Lorenz Studer | Center for Stem Cell Biology, Memorial Sloan Kettering Cancer Center, New York, NY

Parkinson’s disease was first described by James Parkinson, an apothecary and surgeon in London, more than 200 years ago. In his essay, reprinted recently,¹ he defined the disease as “shaking palsy” to stress the fact that patients show aspects of both aberrant movement (such as shaking/tremor) as well as lack or slowness of it. In honor of James Parkinson, the famous French neurologist Jean Martin Charcot was the first to refer to the disorder as Parkinson’s disease,² and to describe in great scientific detail the specific motor as well as autonomic symptoms of the disease (Figure 1).

PD is the second most common neurodegenerative disorder affecting approximately 1 million patients in the US, and causes enormous health and financial burden, estimated at $52 billion of direct and indirect costs to society within the US alone.³ Today, we understand that the key motor symptoms of PD are caused by the loss of dopamine neurons in the midbrain. A healthy individual has about 300,000-400,000 midbrain dopamine neurons⁴ and the loss of greater than 50% of those neurons is thought to trigger symptoms of Parkinson’s disease and bring the patient to seek help from a neurologist. As the brain is estimated to comprise about 50-100 billion neurons, dopamine neurons represent a tiny fraction of total neurons. The rather discrete loss of dopamine neurons in a defined brain region makes PD an attractive target for regenerative medicine, as only a limited number of new neurons might suffice to significantly impact motor function in an individual patient. On the other hand, while motor symptoms are the classic feature of the disease, it is important to stress that PD affects many systems of the human body beyond those relevant to the movement disorder. Early symptoms that often precede movement-related symptoms include a loss of smell, sleep disturbances such as restless leg syndrome and chronic constipation. Some of the feared long-term complications include a progressive cognitive loss which can occur many years after onset of motor symptoms and can lead to PD-associated dementia.⁵ It is important to note that both cognitive loss and peripheral disease symptoms are thought to be mostly independent of the dopamine dysfunction. Therefore, even a permanent “cure” of the dopamine-related movement disorder would likely not result in a true cure of the broader disease.

There is an increasing understanding of specific genetic factors that predispose an individual to PD. Those findings are based on studies of familial forms of the disease implicating more than 20 PD genes and on human genetic studies that define about 90 PD genetic risk loci in sporadic patients.⁶ PD-related genes point to vulnerabilities in mitochondrial and lysosomal function that may underlie disease pathogenesis. Vulnerabilities related to energy demand and protein homeostasis may be particularly acute in midbrain dopamine neurons, given the remarkable size and complexity of their neurite arbors. It is estimated that a single human midbrain dopamine neuron in the substantia nigra has an axonal arbor with a combined length of about 4-5 meters and comprises 1-2.4 million synapses each.⁴ The enormous size of dopamine neurons may also contribute to challenges in protein homeostasis, particularly for synaptic proteins of high abundance such as a-synuclein. The aggregation of a-syn into Lewy bodies is one of the key pathological hallmarks of the disease.⁷

Despite the considerable progress in unraveling genetic and biochemical pathways involved in PD pathogenesis, no disease-modifying therapy is available today that can halt or significantly slow down disease progression. The currently approved therapies are symptomatic and include strategies to replace the neurotransmitter dopamine by supplying L-Dopa pharmacologically. L-Dopa is taken as an oral drug that enters the brain, where it gets converted into dopamine in the remaining dopamine neurons present in the brain. While highly effective at early stages of the disease, as the disease progresses, L-Dopa treatment becomes less and less effective and can trigger significant side effects such L-Dopa-induced abnormal movements (dyskinesia). The use of deep brain stimulation (DBS) represents another currently approved clinical treatment for PD patients that can improve several symptoms of the disease such as tremor or dyskinesia. However, DBS is not suitable for all patients and requires the implantation of hardware in the brain that needs to be serviced throughout life. Furthermore, the procedure can worsen speech-related or psychiatric symptoms in some of the patients. An experimental treatment approach that is not approved yet is gene therapy to either protect the remaining dopamine neurons or to boost their function via delivery of neurotrophic factors or genes that enhance the biochemical function respectively. Finally, there is the long-standing goal of replacing the dopamine neurons degenerated in PD via cell therapy, the main topic of this current paper.

The possibility of dopamine neuron replacement

While a plethora of cell types have been proposed in the past as a potential source of dopamine-producing neurons (ranging from adrenal medulla cells to peripheral neurons coaxed to produce dopamine), none of those non-midbrain cells has shown convincing evidence of long-term survival and dopamine function. In contrast, the use of human fetal midbrain dopamine neurons has offered valuable insights into the potential of achieving dopamine neuron replacement in PD patients.

This approach started in the late 1980s and has been performed since on more than 300 patients worldwide. The results showed convincingly that fetal dopamine neurons can survive when injected into the striatum of PD patients as illustrated by both histological data as well as by functional PET imaging using a radioactive F-Dopa tracer (Figure 2). However, two placebo controlled clinical trials,^10,11 assessing the efficacy of the approach at 12 months post-surgery in a larger set of patients, failed in their primary end point and showed some evidence of efficacy only in younger patients (< 60 years of age).¹⁰ Furthermore, a subset of the patients showed unexpected side effects referred to as graft-induced dyskinesia that – combined with the modest evidence of efficacy – halted most fetal dopamine neuron grafting trials. Nevertheless, some of the treated patients were followed for more extended time periods in studies that suggest that the optimal effects of the treatment may develop only at 2-3 years after transplantation.¹² In fact, a subset of patients was reported to do particularly well after being followed for 10 years and beyond, with several patients off any L-Dopa medication,¹³ which is highly unusual for patients suffering from a chronic progressive disease such as PD. This suggested that the cell replacement approach can work at least in a subset of PD patients. However, it remained unclear which factors predict success regarding patient selection or graft preparation. Furthermore, the use of fetal tissue raised considerable logistical and ethical issues that have prevented the development of this approach for routine use in larger sets of patients.

To find a renewable source of midbrain dopamine neurons, several potential stem cell types were pursued starting around the mid-1990s. On a personal note, I got involved in this endeavor after working with Dr. Christian Spenger, a young Neurosurgeon at the University of Bern, to drive the first human clinical study in Switzerland using human fetal dopamine neurons in 1995. This experience set me on a journey to explore the potential of neural stem cells as a possible source of dopamine neurons. Neural stem cells seemed to be an obvious choice as they are, unlike embryonic stem cells, already committed to a neural fate, and the remaining challenge is “only” generating the correct neuron subtype. Indeed, we were able to demonstrate the production of dopamine neurons from dividing midbrain rat neural precursors and their successful engraftment in a rat model of PD.¹⁴ However, the extent of in vitro expansion via proliferation of the midbrain stem/precursor compartment was relatively modest and did not enable a truly unlimited source of dopamine neurons. Therefore, I got involved in a parallel effort using mouse embryonic stem cells (ESCs) that provided the first true proof-of-concept for generating potentially unlimited numbers of midbrain dopamine neurons, and one of the very first examples of generating a specific neuron subtype from a pluripotent stem cell source.¹⁵ The use of pluripotent stem cells such as mouse ESCs resolved two major challenges, first, the ability to generate near unlimited numbers of differentiated cell types because ESCs (unlike midbrain stem cells) can be extensively expanded without losing fate potential. Second, the differentiation of ESCs offered access to the earliest stages of neural development. This turned out to be critical, as the key factors determining ventral midbrain identity such as FGF8 and SHH act during a very early and a very narrow temporal window that precedes the developmental stage when midbrain neural stem or precursor cells are isolated. Accordingly, the fate of the ESC-derived neural lineages can be readily directed to derive neurons specific to any brain region, while neural stem cells are largely restricted regarding their regional identity, even though they are still capable of committing to neuronal versus glial fate at this stage.

Pluripotent stem cells as a renewable source of dopamine neurons

The rapid progress in directing the differentiation of mouse ESCs was illustrated in multiple follow-up papers in the early 2000s that applied the same technology to many ESC lines, including ESCs derived via nuclear transfer^16,17 or by performing more and more in-depth studies in vivo to demonstrate rescue of Parkinsonian symptoms in various models of PD.¹⁸ Given such rapid progress, there was the expectation that within just a few years, similar results would be obtained using human pluripotent stem cells (hPSCs) that became available upon the isolation of human ESCs¹⁹ and subsequently human iPSCs, following the seminal work of Shinya Yamanaka.^20,21 However, it would take more than 10 years to develop differentiation protocols in hPSCs to generate midbrain dopamine neurons capable of efficient engraftment in mouse, rat, and primate models of PD.²² In a series of papers between 2009 to 2011, we were able to greatly simplify and accelerate the differentiation of human ESC or iPSCs into neural cells using only two small molecule inhibitors to drive neural specification, a widely used protocol termed dual-SMAD inhibition.²³ We had to combine dual-SMAD inhibition with the insight that dopamine neurons are derived from FOXA2+ floor plate precursors²⁴ and the finding that activation of WNT signaling is critical for driving midbrain specification and neurogenic conversion of floor plate precursors to ultimately enable this breakthrough in human dopamine neuron generation.²² We and many other groups went on to show that dopamine neurons derived via our floor plate protocol can restore motor deficits across various PD models, including drug-induced rotation behavior or measures of aberrant movement initiation. We further demonstrated how dopamine neurons achieve functional benefit in the host brain using an optogenetic switch in the grafted neurons. This switch allowed us to selectively turn off activity in grafted dopamine neurons without impacting neurons in the host brain. These studies showed that the functional benefit from the grafted cells is completely dependent on their neuronal activity and activity-dependent dopamine release. We further demonstrated that grafted neurons modulate synaptic input onto the striatum in a manner highly reminiscent of healthy endogenous midbrain dopamine neurons.²⁵ Given those promising proof-of-concept preclinical and mechanistic studies, there was a great impetus to translate those findings from animal studies towards actual clinical trials in human PD patients.

From proof-of-concept to clinical trial

It would take nearly another 10 years to move from the proof-of-concept study²² to a first-in-human clinical trial. A key step on this long journey includes the further optimization of the differentiation protocol under conditions that are suitable for clinical translation. In addition to using clinical grade reagents and cell lines, we further optimized the differentiation protocol to achieve better “on target” and “off target” performance.²⁶ For “on target” performance we tracked makers such as engrailed-1, a transcription factor expressed in most dopamine neurons in vivo, but that was inconsistently expressed in human PSC-derived dopamine neurons. In addition, we optimized “off target” performance by avoiding lineages that were present in fetal preparations such as serotonergic neurons, implicated as the potential culprit in triggering graft-induced dyskinesia.²⁷ Other “off target” cell types that we can largely avoid using our clinical grade protocol include perivascular fibroblast and choroid plexus cell contaminants that have been reported in other dopamine neuron differentiation protocols as potential “off target” cell types.

After optimizing the differentiation protocol, the next step was the production of large batches of dopamine neurons to develop an “off-the-shelf” product. This means that we needed to produce very large numbers of dopamine neurons and define conditions suitable for cryopreservation to be ready for ‘off-the-shelf’ use in preclinical studies and for the actual clinical trial. In fact, we produced nearly 10 billion cells under clinical grade conditions in a series of 4 replicate differentiation batches. Each batch had to pass a set of detailed release criteria that determined the purity and viability of the dopamine neuron preparations. For example, we showed that nearly all the final cells express the marker FOXA2 (Figure 3). FOXA2 is expressed in the dopamine lineage both at the precursors stage and in postmitotic cells. Furthermore, we demonstrated the lack of any remaining pluripotent cells in the final preparation.

Beyond qualifying the in vitro product, it was essential to test the safety and performance of those cells in vivo. We performed detailed good laboratory practice (GLP)-grade studies on tumorigenicity, biodistribution and toxicity across hundreds of mice at an outside, independent contract research organization (CRO). This is required by the regulatory agencies as to assure that results are reported in an unbiased manner by an organization that has no personal or financial gain from the outcome of the study.

In addition, we performed efficacy studies in a cohort of 48 Parkinsonian rats to show that grafting the cryopreserved dopamine neurons fully restores some of the motor symptoms in this model such as drug-induced rotation behavior (Figure 4). Finally, we performed targeting studies in a small number of non-human primates to assure that the clinical injection device proposed for human application is suitable to deliver the cells and to provide appropriate cell doses to the brain. All those studies supported the notion that our product is safe, does not form any tumors upon long-term engraftment or other signs of toxicity and reliably induces functional recovery.²⁸

In parallel, we developed the clinical protocol for our phase I/IIa human study as detailed on www.clinicaltrials.gov (NCT#04802733). We proposed to enroll 12 patients: 5 patients at a lower dose and 7 patients at a higher dose level. The appropriate dose was calculated based on our preclinical studies and is aimed at replacing 100,000 versus 300,000 dopamine neurons respectively on each side of the patient’s brain. We expect that the lower dose is the minimum number of dopamine neurons required to achieve clinical benefit, while the larger dose is closer to providing a patient with a full complement of new dopamine neurons. The target patient population is moderate to severe PD. It includes patients that cannot be properly treated with conventional therapy such as L-Dopa treatment, but do not exhibit major cognitive deficits or psychiatric comorbidities. Patients have to undergo transient immunosuppression as this is an allogenic, off-the-shelf, product. However, we think that no long-term immunosuppression is required, based on the immune status of the brain and the experience with fetal dopamine neuron grafting that showed survival of dopamine neurons for up to 24 years after only transient immunosuppression.⁹

The surgical targeting involves the injection of cells along three tracts with three deposits each on both sides of the brain (Figure 5). The target region is the post-commissural putamen, a brain region that is part of the striatum and commonly represents the most severely, dopamine-depleted brain region in PD patients. We perform the surgery under intraoperative MRI guidance which allows for exquisite control of targeting the desired brain region and helps minimize risks associated with stereotactic injections, such as risk of causing bleeding by avoiding regions with high densities of blood vessels.

After our protocol was cleared by the FDA in late 2020 and by Health Canada in 2021, the trial started with the lower dose cohort of 5 patients who were all dosed by late 2021. Injections of the higher dose cohort of 7 patients started in early 2022 and is expected to be completed in 2022. The main endpoints of the study are safety and feasibility as typical for a Phase I study. In addition, we will look for early signs of efficacy based on clinical rating scales including UPDRS part III and based on F-Dopa PET imaging to look for evidence of graft survival and function at the 1-year and 2-year marks. While we are awaiting the results from this first clinical study, questions remain about optimal patient selection and cell dose. For example, even within the current study, there were slight differences in patient selection criteria among the two surgical sites. While Health Canada followed our initial proposal of targeting moderate to severe PD patients that are older than 50 years of age, clearance by the FDA required us to limit the study to severe PD patients only and individuals who are at least 60 years of age. This is just one example of how different regulatory bodies can impact clinical development in a manner that is difficult to predict and points to fact that both regulatory bodies and investigators have to learn how to best proceed with this new class of therapies.

Other ongoing trials and efforts to coordinate efforts across the globe

The PD cell therapy community has made a significant effort to coordinate efforts among the various groups involved across the globe. To this end, in 2014 we founded G-Force PD, a global organization with members in Europe, North America and Asia that is aimed at streamlining preclinical and clinical development of PD cell therapies.²⁹ For example, G-Force has developed guidelines for clinical trial design that have been adopted by several groups.³⁰ Among the G-force members, two groups have already started clinical studies prior to our current trial. In the US, a single patient was injected using autologous iPSC-derived dopamine neurons in a study led by Kwang-Soo Kim and colleagues.³¹ The resulting data showed feasibility of the approach, albeit the results were not conclusive regarding graft survival or evidence of graft function. The group in Kyoto by Jun Takahashi started their trial even earlier using human PSC-derived dopamine neurons³² from a single iPSC line donor, that is HLA homozygous, and expected to match about 16% of the Japanese population. However, the trial is not restricted to those individuals and all the grafted patients will receive transient immunosuppression similar to that used in our ongoing trial. Neither of those two trials were based on the use of a cryo-preserved, “off the shelf” product, which complicated the QC of the product and led to a protracted timing of those trials. While our current trial is expected to dose all 12 patients within a 12-month period, the Kyoto study has been ongoing for several years with the goal of completing all (n=7) patients.

Next steps – beyond Phase I

Some of the next steps include the production of a commercial-grade dopamine neuron product. This effort is currently ongoing at BlueRock Therapeutics, the company I co-founded in 2017 and that was recently acquired by Bayer. For a commercial-grade product, it is important to further increase lot size by at least 1 to 2 logs in cell number, while defining robust in vitro functional assays that are required by the FDA for qualification of a commercial cell product. Other next steps include the development of a simplified surgical injection device to make loading and cell delivery foolproof for any practicing neurosurgeon. Another important step is the planning of a pivotal phase IIB/phase III study that could lead to the clinical approval of the product for broader use in PD if successful.

In addition, there are also remaining scientific challenges. The current product is suitable for clinical use, but there is the possibility of genetically engineering the human PSC line as to prevent any immune response, and thereby avoiding the need for transient immunosuppression. Further engineering could include modifications to better protect the grafted dopamine neurons from the ongoing disease process such as by lowering alpha-synuclein levels. Another open question is whether cell identity or composition can be further improved to achieve maximal potency and clinical benefit with minimal clinical risk. Yet another interesting challenge is to better understand the in vivo engraftment process and the factors that determine initial graft survival. It is estimated that only about 10% of the dopamine neurons injected ultimately survive long-term in the host brain and the factors limiting survival remain largely unknown. Finally, there is the option of developing a clinical cell replacement strategy that involves not only the striatal target region but places the cells directly into the substantia nigra to achieve a more complete integration of the grafted neurons. All those areas for further improvement could yield the versions 2.0 or 3.0 of the dopamine neuron grafting paradigm in the future to come.

Conclusions and outlook

The idea of dopamine neuron replacement has been pursued for more than 30 years. Today we have access to technologies that allows the production of nearly unlimited dopamine neurons, and there is a consensus that human PSCs are the currently most appropriate choice for those efforts. While our current trial is ongoing and additional clinical trials are expected to start soon, there are already several lessons from that 30-year journey. First, there is a critical and continuing need for basic research to understand the developmental biology underlying human dopamine neuron differentiation and subtype specification and the factors controlling in vivo engraftment. Further refinements of the dopamine neuron product will likely take advantage of our increasing ability to profile dopamine neurons,^33,34 as well as all the other lineages of the human body,^35,36 in detail using single cell-based approaches. Another lesson is the need to understand and streamline the process of translating findings from the bench to the bedside. While this initial effort took nearly 10 years from the first successful proof-of-concept²² study to the actual use in PD patients, it is likely that future efforts will move more quicky as clinical grade cell production and regulatory processes become increasingly streamlined. Accordingly, the work currently performed with dopamine neurons in PD may pave the way for other cell-based therapies in the future. Finally, while it is exciting that regenerative approaches for PD are becoming a reality, it will be important to develop strategies that go beyond the standard paradigm of replacing dopamine neurons. There is a need to think outside the box and to develop the next generation of cell-based strategies that can tackle even more challenging problems such as treating the feared cognitive symptoms in late-stage PD, where a breakthrough could herald treatments for other major neurodegenerative disorders such as Alzheimer’s disease. Just a few decades ago, the now imminent prospect of cell therapy for PD seemed to be science fiction rather than science. Over this coming decades, the community should strive to develop cell-based approaches that can combine cell replacement with genetic engineering to tackle many additional neural disorders and to bring novel treatment options to the millions of patients suffering from those currently intractable disorders.

Declaration of interests: L.S. is a scientific co-founder and paid consultant of BlueRock Therapeutics, a biotech company recently acquired by Bayer that sponsors the ongoing clinical trial of dopamine neuron grafting in PD.

1. Parkinson, J. An essay on the shaking palsy. 1817. J Neuropsychiatry Clin Neurosci 14, 223-236; discussion 222, doi:10.1176/jnp.14.2.223 (2002).
2. Charcot, J.M. Vol. 65 129-156 (New Sydenham Society, 1877).
3. Yang, W. et al. Current and projected future economic burden of Parkinson’s disease in the U.S. NPJ Parkinsons Dis 6, 15, doi:10.1038/s41531-020-0117-1 (2020).
4. Pissadaki, E.K. & Bolam, J.P. The energy cost of action potential propagation in dopamine neurons: clues to susceptibility in Parkinson’s disease. Front Comput Neurosci 7, 13, doi:10.3389/fncom.2013.00013 (2013).
5. Postuma, R.B. et al. MDS clinical diagnostic criteria for Parkinson’s disease. Mov Disord 30, 1591-1601, doi:10.1002/mds.26424 (2015).
6. Blauwendraat, C., Nalls, M.A. & Singleton, A.B. The genetic architecture of Parkinson’s disease. The Lancet. Neurology 19, 170-178, doi:10.1016/S1474-4422(19)30287-X (2020).
7. Spillantini, M.G., Crowther, R.A., Jakes, R., Hasegawa, M. & Goedert, M. alpha-Synuclein in filamentous inclusions of Lewy bodies from Parkinson’s disease and dementia with lewy bodies. Proceedings of the National Academy of Sciences of the United States of America 95, 6469-6473, doi:10.1073/pnas.95.11.6469 (1998).
8. Kordower, J.H. 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 332, 1118-1124, doi:10.1056/NEJM199504273321702 (1995).
9. Li, W. et al. Extensive graft-derived dopaminergic innervation is maintained 24 years after transplantation in the degenerating parkinsonian brain. Proceedings of the National Academy of Sciences of the United States of America 113, 6544-6549, doi:10.1073/pnas.1605245113 (2016).
10. Freed, C.R. et al. Transplantation of embryonic dopamine neurons for severe Parkinson’s disease. N Engl J Med 344, 710-719, doi:10.1056/NEJM200103083441002 (2001).
11. Olanow, C.W. et al. A double-blind controlled trial of bilateral fetal nigral transplantation in Parkinson’s disease. Ann Neurol 54, 403-414, doi:10.1002/ana.10720 (2003).
12. Ma, Y. et al. Dopamine cell implantation in Parkinson’s disease: long-term clinical and (18)F-FDOPA PET outcomes. J Nucl Med 51, 7-15, doi:10.2967/jnumed.109.066811 (2010).
13. Kefalopoulou, Z. et al. Long-term clinical outcome of fetal cell transplantation for Parkinson disease: two case reports. JAMA neurology 71, 83-87, doi:10.1001/jamaneurol.2013.4749 (2014).
14. Studer, L., Tabar, V. & McKay, R.D. Transplantation of expanded mesencephalic precursors leads to recovery in parkinsonian rats. Nat Neurosci 1, 290-295, doi:10.1038/1105 (1998).
15. Lee, S.H., Lumelsky, N., Studer, L., Auerbach, J.M. & McKay, R.D. Efficient generation of midbrain and hindbrain neurons from mouse embryonic stem cells. Nat Biotechnol 18, 675-679, doi:10.1038/76536 (2000).
16. Wakayama, T. et al. Differentiation of embryonic stem cell lines generated from adult somatic cells by nuclear transfer. Science 292, 740-743, doi:10.1126/science.1059399 (2001).
17. Tabar, V. et al. Therapeutic cloning in individual parkinsonian mice. Nat Med 14, 379-381, doi:10.1038/nm1732 (2008).
18. Kim, J.H. et al. Dopamine neurons derived from embryonic stem cells function in an animal model of Parkinson’s disease. Nature 418, 50-56, doi:10.1038/nature00900 (2002).
19. Thomson, J.A. et al. Embryonic stem cell lines derived from human blastocysts. Science 282, 1145-1147, doi:10.1126/science.282.5391.1145 (1998).
20. Takahashi, K. et al. Induction of pluripotent stem cells from adult human fibroblasts by defined factors. Cell 131, 861-872, doi:10.1016/j.cell.2007.11.019 (2007).
21. Takahashi, K. & Yamanaka, S. Induction of pluripotent stem cells from mouse embryonic and adult fibroblast cultures by defined factors. Cell 126, 663-676, doi:10.1016/j.cell.2006.07.024 (2006).
22. Kriks, S. et al. Dopamine neurons derived from human ES cells efficiently engraft in animal models of Parkinson’s disease. Nature 480, 547-551, doi:10.1038/nature10648 (2011).
23. Chambers, S.M. et al. Highly efficient neural conversion of human ES and iPS cells by dual inhibition of SMAD signaling. Nat Biotechnol 27, 275-280, doi:10.1038/nbt.1529 (2009).
24. Kittappa, R., Chang, W.W., Awatramani, R.B. & McKay, R.D. The foxa2 gene controls the birth and spontaneous degeneration of dopamine neurons in old age. PLoS Biol 5, e325, doi:10.1371/journal.pbio.0050325 (2007).
25. Steinbeck, J.A. et al. Optogenetics enables functional analysis of human embryonic stem cell-derived grafts in a Parkinson’s disease model. Nat Biotechnol 33, 204-209, doi:10.1038/nbt.3124 (2015).
26. Kim, T.W. et al. Biphasic Activation of WNT Signaling Facilitates the Derivation of Midbrain Dopamine Neurons from hESCs for Translational Use. Cell Stem Cell 28, 343-355 e345, doi:10.1016/j.stem.2021.01.005 (2021).
27. Politis, M. et al. Serotonergic neurons mediate dyskinesia side effects in Parkinson’s patients with neural transplants. Sci Transl Med 2, 38ra46, doi:10.1126/scitranslmed.3000976 (2010).
28. Piao, J. et al. Preclinical Efficacy and Safety of a Human Embryonic Stem Cell-Derived Midbrain Dopamine Progenitor Product, MSK-DA01. Cell Stem Cell 28, 217-229 e217, doi:10.1016/j.stem.2021.01.004 (2021).
29. Barker, R.A., Studer, L., Cattaneo, E., Takahashi, J. & consortium, G.F.P. G-Force PD: a global initiative in coordinating stem cell-based dopamine treatments for Parkinson’s disease. NPJ Parkinsons Dis 1, 15017, doi:10.1038/npjparkd.2015.17 (2015).
30. Barker, R.A., Parmar, M., Studer, L. & Takahashi, J. Human Trials of Stem Cell-Derived Dopamine Neurons for Parkinson’s Disease: Dawn of a New Era. Cell Stem Cell 21, 569-573, doi:10.1016/j.stem.2017.09.014 (2017).
31. Schweitzer, J.S. et al. Personalized iPSC-Derived Dopamine Progenitor Cells for Parkinson’s Disease. N Engl J Med 382, 1926-1932, doi:10.1056/NEJMoa1915872 (2020).
32. Normile, D. in Science (2018).
33. La Manno, G. et al. Molecular Diversity of Midbrain Development in Mouse, Human, and Stem Cells. Cell 167, 566-580 e519, doi:10.1016/j.cell.2016.09.027 (2016).
34. Kamath, T. et al. Single-cell genomic profiling of human dopamine neurons identifies a population that selectively degenerates in Parkinson’s disease. Nat Neurosci 25, 588-595, doi:10.1038/s41593-022-01061-1 (2022).
35. Tabula Sapiens, C. et al. The Tabula Sapiens: A multiple-organ, single-cell transcriptomic atlas of humans. Science 376, eabl4896, doi:10.1126/science.abl4896 (2022).
36. Eraslan, G. et al. Single-nucleus cross-tissue molecular reference maps toward understanding disease gene function. Science 376, eabl4290, doi:10.1126/science.abl4290 (2022).