A High-Efficiency Induction of Dopaminergic Cells from Human Umbilical Mesenchymal Stem Cells for the Treatment of Hemiparkinsonian Rats

Plasmid Construction and Transfection

To generate the pCMV-HA-Nurr1 plasmid, human Nurr1 cDNA was amplified and inserted into the Bgl II and Xho I sites of the pCMV-HA vector (#K6003-1; Clontech, Mountain View, CA, USA). The constructive plasmid was confirmed by restriction digestion and sequence analysis. After that, HUMSCs at a concentration of 5 × 10⁵ cells were transfected with pCMV-HA or pCMV-HA-Nurr1 by electroporation (Amaxa Biosystems Nucleofector^® Device; Lonza, Cologne, Germany) using Human MSC Nucleofector^™ Kit (Amaxa #VPE-1001; Lonza). Briefly, cells were resuspended in transfection buffer (Amaxa #VPE-1001; Lonza), and then transferred to a sterile cuvette (Amaxa #VPE-1001; Lonza). Subsequently, cells were incubated with 2 μg pCMV-HA-Nurr1 or pCMV-HA and electroporated via a high transfection efficiency program, rinsed with sterile culture medium (21870-076; Gibco), and finally transferred to a culture dish.

Cultivation and Differentiation of HUMSCs

With the written consent of the parents, fresh human umbilical cords (n = 10) from both sexes were obtained after birth and collected in Hank's balanced salt solution (14185-052; Gibco) at 4°C. HUMSCs were obtained and propagated as in a previous study (10), with some modifications (Fig. 1A). Sprague–Dawley rats at the age of 7 days were deeply anesthetized by intraperitoneal injection of 10% chloride hydrate, and their brains were removed and triturated in Ca²⁺/Mg²⁺-free buffer. The dissociated cells were resuspended in 10% fetal bovine serum in Dulbecco modified Eagle medium (DMEM; 12100-046; Gibco) and incubated at 37°C. The medium was collected 5 days later as neuron-conditioned medium (NCM) for the culture of HUMSCs (20). First, HUMSCs were transfected with pCMV-HA or pCMV-HA-Nurr1. After 24 h of cultivation, Nurr1-transfected HUMSCs were separated into four groups: DMEM 9 days; NCM 9 days; NCM 6 days followed by FGF8 (100 ng/ml, 423-F8; R&D Systems, Minneapolis, MN, USA) + Shh (500 ng/ml, 461-SH; R&D Systems) 3 days; and finally, FGF8 + Shh 3 days then NCM 6 days. Vector-transfected HUMSCs were separated into two groups: NCM 6 days later then FGF8 + Shh 3 days and FGF8 + Shh 3 days followed by NCM 6 days.

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Figure 1.

Development of dopaminergic cell differentiation protocols for HUMSCs. (A) The grouping and protocol of in vitro incubation of HUMSCs. Tyrosine hydroxylase-positive populations were generated from undifferentiated HUMSCs via a multistep in vitro differentiation method. Abbreviations: DMEM, Dulbecco's modified Eagle's medium; FBS, fetal bovine serum; FGF, fibroblast growth factor; HUMSC, human umbilical mesenchymal stem cell; NCM, neuron-conditioned medium; Shh, sonic hedgehog. (B) Realtime PCR analyses for Nurr1 expression. The expression of mRNA was assessed after 24 h of transduction. Nurr1 expression was significantly higher in the Nurr1-transduced groups compared with the vector-transduced groups. *Significant difference at p < 0.05 compared with the vector group. (C) Photomicrographs showing the morphology of cultured HUMSCs. (a) HUMSCs incubated in 10% FBS-DMEM for 9 days. (b) Vector-expressing HUMSCs cultured in FGF8 + Shh for 3 days and then NCM for 6 days. (c) Nurr1-expressing HUMSCs cultured in FGF8 + Shh for 3 days and then NCM for 6 days. (d) Nurr1-expressing HUMSCs cultured in NCM for 6 days and then FGF8 + Shh for 3 days. After 9 days of differentiation, morphological changes were observed in Nurr1-expressing HUMSCs by phase contrast microscopy. Undifferentiated cells showed fibroblast-like shape morphology, whereas Nurr1-expressing cells protruded, exhibiting an elongation of neurite-like process (arrows). Scale bar: 100 μm.

RNA Preparation and RT-PCR

Total RNA purification and RT-PCR was performed as described previously (35). The primer sequences were as follows: Nurr1, 5′-AGTATGGGTCCTCGCCTCAA-3′ and 5′-ATTCTCCCGAAGAGTGGTAACTGT-3′; TH, 5′-TGTCCACGCTGTACTGGTTCAC-3′ and 5′-GTCGAAGGCCCGAATCTCA-3′; AADC, 5′-ACAGACTTAACGGGAGCCTTTAGA-3′ and 5′-GTGATAAGCCCTGAATCCTGATG-3′; DAT, 5′-CATAGACGGCATCAGAGCATACC-3′ and 5′-CCGCGTCAATCCAAACAGA-3′; GAPDH, 5′-TGGTATCGTGGAAGGACTCA-3′ and 5′-AGTGGGTGTCGCTGTTGAAG-3′.

Immunofluorescence of Tyrosine Hydroxylase (TH)

HUMSCs and brain sections were fixed with 4% paraformaldehyde (Sigma-Aldrich) in 0.1 M phosphate buffer for 20 min and washed with 0.1 M phosphate buffer (Sigma-Aldrich). They were then treated with a blocking solution [0.05% Triton X-100 (Sigma-Aldrich), 5% normal goat serum (S-1000; Vector Laboratories, Burlingame, CA, USA), and 3% bovine serum albumin (Sigma-Aldrich)] for 30 min to prevent nonspecific antibody–antigen binding. The cells and brain sections were then reacted with primary antibodies (mouse anti-TH, 1:333, MAB318; Chemicon, Temecula, CA, USA) at 4°C for 18 h, washed with 0.1 M phosphate-buffered saline (PBS; Sigma-Aldrich), reacted with secondary antibodies (all from Chemicon) at room temperature for 1 h, and washed again with 0.1 M PBS. Brain sections were reacted with ABC complex (ABC KIT, PK-4000; Vector Laboratories) at room temperature for 1 h, washed with 0.1 M PBS, and finally developed with 3,3′-diaminobenzidine (DAB, Sigma-Aldrich) (5 mg DAB, 3.5 μl of 30% H₂O₂ in 10 ml of 50 mM Tris buffer). HUMSCs were reacted with secondary antibodies (Rhodamine-conjugated goat anti-mouse IgG for TH, 1:250, AP124R; Chemicon) at room temperature for 1 h, washed again with PBS, incubated with 4′,6-diamidino-2-phenylindole dihydrochloride (DAPI; Sigma-Aldrich) to stain the nucleus, and finally observed under a fluorescence microscope. TH⁺ cells were manually counted by researchers blinded to the treatment groups. Quantitative immunocytochemical data represent means and standard errors from more than 200 cells counted in 10 randomly sampled fields of six separate samples.

Western Blotting for TH

Western blot analysis was performed according to a procedure outlined in our previous study (6). Cell membranes were prepared from HUMSCs cultured in NCM, Shh, and FGF8 for varying periods. After resolution on 20% SDS-PAGE (Level Biotechnology), the cell proteins were blotted onto polyvinylidene difluoride membranes (Millipore Corporation, Bedford, MA, USA), which were then washed with Tris buffer (Sigma-Aldrich) with 0.9% NaCl (pH 7.3), immersed in the blocking solution (0.05% Triton X-100, 5% normal goat serum, and 3% bovine serum albumin) for 60 min, washed with Tris-buffered saline again, and reacted with primary antibodies (mouse anti-TH, 1:333, MAB318; Chemicon) at 4°C for 12 to 18 h. After the reaction was completed, the polyvinylidene difluoride membranes were washed with Triton Tris buffer [0.05% Triton and 0.9% NaCl (Sigma-Aldrich) in 50 mM Tris-HCl, pH 7.3], immersed in the blocking solution for 60 min, and then reacted with secondary antibodies (biotin-conjugated goat anti-mouse-IgG, 1:300, AP124B; Chemicon) at room temperature for 1 h. The PVDF membranes containing the reaction products were washed with Tris-buffered salt solution with Tween (Sigma-Aldrich) (TTBS), reacted with ABC complex (PK-4000; Vector Laboratories) at room temperature for 1 h, washed again with TTBS, and finally developed with DAB.

Analysis of Dopamine Concentration by High-Performance Liquid Chromatography (HPLC)

For the measurement of the concentration of dopamine (DA), 3,4-dihydroxyphenylacetic acid (DOPAC), 5-hydroxyindole acetic acid (5-HIAA), homovanillic acid (HVA), and serotonin (5-HT), cell pellet and the culture medium were acidified with perchloric acid (Sigma-Aldrich) and centrifuged at 10,000 x g for 10 min. The supernatant was immediately frozen in liquid nitrogen and stored at −70°C until analysis. Samples were purified using a 0.22-μm nylon filter (Merck Millipore) before being assayed for their dopamine contents by reverse-phase HPLC (Shimazu, Kyoto, Japan) using a 4.6 × 150-mm C18 column (Agilent, Palo Alto, CA, USA). Other conditions were the same as in a previous study (10).

Preparation and Grouping of Hemiparkinsonian Animals

All procedures involving animals were performed in accordance with the institutional animal welfare guidelines of the College of Medicine at the National Yang-Ming University. Adult male Sprague–Dawley rats were obtained from the Laboratory Animal Center at National Yang-Ming University. All experimental rats received the unilateral lesions by the injections of 6 μl 6-hydroxydopamine (6-OHDA; 6 μg/μl in normal saline containing 0.02% ascorbic acid) into the median fore-brain bundle (A/P: −4.3 mm, R/L: +1.6 mm, H: −8.2 mm) and substantia nigra (A/P: −4.8 mm, R/L: +1.5 mm, H: −8.2 mm). After 6-OHDA injection, amphetamine-induced rotational behavior was assessed at 1 to 4 months. For the rotational behavior test, the rats were placed in individual plastic hemispherical bowls from automated rotometer system (MED Associates, Inc., St. Albans, VT, USA) and allowed to habituate for 10 min before being injected with a subcutaneous dose of amphetamine (2.5 mg/kg; Sigma-Aldrich). Left and right full-body turns were counted. Amphetamine-induced net rotation over a period of 60 min, starting 30 min after injection, was enumerated. Animals showing >6 turns per minute ipsilaterally toward the lesioned side after a single dose of amphetamine were considered successful hemiparkinsonian models and were selected for grafting (2,36,37). All behavioral tests were performed in a closed room to avoid any environmental disturbance and assessed by an independent observer blind to the treatments.

One month after 6-OHDA lesion, rats were divided into four experimental groups on the basis of different treatments: (1) parkinsonian group (PD group, n=6), with 6-OHDA lesion-induced hemiparkinsonism and received PBS only in the lesioned striata; (2) rats (n = 6) with 6-OHDA lesion-induced hemiparkinsonism and receiving a suspension of 4 × 10⁵ graft cells that had been transfected with vector alone, and cultured in FGF8 + Shh 3 days then NCM 6 days [referred to as PD + Vector+FGF8 + Shh 3d+NCM 6d (4 × 10⁵) group]; (3) rats (n = 6) with 6-OHDA lesion-induced hemiparkinsonism and receiving a suspension of 1 × 10⁵ graft cells, which had been transfected with Nurr1, and cultured in FGF8 + Shh 3 days followed by NCM 6 days [referred to as PD+Nurr1+FGF8 + Shh 3d+NCM 6d (1 × 10⁵ cells) group]; (4) rats (n = 6) with 6-OHDA lesion-induced hemiparkinsonism and receiving a suspension of 4 × 10⁵ graft cells, which had been transfected with Nurr1, and cultured in FGF8 + Shh 3 days followed by NCM 6 days [referred to as PD + Nurr1 + FGF8 + Shh 3d+NCM 6d (4 × 10⁵ cells) group]. Histological examinations of grafted cryosections were carried out according to procedures outlined in a previous study (10).

Preparation and Transplantation of HUMSC-Derived TH⁺ Cells

The HUMSCs after in vitro differentiation were treated with 1 μg/ml bis-Benzimide (B2883; Sigma-Aldrich) for 24 h to label the cells. Cells were trypsinized at 37°C for 5 min with 0.25% trypsin (15090-046; Gibco), and the dissociated cells were resuspended in PBS. Animals were implanted with a total of 10 μl of PBS containing 1 × 10⁵ or 4 × 10⁵ cells, deposited at two stereotaxic levels (anterior, 1.0 mm; lateral, 3.0 mm; ventral, −6.0 mm and −5.0 mm) in the striatum on the side ipsilateral to the 6-OHDA lesion. The Hamilton syringe with a 22-gauge needle was left in situ for further 10 min to avoid reflux along the inoculation track. Rat hosts did not receive any immunosuppression medications.

Statistical Analyses

Values are expressed as the mean ± standard error of the mean. One-way or two-way analysis of variance was used to compare all means, and Tukey's test was used for the posteriori tests. A value of p < 0.05 was considered statistically significant.

Acquisition of Dopaminergic Phenotype From Nurr1-Expressing HUMSCs Treated with NCM, FGF8, and Shh

HUMSCs were transfected with either the Nurr1-expressing vector (pCMV-HA-Nurr1) or the (empty) (sham) vector (pCMV-HA-vector) serving as a vehicle. Q-PCR analysis showed that Nurr1 expression was increased about 40-fold in Nurr1-transduced HUMSCs after 24-h modification (Fig. 1B). The untreated umbilical mesenchymal stem cells appeared mostly spindle shaped and were closely apposed to each other due to proliferation following treatment with DMEM for 9 days (Fig. 1Ca). After 9 days of differentiation, vector- and Nurr1-expressing HUMSCs all developed smaller cell bodies, but Nurr1-expressing cells exhibited more elongation of neurite-like processes (Fig. 1Cb, c, d). These processes were exceedingly long and had formed cell–cell contacts.

To address whether Nurr1 expression enhancement could enhance in vitro differentiation of HUMSCs into dopaminergic cells, the catecholaminergic marker of the rate-limiting synthesizing enzyme TH in these cells was monitored by immunocytochemical staining. The proportion of vehicle vector cells expressing TH was 2.50 ± 0.31% after treatment with DMEM for 3 days (Fig. 2Aa, B). That percentage was increased to 25.05 ± 0.74% after treatment with NCM for 6 days and Shh + FGF8 for 3 days (Fig. 2Ab, B). Nurr1 expression enhancement alone promoted an increase in the percentage of TH⁺ cells to 29.19 ± 0.60% in the group cultured in DMEM for 3 days. Further incubation of up to 9 days under the same condition (DMEM only) did not increase further the number of TH⁺ cells (Fig. 2Ac, B). In the NCM and FGF8 + Shh groups, the data showed that as the incubation time increased, the number of TH⁺ cells also gradually increased for up to 9 days in the enhanced Nurr1 expression groups. Among those, combined treatments of NCM for 6 days and FGF8 + Shh for 3 days could increase the number of TH⁺ cells to 70.5 ± 4.34% (Fig. 2Ae, B). The success rate in the differentiation of TH⁺ cells increased 2.8-fold compared to the vehicle vector groups that underwent the same procedure. No difference in the proportion of TH-expressing cells was observed between different orders of NCM and FGF8 + Shh (Fig. 2B). Western blotting showed similar results as the analyses of immunocytochemical staining (Fig. 2C). The protein level of TH in the Nurr1-expressing group combined treatment of NCM for 6 days and FGF8 + Shh for 3 days has significantly increased 1.8-fold in contrast to the vehicle vector group.

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Figure 2.

Enhancing Nurr1 expression leads to higher efficiency in the generation of TH-expressing cells from HUMSCs. The differentiation of the Nurr1-transduced HUMSCs was induced by various procedures. (A) Photomicrographs showing TH immunocytochemistry of Nurr1-expressing HUMSCs cultured under different conditions. (B) Histograms showing the percentages of TH⁺ cells induced by different treatments. Nurr1 transduction imparted high generation efficiency of TH⁺ cells when cultured in NCM and FGF8 + Shh for 9 days. *Significant difference at p < 0.05 compared with untreated group. #Significant difference at p < 0.05. The three groups of Nurr1 + NCM 6d, Nurr1 + NCM 3d + FGF8 + Shh 3d, and Nurr1 + FGF8 + Shh 3d + NCM 3d versus the four groups of the Nurr1 + DMEM 3d, Nurr1 + NCM 3d, Nurr1 + FGF8 + Shh 3d, and Nurr1 + DMEM 6d. ♠Significant difference at p < 0.05 compared with the other groups. (C) Western blot showing TH-immunoreactive bands in cell lysates derived from different treatments 9 days postinduction. β-Actin expression was measured as a loading control.

To further characterize the Nurr1-mediated induction of dopaminergic cells, real-time PCR was used to evaluate the expressions of specific genes involved in dopamine synthesis and transport. The mRNA expression of TH, L-aromatic amino acid decarboxylase (AADC), and DA transporter (DAT) were all upregulated in differentiated HUMSCs compared with untreated HUMSCs. The expression level of Nurr1-transduced groups was significantly higher than in the vector-transduced groups (Fig. 3A).

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Figure 3.

Effect of Nurr1, FGF8 + Shh, and NCM can synergistically enhance the generation of dopaminergic neuron-specific genes and the release of dopamine. (A) Real-time PCR analyses for the genes specific to DA synthesis and transport. Histograms demonstrate the relative expressions of TH, AADC, and DAT mRNA in cultures of HUMSCs cultured in FGF8 + Shh for 3 days followed by NCM for 6 days after Nurr1 transduction. The expressions of mRNA of such genes were all significantly increased in differentiated HUMSCs compared with untreated HUMSCs. The expression levels in Nurr1-expressing groups were significantly higher than those in the vector-expressing groups. *Statistical difference at p < 0.05 compared with untreated groups. #Statistical difference at p < 0.05 compared with vector and untreated groups. HPLC quantification of dopamine in cultured medium (B) and cells (C). After stepwise differentiation, dopamine concentrations were evaluated in each condition by reverse-phase HPLC. The results showed more amounts of dopamine in the cell-transduced Nurr1 and cultured in NCM and FGF8 + Shh for 9 days. Results represent the mean ± standard error from four different experiments. *Statistical difference at p < 0.05 compared with the other groups in (B). *Significant difference at p<0.05 compared with untreated group. #Significant difference at p<0.05 between the Nurr1 and vector groups in the same condition in (C).

Since dopamine release is a definitive characteristic for the identification of dopaminergic cells, we evaluated the dopamine concentration by using reverse-phase high-performance liquid chromatography (HPLC). Dopamine was below detection in the medium of untreated HUMSCs cultured in DMEM. The dopamine concentrations of the conditioned media were between 2.28 ± 0.46 and 4.74 ± 0.08 ng/ml in the vector-expressing HUMSCs after a 3- to 9-day differentiation culture (NCM and FGF8 + Shh treatment) of 10⁵ cells in a 100-mm culture dish. Dopamine level was not further augmented in the conditioned media prepared from Nurr1-expressing HUMSCs treated with NCM for 3 or 6 days, and/or FGF8 + Shh for 3 days, compared with the vector-expressing groups. However, dopamine release into the media of HUMSCs treated with FGF8+Shh for 3 days and then NCM for 3 or 6 days yielded 11.39 ± 1.02 and 11.78 ± 1.12 ng/ml/10⁵ cells, respectively (Fig. 3B). Furthermore, dopamine was still detectable in the cell pellets of cells collected following 9 days of differentiation even after release stimulation. In untreated HUMSCs, total DA contents were only 0.013 ± 0.003 ng/ml. In contrast, dopamine concentrations in differentiated cells rose to concentrations of between 1.03 ± 0.27 and 2.58 ± 0.18 ng/ml after a 9-day culture. More importantly, dopamine levels in the Nurr1-expressing groups were significantly higher than those in the vector-expressing groups (Fig. 3C).

Since previous literature reported that serotonin may play as a modulator for the synthesis and release of dopamine (9,13,25,26), we evaluated the serotonin concentration by using reverse-phase HPLC (Fig. 4A, B). Serotonin was hardly detected in the medium of untreated HUMSCs cultured in DMEM. The serotonin concentrations of the conditioned media were between 2.41 ± 1.67 and 10.76 ± 5.65 ng/ml in the vector-expressing HUMSCs after a 3- to 9-day differentiation culture (NCM and FGF8 + Shh treatment) of 10⁵ cells in a 100-mm culture dish. Serotonin level was not further augmented in the conditioned media prepared from Nurr1-expressing HUMSCs treated with NCM for 3 or 6 days, and/or FGF8 + Shh for 3 or 6 days, compared with the vector-expressing groups (Fig. 4C). Furthermore, serotonin was still detectable in the cell pellets of cells collected following 9 days of differentiation even after release stimulation. In untreated HUMSCs, total serotonin contents were only 0.52 ± 0.17 ng/ml. In contrast, serotonin concentrations in the vector-expressing HUMSCs after a 9-day differentiation rose to concentrations of between 7.05 ± 2.60 and 9.36 ± 3.80 ng/ml. However, serotonin concentration in Nurr1-expressing HUMSCs treated with NCM for 6 days and then FGF8 + Shh for 3 days was 6.36 ± 2.20 ng/ml. More interesting, serotonin levels in the Nurr1-expressing HUMSCs pretreated with FGF8 + Shh for 3 days and then NCM for 6 days were significantly higher to 22.00 ± 11.82 ng/ ml (Fig. 4D).

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Figure 4.

Effect of Nurr1, FGF8 + Shh, and NCM can enhance the production of serotonin. (A) Upper panel is representative HPLC chromatograms showing the peaks of DOPAC, DA, 5-HIAA, HVA, and 5-HT that were detected from the medium of the Nurr1-expressing HUMSCs precultured in FGF8 + Shh for 3 days and then NCM for 6 days. (B) Lower panel is the standard solutions containing DOPAC, DA, 5-HIAA, HVA, and 5-HT 10 ng/ml, respectively. DOPAC, 3,4-dihydroxyphenylacetic acid; DA, dopamine; 5-HIAA, 5-hydroxyindole acetic acid; HVA, homovanillic acid; 5-HT, serotonin. The quantitative result of HPLC chromatography experiments measuring serotonin concentration from the medium (C) and cell pellet (D). *Significant difference at p < 0.05 compared with untreated stem cell group. #Significant difference at p<0.05 compared with the Nurr1-expressing HUMSCs precultured in FGF8 + Shh for 3 days and then NCM for 6 days group.

Dopaminergic Cells Differentiated From HUMSCs Exist in Grafted Striatum

At 3 months after transplantation, bis-Benzamide-labeled cells were found in and around the implantation sites (Fig. 5A, B). Anatomical examinations revealed that many cells whose somata and process stained positively for TH were found in 6-OHDA-lesioned striatum (Fig. 5C). Cell migration patterns were followed by bis-Benzimide labeling in 30-μm serial sections. The labeled cells had migrated for approximately 2.8 mm in both directions of the rostrocaudal axis from the implantation site (bregma 1.0). Most of the labeled cells were localized in the region of bregma −1.0 to the region of bregma 1.8, almost throughout the entire striatum (Fig. 5D).

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Figure 5.

Photomicrographs showing the distribution of implanted HUMSCs in hemiparkinsonian rats 3 months after transplantation. Following labeling of the cell nuclei with bis-Benzamide, HUMSCs were microinjected into the striata of hemiparkinsonian rats. The cells survived in the striata 3 months after transplantation. Phase-contrast (A) and same field (B) fluorescence photomicrograph. Scale bar: 1 mm. (C) Existence of TH⁺ cell bodies and its processes (arrows) in the grafted striatum. Scale bar: 50 μm. (D) Line drawings of rat brain demonstrating the extent of HUMSC migration after implantation in the striatum of the rat at the bregma level. Cells of bis-Benzamide-labeled HUMSCs.

Effect of Transplantation on Amphetamine-Induced Rotation

The effects of cell transplantation were evaluated in 6-OHDA-lesioned rats by quantification of rotations in response to amphetamine. Rotational scores were examined at 1, 2, 3, and 4 months post-6-OHDA lesioning. One month after lesioning, the numbers of amphetamine-induced rotations in all groups reached to between 6.40 ± 0.19 and 6.82 ± 0.56 rotations per minute. The PD group, which received injections of PBS in the dopamine-denervated striatum, showed a gradual but significant increase in the numbers of rotations over the observation period. By contrast, rotations in all of the PD animals that received grafts of 4 × 10⁵ vector-transduced cells preconditioned by being cultured in FGF8 + Shh for 3 days followed by NCM for 6 days did not continue to get worse throughout the observation period as in the case of the PD group. Similar patterns were found in all of the PD animals receiving grafts of 1 × 10⁵ of the same preconditioned Nurr1-expressing cells and treated with FGF8 + Shh for 3 days followed by NCM for 6 days. Among the grafted categories, the group that received a graft of 4 × 10⁵ of the preconditioned Nurr1-expressing HUMSCs previously treated by FGF8 + Shh for 3 days followed by NCM for 6 days made a marked recovery in rotational scores (Fig. 6).

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Figure 6.

Time course of amphetamine-induced rotation response. Rotation scores were evaluated at 1 to 3 months posttransplantation. A significant decrease in the number of amphetamine-induced tuning was observed in the rats receiving a graft of 4 × 10⁵ Nurr1-expressing HUMSCs cultured in FGF8 + Shh for 3 days followed by NCM for 6 days at all time points after transplantation. *Significant difference at p < 0.05 compared with the PD group at the same time point. #Significant difference at p < 0.05 compared with the PD + vector + FGF + Shh 3d + NCM 6d (4 × 10⁵ cells) at the same time point. ♠Significant difference at p < 0.05 compared with the PD + Nurr1 + FGF + Shh 3d + NCM 6d (1 × 10⁵ cells) at the same time point.