Twenty two adult female Sprague-Dawley rats (Orient Bio lnc., Seongnam, Korea) weighing 180-210 g were used in this study. All animals were housed individually in cages, with free access to food and water in a climate-controlled room. This study was approved by the Institutional Animal Care and Use Committee of the Asan Institute for Life Science. At the beginning of the study, each animal was anesthetized with Zoletil 50 (Virbac S.A, Carros, France) and Rompun (Bayer, Leverkusen, Germany) at doses of 37.5 mg/kg and 6 mg/kg, respectively, using an intraperitoneal (i.p.) injection. A total of 22 rats were administered unilateral injections of 14 µg 6-OHDA hydrochloride (Sigma, St. Louis, MO, USA) in 4 µL of 0.9% saline with 0.1% ascorbic acid delivered into the right medial forebrain bundle at the following coordinates : anteroposterior -4.4 mm, lateral +1.2 mm relative to the bregma and ventral -7.8 mm from the dura by Paxinos's rat atlas, with the tooth bar set at -2.4 mm. The injections were delivered at a rate of 1 µL/min using a Hamilton syringe (33-gauge) and an automated microsyringe pump (Harvard Apparatus, Holliston, MA, USA). Once the injection was finished, the needle was left in place for 5 minutes to prevent backflow of the solution. Among 22 rats, 18 rats which showed apomorphine-induced rotation exceeded 6 rotations per min after 4 weeks of 6-OHDA injection (success rate 81.8%) were selected for this study. Then, HB-NSC cells were injected in 9 rats (HB-NSC group) and PBS in 9 rats (control group). However, 2 rats were dead during PET study, so the final numbers of experimental animals became 9 in HB-NSC group and 7 in control group.

HB-NSC (San Pedro, CA, USA) were expanded in brain stem-cell growth medium according to the manufacturer's instructions. For formation of homogenous aggregates, polyethylene glycol (PEG) hydrogel microwell arrays with a diameter of 500 µm were produced using micro-fabrication procedures, as reported previously13). The expanded HB-NSC were allowed to form aggregations within the PEG hydrogel microwell arrays. Briefly, HB-NSC at an approximate cell density of 1×10⁶ cells/200 µL were deposited into each microwell array, and then placed in a 5% CO₂ humidified incubator for 30 min to allow the cells to settle. Non-adherent cells were carefully removed by washing with PBS, and the adherent cells were allowed to form aggregations within each microwell. The cell aggregations were retrieved from the PEG hydrogel after 5 days of culture in the brain stem-cell growth medium (Celprogen, San Pedro, CA, USA). The cell aggregates are visible with the naked eyes and the diameter of each aggregate is approximately 200 µm. And then, the cell aggregation were administered in sterilized PBS at a concentration of 8×10⁴ cells/8 µL. We could evaluate the concentration of cell aggregation by cell counting with trypan blue viability staining which was partially harvested from cell aggregation solution and transformed into cell suspension.

The cell aggregations transplanted into the right striatum of the PD rat model (HB-NSC group, n=9) using a 22 G Hamilton syringe and a stereotaxic microinjection device (coordinates : AP +0.6 mm from the bregma, ML +3.5 mm from the midline, DV -4.5 mm, nose bar +2.3 mm) 4 weeks after the 6-OHDA injection. The control group (n=7) received 8 µL of PBS (pH 7.4) injected at the same coordinates. All injections used a rate of 1 µL/min, and the needle was held in place for 5 min after the injection, followed by withdrawal at a rate of 1 mm/min. Postoperatively, all rats received daily Cyclosporine A (Chong Kun Dang Pharm, Seoul, Korea) at 10 mg/kg (i.p.) until the animals were sacrificed.

The stepping tests were performed six times : prior to any treatment, 1 week after 6-OHDA injection, and 2, 4, 6, and 8 weeks after cell transplantation, as previously described24), with slight modifications. Briefly, both hind limbs were firmly held in one hand of the experimenter, while the other hand was used to hold one of the forelimbs. The anterior end of the animal was lowered onto a treadmill (Jeung Do Bio & Plant Co., Seoul, Korea), which was set at a rate of 0.9 m/5 s. The rat's body remained stationary while the unilateral forelimb was allowed to touch the moving treadmill track for >5 seconds while the treadmill was operating. All of the experiments were video recorded to count the number of adjusted steps taken in the backward direction. Every rat performed the stepping test twice during every session, and the data were averaged. The data are expressed as the percentage of contralateral forelimb steps versus ipsilateral forelimb steps.

Apomorphine-induced rotation tests were performed in transparent cylinders. The rotation tests were conducted 1 week after 6-OHDA injection, and 4 and 8 weeks after cell transplantation, as previously described25), with slight modifications. Briefly, as soon as 0.5 mg/kg apomorphine (Sigma; in sterile water, injected subcutaneously) was administered, the rat was harnessed to an automated rotometer (Panlab, Barcelona, Spain) and its rotational behavior was assessed over 45 minutes. Data are expressed as the net average rotations (contralateral-ipsilateral turns) per minute. 6-OHDA-lesioned rats that demonstrated >6 rotations per minute were selected for inclusion in the present study.

All animals were scanned using the microPET Focus 120 system (Siemens Medical Solutions, Inc., Erlagen, Germany), which includes a 12×12 array of lutetium oxyorthosilicate and 96 detector blocks. Glucose metabolic activity was assessed in the control (n=3) and HB-NSC groups (n=5) using [¹⁸F]-FDG at 10 weeks posttransplantation. After fasting for at least 12 h, the rats were injected with 1 mCi [¹⁸F]-FDG in 0.2 mL of sterile saline via the tail vein. One hour after the injection, the animals were scanned for 30 min under isoflurane anesthesia.

Dopamine transporter function was assessed with the same animals in the control (n=3) and HB-NSC group (n=5) using [¹⁸F]-FP-CIT, which binds to the dopamine transporter with high affinity, at 1 or 2 weeks after 6-OHDA injection (PD status before cell transplantation) and at 5 and 10 weeks after cell transplantation. Rats were injected with 1 mCi [¹⁸F]-FP-CIT in 0.4 mL sterile saline via the tail vein, immediately followed by dynamic scanning for 90 minutes.

The [¹⁸F]-FDG PET data were reconstructed using a 3-dimensional (3D) maximum a posteriori (MAP) algorithm, and the [¹⁸F]-FP-CIT data were reconstructed using a 2D ordered-subset expectation maximization16). PET images were analyzed using the ASIPro software (Siemens Preclinical Solutions, Knoxville, TN, USA) to generate a 3D volume of interest that consisted of three regions of interest (ROI). To produce 3D volume of interest, we selected a coronal image which showed highest uptake of striatum, and then, three consecutive ROIs in axial images which show striatum were taken on the selected coronal image base. Each ROI of axial image was composed of 5×5 pixels30). Data are expressed as the percentage of ipsilateral standardized uptake values based on the mean uptake in the 3D volume of interest against the contralateral standardized uptake values. The equation of standardized uptake values is [tissue concentration (MBq/cc)]/[injected dose (MBq)/body weight (g)].

Tissue was harvested 12 weeks after cell transplantation. Rats were transcardially and sequentially perfused with 0.9% saline containing 10000 IU heparin (Hanlim Pharm, Seoul, Korea) and 4% paraformaldehyde in PBS. Extracted brain tissues were postfixed overnight in the same fixative, followed by incubation and dehydration in 30% sucrose until they sank. The IHC and IF procedures were performed as previously described11,17). Serial sections from frozen tissue, including the striatum (anteroposterior +2.5 to 0.0 mm) ,were produced and the brain tissue was cut into 40 µm sections on a sliding microtome (HM450; Sliding electric microtome, Thermo Scientific). The sections were stored in 0.08% sodium azide (Sigma-Aldrich, St. Louis, USA) in PBS at 4℃ until analysis.

For IHC, striatal sections were incubated overnight with primary antibodies (anti-TH monoclonal 1 : 5000, Sigma Aldrich, St. Louis, USA) diluted in PBS (pH 7.4) containing 0.5% BSA (bioWORLD, Dublin, USA). These sections were subsequently incubated for 2 h at room temperature with biotinylated secondary antibodies produced in mouse (Vector Laboratories, Burlingame, CA, USA) and diluted 1 : 200 in PBS containing 0.5% BSA. TH staining was detected by adding diaminobenzidine (R&D System, Minneapolis, MN, USA). Sections were mounted on gelatin-coated slides and dried. Diaminobenzidine-treated tissues were dehydrated in a series of alcohol and clearing agents, and a coverslip was applied. TH-positive cells were observed in the coronal sections of the striatum using a Nikon 80i microscope (Nikon, Tokyo, Japan) at 100× magnification using the NIS-Elements F3.0 software (Nikon, Tokyo, Japan).

For IF, tissue sections were incubated with blocking solution containing 1% BSA, 0.2% Triton X-100 (Sigma Aldrich, St. Louis, USA) and 0.05% sodium azide, rinsed in 0.5% BSA in PBS twice and then incubated with anti-TH monoclonal (1 : 5000, Sigma Aldrich, St. Louis, USA) and anti-human β2 microglobulin polyclonal (1 : 800, Dako, Seoul, Korea) antibodies for 1 h at room temperature. The sections were washed three times and incubated for 90 min with Alexa 546-labeled anti-mouse IgG (1 : 200, Invitrogen, Grand Island, NY, USA) and Alexa 488-labeled anti-rabbit IgG (1 : 200, Invitrogen Grand Island, NY, USA). After washing twice, the sections were mounted onto gelatin-coated slides, dried at 50℃ for 30 min, and cover-slipped with VECTOR mounting (Dako, Seoul, Korea) medium for fluorescence. Fluorescent images were obtained using a confocal microscope (TCS-ST2; Leica, Wetzlar, Germany).

All data are presented as mean±SD. All statistical analyses were performed using SPSS (version 12.0.1; SPSS Inc., Chicago, IL, USA). Behavioral parameters were analyzed using Mann-Whitney U test, and PET data were analyzed by the paired sample t-test for working out the relative importance of each data which was mat-ched one-on-one. A p-value <0.05 was considered significant.

The stepping test showed no significant increase in contralateral paw touches up to 4 weeks posttransplantation; however, at 6 weeks posttransplantation, a significant improvement of 52.9% was measured. At 8 weeks posttransplantation, the increase was 48.9% and this improvement was maintained through to 10 weeks, at which time the increase was 64.2% in the HB-NSC group compared to the control group (Fig. 1A). In apomorphine-induced rotation tests, the HB-NSC group showed a slight decrease in contralateral rotations at week 8, compared to week 4; however, the difference was not statistically significant (Fig. 1B).

To examine glucose metabolism, [¹⁸F]-FDG microPET scans were used. There was no significant decrease in uptake of [¹⁸F]-FDG in either the control or HB-NSC transplantation group, indicating that glucose metabolism was normal, as is characteristic of PD (Fig. 2A). Using [¹⁸F]-FP-CIT to quantify dopamine transporter activity, a gradual increase in radioactivity in the lesion-side striatum was detected in the HB-NSC group and, in particular, there was a statistically significant increase in dopamine trans-porter activity by 10 weeks posttransplantation compared to the pretransplantation state (48.5±3.9% vs. 41.0±2.6%, respectively) (Fig. 2B). These microPET data are consistent with the results of the stepping test in which significant improvement was detected from 6 weeks posttransplantation onward.

IHC and IF analysis revealed the presence of tyrosine hydroxylase (TH)-positive cells in the ipsilateral striatum of the HB-NSC transplantation group (Fig. 3). High-magnification images showed that the TH-positive cells corresponded to the cells positive for the human β2 microglobulin marker (Fig. 3G), which demonstrates HB-NSC origin. The TH-positive cells in the striatum were surrounded by a round margin that indicated the area of the transplantation (Fig. 3B, D, E). These results indicate that the implanted HB-NSC aggregates successfully differentiated into dopaminergic cells within the host striatal tissue. This finding differs from our previous study using single-cell suspensions for injection which demonstrated abnormal parallel staining of dendrites (Fig. 4A, C). Dendrites of the stained cells extended in various directions, suggesting network formation with the neighboring cells (Fig. 4B, D).