Pre-clinical study of induced pluripotent stem cell-derived dopaminergic progenitor cells for Parkinson’s disease

Induced pluripotent stem cell (iPSC)-derived dopaminergic (DA) neurons are an expected source for cell-based therapies for Parkinson’s disease (PD). The regulatory criteria for the clinical application of these therapies, however, have not been established. Here we show the results of our pre-clinical study, in which we evaluate the safety and efficacy of dopaminergic progenitors (DAPs) derived from a clinical-grade human iPSC line. We confirm the characteristics of DAPs by in vitro analyses. We also verify that the DAP population include no residual undifferentiated iPSCs or early neural stem cells and have no genetic aberration in cancer-related genes. Furthermore, in vivo studies using immunodeficient mice reveal no tumorigenicity or toxicity of the cells. When the DAPs are transplanted into the striatum of 6-OHDA-lesioned rats, the animals show behavioral improvement. Based on these results, we started a clinical trial to treat PD patients in 2018. Induced pluripotent stem cell (iPSC) derived dopaminergic neurons are a promising source for cell-based Parkinson’s disease (PD) therapy. Here the authors report a comprehensive pre-clinical evaluation of the safety and efficacy of dopaminergic progenitors derived from a clinical-grade human iPSC line.

T he discovery that the transplantation of fetal midbrain tissues can restore neurological symptoms of Parkinson's disease (PD) patients [1][2][3][4] has stimulated interest in cell therapies for the disease. Human pluripotent stem cells (PSCs), such as embryonic stem cells (ESCs) and induced PSCs (iPSCs), are sources for a stable supply of cells for the therapy 5 . Culture protocols that induce authentic midbrain dopaminergic (DA) neurons from human PSCs have already been established by several research groups, and the DA neurons have been proven safe and effective in rodent 6,7 and primate 8 PD models. These results suggest that PSC-derived DA neurons can contribute to the treatment of PD. However, the use of PSCs for cell-based therapies is such a new challenge that regulatory rules are not universally standardized. Indeed, each country has its own regulations and different criteria for clinical application. Therefore, to develop a global standard cell-based therapy for PD, it is important to share the results of pre-clinical studies and the outcomes of clinical trials 9 .
Currently, two clinical trials using human ESCs are ongoing in Australia (NCT02452723) and China (NCT03119636), and their pre-clinical studies are reported 10,11 . We recently started a clinical trial (JMA-IIA00384, UMIN000033564) in Japan to treat PD patients by using iPSC-derived DA progenitors (DAPs). Because in these therapies the grafted cells survive and function as DA neurons for a long time (possibly until the patient dies), they must undergo more intensive risk management compared to other clinical trials using mesenchymal stem cells 12 , in which the grafted cells function to provide neurotrophic support or immunomodulation but not as DA neurons, and do not survive long. Since 2015, we have continued discussion with the Japanese regulatory agency, Pharmaceuticals and Medical Devices Agency (PMDA), about details of the pre-clinical study. In this report, we show that the DAPs have no tumorigenicity or toxicity and that they improve the abnormal behavior of 6-hydroxydopamine (6-OHDA)-lesioned rats. Thus, we confirm the safety and efficacy of iPSC-derived DAPs for clinical trials.

Results
Manufacturing iPSC-derived DAPs under good manufacturing practice (GMP) conditions. As a starting material, we used a human iPSC line (QHJI01s04) derived from the peripheral blood cells of a human leukocyte antigen (HLA)-homozygous healthy volunteer 13,14 and established for clinical application at the Center for iPS Cell Research and Application, Kyoto University, Japan. Then, we established a clonal master cell bank, MCB003, for our project. We have preserved the iPSCs in hundreds of frozen vials as one lot. To treat a patient, we thaw one of the vials, differentiate the cells into DAPs, and transplant them without freezing. In this pre-clinical study, we used multiple vials to confirm reproducibility of the results.
We modified our culture protocol to induce DAPs from human iPSCs 8,15 to meet the GMP-grade culture condition ( Fig. 1a-d, Supplementary Table 1). Briefly, human iPSCs (MCB003) were seeded onto 6-well plates coated with laminin 511-E8 fragment and cultured for 12 days, and then CORIN+ 16 cells were isolated by fluorescence-activated cell sorting (FACS). The sorted cells were cultured in 96-well plates as cell aggregates until day 30 and then used for the transplantation. In this culture condition, all reagents including an anti-CORIN antibody were at GMP grade. A clinical-grade cell sorter with a disposable fluid tube was equipped to mitigate the risk of cross-contamination.
Characterization of iPSCs (MCB003) in vitro. In the process of inducing DAPs, quality control check points were placed at the stage of MCB003, pre-and post-FACS sorted cells, and the final product (Fig. 1e). The quality standards of MCB003 include the morphology of the colonies, the expression of pluripotent markers such as TRA-1-60, TRA-2-49, and SSEA-4, and sterility. We confirmed that almost all cells expressed pluripotent cell markers (Supplementary Table 2).
Characterization of the final product in vitro. We conducted the DAP induction process in 25 randomly chosen vials from MCB003 (one time for each vial). Before sorting on day 12, the percentages of TRA-1-60+ and CORIN+ cells were 0.0% (n = 24) and 31.4 ± 12.7% (n = 25), respectively. After sorting, the percentage of CORIN+ cells were 93.2 ± 2.1% (n = 25), confirming purity (Table 1, Supplementary Fig. 1a, b). The in-process testing for the manufacturing of DAPs is described in Supplementary Table 3.
The quality standards of the final product were determined to confirm that the cells were mainly composed of DAPs and included no undifferentiated cells. For this purpose, the expression of markers for DAPs (FOXA2 and TUJ1), iPSCs (OCT3/4 = POU5F1, LIN28, and TRA-2-49), and proliferating early neural progenitors (SOX1, PAX6, and KI67) were examined by flow cytometry and quantitative reverse transcription PCR (RT-qPCR). Flow cytometry on day 26 revealed that the percentage of DAPs (FOXA2+TUJ1+) was 92.3 ± 4.0% (n = 25; Table 1 and Supplementary Fig. 1c, d). Immunostaining also showed that most cells expressed DAP markers such as LMX1A and FOXA2 (Fig. 1f), and a smaller population expressed more mature DA markers such as NURR1 and TH (Fig. 1g).
To further confirm that there were no residual iPSCs, day-26 spheres were cultured in a condition suitable for iPSCs for 2 weeks. In this experiment, we mixed 0.001 to 1.0% of iPSCs with 2 × 10 5 dissociated cells from the day-26 spheres and found that even 0.001% was enough to form visible iPSC colonies. Importantly, day-26 spheres without mixture did not show any colony formation ( Supplementary Fig. 1i). Taken together, these results indicated that the final product contained no or minimal (<0.001%) undifferentiated iPSCs.
N\ext, we examined if the DAPs could differentiate into functional DA neurons. To evaluate the production of dopamine, day-26 spheres were cultured until day 56 on L-ornithine/laminin/ fibronectin-coated plates, and dopamine release in response to high KCl stimulation was measured by liquid chromatography with tandem mass spectrometry (LC-MS/MS) (n = 5, Fig. 1j). The amount of dopamine was 3.30 ± 2.72 pmol μg −1 DNA in the cultured media, 0.47 ± 0.45 pmol μg −1 DNA in low KCl, and 1.46 ± 0.65 pmol μg −1 DNA in high KCl. Upon electrophysiological analyses, we found 67% of examined neurons showed spontaneous action potentials (Fig. 1k, l) and 89% of neurons showed induced action potentials (n = 18, Fig. 1m). The resting membrane potential was −49 ± 15 mV.
Genomic, epigenetic, and single-cell analyses. The results above suggest that there is no tumorigenic component in the final product. However, genomic and epigenetic changes in the cells may affect cell behavior after transplantation. To examine this possibility, genomic and epigenetic stability during the differentiation was examined, and the final products were subjected to tumorigenicity studies. We repeated DAP induction six times and compared the results of whole-genome sequencing (WGS) and whole-exome sequencing (WES) obtained from original peripheral blood cells, undifferentiated iPSCs, and differentiated cells on days 12 and 26.
We extensively investigated mutations in the 686 cancerrelated genes listed in the Catalog of Somatic Mutations In Cancer (COSMIC) Census (COSMIC version 83). We also   (Table 2). WES revealed single-nucleotide variants (SNVs) in three genes, but this discovery was not consistently observed and thought to be a false positive after considering the results of the amplicon sequencing (Supplementary Tables 5, 6). In addition, there were no residual plasmids or copy number variations (CNVs) in all samples tested.
Regarding epigenetic modifications, we examined the methylation ratio at the transcriptional start site of 73 cancer-related genes. Our final products showed lower methylation levels than those of tissues with brain malignant tumor glioblastoma multiforme and similar levels with normal cells and fetal ventral mesencephalon ( Fig. 2a and Supplementary Fig. 2).
To examine the variation of the gene expressions among the six independent preparations, we performed a single-cell RT-qPCR analysis. In each cell preparation, iPSCs on day 0, CORIN+ cells on day 12, and the final products on day 26 showed similar expression patterns in t-distributed stochastic neighbor embedding (tSNE) plots (Fig. 2b). In addition, there was no difference in the expression of genes related to DA differentiation (Fig. 2c, Supplementary Data 1). These results support the reproducibility of the cell manufacturing process.
Tumorigenicity, toxicity, and biodistribution studies. The same final products used for the genomic and epigenetic analyses were subjected to tumorigenicity studies. We repeated DAP induction six times, and each DAP population was injected into the right striatum, which is the same target used in the clinical trial, of immunodeficient NOG (NOD.Cg-Prkdc scid Il2rg tm1Sug /ShiJic) mice. In this study, the toxicity and biodistribution of the cells were also examined. After discussion with the PMDA, we observed the animals as long as possible, namely for a life-long period. Initially, we grafted day-30 spheres (2 × 10 5 cells per mouse, maximum dose) into 80 (males: 40, females: 40) and 50 mice (males: 25, females: 25) as the cell product-transplanted group and saline-injected control group (Supplementary Table 7), respectively, and observed the groups until the number of surviving male or female mice was 30 (transplantation group) or 20 (control group). At 52 weeks after transplantation (59-60 weeks old), the number of male mice in the control group was 19, at which point we stopped the observation. At this time, 28 males and 27 females survived in the transplantation group, and 19 males and 22 females survived in the control group. A survival curve of the animals showed no significant difference between groups (log-rank test, p = 0.3208, Fig. 3a). No changes were attributable to the transplantation in the mice's general conditions, body weight, food consumption, pharmacological conditions (modified Irwin test), ophthalmological findings, urinalysis, hematology and blood chemistry data, necropsy, or organ weight. Hematoxylin-eosin (H-E) staining of 5-μm-thick brain slices revealed no proliferative or malignant findings, and immunostaining for tyrosine hydroxylase showed that DA neurons had survived ( Fig. 3b-e). In 23 (12 males and 11 females) of the 55 mice in the transplantation group, tubular cell clusters were found in the grafts and expressed pan-cytokeratin and   Fig. 3a, b). The cell clusters were very small (<0.5 mm in diameter), not frequent (mean 1.4 clusters per graft), and not proliferating (no expression of KI67). In addition, they showed no effect on the survival or neurite extension of surrounding DA neurons. These results verified no tumorigenicity of the final products. Toxicity and biodistribution were also investigated using the same animals (see "Methods"), no abnormal findings were observed in the toxicity study, and grafted human cells (KU80+) were found only in the brain and not in systemic organs.
Teratoma formation assay in subcutaneous space. We further examined the existence of residual iPSCs in the final products by injecting day-30 spheres into the subcutaneous space of NOG mice with Matrigel, which is reported to be the most sensitive method to detect teratoma formation by residual undifferentiated iPSCs 17 . As a spike control, 0.001-10% of undifferentiated iPSCs (MCB003) were mixed into the day-30 spheres. As a positive control, another cell line of iPSCs (201B7, established by a retroviral vector with c-Myc 18 ) and HeLa cells were used. When 600,000 HeLa cells or 201B7 cells were injected, subcutaneous tumors became detectable at around 10 weeks. In contrast, there was no tumor formation for 26 weeks in the DAP group, DAP with spiked MCB003 group, or even MCB003 group (Fig. 3f). Histological analysis revealed that KU80+ human cells survived in the DAP groups. On the other hand, no KU80+ cells were detected in the MCB group, suggesting that undifferentiated MCB003 cells cannot survive in the subcutaneous space ( Fig. 3g-o). Similarly, MCB003 cells did not form any tumors in the mouse striatum, while 201B7 cells formed large teratomas ( Supplementary Fig. 3c). When MCB003 cells were injected into the testes of NOG mice, however, they formed typical teratomas, indicating that they were viable and pluripotent ( Supplementary  Fig. 3d). These results suggested that undifferentiated MCB003 cells do not remain in day-30 spheres, and that even if they did, the risk of teratoma formation is negligible.
For the clinical trial, we have developed a long-thin needle that is attachable to a stereotaxic frame. To examine the usability of the needle, MCB003-derived day-30 spheres (1.5-2.0 million cells per monkey) were transplanted into the left putamen of MPTP (1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine)-treated monkeys (n = 3). Histological analysis 6 months after the transplantation revealed that the grafted DA neurons survived without any adverse effects, including hemorrhage or inflammation ( Fig. 4i-n). The results of the in vivo pre-clinical studies are summarized in Supplementary Table 8.

Discussion
For the clinical application of a cell-based therapy, a 2 × 2 matrix (Safety, Efficacy × Cellular and Non-cellular components; Supplementary Table 9) is recommended for quality control of the final product. In our case, the active cellular component is iPSCderived DAPs. We confirmed that 92.3% of the total cells are FOXA2+TUJ1+ DAPs on culture day 26. We also confirmed that some of the survived human cells in rat were positive for GFAP, which suggests glial progenitors were included in the grafted cells with DAPs. The DAPs became mature DA neurons that produced dopamine and improved the abnormal behaviors of 6-OHDA-lesioned rats. Two previous clinical trials for PD using PSCs have transplanted ESC-derived neural progenitor cells (NCT02452723) and ESC-derived DA neurons (NCT03119636). These pre-clinical studies are summarized in Table 3.
There are three cellular components that should not be contained in the final product. The first is undifferentiated iPSCs, and we confirmed their absence by four methods in vitro. The second is early neural stem cells. Previously, we found that SOX1+ PAX6+ cells exist in the early stage of differentiation and that these cells form rosettes and proliferate in the brain 19 . The expression of CORIN, however, is never common with SOX1 or PAX6 expression. As a result, we can eliminate SOX1+PAX6+ cells by sorting CORIN+ cells. The third is transformed cells. The genomic instability of iPSCs and their derivatives may lead to tumorigenicity 20,21 . However, the need for genomic analysis is controversial, because genomic abnormalities do not always cause tumors. In addition, there is no standard gene list for which we should investigate. In this situation, we decided to investigate the genes listed in COSMIC Census and the cancer-related gene list made by the PMDA as well as familial disease-related genes listed in a HGMD PRO database. We performed WGS and WES of both iPSCs and the final products, but detected no aberrations in these genes. Epigenomic changes in iPSCs are another possible  22 . From our results of a methylation array, iPSCs and the final products were not hypermethylated compared with malignant brain tumor tissue and instead showed a methylation rate consistent with normal cells. Tumorigenicity studies in vivo are also not standardized. In previous pre-clinical studies, the observation periods in nude rats and SCID (severe combined immunodeficiency) mice were 9 months (Table 3) 10,11,23 . In the case of spinal cord injury, ESCderived oligodendrocyte progenitors (two million cells) were injected into the spinal cord of SCID mice, and the histological analysis was performed 12 months later 24 . In the present study, we used NOG mice, in which not only T cell and B cell activities but also NK cell activity is reduced, thus reducing the immune  BRN3a  CALB1  CORIN  DAT  EN1  EN2  FN1  FOXA2   GAPDH  GATA6  GBX2  GFAP  GLI1  GSC  HES1  HES5  HOXA2  HOXAB4  KCNJ6  LMX1A  LMX1B   LRP4  LRTM1  MAP2  MKI67  NANOG  NCAM  NKX2-1  NKX6-1  NR4A2  PAX2  PAX6  PITX3  POU5F1   SHH  SNCA   WNT5A  ZBTB16  Preparation   SOX1  SOX17  SOX2  T  TBR  TH  TPH2  TUBB3  VIM  VMAT2  response 25 . The observation period was 52 weeks, which approximates the mouse lifespan. Because we found no proliferating cells or abnormal findings in the grafts, 52 weeks (or 12 months) seem to be long enough for tumorigenicity study. Of note, before starting the pre-clinical study, we consulted the PMDA about the study design. The PMDA requested we use NOG mice, which is the least immunogenic animal model, is sensitive to tumor development 17,26 , and offers a long observation period. The PMDA also requested we evaluate at least 10 animals per group in the histological analysis. Major discussion points with the PMDA and our solutions are summarized in Supplementary Table 10. Regarding non-cellular components, we need to use clinical-or GMP-grade materials and reagents. We have made a GMP-grade anti-CORIN antibody and carefully confirmed that its remnant on cells was negligible. Regarding donor eligibility for iPSCs, a medical interview and blood exam for contamination were conducted. For other materials, γ-ray irradiation and viral elimination were applied to meet Japanese standards for biological raw materials. In addition, following guidelines of the International Council for Harmonization of Technical Requirements for Pharmaceuticals for Human Use (ICH) Q5A, we confirmed that the clinical-grade iPSCs (MCB003) were not contaminated with pathogenic viruses (Supplementary Table 11).
Unexpectedly, choroid plexus epithelia-like cells were observed in 23 of 55 mice with transplantation of the final product. In all cases, the cell clusters were microscopic (<0.5 mm), not frequent, not proliferating, and asymptomatic. Because bone morphogenetic protein (BMP) signaling is essential for developing choroid plexus tissue 27 , we modified our differentiation protocol to extend the period of the BMP inhibition ( Supplementary Fig. 4a). Accordingly, we successfully reduced the expression of TTR, a marker for the choroid plexus, in the final product, without any changes of other DA markers ( Supplementary Fig. 4b, c). Transplantation to nude rat resulted in TTR+ cells appearing in two out of seven rats without the BMP inhibitor, but in no rats out of six rats with the BMP inhibitor ( Supplementary Fig. 4d). We also confirmed that extension of the BMP inhibition can reduce the risk of inducing TTR+ epithelium-like cells in the brain.
In conclusion, the safety and efficacy of clinical-grade cell products were confirmed in this study. Based on these results, we received approval to start a clinical trial for PD from the PMDA and the institutional regulatory board of Kyoto University. The trial is ongoing.

Methods
Establishment of iPSC stock and manufacturing of DAPs. This study was approved by the ethics committee of Kyoto University Hospital and Center for iPS cell Research and Application (CiRA), Kyoto University, Kyoto, Japan. After informed consent was obtained from an HLA-homozygous volunteer donor, an iPSC stock for clinical use was established by reprogramming the donor's peripheral blood with episomal plasmid vectors expressing OCT3/4, SOX2, KLF4, L-MYC, LIN28, mp53DD, and EBNA1 at a cell processing center (Facility for iPS Cell Therapy, CiRA). The peripheral blood cells were isolated by using Ficoll-Paque PREMIUM (GE Healthcare), and 1.2 × 10 7 mononuclear cells were cultivated with StemFit AK03 without solution C (contains basic fibroblast growth factor) media (Ajinomoto) with 50 ng mL −1 interleukin-6 (IL-6), 50 ng mL −1 stem cell factor, 10 ng mL −1 thrombopoietin, 20 ng mL −1 Flt-3 ligand, 20 ng mL −1 IL-3, and 10 ng mL −1 granulocyte colony-stimulating factor (all WAKO) in four wells of a 24-well plate (3 × 10 6 cells per well). After 7 days of cultivation, the vectors were induced in 5 × 10 6 dissociated cells by a Nucleofector 4D electroporation system (Lonza), and the cells were replated on laminin 511-E8 fragment (iMatrix, Nippi)-coated 6-well plates (1.67 × 10 5 cells per well) in the same media as the mononuclear cells. One milliliter per well of StemFit AK03 media (Ajinomoto) was added 3, 5, and 7 days after the induction, and 9 days onward StemFit AK03 media were exchanged every 3 days. After 3 weeks of cultivation, single-cell-derived colonies became visible, and we picked up 15 of them manually. Each colony was dissociated with TrypLE Select CTS (Thermo Fisher), and all the cells were transferred to an iMatrix-coated 12-well plate and defined as passage 1 (P1). The P1 cells were passaged at 1.4 × 10 3 cells cm −2 every 8-12 days, and 30 vials of a primary cell stock (PCS) were frozen at P4. Two out of fifteen PCS clones were identified as iPSCs by ESC-like morphology, the absence of residual plasmids, confirmation of a normal karyotype, the expression of surface markers of undifferentiated iPSCs, and efficient neural differentiation. We selected the clones with the best efficiency for DA differentiation for the transplantation experiments. In order to confirm that the iPSCs were plasmid free by a longer culture, one vial of the PCS was thawed (P5) and expanded to 70 vials of secondary cell stock (SCS, P9). After thawing the SCS, we confirmed that there was no residual plasmids (tested at P9, P12, and P19), that they expressed undifferentiated markers (TRA-1-60, SSEA-4, and TRA-2-49; tested at P15), and had normal karyotypes (46, XY; tested at P12 and P13). One vial of the SCS was thawed and passaged two times, and then 384 frozen vials of MCB (P12) were stored. All freezing procedures were performed by using a programmed freezer. iPSCs were dispensed at 2 × 10 5 or 5 × 10 5 cells per tube in 200 or 500 μL of Stem Cell Banker GMP grade (TAKARA Bio, CB045), frozen to −80°C at −1°C min −1 , and stored in the gas phase of a liquid nitrogen tank.
Frozen MCB was differentiated after two passages of maintenance culture on iMatrix in StemFit AK03 medium was conducted. To manufacture the DAPs, the iPSCs were dissociated into single cells after 10-min incubation with 0.5× TrypLE select CTS (Thermo Fisher) and plated onto iMatrix-coated 6-well plates at a density of 5 × 10 6 cells well −1 in differentiation media containing Glasgow's minimum essential medium (Thermo Fisher) supplemented with 8% γ-rayirradiated knockout serum replacement (Thermo Fisher), 0.1 mM MEM (minimum essential medium) non-essential amino acids (Thermo Fisher), 1 mM sodium pyruvate (Sigma-Aldrich), and 0.1 mM 2-mercaptoethanol. A ROCK inhibitor, Y-27632 (final 10 μM), was added when the cells were dissociated, and the day of dissociation was defined as differentiation day 0. The media were changed daily, and 100 nM LDN193189 was added from days 0 to 12, 500 nM A83-01 was added from days 0 to 6, 100 ng mL −1 fibroblast growth factor 8, and 2 μM purmorphamine were added from days 1 to 6, and 3 μM CHIR99021 was added from days 3 to 12. All materials and reagents used in this study are listed in Supplementary Table 12. On differentiation days 12 and 13, the cells were dissociated using 0.5× TrypLE select CTS, stained by phycoerythrin-conjugated anti-CORIN antibody (final concentration 100 ng mL −1 , Sumitomo Dainippon Pharma) for 20 min, and suspended with the sorting buffer containing phosphatebuffered saline (PBS)(−), 2% γ-ray-irradiated fetal bovine serum, 20 mM Dglucose, 80 μg mL −1 gentamicin, and 10 μM Y-27632. Data analysis and cell sorting were performed with a FACSAria III or an Influx cell sorter (BD Biosciences). A 100-μm ceramic nozzle with a sheath pressure of 20 or 26 psi and an acquisition rate of~5000 events s −1 were used for the cell sorting. The positive gate was set so that <0.1% of cells were positive in unstained samples or samples stained by an isotype control antibody. Sorted CORIN+ cells were plated onto PrimeSurface® U-shaped 96-well plates (Sumitomo Bakelite) at a density of 2-3 × 10 4 cells well −1 to make aggregate spheres in neural differentiation media containing Neurobasal medium with γ-ray-irradiated B27 supplement (without Vitamin A), 2 mM Lglutamine, 10 ng mL −1 glial cell-derived neurotrophic factor, 200 mM ascorbic acid, 20 ng mL −1 brain-derived neurotrophic factor, and 400 μM dibutyryl cAMP. Thirty micromolars of Y-27632 and 80 μg mL −1 gentamicin were added at the initial plating after sorting, and the media were changed every 3 days until shipment or evaluation. The cell number of each aggregate sphere was calculated by the result of the flow cytometric analysis at day 26, and the number of spheres for animal transplantation was estimated by the cell number. At differentiation day 30, the aggregate spheres were collected in custom-made cell containers (JMS), washed with saline four times, and stored at 5°C until shipment. A step-by-step protocol describing the DAP differentiation can be found at Protocol Exchange 28 .  Immunostaining. For in vitro studies, spheres were fixed with 4% paraformaldehyde and sliced at 10 μm thickness by using a cryostat (CM-1850, Leica Biosystems). Double-or triple-labeled immunostaining was performed after permeabilization, antigen retrieval if needed, and blocking with 0.1% Triton X-100 and 2% skim milk. The stained samples were visualized by using a fluorescence microscope (BZ-X710; Keyence) and a confocal laser microscope (Fluoview FV1000D; Olympus). The primary and secondary antibodies are listed in Supplementary Table 13.
Single-cell analysis. Single-cell complementary DNA (cDNA) preparation and gene expression analysis were performed following the manufacturer's protocol. A cell suspension of iPSCs (day 0), intermediate cells after sorting (days 12 and 13), or DAPs (day 26) at 300 cells μL −1 were used. C1 Suspension Reagent (Fluidigm) was added to the cell suspension at a ratio of 40%. Six microliters of cell suspension mix were loaded on a C1 Single-Cell AutoPrep IFC microfluidic chip (Fluidigm) designed for cells 10-17 μm in size, and the chip was then processed on a Fluidigm C1 instrument using the "STA: Cell Load (1862×)" script. Live and single cells were confirmed under an inverted microscope. Then, the "STA: Preamp (1862×)" script was executed, which includes lysis, reverse transcription, and 18 cycles of PCR. When the run had finished, the amplified cDNA was harvested in a total of 28 μL C1 Harvesting Reagent. The amplified cDNA from single cells and DNA primers were then loaded onto a Biomark chip (Fluidigm) for expression profiling with a panel of qRT-PCR assays (Biomark HD, Fluidigm). The chip was then loaded onto Biomark HD to analyze gene expression levels. The expression data were processed and analyzed using the Singular Analysis Toolset version 3.6.2 (Fluidigm). The gene expression profiles of the analyzed cells were visualized using tSNE. A spectrum of gene expressions was visualized by violin plots. The sequences of the DNA primers are listed in Supplementary Table 14.  For the methylation analysis, the bisulfite conversion of 500 ng genomic DNA was performed using the EZ DNA Methylation Kit (Zymo Research), followed by methylation profiling using the Infinium MethylationEPIC BeadChip Kit (Illumina) according to the manufacturer's protocols. After exporting the methylation data by using GenomeStudio (V2011.1) and normalizing the beta values by using BMIQ 42 , we calculated the beta values of 73 genomic blocks at the transcription start sites of cancer-related genes as described previously 43 and visualized them using R (https://www.R-project.org) with ggplot2 44 . As a reference, 53 methylation datasets of normal tissues (in GSE31848) and 11 methylation datasets of glioblastoma multiforme (downloaded from TCGA, Sentrix_ID: 9976861010) were also visualized.
Residual plasmids were tested by quantitative PCR with TaqMan™ Gene Expression Master Mix (Thermo Fisher). Quantitative PCR was done to amplify exogenous genes (KLF4, SOX2, L-MYC, LIN28, Trp53DD, OCT3/4, and EBNA1) in Fig. 4 Results of the efficacy study. a Rotational assays of methamphetamine-injected rats. Two-way ANOVA and Sidak's multiple comparison test, adjusted p value: *** = 0.0002 and ****<0.0001. b-e Representative images of the brain of a rat (number of animals = 8 for cell transplantation and 6 for saline injection) after transplantation and stained for b TH, c HNA (green) and TH (magenta), d TH (green) and FOXA2 (magenta), and e HNA (green) and GFAP (magenta). Bars in b = 1 mm, c = 50 μm, and d, e = 100 μm. R = right side of brain. f-h Representative images of the brain of a rat after transplantation and DAB stained for TH. g, h Magnified images of the boxes in f. Bars in f = 1 mm and g, h = 100 μm. i-k A magnetic resonance imaging (i) of the transplanted monkey and representative images (j, k) of the graft stained for TH. Arrowhead in i shows the grafts. Bars in j = 1 mm and k = 50 μm. l-n Representative H-E staining of the brain of a monkey after transplantation (m is a magnification of l, and n is a magnification of m). Bars in l = 5 mm, m = 1 mm, n = 200 μm. b-n Number of cell preparations = 2 and number of animals = 3. the plasmid using iPSC genome DNA and real-time PCR Thermal Cyclers (StepOnePlus; Thermo Fisher). The DNA quantity was normalized to the copy number of endogenous FBX15. The DNA genomes of more than 25,000 cells were used as the template for the PCRs based on the copy number of endogenous FBX15. The probes and primers are as follows: KLF4 probe, forward and reverse: Hs00358836_m1 (Thermo Fisher) Animal studies. All animals were cared for and handled according to the Guidelines for Animal Experiments of Kyoto University and that of Shin Nippon Biomedical Laboratories (Kagoshima, Japan), and all animal experiments were certified by an ethics committee at Kyoto University and Shin Nippon Biomedical Laboratories.
Tumorigenicity/toxicity/biodistribution study. Seven-eight-week-old male and female NOG mice (CLEA Japan) were used for the transplantation. Cells were transplanted stereotactically, and the coordinates of the striatum were calculated with reference to the bregma: anterior (A), +0.5 mm; lateral (L), −2.0 mm; ventral (V), −3.0 mm; and tooth bar (TB), 0 mm. Two hundred thousand cells in 0.6-2.8 μL were injected through a 24 G needle (Hamilton 7105KH). Fifty-two weeks after the injection, the mice were euthanized with isoflurane and pentobarbital and perfused transcardially with PBS. The brains were immersion fixed by 10% formalin, paraffin embedded, and sliced with a microtome at 5 μm thickness throughout the graft. One of every 10 sections was used for H-E staining, and the other sections were used for immunohistochemisty. Other systemic organs (Supplementary Table 15) were also immersion fixed by 10% formalin and paraffin embedded and sliced with a microtome at 5 μm thickness. One of the sections was used for H-E staining, and the others were used for immunohistochemistry by anti-TH, anti-KU80, anti-KI67, anti-cytokeratin, and anti-TTR antibodies (Supplementary Table 13). Toxicity and biodistribution analyses were combined with a tumorigenicity test of the brain injection in NOG mice. The test items evaluated are listed in Supplementary Table 15.
Teratoma formation study (subcutaneous and testis injection). Five-nineweek-old male NOG mice were used. Two hundred fifty or 500 μL of dissociated single-cell solutions containing 6 × 10 5 cells mixed with ice-cold Matrigel (BD 356237) were injected by using a 22 G needle (Hamilton 750N) into the subcutaneous space of the upper back. The DAPs were spiked with 0.001-10% (6 × 10 0 -6 × 10 4 ) undifferentiated iPSCs by the stepwise dilution method, and the cell concentration was confirmed to be within ±15% of the target concentration. The subcutaneous tumor size was measured by calipers, and the tumor volume was calculated via the following formula: Volume ðmm 3 Þ ¼ Long diameter ðshort diameterÞ 2 =2: After 26 weeks of observation, the subcutaneous tissue was fixed by 10% formalin, and all tissues were paraffin embedded and sliced at 5 μm thickness for staining. Animals in the positive control group (HeLa cells and iPSC line 201B7) were observed for 16 weeks or until the subcutaneous tumor volume was over 1500 mm 3 .
Ten microliters (6 × 10 5 cells) of dissociated cells were injected into the right testis with a 22sG needle (Hamilton 702N). After 16 weeks of observation, the testes were extracted and fixed by FSA solution (formalin-sucrose-acetic acid). All fixed tissues were paraffin embedded and sliced at 5 μm thickness for staining.