Autologous mesenchymal stem cell–derived dopaminergic neurons function in parkinsonian macaques

A cell-based therapy for the replacement of dopaminergic neurons has been a long-term goal in Parkinson’s disease research. Here, we show that autologous engraftment of A9 dopaminergic neuron-like cells induced from mesenchymal stem cells (MSCs) leads to long-term survival of the cells and restoration of motor function in hemiparkinsonian macaques. Differentiated MSCs expressed markers of A9 dopaminergic neurons and released dopamine after depolarization in vitro. The differentiated autologous cells were engrafted in the affected portion of the striatum. Animals that received transplants showed modest and gradual improvements in motor behaviors. Positron emission tomography (PET) using [¹¹C]-CFT, a ligand for the dopamine transporter (DAT), revealed a dramatic increase in DAT expression, with a subsequent exponential decline over a period of 7 months. Kinetic analysis of the PET findings revealed that DAT expression remained above baseline levels for over 7 months. Immunohistochemical evaluations at 9 months consistently demonstrated the existence of cells positive for DAT and other A9 dopaminergic neuron markers in the engrafted striatum. These data suggest that transplantation of differentiated autologous MSCs may represent a safe and effective cell therapy for Parkinson’s disease.

Cell-based therapies are expected to replace the missing dopaminergic neurons and to restore the motor function in patients with Parkinson’s disease (PD) (1). Early studies on cell-based therapies used fetal midbrain tissue containing dopaminergic neurons as a cell source and suggested potential therapeutic effects in PD (for review, see refs. 2, 3). However, limited availability and ethical considerations relating to the use of fetuses pose limitations for practical use. Bone marrow–derived mesenchymal stem cells (MSCs), a type of adult stem cells, have trophic effects (4) and a differentiation spectrum that crosses oligolineage boundaries (5), offering the potential for use in autologous cell therapy, with low risk of tumorigenesis (6). The MSCs have been already tested for cell therapy in PD model rodents (7–9) and even in patients with PD (10). However, they have shown poor performance for restoration of motor function, potentially due to limited spontaneous differentiation (11) or facilitated apoptosis (12, 13) of MSCs. Recent studies of fetal midbrain graft have suggested that better outcomes could be obtained if the graft consisted of well-differentiated A9 dopaminergic neurons (14–16), the most severely damaged neuronal type in PD (17). Therefore, differentiation of MSCs into desired cells, such as A9 dopaminergic neurons, would probably provide effective functional restoration in PD.

Recently, it was shown that MSCs could be artificially directed to differentiate into several specialized cell types, including those in nervous tissues (18–21). Previously, we reported that dopamine-producing cells could be induced from MSCs (MSC-DP cells) by introduction of a Notch1 intracellular domain–containing (NICD-containing) plasmid, followed by cytokine stimulation with bFGF, forskolin, ciliary neurotrophic factor (CNTF), and glial cell line–derived neurotrophic factor (GDNF) (20, 21). The differentiated cells were positive for markers of dopaminergic neurons, such as tyrosine hydroxylase (TH) and the dopamine transporter (DAT), and had an ability to release dopamine after depolarization by potassium stimulation. When rat and human MSC-DP cells were transplanted into the striata of PD model rats, integration of TH⁺ and DAT⁺ cells and functional recovery in motor behaviors were confirmed (20). Subsequent development of a spermine-pullulan–mediated reverse transfection method allowed us to induce MSC-DP cells more safely and efficiently than before from MSCs of macaque monkeys (21), an animal species frequently used for preclinical trials of PD (22–27).

To test the scalability of MSC-DP cell–based therapy in primates in this study, monkey MSC-DP cells were characterized in detail using specific markers and evaluated for their longitudinal effects after they were engrafted into hemiparkinsonian monkeys using behavioral tests and positron emission tomography (PET). The MSC-DP cells, prepared autologously from the bone marrow of each test animal, expressed cell makers not only for antigens that have been previously described (20, 21), such as β tubulin III (Tuj1), microtubule-associated protein 2 (MAP-2), TH, and DAT, but also for those specific to the A9 subtype, namely, G protein–coupled inward rectifying current potassium channel type 2 (GIRK2) (15) and forkhead box protein A2 (FOXA2) (28). The effect of transplantation was evaluated for up to 9 months based on motor behaviors of affected hand movements; PET scans using ¹¹C-CFT, which specifically labels DAT; and postmortem histology. Tumorigenicity was also estimated from the results of blood tests and PET scans. The preclinical data obtained thus may extend the applicability of the current autologous cell system as a therapy for PD.

Evaluation of MSC-DP cells. Cynomolgus monkey MSCs drastically changed their morphology following induction, as reported previously (21): naive MSCs initially showed fibroblast-like mesenchymal cell features (Figure 1A), while induced cells showed a neuron-like morphology with neurite-like processes (Figure 1B). By immunohistochemistry, naive MSCs were negative for neuronal markers, as reported previously (21), but induced cells were positive for neuronal markers, Tuj1 (Figure 1C) and MAP-2 (Figure 1D); dopaminergic neuron markers, TH and DAT (Figure 1, E and F); the A9 dopaminergic neuron marker, GIRK2 (Figure 1G); and a marker of floor plate–derived cells, FOXA2 (Figure 1H). We also confirmed that these cells were positive for sodium channels (Figure 1I), a marker of differentiated neurons.

Figure 1

Monkey bone marrow MSCs and MSC-DP cells. (A) Morphological changes were evident in cynomolgus naive MSCs (phase-contrast microscopy) and (B) MSC-DP cells (phase-contrast microscopy). The MSC-DP cells possessed neurite-like processes. Immunocytochemistry of MSC-DP cells showed that the cells were immunoreactive for the neuronal markers (C) Tuj1 and (D) MAP-2; for the markers of dopaminergic neurons (E) TH, (F) DAT, (G) GIRK2, and (H) FOXA2; and for the marker of neurons, (I) sodium channel (SCN). DAPI was used for counterstaining of nuclei. Scale bar: 30 μm. (J) Results of RT-PCR in naive MSCs, MSC-DP cells, and tissue samples from the SNc and VTA. Naive MSCs expressed no GIRK2, FOXA2, and CALB1 mRNA, while the MSC-DP cells expressed GIRK2 and FOXA2 mRNA. SNc tissue samples from an embryo and an adult cynomolgus monkey also contained high levels of GIRK2 and FOXA2 mRNA but only low levels of CALB1 mRNA, while the VTA contained high levels of CALB1 mRNA and only very low levels of GIRK2 and FOXA2 mRNA. NC, negative control. (K) Percentages of cells immunoreactive for TH, DAT, GIRK2, and FOXA2 in MSC-DP cells.

To confirm whether these cells have an ability to produce and release dopamine, we measured the secretion of dopamine by HPLC. The amount of dopamine in the culture supernatant was measured following application of high K⁺ depolarizing stimuli, which resulted in release of 1.04 ± 0.4 pM dopamine per 10⁶ cells (Table 1); by contrast, naive cynomolgus monkey MSCs showed no detectable dopamine release. These results are consistent with those of our previous studies: the amount of dopamine release was comparable to the amounts in rats (1.1 pM/10⁶ cells) (20) and monkeys (0.9 ± 0.2 pM/10⁶ cells) (21).

Table 1

Dopamine-producing capacity of MSC-DP cells and the number of engrafted cells

We further investigated the expression of markers specific for A9 dopamine neurons using RT-PCR. The MSC-DP cells expressed GIRK2 and FOXA2 mRNA but not CALB1 mRNA (GIRK2⁺/FOXA2⁺/CALB1^–) (Figure 1J). When control tissues obtained from a cynomolgus embryo and an adult animal were analyzed, both showed that the substantia nigra pars compacta (SNc) was strongly positive for GIRK2 and FOXA2 and weakly positive for calbindin, while the ventral tegmental area (VTA) was weakly positive for GIRK2 and FOXA2 but strongly positive for calbindin (Figure 1J). This distinct pattern of GIRK2/FOXA2/calbindin expression in the SNc and VTA is consistent with those reported in other species, including rodents and humans (15, 28, 29). We also evaluated the induction efficiency of MSC-DP cells by quantitative immunocytochemistry. Fifty to seventy-five percent of MSC-DP cells were positive for TH, DAT, GIRK2, and FOXA2 (Figure 1K). These findings indicated that the current method efficiently produced MSC-DP cells from the MSCs, which had properties similar to those of the A9 dopamine neurons in the model species.

Behavioral analysis of motor symptoms. The clinical rating scores (CRSs) for parkinsonian animals are shown in Figure 2A. The CRS revealed a significant interaction effect between groups and time after transplantation (F distribution [F_4,32] = 3.07, P < 0.05). Thus, we further tested for an effect of time, separately in each of the groups in our study (engrafted or sham), by 1-way ANOVA. The CRS in the MSC-DP–engrafted group showed a marginal effect of time (F_4,16 = 2.95, P = 0.055). In a post-hoc comparison of scores at each time point after engraftment with those at baseline, significant improvements in the CRS were observed at 8 months after engraftment (Dunnett’s multiple comparison, P < 0.05). This time effect was not observed in the sham group either by 1-way ANOVA or post-hoc analysis.

Figure 2

Behavioral assessment. (A) CRSs and (B) hand-reach scores were plotted against time for MSC-DP cell–engrafted (MSC-DP) and sham-operated (Sham) groups. *P < 0.05 compared with baseline in the MSC-DP–engrafted group, Dunnett’s multiple comparison test.

Hand-reach scores showed similar time courses (Figure 2B). The scores for the affected hand showed significant group and time interaction (F_4,32 = 2.83, P < 0.05). One-way ANOVA for the repeated measures of hand-reach scores for the affected hand of the MSC-DP–engrafted group revealed a significant effect of time (F_4,16 = 5.62, P < 0.001). A post-hoc comparison of scores at each time point after engraftment with those at baseline revealed significant improvements in hand-reach scores at 4 months (Dunnett’s multiple comparison, P < 0.05) and 8 months (P < 0.01) after engraftment. This effect of time was not observed in the sham group. Therefore, the CRSs and hand-reach scores suggested that the engraftment of MSC-DP cells modestly improved motor behaviors in parkinsonian animals.

Despite these improvements in MSC-DP–grafted animals, spontaneous activities of animals were not affected by any of group (sham vs. MSC-DP engrafted), time (before engraftment and 4 months and 8 months after engraftment), or interaction among these variables (2-way ANOVA with repeated measures, Supplemental Figure 1; supplemental material available online with this article; doi: 10.1172/JCI62516DS1). Animals in the engrafted group tended to show higher spontaneous activities than sham-operated animals at 4 and 8 months after engraftment; however, no statistically significant effect of group was observed at any time point in post-hoc analysis (Bonferroni corrected, P > 0.05). Within-subject and between-subject data were highly variable, consistent with a previous report in this type of parkinsonian animal model (30). No dyskinesia-like abnormal movements were observed in any MSC-DP cell–engrafted animals during the observation period.

¹¹C-CFT PET. Voxel-based analysis of ¹¹C-CFT binding potential (BP_ND) images disclosed a significant effect of time in a cluster extending into the dorsal posterior putamen in the engrafted group (Figure 3C) (family-wise error rate [FWE] corrected, P < 0.05; Table 2). The maximum of this cluster was located (x = 11.5 mm, y = –8.0 mm, z = 3.5 mm) in a standard macaque brain space of the Montreal Neurological Institute (MNI) (31) and was safely within the area targeted when engrafting MSC-DP cells into the striatum (Figure 3A). Using this cluster as a region of interest (ROI), we obtained BP_ND values for this ROI across all animals, groups, and time points. The BP_ND values obtained are presented in Figure 3D. Two-way ANOVA with repeated measures (Figure 3D) revealed significant effects of an interaction between group and time (F_4,24 = 4.3, P < 0.01). Post-hoc analysis showed that the BP_ND at 7 days after engraftment was higher than that in the sham-operated group (Bonferroni multiple comparison, T = 4.56, P < 0.001), as shown in Figure 3D. In particular, animal mon0703, who received the graft with the largest amount of MSC-DP cells (Table 1), showed the highest BP_ND (0.59) in the engrafted striatum at 7 days after engraftment, followed by animal mon0710, who showed a BP_ND of 0.43 at 7 days after engraftment (Figure 3B).

Figure 3

Engrafting and neuroimaging of MSC-DP–engrafted animals. (A) Twelve target points (white dots) for engraftment in putamen in the coronal sections of standard space of cynomolgus macaque. Scale bar: 10 mm. (B) Representative 11C-CFT PET images of MSC-DP–engrafted animals. Seven days after engraftment (engraft), animal mon0703 showed the highest 11C-CFT BP_ND on the MPTP-treated side of striatum (yellow arrow), followed by animal mon0710 (white arrow). Animal mon0710 showed low signal (green arrow) in a T1-weighted MRI after engraftment, which diminished in later scans. Note that the BP_ND in the non-MPTP–treated (and nonengrafted) side of striatum was very high (white arrowheads) and not different by engraftments. (C) Significant effect of time on 11C-CFT BP_ND (P values range from yellow [P < 0.00] to red [P < 0.05]) overlaid on the study-specific MRI template in MNI space. A cluster with FWE-corrected P < 0.05 was located in the dorsal posterior putamen in the engrafted striatum. See also Table 2. (D) Time course of 11C-CFT BP_ND in the cluster (in C) and contralateral equivalent region. *P < 0.05 compared with sham, Bonferroni corrected. (E) Time course of 11C-CFT BP_ND values in the cluster (in C), fitted by the 1-hit (exponential) model of neurodegeneration. The dashed line and gray area indicate the mean and SEM for the baseline BP_ND. *P < 0.05 between the baseline BP_ND and the plateau of the fitted model. (F) The DArc and the 11C-CFT BP_ND values at 7 days and 1, 3, and 7 months after engraftment.

Table 2

Voxel-based statistical results of ¹¹C-CFT BP_ND in repeated-measure ANOVA

The time-dependent decline in ¹¹C-CFT binding in the engrafted striatum (Figure 3D) allowed us to analyze the kinetics of ¹¹C-CFT binding in detail. Because recent studies have suggested that MSCs are susceptible to senescent (12) and apoptotic changes (13), we supposed that this decline was due to degeneration of engrafted MSC-DP cells. ¹¹C-CFT binding is known to be correlated with the density of dopamine neurons or terminals rather than any physiological (or functional) variation in dopamine release. The rate of ¹¹C-CFT binding reduction, calculated based on the 1-hit model of neurodegeneration (32), was 0.30 months (~10 days) as a half-life period (Figure 3E). This rate of reduction was slightly slower than that for engrafted naive rodent MSCs (~3 days; Supplemental Figure 2) based on previously described data (13). We further tested whether this degenerative process would affect all grafted cells. The plateau of the 1-hit model was significantly higher than the baseline (before engraftment) ¹¹CFT binding level (baseline BP_ND = 0.087 ± 0.028 vs. BP_ND at plateau = 0.17 ± 0.029; Welch’s corrected T₁₃ = 2.04, 1-tailed P < 0.05), suggesting that a small portion of the grafted MSC-DP cells survive and integrate for more than 7 months. This is in contrast to findings in naive MSCs engrafted into rodents, which showed a plateau of the reduction curve returned to the baseline (Supplemental Figure 2), indicating that all naive MSCs may eventually degenerate when transplanted into brain.

We also estimated how the dopamine-releasing capacity (DArc) of grafts related to ¹¹C-CFT binding after engraftment (Figure 3F). The dopamine-releasing capacity was calculated by multiplication of dopamine release by K⁺ concentration (pM per 10⁶ cells) in vitro, as measured by HPLC, and the total number of cells engrafted in each animal (10⁶ cells). This graft DArc (pM/graft) significantly predicted the ¹¹C-CFT BP_ND obtained from the cluster of engrafted striatum scanned at 7 days after engraftment (linear regression, BP_ND = 5.1 × 10^–3 × DArc + 0.31, F_1,3 = 10.3, P < 0.05; Figure 3F) but not those obtained at later time points. Although dopamine release and DAT expression are different measures, both are a common and specific attribute of well-differentiated dopamine neurons (33). Therefore, the findings indicate that the DAT expression at the earliest time point after transplantation is coupled to the dopaminergic capacity of the engrafted cells, while other factors may have degraded this coupling at later time points.

Tests for tumorigenicity and general condition of animals with MSC-DP cell engraftment. There were no abnormal changes in the results of blood tests, including red and white blood cell counts, and blood chemical tests in any of the MSC-DP– and sham-treated animals tested (Supplemental Table 1). There was also no abnormal elevation in the levels of the tumor markers carcinoembryonic antigen (CEA), tissue polypeptide antigen (TPA), sialyl Lewis X antigen (SLX), neuron-specific enolase (NSE), and basic fetoprotein (BFP) (Supplemental Table 2).

¹⁸F-FDG PET scans revealed normal uptake of ¹⁸F-FDG in the engrafted striatal region of animals in the engrafted group (Figure 4). In MRI scans, 2 animals (mon0710 and mon0049) showed subtle hemorrhagic changes, as suggested by low signals in the engrafted region of the right striatum in T1-weighted MRI images at 7 days after engraftment (Figure 3B); however, no significant signal changes were observed in later MRI scans. Therefore, no enlargement of or tumor formation by the engrafted tissues was suspected, at least at the macroscopic level, up to 8 months after transplantation of MSC-DP cells. These findings were confirmed by histological evaluations, as described below.

Figure 4

Evaluation of tumorigenicity of MSC-DP–grafted animals. (A) SUV images of ¹⁸F-FDG PET scans, obtained 8 months after engraftment, are shown for each animal in the engrafted group. No apparently high uptake of ¹⁸F-FDG was found in any of MSC-DP cell–engrafted animals. (B) Immunostaining with Ki-67 is shown in a section of MSC-DP cell–engrafted striatum. Cells positive for Ki-67 (green) scarcely existed in the dorsal-posterior putamen. Scale bar: 100 μm. (C) Quantitative analysis of the ratio of Ki-67⁺ cells to the total number of cells in 3 groups of striatum (MSC-DP grafted, sham operated, nonaffected). See also Supplemental Tables 1 and 2 for the results of blood tests for biochemical and tumor markers.

Immunohistochemical study. Consistent with the ⁸F-FDG PET scans and MRI data, no tumor formation was observed in either group by H&E staining (data not shown). We further evaluated the proliferative activity of MSC-DP cells engrafted into the striatum by immunostaining using a Ki-67 antibody. Although, we found a small number of Ki-67⁺ cells in the MSC-DP cell–engrafted striatum (Figure 4B), the proportion of Ki-67⁺ cells was <2.5%, and there was no significant difference between MSC-DP cell–engrafted and control striata (Figure 4C). Moreover, the Ki-67⁺ cells existed solitarily, and none of them formed tumor-like masses (Figure 4B).

We then performed triple staining of striatal sections using markers, including those for A9 and A10 dopaminergic neurons. In the transplanted region of MSC-DP cell–engrafted striata, cells positive for DAT and GIRK2 but not for calbindin were clearly observed (Figure 5, A–F), but such cells were not found in any of sham-operated striata. The staining pattern of A9-type dopaminergic neurons was confirmed by analyzing the midbrain of a normal control adult animal with the same triple staining (Supplemental Figure 3): the A9-type dopaminergic neurons in the SNc showed a pattern of DAPI⁺/DAT⁺/GIRK2⁺/calbindin^– (Supplemental Figure 3A), while the A10-type neurons in the VTA had a pattern of DAPI⁺/DAT⁺/GIRK2^–/calbindin⁺ (Supplemental Figure 3B). We also confirmed that most of the DAT⁺ cells in the engrafted striata were positive for TH (Figure 5, G–J) but not for a glial marker, glial fibrillary acidic protein (GFAP) (Figure 5, K–N). Taken together, these findings indicate that the transplanted MSC-DP cells survived and maintained the characteristics of A9-type dopaminergic neurons for at least 9 months after transplantation.

Figure 5

Immunohistochemistry of the MSC-DP–engrafted striatum. In the MSC-DP–grafted striatum, the cell bodies positive for DAT and GIRK2 (arrows in B and C, respectively) but not for calbindin (arrow in D) were found in the area close to the MSC-DP cell–engrafted area (white dots in F) in the dorsal posterior putamen. (A) DAPI was used for counterstaining of nuclei. (E) A merged image for DAT, GIRK2, and DAPI is shown in green, red, and blue, respectively. Most of the DAT⁺ cells were also positive for TH (arrows in G–J) but not for GFAP (arrows in K–N), while the GFAP⁺ cells with round-shaped nuclei, putative astroglial cells, were not positive for DAT (arrowheads in K–N). Scale bar: 100 μm (A–E and G–N); 100 mm (F).

Quantitative analysis revealed that the number of cells positive for NeuN tended to be higher in the MSC-DP–engrafted striata than in sham and nonaffected (i.e., non–1-methyl-4-phenyl-1,2,3,6-tetrahydoropyridine–treated [non-MPTP–treated]) striata, but no statistically significant differences were found among the 3 groups of striata (F_3,112 = 1.12, P > 0.05; Figure 6, A and B). The number of TH⁺ cell bodies in the engrafted region was higher in the MSC-DP group than in the sham and nonaffected groups, with statistical significance (F_2,88 = 5.99, P < 0.005, Tukey’s multiple comparison P < 0.05; Figure 6, A and C). In contrast, the number of TH⁺ axon terminals was higher in the MSC-DP group than in the sham group, with statistical significance (F_2,119 = 3.93, P < 0.05, Tukey’s multiple comparison P < 0.05; Figure 6, A and D), suggesting that the TH⁺ cell bodies observed in the engrafted striatal group integrated with host striatal neurons by forming new synaptic connections. Similarly, the number of DAT⁺ cells was also higher in the MSC-DP group than in the sham and non-affected striatum groups, with statistical significance (F_2,74 = 16.78, P < 0.0001, Tukey’s multiple comparison P < 0.0001; Figure 6, A and E).

Figure 6

Quantitative analysis of immunohistochemistry of the dorsal posterior striatum. (A) Immunohistochemical sections of the dorsal posterior striatum for NeuN, TH⁺ cells, TH⁺ terminals, and DAT⁺ cells in MSC-DP cell–engrafted animals (MSC-DP), in the sham-operated striatum (Sham), and in the non-MPTP–treated side of the striatum (nonaffected) of sham animals. Scale bar: 100 μm. (B) Cell counts of NeuN⁺ and (C) TH⁺ neuronal cell bodies, (D) TH⁺ axon terminals, and (E) DAT⁺ neurons in the dorsal posterior striatum were plotted for each group. *P < 0.05, **P < 0.01, ***P < 0.001, Bonferroni-corrected multiple comparisons.

Early transplantation trials for PD treatments used fetal midbrain tissue as a cell source. Successful results were first reported in studies using macaques in the late 1980s (22–24). Subsequent open-label trials in patients with PD also showed that engrafted fetal neurons can survive, appropriately differentiate, and provide striatal dopamine release as measured by PET and showed clinical improvements (34–36), with only one exception (37). Although later double-blind placebo-controlled trials reported no significant clinical benefits (38, 39), successful cell replacement therapy in PD would probably be achieved with more sophisticated cell preparation, surgical and patient selection procedures (2, 3). In particular, optimization of cell preparation has been an issue when using fetal tissue because of its limited availability and accessibility; thus, there is an urgent need to develop alternative cell sources (40). In addition, recent basic studies based on fetal tissue graft systems have suggested several potential refinements. For example, whether or not the graft contains A9 dopaminergic neurons may be a determinant factor for achieving synaptic formation with host tissues and better behavioral recovery (16). Others have also suggested that expression of DATs in the graft cells alleviates dyskinesias after graft (41). Therefore, proof-of-concept studies using fetal tissue grafts have established the direction of cell-based therapies, including those using stem cells (2, 3, 40). Particular concerns when applying stem cells are functionality and safety issues after graft, both of which need to be overcome before clinical trials can be commenced (40). From these standpoints, we examined whether the MSC-DP cells could safely function in primate PD model.

Our results can be summarized into the four points. First, the MSC-DP cells induced from macaque MSCs were confirmed to express markers for Tuj1, MAP-2, TH, and DAT and to release dopamine in vitro, as shown previously (21). Moreover, they expressed GIRK2 and FOXA2, specific markers of A9 dopaminergic neurons, but not calbindin, a marker of A10 neurons. Second, when MSC-DP cells were autologously engrafted into the striatum of hemiparkinsonian macaques, motor behaviors, as assessed by CRS and the hand-reach task, gradually improved without accompanying dyskinesia. PET scans detected distinct binding of ¹¹C-CFT in the engrafted striatum initially, followed by an exponential decrease in ¹¹C-CFT binding. Kinetic analysis suggested that a portion of the MSC-DP graft showed levels of ¹¹C-CFT binding above the baseline over a period of 7 months. Third, immunohistochemical analysis at 9 months confirmed the increase of DAT⁺/TH⁺/GIRK2⁺ cells and the TH⁺ axon terminals in the engrafted striatum. Fourth, no tumorigenicity was suspected by peripheral blood tests, blood tumor markers, ¹⁸F-FDG PET scans, and histology. Therefore, MSC-DP cells are likely to be nontumorigenic and able to remain in the transplanted region until at least 9 months after engraftment, although not in large quantities, thereby modestly restoring dopaminergic function and motor behaviors in vivo.

To the best of our knowledge, this study is the first to show restoration of dopaminergic function and motor behaviors in parkinsonian primate animals following engraftment of MSC-derived cells. It is notable that kinetic analysis of ¹¹C-CFT binding and histology results suggested that at least a portion of MSC-DP cells survived in situ for over 9 months. This is in contrast to naive MSCs, which have much shorter survival times in the brain and disappeared within 3 months in rodent studies; one possible reason for this could be senescent or apoptotic changes (12, 13). The long-term survival of MSC-DP cells may have been accompanied by gradual integration into or reinnervation of the host striatal tissues. As shown in Figure 6D, increased numbers of TH⁺ terminals were observed in engrafted animals compared with control animals, suggesting formation of new synaptic connections between the engrafted cells and host tissues; this may at least in part underlie the motor recovery observed at a later time after transplantation of MSC-DP cells. The fact that in vitro DArc was correlated with ¹¹C-CFT binding only at early time points after engraftment also supports the hypothesis that other mechanisms, such as integration of MSC-DP cells, into host tissues may be critical for full restoration of motor function.

A notable feature of the current MSC-DP cells is their similarity to A9 dopaminergic neurons. Previously, we reported that dopaminergic neuron-like cells are inducible from MSCs by genetic introduction of NICD, followed by cytokine treatment with bFGF, forskolin, CNTF, and GDNF (20, 21). This report further characterized the MSC-DP cells and found that they were double positive for FOXA2 and GIRK2, both specific markers of differentiated A9 dopaminergic neurons. In particular, FOXA2 was recently identified as a transcription factor required for specifying and maintaining the dopaminergic neuron phenotype (28, 42) and is considered to be expressed exclusively in floor plate–derived dopaminergic neurons (43). We confirmed the coexpression of FOXA2 and GIRK2 in SNc tissue that contains A9 dopaminergic neurons but not in the VTA, which contains A10 dopaminergic neurons. GIRK2/FOXA2 double-positive cells have not been induced from other stem cell sources using existing protocols (44–46), except for one that manipulates both the sonic hedgehog and WNT signaling pathways (43, 47). Recent studies of fetal tissue engrafting in patients with PD or PD model rats have shown that A9 dopaminergic neurons are determinants of successful functional recovery (16, 48) and decreased dyskinesia (49) and that they are associated with well-organized synaptic connectivity (16). Overall, the results of our study support a recent hypothesis that well-differentiated stem cells offer benefits for both survival and functional integration with host neural tissues (47).

There are 2 points that favor the suitability of MSC-DP cell transplantation for clinical application. First, the current strategy rests on the autologous cell system. This offers advantages over allograft systems, such as fetal tissue engrafting, in terms of the ethical, social, and political implications. MSCs can be easily collected autologously from the patients’ own bone marrow aspirates. Aspiration of bone marrow by itself is widely applied in the field of hematology, and infusion of bone marrow from HLA-matched donors is also commonly performed for treatment of leukemia. Moreover, bone marrow–derived MSC transplantation into brain has been tried clinically without any safety problems (10). The autologous system also has the advantage of not requiring immunosuppression, as compared with allograft systems. Second, no tumorigenicity was suspected up to 9 months after engraftment in primates. Adult stem cells, including MSCs, are known to have less tumorigenic proliferative activity than other cell sources, such as embryonic stem cells. Moreover, the MSC-DP cells expressed MAP-2, a marker of postmitotic neurons (50), suggesting that they possess minimal risk of tumor formation. A previous study in which MSC-DP cells were engrafted into rodents consistently showed no evidence of tumorigenicity during a 14-week period (20). Schwann cells derived from MSCs also showed no tumorigenicity during a 1-year observation period in our previous study in primates (18). The differentiation technique for converting naive MSCs to MSC-DP cells involved NICD gene introduction by transfection of a plasmid gene but not by retrovirus- or lentivirus-mediated gene transfer, which is known to modify the nuclear genome. The introduced plasmid NICD gene does not appear to have a prolonged effect on the cell cycle, because the introduced NICD gene did not remain in MSCs for a long time (for no longer than 2 passages) (51).

Although our study showed a potential therapeutic effect of MSC-DP cells in a PD model, one may argue that cells pushed to full maturity in vitro would not be the best cell source for grafting (2). For example, grafted dopaminergic neurons obtained from fetal brain are known to survive poorly if their maturation has passed a narrow optimal time window at the time of harvesting (52). Such an argument might be applicable to our results in that the rapid loss of ¹¹C-CFT binding over time may have been a consequence of the use of differentiated MSCs, namely MSC-DP cells. However, this is still an open question, particularly with respect to stem cells; the relationship between in vitro maturity and survival after graft is not simple and may also depend on other factors, such as the types of cell sources and in vitro preparation. ES grafts with very immature cells seem to cause tumorigenicity, while relatively immature cells, such as neural precursors/neuroblasts, suffer from poor cell survival (40). But more highly differentiated DA neurons recently engineered from ES cells showed good survival (47). Undifferentiated MSCs are known to die very rapidly, within several days after being engrafted into the brain, and this seems to be due to facilitated senescence (12) or programmed cell death of the MSCs (13). Therefore, we have followed the working hypothesis, testing well-differentiated cells derived from MSCs. To address the issue of optimal maturity, cell marker–based subtyping should also be standardized in future studies (33).

A few issues suggest the need for some caution when interpreting our results. First, in this study, we did not label the MSC-DP cells by tagging them with, for example, green fluorescent protein in order to trace the cells after transplantation. This was because we avoided using any extrinsic factors that may pose a safety concern or affect cell survival. As a result, the dopaminergic neuron-like cells found in the striatum may not truly have originated from the grafted MSC-DP cells. However, this is unlikely because the striatum in primates has only a few dopaminergic neurons under normal circumstances: TH⁺ cells number <1% of striatal neurons (53) and are preferentially scattered near the white matter–ventral striatum border. In our case, the TH⁺/DAT⁺/GIRK2⁺ cells were located only in an area near the engrafted area in the dorsal striatum (Figure 5), and the number of the cells was larger than that in sham and nonaffected striata (Figure 6, C and E). Second, regarding PET ligands, a well-known ligand for DA, ¹⁸F-DOPA was not used in this study, because it is a metabolic ligand for amino acids and may reflect not only dopamine turnover in dopaminergic neurons, but also uptake of amino acid precursors in tumors (54) or inflammation (55). Therefore, ¹¹C-CFT was carefully chosen as an in vivo surrogate marker for tracking MSC-DP cells, because it is a highly specific ligand for DAT, which is specifically expressed in dopaminergic neurons and was confirmed to be present in the current MSC-DP cells in vitro. The high uptake of ¹¹C-CFT was not found in any of the sham-operated animals, even though they received PBS injections, which may induce tissue damage or inflammation. Third, the study does not clarify the complete mechanism underlying the motor recovery in the current animals. PET findings revealed increased DAT expression only in the early phase, while behavioral data showed significant recovery only in the later phase. As described above, we believe the discrepancy is explained by a time delay required for the remaining MCS-DP cells to achieve synaptic connections with the host tissues. This speculation should be clarified in the future study, since the delayed motor recovery found here seems to be common in trials of cell replacement therapies in PD. For example, fetal tissue grafting improved motor symptoms at 6 to 9 months after grafting in selected patients with PD (39). ES cell–derived well-differentiated DA neurons also restored motor behaviors 3–5 months after grafting in an animal model (47). Finally, these data do not show convincing evidence that benefits were truly results of an in vitro process differentiating MSCs into A9 DA neurons. We cannot completely exclude the possibility that undifferentiated MSCs, contaminated in the graft and not positive for A9 markers (25%–50% of cells; Figure 1K), might have contributed to the observed benefits, for example, by way of trophic effects. Therefore, future studies should directly compare the MSC-DP cell with naive MSC transplantation.

In conclusion, this study indicates that the autologous MSC-differentiation system may be safe and potentially effective for restoring motor dysfunction in hemiparkinsonian nonhuman primate animals. Although further studies are needed to improve the viability of MSC-DP cells and net performance for functional recovery, the current system may expand the availability of cell sources for cell-based therapies for patients with PD.

View Supplemental data

We thank Noboru Teramoto, Hajime Fukuda, Hiroshi Koshino, Kyoko Shioya, Keiko Tokuda, and Akihiro Kawasaki for their technical help and Tsuyoshi Tahara and Hideki Mochizuki for their helpful comments. This study was supported by the Program for Promotion of Fundamental Studies in Health Sciences of the National Institute of Biomedical Innovation (NIBIO, 05-06, 10-05).

Address correspondence to: Takuya Hayashi, Functional Probe Research Laboratory, Center for Molecular Imaging Science, RIKEN, 6-7-3 Minnatojima-minamimachi, Chuo-ku, Kobe, Hyogo 650-0047, Japan. Phone: 81.78.304.7121; Fax: 81.78.304.7123; E-mail: takuya.hayashi@riken.jp. Or to: Mari Dezawa, Department of Stem Cell Biology and Histology and Department of Anatomy and Anthropology, Tohoku University Graduate School of Medicine, 2-1 Seiryo-machi, Aoba-ku, Sendai, Miyagi 980-8575, Japan. Phone: 81.22.717.8025; Fax: 81.22.717.8030; E-mail: mdezawa@med.tohoku.ac.jp.

Conflict of interest: The authors have declared that no conflict of interest exists.

Reference information: J Clin Invest. 2013;123(1):272–284. doi:10.1172/JCI62516.

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