The human iPSC line, 201B7 (Takahashi et al., 2007), was obtained from Kyoto University via RIKEN BioResource Research Centre in accordance with the relevant guidelines and regulations. The iPSCs were cultured on mitomycin C-treated SNL murine fibroblast feeder cells in human iPSC medium, according to a previous report (Takahashi et al., 2007). All experimental procedures were approved by the Juntendo University School of Medicine Ethics Committee and all experiments with human iPSCs were performed in accordance with relevant guidelines and regulations.

Differentiation into mDA neurons from human iPSCs was performed as described previously (Imaizumi et al., 2015; Matsumoto et al., 2016; Fujimori et al., 2017; Ishikawa et al., 2018; Yamaguchi et al., 2020) with minor modifications. The iPSCs cultured on feeder cells were treated with 3 μM aSB431542 (Tocris Bioscience, Avonmouth, UK), 3 μM dorsomorphin (Sigma-Aldrich, St. Louis, MO, USA), and 3 μM CHIR99021 (REPLOCELL, Yokohama, Japan) on day 0 in the human iPS medium. The medium was replenished with the three chemicals every day for 5 days for differentiation into the three germ layers with a chemically transitional EB-like state (CTraS) (Fujimori et al., 2017). To generate non-CTraS-mediated mDA progenitors for evaluating tumorigenesis, iPSCs were cultured for 5 days in the absence of these three small molecules. On day 5, the iPSC colonies were detached from the feeder layers using a dissociation solution (REPLOCELL) and enzymatically dissociated into single cells using TrypLE Select (Life Technologies, Carlsbad, CA, USA) at 37°C for 5–7 min. The dissociated and filtered (40 μm) cells were cultured in a suspension at a density of 1 × 10⁴ cells/mL in the neurosphere medium in 4% O₂, to form primary neurospheres. The neurosphere medium is a KBM Neural Stem Cell medium (KOHJIN BIO, Saitama, Japan) supplemented with 1% penicillin–streptomycin (Life Technologies), 2% B27 supplement (Life Technologies), 20 ng/mL basic fibroblast growth factor (bFGF; Pepro Tech, Rocky Hill, NJ, USA), 2 μM SB431542 (Tocris Bioscience), and 5 μM Y27632 (Wako, Osaka, Japan). For ventral midbrain specification, 3 μM CHIR99021 and 2 μM purmorphamine (Millipore, Burlington, MA, USA) were added to the culture medium on day 8. For forebrain specification, we added 3 μM IWR-1-endo (Millipore) on day 5, and 2 μM purmorphamine on day 8. For spinal cord specification, we added 3 μM CHIR and 1 μM retinoic acid (Sigma-Aldrich, St. Louis, MO, USA) on day 5 and 2 μM purmorphamine on day 8. On day 19, the corrected neurospheres were centrifuged for 5 min at 200× g and the supernatant was separated. Further, the neurospheres were dissociated using TrypLE Select and filtered using the same procedures as performed on day 5. Some dissociated neurospheres on day 19 were passaged in suspension at a density of 5 × 10⁴ cells/mL in the neurosphere medium with 3 μM CHIR99021 and 2 μM purmorphamine, and cultured for another 7–10 days, to form secondary neurospheres. For the in vivo transplantation experiments, dissociated neurospheres were used. For in vitro neural differentiation, the dissociated neurospheres were plated onto 6-well plates or 8-well chamber slides coated with poly-L-ornithine (Sigma-Aldrich) and fibronectin (Corning, NY, USA). These cells were cultured in the neuron medium consisting of KBM Neural Stem Cell medium supplemented with 2% B27 supplement, 20 ng/mL brain-derived neurotrophic factor, (BDNF) (BioLegend, San Diego, CA, USA), glial cell-derived neurotrophic factor (GDNF) (PEPROTECH Inc., Rocky Hill, NJ,USA), 200 μM ascorbic acid (Sigma-Aldrich, St. Louis, MO, USA), 0.5 mM dibutyryl-cyclic adenosine monophosphate (Nakalai Tesque, Kyoto, Japan), 1 ng/mL transforming growth factor β3 (TGF-β3; BioLegend), and 10 μM DAPT (Sigma-Aldrich). Cells were cultured for 17 days in a humidified atmosphere containing 5% CO₂. CHIR (3 μM) was added to this medium only on day 19. Half of the medium was changed every 2 days.

Total RNA was isolated using the RNeasy mini kit (QIAGEN, Hilden, Germany) with DNase I treatment, and cDNA was generated using a ReverTraAce qPCR RT kit (TOYOBO, Osaka, Japan). The qPCR analysis was performed with SYBR premix Ex Taq II (Takara Bio, Shiga, Japan) on a ViiA™ 7 real-time PCR system (Thermo Fisher Scientific, Waltham, MA, USA). Values were normalized to ACTB and analyzed using the comparative (ΔΔCt) method. Primers used in this study are listed in Supplementary Table S1.

Male CB17-SCID mice (8-week-old) were purchased from Charles River Laboratories (Yokohama, Japan). Animals were cared for in accordance with the Guiding Principles for the Care and Use of Animals approved by the Ethics Review Committee for Animal Experimentation of Juntendo University School of Medicine, and we performed all mouse experiments in accordance with the approved protocol. All experiments were carried out in compliance with the ARRIVE guideline and the relevant guidelines and regulations. The mice were stereotaxically injected 6-OHDA (Sigma-Aldrich) into the right striatum to generate a Parkinsonian model (Lundblad et al., 2004). Each mouse received two unilateral injections of 6 μg 6-OHDA in 2 μL of saline with 0.02% ascorbic acid.

The coordinates were calculated with reference to bregma (in mm from bregma): AP +1, ML −2.1, DV −3.2, and AP +0.3, ML −2.3, DV −3.2 (Bateup et al., 2010). Three weeks after the 6-OHDA injection, the transplantation group received cell transplantation via a stereotactic injection of 2 × 10⁵ dissociated neurospheres in 2 μL KBM Neural Stem Cell medium through a 22G needle into the right striatum at the following coordinates (in mm from bregma): AP +0.5, ML +2.0, DV −3.8 (Steinbeck et al., 2015). Eleven (of 18) model mice were transplanted with primary neurospheres and seven were transplanted with secondary neurospheres. The sham group received injections of the same amount of saline into the same coordinates. Sixteen weeks after transplantation, the animals were euthanised with pentobarbital and perfused transcardially with 10% formalin (Wako). The brains were removed immediately and saturated with phosphate-buffered saline (PBS) containing 30% sucrose. The brains were cut using a cryostat (CM-1850; Leica Biosystems) at 30 μm thickness and mounted for the immunofluorescence study.

The apomorphine-induced rotational behavior assay was performed 1 week before and every 4 weeks after transplantation. A dose of 0.5 mg/kg of apomorphine (Wako) was injected subcutaneously, and the rotations were recorded for 30 min (Akerud et al., 2001; Han et al., 2015; Zuo et al., 2017). The animal behavior was automatically calculated using DAQ PL3508 PowerLab 8/35 ADInstruments Australia (Software LabChart ver.8 ADInstruments Australia). Only mice that rotated seven or more rotations per minute 2 weeks after 6-OHDA injection were defined as PD model mice.

For in vitro studies, cells were fixed with 4% paraformaldehyde for 15 min at 20–24°C. The fixed cells were blocked with 5% normal foetal bovine serum and 0.3% Triton X-100 for 1 h at 20–24°C, and incubated with primary antibodies diluted with blocking solution overnight (15–20 h) at 4°C. After reaction with the primary antibodies, the samples were washed with PBS and incubated with species-specific secondary antibodies conjugated with Alexa Fluor 488, 594, and 647 (1:500, Thermo Fisher Scientific) for 1 h at 20–24°C. These samples were mounted on slides with mounting medium containing DAPI (Vector Laboratories Inc., Burlingame, CA, USA). For in vivo studies, brain sections were blocked with 1% normal horse serum and 0.05% Triton X for 1 h at 20–24°C and then incubated with primary antibodies diluted with blocking solution overnight (15–20 h) at 4°C using the free-floating method. The samples were washed with PBS and incubated with species-specific secondary antibodies conjugated with Alexa Fluor 488, 594, and 647 (1,500) for 1 h at 20–24°C. These samples were mounted on slides with mounting medium containing DAPI (Vector Laboratories Inc.). These images were examined using laser scanning confocal microscopy (TCS-SP5; Leica Biosystems), an LSM-710 confocal laser-scanning microscope (Carl Zeiss, Jena, Germany) and a BZ-9000 fluorescence microscope (Keyence, Osaka, Japan). The in vitro positivity rate was calculated using ImageJ/Fiji (version 1.53c). The cell positive rate in the brain graft was calculated from the average of 1–3 different fields at a magnification of 40× using sections in which the graft remains as large as possible. Antibodies used in this study are listed in Supplementary Table S2.

Statistical significance between the two samples was determined using the Student’s t-test (SPSS Version. 25.0; SPSS, Inc., Chicago, IL, USA). The data were considered statistically significant at p < 0.05 and are shown as mean ± standard error of the mean.

The iPSCs derived from a healthy volunteer were differentiated into mDA progenitors, according to our previous reports (Imaizumi et al., 2015; Matsumoto et al., 2016; Fujimori et al., 2017; Yamaguchi et al., 2020) as summarized in Figure 1A. Human iPSCs were treated with SB, DM, and CHIR for 5 days to develop the CTraS condition, which expresses the genes specific to the three germ layers and the embryoid bodies in vitro. The dissociated cells were then transferred to a floating culture to form neurospheres from each single neural stem cell (Matsumoto et al., 2016; Fujimori et al., 2017). These neurospheres were formed with purmorphamine (a sonic hedgehog agonist) and CHIR to provide the ventral midbrain regional identity as mDA progenitors (Imaizumi et al., 2015; Yamaguchi et al., 2020). To verify the ventral midbrain regional identity of the neurospheres, we performed qPCR analysis using region-specific markers on day 19. We used neurospheres treated with IWR-1-endo, a Wnt antagonist that provides a forebrain specification; forebrain spheres and neurospheres treated with RA provide posterior hindbrain and spinal cord specification, as spinal-cord spheres. FOXG1, a frontal cortex marker, and SIX3, a forebrain marker, were expressed in IWR-1-endo-treated forebrain neurospheres, but not in purmorphamine and CHIR-treated midbrain neurospheres. EN1, a floor plate midbrain marker, was expressed only in midbrain neurospheres. HOXB4, a hindbrain marker, was expressed in CHIR and RA-treated spinal cord neurospheres, but not in the midbrain or forebrain neurospheres (Figure 1B). These results indicate that neurospheres differentiated from iPSCs acquired ventral midbrain regional identity using purmorphamine and CHIR.

On day 19, the midbrain neurospheres were dissociated and plated onto culture dishes for terminal differentiation into dopaminergic neurons. Immunostaining of neural progenitor cells after one day in adhesive culture (day 20) confirmed that the cells were highly positive for mDA progenitor markers, FOXA2, LMX1A, and NURR1 (Figure 1C). The positivity rates for these markers in all the cells were 79.2 ± 2.11%, 88.2 ± 2.63%, and 98.0 ± 0.79%, respectively (Figure 1D). We also confirmed the expression of EN1 and these markers using qPCR (Supplementary Figures S1A–D). Additionally, 95.4 ± 1.65% of the progenitor cells were positive for the floor plate surface marker CORIN (Figures 1E,F), which has been used for sorting to enrich midbrain progenitors in the ongoing clinical trial by Takahashi and colleagues (Doi et al., 2014; Kikuchi et al., 2017; Doi et al., 2020; Takahashi, 2020). ALCAM (a central nervous system microvascular endothelium marker), and LRTM1 (a ventral midbrain marker), which have been reported as mDA progenitor markers for transplant therapy (Bye et al., 2015; Samata et al., 2016), were positive in most of the progenitors; 94.2 ± 1.56% positive for ALCAM, 94.5 ± 2.13% positive for an LRTM1 antibody from Abcam (Ab121409) and 99.8 ± 0.11% positive for another LRTM1 antibody from R and D (MAB10046) (Figures 1E,F). The expression of these mDA markers was also validated using qPCR (Supplementary Figures S1E–G). Recently, Xu et al. (2022) proposed other mDA progenitor markers, CLSTN2 and PTPRO, and successfully enriched mDA neurons by sorting, using CLSTN2-tdTomato and PTPRO-tdTomato knock-in hiPSCs due to the lack of commercially available antibodies (Xu et al., 2022). We also confirmed the expression of CLSTN2 and PTPRO using qPCR (Supplementary Figures S1H,IH,I). As expected, these results indicate that midbrain neurospheres differentiating from iPSCs contained mDA neural progenitors.

To determine the ability of mDA progenitors to differentiate into mDA neurons in vitro, the differentiated neurons were evaluated using immunostaining and qPCR analysis on day 36 (Figure 1G; Supplementary Figure S1J–O). The cells were differentiated into neurons (β3-tubulin+, a neuron marker/nucleus, 81.9 ± 1.27%) and enriched with mDA neurons (TH+/β3-tubulin+, 61.0 ± 9.23%). Most of the TH+ cells also expressed mDA markers such as NURR1 and PITX3, and mature mesencephalic dopaminergic neuron markers (Figure 1G). Among the TH-positive neurons, 87.8 ± 1.95% were positive for GIRK2, an A9 mDA neuron marker, and 19.3 ± 2.05% were positive for Calbindin, an A10 mDA neuron marker (Figures 1G,H). These results indicate that the neurosphere-based mDA neuron induction method efficiently induced ventral mDA progenitors from iPSCs without fluorescence-activated cell sorting (FACS), and the progenitors differentiated into mature mDA neurons, which were predominantly A9 neurons in vitro.

To evaluate the therapeutic potential for cell replacement therapy of mDA progenitors derived from iPSCs by our neurosphere-based differentiation method without FACS enrichment, we transplanted mDA progenitors into 6-OHDA-induced PD model mice. We injected 6-OHDA in the right striatum of CB17 severe combined immunodeficient (SCID) mice, and the motor symptoms of each mouse were evaluated by apomorphine-induced rotational behavior to confirm whether 6-OHDA disrupted striatal function and sufficiently induced PD symptoms. Three weeks after the 6-OHDA injection, we transplanted in vitro dissociated neurospheres on day 19 into the 6-OHDA-lesioned striatum (2 × 10⁵ cells in 2 μL), and injected saline in the sham group. Immunostaining with an SC-121 antibody, a human cytoplasmic marker, at 16 weeks after transplantation revealed that the transplanted cells survived and displayed neurite outgrowth in the striatum from the graft cells (Figures 2A,B).

To examine the composition of the graft cells, additional immunostaining was performed using mDA neuronal markers. The percentage of TH positive dopaminergic neurons was 14.0 ± 0.97% among human nuclear antigen (HNA, a human nuclear marker)-positive transplanted cells (Figures 2C,D). Among these HNA and TH double-positive neurons, 33.3 ± 5.68% were positive for the dopamine transporter (DAT), and 31.2 ± 5.23% were positive for aldehyde dehydrogenase 1A1 (ALDH1A1, an A9 mDA marker) (Figures 2E,F). These iPSC-derived dopaminergic neurons were also positive for mature mDA markers such as GIRK2 and Calbindin (Figure 2G). These results indicate that some transplanted iPSC-derived mDA progenitor cells have differentiated into mature dopaminergic neurons in the 4 months following transplantation. To evaluate the maturity of the graft cells, we immunostained brain sections with early mDA markers, FOXA2 and NURR1. The percentage of FOXA2+, early mDA progenitors, was 33.0 ± 2.7% and the percentage of NURR1+, postmitotic mDA progenitors, was 90.7 ± 1.3% in HNA-positive graft cells. Thus, the majority of the grafted cells were in the lineage of mDA neurons, and 14% of cells developed into mature mDA neurons. However, other cells remained at the progenitor stage (Figures 2C,D). The graft included 3.7 ± 0.49% of Ki67-positive proliferating cells and most of the Ki67-positive cells co-expressed FOXA2 (Ki67+/FOXA2+, 92.1 ± 2.06%), indicating that they were early mDA progenitors. The percentage of 5-HT+ serotonergic neurons was 4.7 ± 0.58%. These results indicate that transplanted mDA progenitors differentiated into ventral mDA neurons in the striatum of PD model mice with efficiencies similar to those reported earlier (Doi et al., 2014; Bye et al., 2015; Samata et al., 2016; Doi et al., 2020; Xu et al., 2022).

Recent single-cell analyses of transplanted ESC- and iPSC-derived ventral midbrain progenitor cells into rodent PD models have shown that the cells differentiate into neurons, astrocytes, and vascular leptomeningeal-like cells (VLMCs) (Tiklova et al., 2020; Xu et al., 2022). In the present study, we examined the expression of oligodendrocyte, astrocyte and VLMC markers by fluorescent immunohistochemistry to explore the possibility that our transplanted cells might differentiate into non-neuronal cells. A few grafted cells were positive for astrocyte markers, such as glial fibrillary acidic protein (GFAP) and S100β (Figure 2H). However, we found no evidence that grafted cells differentiate into oligodendrocytes and oligodendrocyte progenitor cells (OPCs) by staining against Olig2 and platelet-derived growth factor receptor (PDGFR) (Supplementary Figure S3). The presence of VLMC was assessed using a COL1A1 antibody that recognizes both human and rodent COL1A1, as well as a human-specific COL1A1 antibody (hCOL1A1). Within the grafts, we observed graft-derived human COL1A1-positive cells (Figure 2I, arrows) and infiltration of mouse-derived COL1A1-positive cells (Figure 2I, arrowheads). These results indicate that our transplanted mDA progenitors have similar differentiation potential to previously reported ESC- and iPSC-derived ventral midbrain progenitor cells.

To evaluate the function of grafted mDA neurons, we performed a behavioral analysis of transplanted PD model mice using apomorphine-induced rotation every 4 weeks after transplantation. The 6-OHDA-lesioned PD mouse model rotates around to the left on being injected with apomorphine, due to the loss of dopaminergic neurons in the right striatum (Akerud et al., 2001; Grealish et al., 2010). A reduction in the number of rotations induced by apomorphine indicates the differentiation of dopaminergic neurons after the successful transplantation of dopaminergic progenitor cells, thus improving PD symptoms.

A decrease in the number of rotations was observed in both cell-transplanted and sham-operated mice. However, 12 weeks after surgery, the number of rotations was significantly lower in the cell-transplanted mice than those in the sham-operated mice, presumably due to intrinsic factors which restored motor function (4.34 ± 0.90 times/min in transplanted mice versus 7.73 ± 1.10 times/min in sham mice). Furthermore, this significant improvement in motor function by the transplanted cells was confirmed until at least 16 weeks after transplantation (2.67 ± 0.59 times/min in transplanted mice versus 6.00 ± 0.95 times/min in sham mice; Figure 3A). Since we used mDA progenitors from primary neurospheres and secondary neurospheres for transplanted cells, the separate graphs for primary neurosphere- and secondary neurosphere-derived cells was shown in Figure 3B. Despite the reduced number of samples in each group, there is still a consistent general trend, although there has been a lower detection of statistically significant differences. Unexpectedly, pre-transplant symptoms tended to be stronger in mice implanted with secondary neurospheres and weaker in those implanted with primary neurospheres. However, there was no statistically significant difference between these three groups, and secondary neurospheres showed a trend toward improvement compared to the Sham group. Thus, we concluded that grafted mDA neurons functioned in PD model mice.

We used the CTraS method to induce the mDA progenitor cells transplanted in this study. In this method, the treatment of undifferentiated iPSCs with three different inhibitors (SB, DM, and CHIR) accelerates the differentiation of subsequent cells (Fujimori et al., 2017). In this study, 18 PD model mice were transplanted with CTraS-mediated mDA progenitors and no tumor formation was observed. To evaluate the inhibitory effect of tumorigenesis using the CTraS method, we injected the same amounts of non-CTraS-mediated mDA progenitors, induced in the same protocol except iPSCs were not treated with the three inhibitors on days 0–5, into six PD model mice; three mice were analyzed at 8 weeks and the remaining three at 16 weeks after transplantation. At 8 weeks, one of three mice showed remarkable proliferation of transplanted cells which were considered to be the tumor (Figure 4A); a large number of the grafted cells in the other two mice were still Ki67-positive immature cells. The ratio of immature cells (Ki67+/HNA+, 69.4 ± 15.4%) was significantly higher than that of CTraS-mediated graft cells (17.2 ± 5.89%, Figures 4B,C). At 16 weeks, no tumor was found in the three mice; however, in one mouse, the transplanted cells were found outside the striatum. Although the sample size excluding mice with cells transplanted to different locations was too small for statistical analysis, many cells remained immature (43.1%) compared to those in CTraS-mediated graft cells (3.73 ± 0.49%, Figures 4D,E). These results indicate that this CTraS-based mDA neuron differentiation method potentially improves the safety of cell transplantation therapy in PD.