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• W3128463973 abstract "•Derivation from human ESCs of a cryopreserved dopamine progenitor cell product, MSK-DA01•MSK-DA01 cells rescue motor deficits in Parkinsonian rats•MSK-DA01 had no adverse effects in biodistribution, toxicology, and tumorigenicity studies•MSK-DA01 cells differentiate predominately into dopamine neurons in the host striatum Parkinson’s disease is characterized by the loss of dopaminergic neurons in the substantia nigra leading to disabling deficits. Dopamine neuron grafts may provide a significant therapeutic advance over current therapies. We have generated midbrain dopamine neurons from human embryonic stem cells and manufactured large-scale cryopreserved dopamine progenitors for clinical use. After optimizing cell survival and phenotypes in short-term studies, the cell product, MSK-DA01, was subjected to an extensive set of biodistribution, toxicity, and tumorigenicity assessments in mice under GLP conditions. A large-scale efficacy study was also performed in rats with the same lot of cells intended for potential human use and demonstrated survival of the grafted cells and behavioral amelioration in 6-hydroxydopamine lesioned rats. There were no adverse effects attributable to the grafted cells, no obvious distribution outside the brain, and no cell overgrowth or tumor formation, thus paving the way for a future clinical trial. Parkinson’s disease is characterized by the loss of dopaminergic neurons in the substantia nigra leading to disabling deficits. Dopamine neuron grafts may provide a significant therapeutic advance over current therapies. We have generated midbrain dopamine neurons from human embryonic stem cells and manufactured large-scale cryopreserved dopamine progenitors for clinical use. After optimizing cell survival and phenotypes in short-term studies, the cell product, MSK-DA01, was subjected to an extensive set of biodistribution, toxicity, and tumorigenicity assessments in mice under GLP conditions. A large-scale efficacy study was also performed in rats with the same lot of cells intended for potential human use and demonstrated survival of the grafted cells and behavioral amelioration in 6-hydroxydopamine lesioned rats. There were no adverse effects attributable to the grafted cells, no obvious distribution outside the brain, and no cell overgrowth or tumor formation, thus paving the way for a future clinical trial. Parkinson’s disease (PD) is one of the most common neurodegenerative diseases worldwide. Its cardinal features include motor symptoms such as bradykinesia, rigidity, resting tremor, and postural instability, though nonmotor symptoms are often also present. The current mainstay of pharmacologic therapy consists of augmentation of dopamine levels in the brain via dopamine supplements or agonists or by inhibiting dopamine degradation. Treatment is symptomatic but not long-lasting, and it has no neuroprotective effect. Cell therapy with grafts of human fetal tissue from the ventral mesencephalon have been carried out successfully, with multiple reports of long-term benefits. But placebo-controlled double-blind studies in the United States have failed to reach their primary endpoints (Freed et al., 2001Freed C.R. Greene P.E. Breeze R.E. Tsai W.-Y. DuMouchel W. Kao R. Dillon S. Winfield H. Culver S. Trojanowski J.Q. et al.Transplantation of embryonic dopamine neurons for severe Parkinson’s disease.N. Engl. J. Med. 2001; 344: 710-719Crossref PubMed Scopus (1942) Google Scholar; Olanow et al., 2003Olanow C.W. Goetz C.G. Kordower J.H. Stoessl A.J. Sossi V. Brin M.F. Shannon K.M. Nauert G.M. Perl D.P. Godbold J. Freeman T.B. A double-blind controlled trial of bilateral fetal nigral transplantation in Parkinson’s disease.Ann. Neurol. 2003; 54: 403-414Crossref PubMed Scopus (1228) Google Scholar). Nonetheless, graft survival was shown by fluorodopa PET scans (Freed et al., 2011Freed C.R. Zhou W. Breeze R.E. Dopamine cell transplantation for Parkinson’s disease: the importance of controlled clinical trials.Neurotherapeutics. 2011; 8: 549-561Crossref PubMed Scopus (41) Google Scholar) and autopsy studies (Kordower et al., 1995Kordower J.H. Freeman T.B. Snow B.J. Vingerhoets F.J.G. Mufson E.J. Sanberg P.R. Hauser R.A. Smith D.A. Nauert G.M. Perl D.P. et al.Neuropathological evidence of graft survival and striatal reinnervation after the transplantation of fetal mesencephalic tissue in a patient with Parkinson’s disease.N. Engl. J. Med. 1995; 332: 1118-1124Crossref PubMed Scopus (773) Google Scholar; Li et al., 2016Li W. Englund E. Widner H. Mattsson B. van Westen D. Lätt J. Rehncrona S. Brundin P. Björklund A. Lindvall O. Li J.Y. Extensive graft-derived dopaminergic innervation is maintained 24 years after transplantation in the degenerating parkinsonian brain.Proc. Natl. Acad. Sci. U S A. 2016; 113: 6544-6549Crossref PubMed Scopus (172) Google Scholar), and subgroup analysis suggested benefit in some patients (Freed et al., 2011Freed C.R. Zhou W. Breeze R.E. Dopamine cell transplantation for Parkinson’s disease: the importance of controlled clinical trials.Neurotherapeutics. 2011; 8: 549-561Crossref PubMed Scopus (41) Google Scholar). Although the clinical results of fetal tissue transplantation were variable, there were enough successes (up to complete cessation of anti-PD therapy in successfully grafted patients) to warrant improved trial design and development of a new European Union-funded study. This new trial, TRANSEURO (NCT01898390), has just finished accrual of 11 patients, randomly selected from a large observational cohort to receive human fetal grafts (Barker et al., 2019Barker R.A. Farrell K. Guzman N.V. He X. Lazic S.E. Moore S. Morris R. Tyers P. Wijeyekoon R. Daft D. et al.TRANSEURO consortiumDesigning stem-cell-based dopamine cell replacement trials for Parkinson’s disease.Nat. Med. 2019; 25: 1045-1053Crossref PubMed Scopus (67) Google Scholar). Although much will be learned from this study, fetal tissue-based studies in humans are fraught with enormous logistical hurdles and ethical concerns. Human embryonic stem cell (hESC) or induced pluripotent stem cell (iPSC) derived dopamine neurons provide several advantages over the use of human fetal tissue. They are readily expandable at the undifferentiated state to generate large-scale cell banks. Robust differentiation protocols can then be applied to yield large cryopreserved lots of differentiated cells that are subjected to stringent quality control measures after development of the critical release criteria. Cell composition and phenotype of ESC or iPSC-derived dopamine cells can be further validated pre-grafting. Ethical and logistical hurdles are also significantly reduced in comparison with the use of fetal tissue. There are at least two patients known to have received iPSC-derived dopamine cells, a patient in a recent trial in Japan (Cyranoski, 2018Cyranoski D. ‘Reprogrammed’ stem cells implanted into patient with Parkinson’s disease.Nat. News. 2018; https://www.nature.com/articles/d41586-018-07407-9Crossref Google Scholar) and a patient in the United States on a compassionate basis (Cyranoski, 2018Cyranoski D. ‘Reprogrammed’ stem cells implanted into patient with Parkinson’s disease.Nat. News. 2018; https://www.nature.com/articles/d41586-018-07407-9Crossref Google Scholar; Schweitzer et al., 2020Schweitzer J.S. Song B. Herrington T.M. Park T.Y. Lee N. Ko S. Jeon J. Cha Y. Kim K. Li Q. et al.Personalized iPSC-derived dopamine progenitor cells for Parkinson’s disease.N. Engl. J. Med. 2020; 382: 1926-1932Crossref PubMed Scopus (119) Google Scholar). Preclinical data have also been reported (Doi et al., 2020Doi D. Magotani H. Kikuchi T. Ikeda M. Hiramatsu S. Yoshida K. Amano N. Nomura M. Umekage M. Morizane A. Takahashi J. Pre-clinical study of induced pluripotent stem cell-derived dopaminergic progenitor cells for Parkinson’s disease.Nat. Commun. 2020; 11: 3369Crossref PubMed Scopus (88) Google Scholar). We have previously demonstrated the in vitro derivation of midbrain dopamine (mDA) neurons (Perrier et al., 2004Perrier A.L. Tabar V. Barberi T. Rubio M.E. Bruses J. Topf N. Harrison N.L. Studer L. Derivation of midbrain dopamine neurons from human embryonic stem cells.Proc. Natl. Acad. Sci. U S A. 2004; 101: 12543-12548Crossref PubMed Scopus (831) Google Scholar), then developed an enhanced specification approach based on floor-plate derivation of authentic mDA neurons that express key lineage-defining transcription factors such as FOXA2, LMX1A, and PITX3 as well as tyrosine hydroxylase (TH) upon maturation. Grafted cells survived, integrated into the host tissue, and rescued the behavioral abnormalities of Parkinsonian rodents (Kriks et al., 2011Kriks S. Shim J.W. Piao J. Ganat Y.M. Wakeman D.R. Xie Z. Carrillo-Reid L. Auyeung G. Antonacci C. Buch A. et al.Dopamine neurons derived from human ES cells efficiently engraft in animal models of Parkinson’s disease.Nature. 2011; 480: 547-551Crossref PubMed Scopus (1226) Google Scholar). Encouraged by these results, we elected to translate our findings toward a clinical trial. In a first step, we further enhanced the robustness of the differentiation and adapted the dopamine neuron differentiation conditions toward clinical compatibility under near current good manufacturing practice (cGMP). The resulting dopamine neuron differentiation protocol and its developmental rationale are presented in the companion article in this issue of Cell Stem Cell (Kim et al., 2021Kim T.W. Piao J. Koo S.Y. Kriks S. Chung S. Betel D. Choi S.J. Mosharov E.V. Irion S. Tomishima M. et al.Biphasic activation of WNT signaling enhances the derivation of midbrain dopamine neurons for translational use.Cell Stem Cell. 2021; 28 (this issue): 343-355Abstract Full Text Full Text PDF Scopus (21) Google Scholar). Building on those results, we report here the large-scale manufacture of cryopreserved clinical-grade cells, named MSK-DA01. We also present the design and results of toxicity, biodistribution, tumorigenicity, and efficacy studies in rodents. The initial step in the transition of the protocol of dopamine neuron differentiation to clinical compatibility entailed the elimination of the mouse fibroblast feeders and the knockout serum replacement product (KSR) in the original protocol to feeder-free conditions using Essential 8 Medium for expansion and Neurobasal Medium for differentiation. However, those changes resulted in a near complete loss of TH+ mDA neurons co-expressing FOXA2 and in the low expression of additional dopamine and midbrain markers such as EN1. As detailed in the accompanying article (Kim et al., 2021Kim T.W. Piao J. Koo S.Y. Kriks S. Chung S. Betel D. Choi S.J. Mosharov E.V. Irion S. Tomishima M. et al.Biphasic activation of WNT signaling enhances the derivation of midbrain dopamine neurons for translational use.Cell Stem Cell. 2021; 28 (this issue): 343-355Abstract Full Text Full Text PDF Scopus (21) Google Scholar), we addressed this problem by modifying timing and concentration of the WNT agonist CHIR99021 with treatment at 0.7 μM from days 0–3, at 7.5 μM from days 4–9, and at 3 μM from days 10–11 to dynamically control WNT signaling levels during differentiation (“Chir-Boost” protocol). This strategy improved rostro-caudal patterning of the SHH-induced ventral FOXA2+ population with minimal contamination of anterior diencephalic or posterior hindbrain lineages or non-neural contaminant and led to the successful derivation of authentic mDA neurons with very low run-to-run variability. We next developed standard operating procedures (SOPs) for the new differentiation conditions and transiently embedded members of our team in the institutional GMP facility (Cell Therapy and Cell Engineering Facility [CTCEF]) to achieve a seamless transfer of the SOPs to the facility staff and to initiate the next steps. Master banks of undifferentiated WA09 (H9) cells (passage 28) were obtained from the WiCell Research Institute. The cells were manufactured under cGMP conditions to establish working cell banks (WCBs) in E8 medium that were further certified by Waisman Biomanufacturing. The cells passed a battery of tests, including cell authentication by STR, karyotype, hESC marker expression, adventitious virus, and mouse antibody testing (Table S1). After transfer to the CTCEF, the cells were thawed and expanded for two or three passages prior to differentiation. Cells passed quality control analysis for morphology, cell identity (NANOG expression ranged from 95.8% to 98.1% by flow cytometry; criteria set at ≥90%), viability (ranging from 83.8% to 90.8% [criteria ≥ 70%] by AO/PI staining), sterility, and mycoplasma tests. For manufacturing of dopamine neuron progenitors (MSK-DA01), we first identified and performance-tested lots of all the reagents required. After completing the engineering run of our process in the CTCEF to evaluate product yield and quality, we manufactured four lots of MSK-DA01. Three of the four lots yielded at least 2 billion cells (2.0 × 109 to 2.8 × 109 cells), stored in >200 vials each. Only lot 3 was more limited in size (1.1 × 109 cells), as some of the cells were used for extended culture to allow adventitious agent testing. An overview of experimental design and quality assurance is shown in Figure 1A. The differentiation process at the CTCEF was monitored at several time points using in-process analyses of cell morphology, sterility, and viability at day 11 and day 16. For cell identity, gene expression of a list of key markers (41 genes plus 1 internal control gene [ACTB]; Kim et al., 2021Kim T.W. Piao J. Koo S.Y. Kriks S. Chung S. Betel D. Choi S.J. Mosharov E.V. Irion S. Tomishima M. et al.Biphasic activation of WNT signaling enhances the derivation of midbrain dopamine neurons for translational use.Cell Stem Cell. 2021; 28 (this issue): 343-355Abstract Full Text Full Text PDF Scopus (21) Google Scholar) was measured at days 0, 11, and 16 and extended to day 21 (Figure S1A). The list consisted of genes involved in mDA development (such as FOXA2, LMX1A, EN1, and TH) (Ferri et al., 2007Ferri A.L.M. Lin W. Mavromatakis Y.E. Wang J.C. Sasaki H. Whitsett J.A. Ang S.L. Foxa1 and Foxa2 regulate multiple phases of midbrain dopaminergic neuron development in a dosage-dependent manner.Development. 2007; 134: 2761-2769Crossref PubMed Scopus (218) Google Scholar; Simon et al., 2001Simon H.H. Saueressig H. Wurst W. Goulding M.D. O’Leary D.D.M. Fate of midbrain dopaminergic neurons controlled by the engrailed genes.J. Neurosci. 2001; 21: 3126-3134Crossref PubMed Google Scholar; Deng et al., 2011Deng Q. Andersson E. Hedlund E. Alekseenko Z. Coppola E. Panman L. Millonig J.H. Brunet J.F. Ericson J. Perlmann T. Specific and integrated roles of Lmx1a, Lmx1b and Phox2a in ventral midbrain development.Development. 2011; 138: 3399-3408Crossref PubMed Scopus (80) Google Scholar) and genes marking possible cell impurities during rostral-caudal patterning (such as FOXG1 for forebrain, HOXB2 for hindbrain) or during differentiation (such as POU5F1 for pluripotent stem cells [PSCs] or PAX6 for early neuroepithelial, rosette-stage neural stem cells [NSCs]). A previous small-scale lot (NB072715SZ), shown to function in vivo, served as a reference. The data demonstrated high expression of genes involved in mDA development and minimal expression of non-mDA fates. POU5F1 expression was undetectable as of day 11 by immunohistochemistry (IHC) (data not shown). At the end of differentiation at day 16, the cells passed sterility, pyrogen, and mycoplasma testing and were cryopreserved using STEM-CELLBANKER and a controlled rate freezer into 8 million cell vials. Viability after thaw ranged from 79.6% to 87.2% (criteria > 70%; n = 6 vials/lot). Additional testing on cryopreserved lots involved markers nominated as release criteria at day 16, including gene expression for LMX1A and POU5F1 (Figure 1B) and gene expression for TH and PITX3 using qRT-PCR at day 21, 5 days after further differentiation of cryopreserved day 16 product (Figure 1B). IHC for co-expression of FOXA2 and TH co-expression was also carried out at day 21 (Figure S1B). We established validated intracellular flow cytometry assays for expression of key proteins of thawed cells, including FOXA2 (criteria > 85%), PAX6 (criteria < 5%), and NANOG (criteria < 0.2%). Data on thawed cells from MSK-DA01 showed a high percentage (90.1%–94.4%) of FOXA2, a floor-plate marker that is maintained throughout the development of mDA neurons, very low percentage of PAX6+ (0.1%–0.2%), and undetectable NANOG+ cells (Figure S1C). We did not observe significant differences among the lots or loss of marker expression in long-term cryopreserved cells. As contamination with undifferentiated PSCs is a major concern for any PSC-based products, in addition to our standard flow cytometry-based release assay for NANOG and qRT-PCR-based assay for POU5F1, we tested carefully for POU5F1+ cells in our product at day 16 using several orthogonal assays. MSK-DA01 was tested alone or in samples spiked with WA09-hPSCs (human PSCs) in a dilution series ranging from 0.001% to 1% undifferentiated PSCs to determine the limit of detection of the various assays. First, we used an automated high-content microscope (Operetta) to detect and quantitate immunostained POU5F1+ cells (Figure S1D). The quantified percentage of POU5F1+ cells is highly similar to the percentage of spiked PSCs (Figure S1E). The automatedly detected POU5F1+ cells were subjected to validation by manual observation, and false-positive signals were excluded. At the lowest spiked percentage tested (0.001% PSC spike), 5 POU5F1+ cells were identified out of 191,570 cells (0.0026%), confirming the sensitivity of the analysis. Using high-content imaging, we could not find evidence for the presence of any POU5F1 cells in MSK-DA01. For qRT-PCR-based detection of POU5F1 mRNA, we observed a 2,000-fold decrease in expression from undifferentiated cells to MSK-DA01. However, there was evidence for persistent low POU5F1 mRNA expression compared with negative control, which is not consistent with the absence of POU5F1+ cells in MSK-DA01 described above. We then suspected that our release POU5F1 primer set (Figure 1B) may have detected known POU5F1 pseudogenes, which may limit sensitivity (Liedtke et al., 2007Liedtke S. Enczmann J. Waclawczyk S. Wernet P. Kögler G. Oct4 and its pseudogenes confuse stem cell research.Cell Stem Cell. 2007; 1: 364-366Abstract Full Text Full Text PDF PubMed Scopus (189) Google Scholar). Therefore, we implemented an additional primer sets optimized to avoid pseudogene detection (Liedtke et al., 2007Liedtke S. Enczmann J. Waclawczyk S. Wernet P. Kögler G. Oct4 and its pseudogenes confuse stem cell research.Cell Stem Cell. 2007; 1: 364-366Abstract Full Text Full Text PDF PubMed Scopus (189) Google Scholar) using both ddPCR (data not shown) and qRT-PCR assays. We analyzed POU5F1 expression in a set of samples including MSK-DA01, fibroblasts (negative control), and 100%, 1%, 0.1%, 0.01%, 0.001%, 0.0001%, and 0.00001% hPSCs spiked into MSK-DA01 (Figure S1F). The data suggest that the limit of detection for this assay reaches at least low as 0.001%. MSK-DA01 showed levels not significantly different from the fibroblast negative control, confirming that POU5F1 mRNA was undetectable in MSK-DA01. We next tested each of the four lots of MSK-DA01 for in vivo survival and mDA differentiation. We injected 400,000 cells (n = 4–6 mice per lot) unilaterally into the striatum of severe combined immunodeficient/nonobese diabetic NSG (NOD.Cg-PrkdcscidIl2rgtmWjl/SzJ) mice and assessed for survival and phenotypes of grafted cells 3 weeks later. We observed robust human cell survival and formation of well-defined grafts in the striatum, at the site of injection. The human dopamine neurons in the graft were identified by co-expression of human cytoplasmic marker (STEM121) and TH (Figure S1G). FOXA2 was expressed in the majority of human cells (84.3% ± 10.3%). TH+ cells in the graft were co-labeled with human nuclear antigen (hNA) and FOXA2 (Figure 1C). Most human cells also expressed NURR1 and PITX3 (Figure 1D; Figure S1H), which are critical for the development and maintenance of mDA neurons (Kadkhodaei et al., 2009Kadkhodaei B. Ito T. Joodmardi E. Mattsson B. Rouillard C. Carta M. Muramatsu S. Sumi-Ichinose C. Nomura T. Metzger D. et al.Nurr1 is required for maintenance of maturing and adult midbrain dopamine neurons.J. Neurosci. 2009; 29: 15923-15932Crossref PubMed Scopus (247) Google Scholar; van den Munckhof et al., 2003van den Munckhof P. Luk K.C. Ste-Marie L. Montgomery J. Blanchet P.J. Sadikot A.F. Drouin J. Pitx3 is required for motor activity and for survival of a subset of midbrain dopaminergic neurons.Development. 2003; 130: 2535-2542Crossref PubMed Scopus (246) Google Scholar; Smidt et al., 2004Smidt M.P. Smits S.M. Bouwmeester H. Hamers F.P. van der Linden A.J. Hellemons A.J. Graw J. Burbach J.P. Early developmental failure of substantia nigra dopamine neurons in mice lacking the homeodomain gene Pitx3.Development. 2004; 131: 1145-1155Crossref PubMed Scopus (261) Google Scholar). No grafted human cells expressed NKX2.1, indicating the cells did not differentiate into hypothalamic fates (Ohyama et al., 2005Ohyama K. Ellis P. Kimura S. Placzek M. Directed differentiation of neural cells to hypothalamic dopaminergic neurons.Development. 2005; 132: 5185-5197Crossref PubMed Scopus (74) Google Scholar). POU5F1, a marker of PSC was absent while PAX6 was present very rarely in short-term grafts as well as in vitro assays, indicating that the cells will unlikely form teratoma or neural rosettes in the brain. There were no significant differences in graft volume and percentage of TH-expressing cells among the four lots of MSK-DA01 3 weeks after grafting. We selected lot 2 to perform all studies, as this lot resulted in the highest average graft volume (0.187 mm3) and percentage of TH+ cells (15.91%) at 3 weeks post-grafting. As cells are mDA progenitors when grafted, they are expected to complete their maturation in vivo over time. We next proceeded to perform a long-term efficacy study, with a goal of analyzing the impact of MSK-DA01 on animal behavior in 6-hydroxydopamine (OHDA)-lesioned nude rats (NIH-Foxn1rnu) as well as graft survival and differentiation. We performed the study in our lab under near good lab manufacturing practice (GLP) conditions, as adequate expertise was not available at any external GLP-certified location. We induced hemiparkinsonism in nude rats by stereotactic injection of 6-OHDA in the median forebrain bundle (Kriks et al., 2011Kriks S. Shim J.W. Piao J. Ganat Y.M. Wakeman D.R. Xie Z. Carrillo-Reid L. Auyeung G. Antonacci C. Buch A. et al.Dopamine neurons derived from human ES cells efficiently engraft in animal models of Parkinson’s disease.Nature. 2011; 480: 547-551Crossref PubMed Scopus (1226) Google Scholar). Amphetamine-induced rotational testing was performed at 4 and 6 weeks (±1 week). Rats exhibiting more than six rotations per minute were selected for inclusion in the efficacy study. Forty-four rats were separated randomly into a cell group (n = 28, male/female ratio [M/F] = 1:1) and a vehicle group (n = 16, M/F = 1:1). Freshly thawed MSK-DA01 cells (day 16) passed an empirical pre-established viability threshold (>70%) and were suspended at a concentration of 100,000 ± 10,000 cells/μL. Animals received 400,000 cells or vehicle in 4 μL via stereotactic injection at four deposits along a single track in the striatum. Cell viability showed some decrease at the end of grafting (less than 5 h from thaw time) but remained above 70% (Figure S2A). There was no correlation between graft volumes and the time of injection after initial cell thawing (r = −0.2797, p = 0.1577). We replaced one female in the cell group because of a premature withdrawal of the injection needle during the procedure, resulting in a total of 29 rats receiving graft injections (14 male and 15 female rats). Animals underwent behavioral testing at 2, 4, 5, 6.5, and 8 months (±1 week) post-grafting (Figure 2A). There was a significant decrease in rotation scores at 5 months post-grafting. Rotations per minute were down to −1.4 (median for all rats) at 8 months after transplantation compared with a median of +14.6 in the vehicle group. Animals were euthanized and their brains analyzed by independent pathologists at a contractor site (Charles River Laboratories). In the vehicle group, there were no human cells and no dopaminergic innervation in the lesioned striatum. Grafted animals showed human TH+ cells at the injection site (Figure 2B). Stereological estimates (physical dissector) of the total number of human cells showed a median of 0.53 million cells in male rats and 0.46 million cells in female rats (Figure 2C). Graft volume analysis showed a median of 16.60 mm3 in males and 14.62 mm3 in females, a difference that was not statistically significant (Figure 2D). There was a strong correlation and linear regression between human cell number and graft volume in all animals (Figure 2E). Large well-differentiated human dopamine neurons were observed in the graft (Figure 2F), extending their processes into the surrounding tissues (Figure 2G; Figure S2F). Human dopamine neurons (hNA+TH+) amounted to an average of 49,250 cells in males and 42,480 cells in females (Figure 2H). Two male rats had small grafts (23,484 and 115,092 surviving human cells, respectively) and a correspondingly small number of TH cells (1,250 and 2,500 cells); these animals were also the only two rats that did not show significant behavioral recovery, suggesting that fewer than 2,500 grafted human TH+ cells cannot support recovery of motor asymmetry in rats. As expected, the number of human TH+ cells correlated with the total number of human cells in all animals (Figure S2B). The percentage of TH+ cells in hNA+ cells was not different between genders (Figure S2C). To evaluate the proliferation of grafted cells, we performed IHC for hNA and Ki67 (Figure 2I). The percentage of Ki67 in hNA+ cells was very low in both male (average at 0.61%) and female (average at 0.68%) animals (Figure 2J). There was no obvious correlation between the percentage of Ki67+ cells and the number of hNA+ cells (Figure S2D) or graft volume (data not shown). About 67.78% hNA+Ki67+ cells expressed FOXA2 (Figure 2I). IHC for 5-hydroxytryptamine (5-HT) and glutamic acid decarboxylase (GAD) was negative (Figures 2K and 2L), suggesting the absence of human serotonergic or GABAergic neurons, respectively. Two female grafted rats developed flank masses at 22 and 32 weeks after transplantation but remained well. The rats were euthanized at the scheduled time and underwent a necropsy. The masses were identified histologically as mammary fibroadenomas (Figure S2E) and had no human cells (hNA) (data not shown); they were ruled as spontaneous benign tumors, known to occur in laboratory rats (Percy and Barthold, 2001Percy D. Barthold S. Pathology of Laboratory Rodents and Rabbits. Iowa State University Press, 2001Google Scholar). In addition, four rats had to be terminated earlier than scheduled (Table S2). Those rats were not included in the data analysis described above, because of the discrepancy in the endpoints. However, full necropsy was performed independently by the Veterinary Pathology Services at our institution, including histological evaluation of an extensive list of organs (>30). The cause of death of three animals was infection in multiple organs, while one male rat had trauma due to an attack by its cage mate; all deaths were ruled unrelated to the grafts. Two of these animals, which were from the graft group, exhibited surviving human TH neurons in the striatum (Figure S2F), with absent POU5F1 (Figure S2G), a small number of Ki67 cells (Figure S2H), and no evidence of abnormal graft histology (Figure S2I). We conducted large-scale biodistribution, toxicology, and tumorigenicity studies under GLP conditions. The studies were designed by our team and conducted at an outside contracted facility (WuXI-Apptec, St. Paul, MN). MSK-DA01 cells were grafted into 6- to 8-week-old normal NSG mice. We used two doses for the biodistribution (n = 10 per dose group, M/F = 1:1) and toxicology studies (n = 20 per dose group, M/F = 1:1): a lower dose of 200,000 cells in 2 μL and a higher dose of 400,000 cells in 4 μL; a third group received vehicle only. An additional mouse group (n = 10, M/F = 1:1) was added to the study as a potential source of replacement should any mouse die or require termination. Our target dose in rats was 400,000 cells, and we recognize that this dose and volume are too large for a mouse brain. However, to maximize our chances to reveal a potential safety signal, we used the maximally feasible dose of 400,000 cells. The animals were separated randomly, and there was no significant difference in weight among all the groups in both studies. On each day of grafting, MSK-DA01 cells were thawed and reconstituted to 100,000 ± 10,000 cells/μL. Cell viability at initial thaw, pre-transplantation, and post-transplantation exceeded our preset threshold of >70% (Figure S3A). A few animals did not have any graft, probably for technical reasons, and were not included in the analysis (see STAR Methods; Table S4). Biodistribution was assessed in an array of tissues collected on days 30 and 180, by qPCR for human-specific Alu repeat sequences. The collected tissues included liver, blood, lungs with mainstem bronchi, bone marrow, axillary lymph nodes, brain near the injection site, spinal cord, spleen, heart, kidneys, and any visible abnormality. Sensitivity and specificity of the qPCR assay were determined prior to the start of the study. The limit of quantification (LOQ) was found to be 0.303 genome copies for all samples, with the exceptions that LOQ was 1.51" @default.
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• W3128463973 title "Preclinical Efficacy and Safety of a Human Embryonic Stem Cell-Derived Midbrain Dopamine Progenitor Product, MSK-DA01" @default.
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