) for direct recording of the potential. EP was defined as the voltage difference between scala media and a pool of isotonic saline on the neck muscles. The micropipette was introduced into the scala media via 30- to 50-m holes drilled through the otic capsule of the three cochlear turns. We first measured the EP in the apical turn (T3), followed by the middle turn (T2) and basal turn (T1). This procedure minimizes the trauma of inserting the micropipette into one turn and causing a reduction of the EP in other turns (Schmiedt et al. 2002; Lang et al. 2002).

The inner ears were fixed for 68h with 4% paraformaldehyde and then decalcified with EDTA. Tissues were embedded in PARAPLAST@ for paraffin sectioning. Deparaffinized and rehydrated sections were immersed in blocking solution for 20min and then incubated overnight at 4C with a primary antibody diluted in phosphate-buffered saline (pH7.4). The primary antibodies used in this study were rabbit anti-GFP (1:200, A11122) or mouse anti-GFP (1:100, A11120) (Molecular Probes, Eugene, OR), rat monoclonal antibody to mouse-specific brain membrane (1:50, M2, Developmental Studies Hybridoma Bank, Iowa City, IA), rabbit anti-bcl2 (1:200, sc492) (Santa Cruz, Santa Cruz, CA), anti-mouse CD45R (1:200, sc19597; Santa Cruz), mouse anti-neurofilament 200 (1:200, Clone N52, N0142; Sigma, Atlanta, GA) and mouse anti-glial fibrillary acidic protein (GFAP; 1:200, MAB360; Chemicon, Temecula, CA). An antigen retrieval treatment was used for immunostaining with mouse brain membrane-specific antibody M2.

The antibodies employed in this study have been widely used and are well characterized. The rabbit anti-GFP polyclonal antiserum was raised against GFP isolated directly from the jellyfish, Aequorea victoria, and has been used for detection of native GFP, GFP variants, and most GFP fusion proteins (Chalfie et al. 1994; Senut et al. 2004). No staining was seen when rabbit anti-GFP antibody was applied to tissues from ears injected with wild-type D3 ESCs. The rat monoclonal antibody M2 was raised against a mouse-specific glial and neuronal cell membrane glycoprotein (Lagenaur and Schachner 1981). The M2 antibody recognizes a 45-kDa band in Western blots and has been used widely as a marker to identify mouse neural cells in host tissues after xenogeneic transplantation (Eriksson et al. 2003; Gates et al. 1998). Anti-bcl2 reacts with bcl2 of mouse, rat, and human origin by Western blotting, immunoprecipitation, and immunohistochemistry and does not cross-react with other apoptosis-associated proteins (Thomas-Mudge et al. 2004; Weisleder et al. 2004). This antibody recognizes a single band around 29kDa in Western blots (manufacturers technical information from Santa Cruz). CD45R (RA3-6B2) is a rat monoclonal IgG2a antibody raised against an extracelluar domain of the transmembrane glycoprotein CD45 and is expressed broadly among hematopoietic cells including macrophages and microglia (Bhave et al. 1998). Monoclonal anti-neurofilament 200 reacts with a single 200-kDa band in both alkaline phosphatase dephosphorylated and untreated preparations of rat spinal cord (manufacturers technical information from Sigma) and specifically stains nerve fibers in the inner ear (Lang et al. 2006; Wise et al. 2005). The GFAP monoclonal antibody recognizes a 50-kDa band by immunoblotting (manufacturers technical information from Chemicon) and has been used extensively to label astrocytes and neoplastic cells of glial lineage in the central nervous system (McLendon and Bigner 1994; Kasischke et al. 2006; Ward et al. 2004). Control staining for all primary antibodies included omission or substitution with similar dilutions of non-immune serum of the appropriate species. No specific staining was detected in any of the control experiments.

Secondary antibodies were biotinylated, and binding was detected with fluorescent (FITC)-conjugated avidin D (1:100; Vector, Burlingame, CA). The procedure for detection of a second antigen with double labeling was the same as for the first antigen but substituting Texas red conjugated avidin D (1:100; Vector) for visualization. Nuclei were counterstained with propidium iodide or bis-benzimide.

The sections were examined with either a Zeiss LSM5 Pascal confocal microscope (Carl Zeiss Inc., Jena, Germany) or a Zeiss Axioplan microscope equipped with a 100-W mercury light source. The captured images were processed using Image Pro Plus software (Media Cybernetics, MD), Zeiss LSM Image Browser Version 3,2,0,70 (Carl Zeiss Inc.) and Adobe Photoshop CS.

Five to six sections approximately 50m apart from each other from the mid-modiolar region were used for cell counts. The observed sections included all three cochlear turns and five vestibular organs. The surviving ESCs were identified by direct fluorescent microscopy for GFP or labeling with M2 antibody in combination with the nuclear marker bis-benzimide. All data are reported as meanSEM. Statistical comparisons of the number of surviving ESCs in EPI model compared to in the LPI model as well as the percentage of GFAP-positive ESCs in RC versus the perilymphatic space were obtained using the Students t test (SPSS, Chicago, IL). A value of p<0.05 was considered statistically significant.

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Transplantation of Mouse Embryonic Stem Cells into the ...