Induced pluripotent stem cell–related approaches to generate dopaminergic neurons for Parkinson’s disease

Abstract

The progressive loss of dopaminergic neurons in affected patient brains is one of the pathological features of Parkinson’s disease, the second most common human neurodegenerative disease. Although the detailed pathogenesis accounting for dopaminergic neuron degeneration in Parkinson’s disease is still unclear, the advancement of stem cell approaches has shown promise for Parkinson’s disease research and therapy. The induced pluripotent stem cells have been commonly used to generate dopaminergic neurons, which has provided valuable insights to improve our understanding of Parkinson’s disease pathogenesis and contributed to anti-Parkinson’s disease therapies. The current review discusses the practical approaches and potential applications of induced pluripotent stem cell techniques for generating and differentiating dopaminergic neurons from induced pluripotent stem cells. The benefits of induced pluripotent stem cell-based research are highlighted. Various dopaminergic neuron differentiation protocols from induced pluripotent stem cells are compared. The emerging three-dimension-based brain organoid models compared with conventional two-dimensional cell culture are evaluated. Finally, limitations, challenges, and future directions of induced pluripotent stem cell–based approaches are analyzed and proposed, which will be significant to the future application of induced pluripotent stem cell–related techniques for Parkinson’s disease.

Introduction

Neurodegenerative diseases, including Alzheimer’s disease (AD), Parkinson’s disease (PD), and Huntington’s disease, are pathologically characterized by a progressive loss of structure and function of neurons in the brain. There is a lack of disease-modifying medications that could halt or reverse the neurodegenerative processes (De Gioia et al., 2020). Stem cell therapy is a promising therapeutic option for treating neurodegenerative diseases. The strategies include the regeneration of neural tissue, stabilizing the neuronal networks, providing neurotrophic support, and alleviating neurodegeneration at different neuronal circuitry levels (De Gioia et al., 2020). Stem cells are characterized by their distinct capacity to proliferate, self-renew, and differentiate into various cell lineages, which can be classified into embryonic stem cells (ESCs), induced pluripotent stem cells (iPSCs), mesenchymal stem cells (MSCs), and neural stem cells (NSCs) (Sivandzade and Cucullo, 2021). Among these stem cells, human ESCs and iPSCs are commonly used in stem cell therapy for managing neurodegenerative diseases (Blau and Daley, 2019). iPSCs are generated from adult somatic cells via reprogramming protocols (Fernandopulle et al., 2018). iPSCs can be differentiated into any type of cell in the body, forming a potential source of personalized cells for research and therapy. Before the development of reprogramming protocols to induce somatic cells into a pluripotent state, pluripotent cells had to be derived from embryos, which hindered the convenient availability and raised ethical concerns (Takahashi and Yamanaka, 2006). ESCs are derived from the inner cell mass of blastocysts with indefinite self-renew capacity and can differentiate into almost all cell types in the brain, offering a promising therapeutic approach for managing chronic neurodegenerative diseases (Sivandzade and Cucullo, 2021). ESCs and iPSCs are being used in large quantities and high purity as invaluable cell sources for potential cell replacement therapy related to neurodegenerative diseases.

Human ESCs (hESCs) have become an attractive candidate in PD cell therapy due to their ability to yield defined lineages such as midbrain dopaminergic neurons (Kim et al., 2020a). A previous preclinical study has shown that in rat PD models, the hESC-derived midbrain dopaminergic neurons were comparable in efficacy (reflected by long-term survival and functionality) to human fetal tissue grafts in rescuing dopamine (DA) deficiency (Grealish et al., 2014). The hESC-derived dopaminergic neurons also appeared to be morphologically identical to fetal ventral midbrain-derived dopaminergic neurons and expressed the same markers (Grealish et al., 2014). Grealish et al. (2014) showed that similar to fetal ventral midbrain-derived dopaminergic neurons, hESC-derived dopaminergic neurons were also able to extend their axons over long distances and achieve target-specific innervation. The axonal outgrowth capacity shown by hESC-derived dopaminergic neurons appears to be sufficient for use in humans (Grealish et al., 2014). These findings show that hESC-derived dopaminergic neurons can survive, mature, and also restore DA neurotransmission in the striatum, supporting the remarkable potential of hESCs in cell replacement therapies for PD.

However, while the differentiation of hESCs holds promise for regenerative medicine, there are also ethical considerations associated with the use of human embryos for research purposes. Furthermore, compared with traditional methods to acquire neurons from ESCs, there are multiple advantages to generating neurons from iPSCs. The iPSC techniques offer a powerful platform to model human disease within a dish with a patient’s cells from a biopsy or blood samples, allowing the study of disease mechanisms and therapy development on a case-by-case basis (Lin et al., 2019). This is especially significant in studies on neurodegenerative diseases, where it is impossible to obtain neurons from patient brains. The iPSC-derived neuron models are more physiologically accurate, having the ability to emulate the cellular structure and architecture as well as the cellular interactions that would be present in the actual environment (Du and Parent, 2015). Furthermore, iPSCs can be generated and cultured in large quantities, providing a consistent and reliable source of cells for research and clinical application (Lin et al., 2019).

In this review, we summarize dopaminergic neuronal differentiation protocols to generate dopaminergic neurons from patient cell-derived iPSC and ESC, which can add to PD pathogenesis and therapies. The protocols to generate dopaminergic neurons from other cell types have also been discussed.

Search Strategy

Studies mentioned in this review, published all years were searched on the PubMed database using these keywords: (“dopaminergic neurons” OR “dopamine neurons”) AND differentiation AND (“stem cells” OR “induced pluripotent stem cells” OR “embryonic stem cells”) AND (protocol OR methodology OR technique) AND “ventral midbrain.”

Overview of Neural Differentiation from Embryonic Stem Cells and Induced Pluripotent Stem Cells

The process of neural differentiation from ESCs and iPSCs involves a series of steps to mimic the physiological developmental processes that occur during embryonic development. These steps involve supplementing the culture medium with specific growth factors and signaling molecules to emulate an appropriate growth environment that gives rise to neuronal differentiation (Yu et al., 2007). Typically, ESCs and iPSCs are treated with different factors to differentiate into neural progenitor cells (NPCs) in neural rosette format, which can be subsequently differentiated into specific types of neurons or glial cells with different protocols (Figure 1).

Figure 1.

Neuronal differentiation from iPSCs.

iPSCs can be induced from adult somatic cells, while ESCs can be derived from the inner cell mass of blastocysts. Both iPSCs and ESCs can be differentiated into various types of neurons and glial cells for research on neurodegenerative diseases. Neural differentiation of iPSCs and ESCs includes a series of steps to mimic the physiological developmental processes that occur during embryonic development. Typically, both iPSCs and ESCs are treated with different signaling molecules and/or growth factors to differentiate into NPCs in a neural rosette format, which can be subsequently differentiated into specific types of neurons, including cortical neurons, motor neurons, dopaminergic neurons, cholinergic neurons, glutamatergic neurons, GABAergic neurons, serotonergic neurons and glial cells, including astrocytes, oligodendrocytes and microglia, with different protocols. Arrows refer to differentiation processes. Created with Science Slides 2016 and Photoshop 6.0. ESCs: Embryonic stem cells; iPSCs: inducible pluripotent stem cells; NPCs: neural progenitor cells.

Factors and pathways involved in neural differentiation from embryonic stem cells and induced pluripotent stem cells

There are various protocols for neuronal differentiation from ESCs and iPSCs, which involve the modulation of many signaling pathways to drive the differentiation process. These protocols can be used either independently or in tandem to derive neuron generation (Swistowski et al., 2010; Zhang et al., 2014). There are many potential pathways involved in the process of iPSC or ESC differentiation, including the suppressor of mothers against decapentaplegic (SMAD) transcription factor (Zhang et al., 2014), the wingless-related integration site (Wnt) (Zhang et al., 2014), the fibroblast growth factor (FGF) (Zhang et al., 2014) and the retinoic acid pathways (Fu et al., 2020). Alternatively, iPSCs can be induced to differentiate into mature neurons via genetic modifications to alter the expression levels of certain key transcription factors (Solomon et al., 2021).

Small molecule induced differentiation

The development of the nervous system involves several specific signaling pathways, including SMAD-transforming growth factor-β (TGF-β), Wnt, and FGF pathways. Wnt components are secreted morphogens that mediate embryonic patterning, cell specification, neuronal migration and polarization, dendrite development, and synapse formation (Rosso and Inestrosa, 2013). The FGF pathway includes a family of multifunctional proteins to orchestrate the pluripotency process by controlling the levels of subsets of genes expressed by pluripotent blastula cells and mediate essential biological processes in mammalian cell development such as cell survival, proliferation, differentiation, and migration (Mossahebi-Mohammadi et al., 2020). Meanwhile, SMADs are critical components of the TGF-β pathway to promote the differentiation of non-neuronal lineage from ESCs or iPSCs (Gordeeva, 2019). Therefore, the usage of small molecules to modulate SMAD, Wnt and FGF signaling pathways have been used to direct neuronal differentiation from iPSC and ESC. The use of small molecules to induce neural differentiation has its strengths and weaknesses. Small molecules have better cell membrane permeability, are easier to be synthesized and used, and can be used for mass production (Kim et al., 2020b). However, small molecules have their limitations of being inefficient and may be unevenly distributed in both media and cells because different iPSC or ESC clones may react differently to small molecules, even if iPSC or ESC clones are generated from the same origin (Fernandopulle et al., 2018). As a result, the resulting differentiated cells can differ in minute ways, leading to variability. This further escalates with longer-term cell culture studies, causing batch-to-batch variations and complicating downstream analysis. The application of small molecules is also tedious and time-consuming. Small molecules have been used in various studies for iPSC and ESC differentiation, and the timeline is extended from 13 to 70 days (Kikuchi et al., 2017; Fernandopulle et al., 2018; Telias, 2023). This increases the laboratory expenses and enhances the protocol complications.

Transcription factors induced differentiation

The overexpression of certain transcription factors can promote neuronal differentiation from iPSCs. Transcription factors play critical roles in driving the differentiation of stem cells into specific lineages, including neurons. This process avoids many challenges and complications in small molecule-mediated neuronal differentiation (Fernandopulle et al., 2018). The transcription factor–mediated neuronal differentiation from iPSCs provides a valuable tool for modeling neurodevelopmental and neurodegenerative disorders in vitro. By recapitulating disease-relevant phenotypes in patient-derived neurons, researchers can gain insights into disease pathogenesis and screen for potential therapeutic compounds in a more physiologically relevant context. To reprogram iPSCs via overexpressing key neuronal transcription factors allows for higher efficiency and purity and a faster timescale than small molecule-mediated neuronal differentiation. It has been reported that the overexpression of the master neuronal transcriptional regulator neurogenin-2 (NGN2) leads to a rapid and one-step differentiation of iPSCs to mature glutamatergic cortical neurons (Zhang et al., 2013). Similar results were obtained in the study conducted by Busskamp et al. (2014), which demonstrated that overexpression of NGN2 could promote the differentiation of iPSCs into synaptically mature neurons in 2 weeks. Differentiation efficiency and purity are greater than 90% in both studies (Zhang et al., 2013; Busskamp et al., 2014). However, there are also limitations of genetic modification approaches such as the need for artificial genetic modifications to modulate gene expression, which will limit its applications for clinical usages and drug screening researches (Kim et al., 2020b). These genetic modifications may also result in some indefinite downstream effects that may cause cell activity changes and compromise the approach validity.

However, during neuronal differentiation from ESCs or iPSCs, not all cells are fully converted into neurons. Several methods have been developed to selectively eliminate non-converted or undifferentiated cells to enhance the efficiency of neuronal differentiation. ROCK inhibitors, such as Y-27632, are used to increase the survival of ESCs/iPSCs after passaging, However, they can help reduce apoptosis in differentiated cells while selectively affecting undifferentiated cells, which can be used to eliminate undifferentiated cells during ESCs or iPSCs differentiation (Watanabe et al., 2007). Cell sorting approaches, such as fluorescence-activated cell sorting, can be used to identify and select differentiated neuronal cells expressing neuronal markers, while eliminating cells that express undifferentiated markers (Doi et al., 2014). It was reported that undifferentiated TRA-1-60 antibodies could be conjugated with cytotoxic agents to selectively kill the undifferentiated cells while leaving the differentiated neurons intact (Schuldiner et al., 2003). The expression of suicide genes under the control of a pluripotency-specific promoter can also help remove undifferentiated cells (Gysel et al., 2023). Furthermore, specific compounds, such as PluriSIn, which can selectively inhibit the growth of undifferentiated pluripotent cells via targeting specific signaling pathways for pluripotency, can kill undifferentiated cells and spare differentiated neurons (Ben-David and Benvenisty, 2012). Other strategies to use cytotoxic drugs and apoptosis inducers as well as hypoxic conditions have been reported to selectively remove undifferentiated cells (Cai et al., 2009; Forristal et al., 2010; Pera and Tam, 2010). By using a combination of these methods or tailoring them to specific protocols, the purity of neuronal populations derived from ESCs or iPSCs can be increased with reduced contamination from undifferentiated cells.

Multiple protocols of dopaminergic neuron differentiation

Numerous research papers have explored different approaches for differentiating iPSCs or ESCs into dopaminergic neurons, each with unique advantages and limitations (Table 1). The anterior-posterior patterning is a critical aspect of the differentiation of dopaminergic neurons from ESCs (Birtele et al., 2022). The anterior–posterior patterning involves a complex interplay of signaling pathways, transcription factors, and epigenetic modifications, ultimately guiding the formation of dopaminergic neurons (Birtele et al., 2022). The Wnt, FGF, and sonic hedgehog (SHH) signaling pathways are vital to promote midbrain dopaminergic neuron identity (Cutts et al., 2016). Transcription factors such as LIM homeobox transcription factor 1 alpha, fork-head box a2, and NGN2 are essential for specifying dopaminergic neuron fate (Tian et al., 2021). Furthermore genetic factors such as nuclear receptor related-1 (Nurr1), ladybird homeobox 1 (LBX1), nescient helix-loop-helix 1 (NHLH1), and nuclear receptor subfamily 2 group F member-1/2 (NR2F1/2) can significantly enhance dopaminergic neuron differentiation from iPSCs or ESCs (Table 1; Gomez Ramos et al., 2024). The treatments of iPSCs or ESCs with small molecular SMAD inhibitors, Wnt/SHH pathway activators, neurotrophic factors, growth factors, hormones and neurotransmitters can induce dopaminergic neuron differentiation with distinct yield of dopaminergic neurons within different timeline (Table 1; Poulsen et al., 1994; Shin et al., 2004; Mimeault et al., 2007; Chambers et al., 2009; Swistowski et al., 2010; Sundberg et al., 2013; Wattanapanitch et al., 2014; Zhang et al., 2014; Popova et al., 2017; Suzuki et al., 2017). The protocols to generate dopaminergic neurons can have some similarities to those for diencephalic cell generation (Tofoli et al., 2019). The most significant difference in protocols for dopaminergic and diencephalic differentiation lies in the distinct growth factors and signaling pathways used at various stages (Tofoli et al., 2019). Dopaminergic neuron differentiation heavily relies on SHH signaling, while diencephalic differentiation often involves retinoic acid (Tofoli et al., 2019). Understanding the differences in these protocols is crucial to further optimizing differentiation efficiency, improving the yield of functional dopaminergic neurons, and enhancing their potential applications in disease modeling and regenerative medicine.

Table 1.

Main research papers for human dopamine neuronal differentiation from progenitor cells

Progenitor cell type	Method	Timeline	Target yield	Reference
Human iPSC	Small molecule induction	~20 d	~90%	Zhang et al., 2014
Human iPSC	Small molecule induction	~26 d	~80%–99%	Kikuchi et al., 2017
Human iPSC	Small molecule induction	~21 d	~30%	Suzuki et al., 2017
Human iPSC	Small molecule induction	~40 d	~35%	Swistowski et al., 2010
Human iPSC	Small molecule induction	~30 d	~30%–75%	Sundberg et al., 2013
Human iPSC	Small molecule induction	~35 d	~45%	Xi et al., 2012
Human ESC	Small molecule induction	~20 d	~90%	Zhang et al., 2014
Human ESC	Small molecule induction	~25 d	~80%	Kriks et al., 2011
Human ESC	Small molecule induction	~30 d	~30%–75%	Sundberg et al., 2013
Human ESC	Small molecule induction	~50 d	~12%	Cooper et al., 2010
Human ESC	Small molecule induction	~42 d	~11%	Nolbrant et al., 2017
Human ESC	Small molecule induction	~11 d	~65%	Kim et al., 2021
Human ESC	Small molecule induction	~45 d	~80%	Kirkeby et al., 2017
Human ESC	Small molecule induction	~10 d		Kee et al., 2017
Human ESC	Small molecule induction	~56 d	~70%	Nishimura et al., 2023
Human ESC	Small molecule induction	~35 d	~45%	Xi et al., 2012
Human ESC	Genetic engineering induction	~30 d	~69%	Maimaitili et al., 2023
Human ESC	Small molecule induction	~14 d	~60%–80%	Alekseenko et al., 2022

ESC: Embryonic stem cell; iPSCs: induced pluripotent stem cell.

Small molecules to induce dopaminergic neuron differentiation

Small molecules have been shown to differentiate stem cells into dopaminergic neurons by modulating signaling pathways and processes at a reasonable cost, with great scalability and reproducibility compared with genetic engineering. So far, multiple small molecules have been employed for dopaminergic neuronal differentiation.

SMAD signaling inhibitors

SMAD inhibition is a well-known method for directing dopaminergic neural differentiation of iPSCs (Wattanapanitch et al., 2014). Noggin and SB431542 are two common inhibitors for SMAD signaling. Noggin represses the endogenous bone morphogenetic protein signals and regulates the trophoblast fate upon dopaminergic neuronal differentiation (Chambers et al., 2009). SB431542 inhibits the TGF-β pathway to downregulate SMAD signaling and enhance dopaminergic neural differentiation (Wattanapanitch et al., 2014). Noggin and SB431542 can synergistically induce iPSC to differentiate into neural progenitors, which can further differentiate into other neural lineages (Wattanapanitch et al., 2014). Moreover, combinative Noggin and SB431542 inhibitions can directly induce the conversion of human ESC into dopaminergic neurons under adherent culture conditions (Chambers et al., 2009).

Wnt/SHH pathway activators

The floor plate (FP) neuronal precursors with dopaminergic neuron differentiation potential can be generated by combinative activating the Wnt and SHH signaling pathway, and these FP neuronal precursors can be further specified into dopaminergic neurons (Zhang et al., 2014). Meanwhile, small molecules like CHIR99021 and purmorphamine have been reported to stimulate the Wnt and/or SHH signaling pathway to promote the generation of FP precursors and dopaminergic neuron differentiation (Zhang et al., 2014).

Other small molecules and factors

Once FP precursors are generated, various small molecules can be used in tandem to direct their differentiation toward dopaminergic neurons. For this step, many papers have used a combination of small molecules such as brain-derived neurotrophic factor (BDNF), glial cell line-derived neurotrophic factor (GDNF), TGF-β3, ascorbic acid (AC), and cyclic adenosine monophosphate (cAMP) (Zhang et al., 2014). These molecules were typically used in later stages of differentiation, directing neurospheres or NPCs derived from iPSCs to dopaminergic fate. The AC is a cofactor that is involved in numerous enzymatic processes essential to cell growth and maintenance. However, AC also plays a role in dopaminergic neuron differentiation, and neurons treated with AC show a 10-fold increase in dopaminergic neuron yield. Studies show that AC mediates the process by various downstream effectors, including bone morphogenetic protein 7, erythropoietin, and various genes involved in cell adhesion and development (Shin et al., 2004). Various hormones and neurotransmitters have been shown to induce or regulate neuronal differentiation by increasing cellular cAMP levels. Elevated cAMP levels can activate protein kinases and ultimately lead to neuronal differentiation and synaptogenesis (Sánchez et al., 2004).

Neurotrophic factors are crucial in neuron development, differentiation, and synaptogenesis. In dopaminergic neuron differentiation processes, both BDNF and GDNF have been used to facilitate the differentiation process. BDNF is closely linked to 5-HT neurons and regulates protein synthesis, neuronal growth, and dendrite maturation (Popova et al., 2017). Additionally, improper regulation of BDNF in terms of metabolism, transport, or signaling has been linked to various neurological diseases (Popova et al., 2017). GDNF is regarded as a key factor in the maintenance, development, and growth of dopaminergic neurons, as well as synaptogenesis and function of neurons (Popova et al., 2017). TGF-β3 is a growth factor that regulates the survival and development of dopaminergic neurons. Studies show that TGF-β3 is essential for the growth and survival of dopaminergic neurons, with experimental proof showing insufficient TGF-β3 levels can inhibit neuronal growth by a 3- to 5-fold decrease (Poulsen et al., 1994).

Genetic engineering to induce dopaminergic neuron differentiation

Genetically modified ESCs and iPSCs can also be utilized to induce dopaminergic neuronal differentiation. The Nurr1 is a transcription factor that plays a crucial role in the differentiation, maturation, and maintenance of midbrain dopaminergic neurons (Saucedo-Cardenas et al., 1998). Overexpression of the Nurr1 gene significantly promoted the differentiation of ESCs into dopaminergic neurons, evidenced by the increased proportions of tyrosine hydroxylase (TH) positive neurons (a marker for dopaminergic neurons) (Kim et al., 2002). Meanwhile, small molecules, such as SHH and FGF8, have been reported to enhance Nurr1-induced differentiation of ESCs into dopaminergic neurons (Kim et al., 2002). Additionally, other transcription factors play crucial roles in regulating cell differentiation and specification to modulate targeted gene expressions via binding to the genomic regulatory regions of targeted genes, such as enhancers and promoters (Spitz and Furlong, 2012). By performing transcriptomic and epigenomic analysis, Ramos et al illustrate that the midbrain dopaminergic neuron differentiation can be promoted by increased expression of three transcriptional factors, namely LBX1, NHLH1, and NR2F1/2 (Gomez Ramos et al., 2024). Among these transcriptional factors, LBX1 and NHLH1 were demonstrated to enhance midbrain dopaminergic neuron specification and NR2F1/2 was found to improve neuronal activity (Gomez Ramos et al., 2024).

Molecular events during induced pluripotent stem cell differentiation

During the differentiation of iPSCs into dopaminergic neurons, various molecular events occur during the transition of cells from a pluripotent state to a more specialized dopaminergic lineage.

Alterations of gene expression profiles

During the differentiation of iPSCs into dopaminergic neurons, the alterations of specific genes associated with dopaminergic neuron development and pluripotency have been observed. Previous studies have shown that the gene expression levels of TH, DA active transporter, and G protein-activated inward rectifier potassium channel 2 that reflected the vulnerable A9 population of substantia nigra neurons, are all elevated in the iPSCs-derived dopaminergic neurons (Rakovic et al., 2022). In contrast, the expression levels of octamer-binding transcription factor 4 (OCT4), which are relevant to stem cell pluripotency, are suppressed in neuronal stem cells (Alekseenko et al., 2022).

Activation of signaling pathways

Various signaling pathways play crucial roles in iPSCs differentiation into dopaminergic cells, such as the Wnt, SHH, and FGF signaling pathways (Brodski et al., 2019). Wnt signaling is crucial in dopaminergic progenitor cell proliferation and differentiation during neuronal development (Rotherham et al., 2019). The differentiation of iPSCs into midbrain dopaminergic neurons requires a high-activity form of SHH, which facilitates dopaminergic differentiation and promotes the survival of differentiated dopaminergic neurons (Cooper et al., 2010). A previous study has suggested that the activation of the bone morphogenetic protein/SMAD pathway can promote iPSC differentiation into midbrain dopaminergic neurons via the Wnt signaling pathway (Cai et al., 2013). FGF8 has been shown to be vital to the generation and functionality of dopaminergic neurons from iPSCs (Lim et al., 2015).

Epigenetic modifications

Epigenetic modifications have been shown to be important in the regulation of the fate of stem cells (Srinageshwar et al., 2016). The epigenetic modifications are vital to regulating gene expression patterns during iPSC differentiation and also contribute to the stabilization of dopaminergic neuron identity (Yang et al., 2024). DNA methylation and histone modifications can activate or repress the expression of multiple genes involved in dopaminergic neuron development and functions (Vargas-Romero et al., 2019). Thus, by stabilizing specific epigenetic marks associated with dopaminergic cell identities, such as methylated DNA regions and specific histone modifications, the differentiation process can be facilitated to produce and maintain dopaminergic neurons more effectively.

Morphology development and functional maturation

Fully differentiated dopaminergic neurons exhibit functional properties of characteristic of mature neurons (Barker et al., 2015). Using calcium imaging and electrophysiology, the maturation and spontaneous synaptic activity of mature dopaminergic neurons can be observed (Hartfield et al., 2014). After the differentiation of iPSCs into dopaminergic neurons, cells undergo morphological changes, extending their neurites and forming synaptic connections with other neurons (Gilmozzi et al., 2021). Whole-cell patch clamp experiments have also revealed that iPSC-derived dopaminergic neurons give rise to currents mediated by voltage-gated sodium and potassium channels, have high degrees of synaptic activity, and can fire trains of spontaneous action potentials (Rakovic et al., 2022). Mature dopaminergic neurons can also be shown to synthesize, release and take up DA (Hartfield et al., 2014).

Application of Stem Cells–Derived Dopaminergic Neurons

The ability to derive mature dopaminergic neurons from iPSCs opens new avenues for PD toward disease modeling and validation, drug screening and therapeutic development.

Parkinson’s disease models

Neuronal differentiation models have been widely used in research on neurological disorders, including AD (Essayan-Perez et al., 2019), PD (Cooper et al., 2010; Kriks et al., 2011; Kikuchi et al., 2017), Huntington’s disease (Mattis and Svendsen, 2017), and amyotrophic lateral sclerosis (Lee and Huang, 2017). The use of iPSCs in PD disease models has brought us many benefits, including the generation of better and more relevant patient-specific cellular models (Engle et al., 2018). Furthermore, the advancement in iPSC techniques promised large and replicable amounts of human patient neurons for study, especially with neurological diseases which often have low primary tissue availability.

Before the advent of iPSCs derived from patients, animal models, like primary rodent neurons were used as the main cellular model for studying neurobiology, which was not always well translatable to human subjects. The iPSCs-derived human neurons serve as reliable in vitro human disease models that can more accurately characterize pathophysiological mechanisms underlying neurodegeneration (Xu et al., 2023). For instance, the protein Parkin, encoded by the PARK2 gene, is crucial in maintaining mitochondrial homeostasis and is implicated in PD (Kazlauskaite et al., 2014). PD patient iPSC-derived dopaminergic neurons with PARK2 mutations present enhanced oxidative stress, dysregulated α-synuclein accumulation, and disturbed mitochondrial morphology and functions (Chung et al., 2016). Similar patterns were observed in human isogenic iPSCs-derived dopaminergic neurons with PARK2 knockout (Bogetofte et al., 2019). These studies provide strong scientific evidence for the effects of Parkin dysfunction and disease-related pathways in neurodegeneration in PD. Recently light-inducible protein aggregation system and optogenetics-assisted α-synuclein aggregation induction system were developed to rapidly induces α-synuclein aggregates and toxicity in animal model and PD patient derived hiPSC-midbrain dopaminergic neurons and midbrain organoids, which significant to disease modeling and anti-PD drug screening applications (Berard et al., 2022; Kim et al., 2023).

Anti-Parkinson’s disease therapies

The patient iPSC-derived DA neurons can be used to do high-throughput drug screening study to identify compounds that modulate neuronal function, promote cell survival, or protect against disease-related toxicity, which can add to anti-PD strategies (Essayan-Perez et al., 2019). iPSC-derived dopaminergic neurons can also be used to investigate the mechanism of studied drugs, and optimize more potent and safer drugs for PD therapy. Another goal of the iPSC technique is to develop cell replacement therapies for PD. The transplantation of iPSC-derived dopaminergic neurons into PD patient brain has been shown to have a better physiologically relevant delivery and response as opposed to treatment with drugs (Hiller et al., 2022). Clinical trials involving the transplantation of cells into PD patient brains are becoming more prevalent focusing on iPSCs as a source of dopaminergic neurons (Daadi et al., 2024). Cells can be transplanted at the stage of immature progenitors and can survive, differentiate, mature, and extend their neurites to form synaptic connections with host neurons in the grafted region (Morizane, 2023). It was reported that in non-human primate models of PD, the transplantation of iPSC-derived dopaminergic neurons could promote functional recovery and lead to the survival of approximately 20,000 TH-positive neurons in animal brains (Osborn T., 2020). In rat models, transplantation of PD patient iPSC-derived dopaminergic neurons brought about an improvement in motor function (Hargus et al., 2010). These studies highlight the potential of iPSC-derived dopaminergic neuron transplantation as a new effective therapeutic strategy for PD which can potentially alleviate patient symptoms and improve the quality of life of PD patients.

Physiological neural development

The development of representative models by iPSC-differentiated neurons allows the investigation of the underlying mechanisms behind physiological neural development and functions. A previous study has utilized iPSC-differentiated neurons to study gene expression profiles of developing neurons and identify key genes involved in development processes (Belinsky et al., 2014). The iPSC techniques can also add to research on transcriptome signatures and signaling pathways in distinct types of neurons and help understand the distinct molecular events and signatures in different neuron subtypes (Solomon et al., 2021).

Dopaminergic Neuronal Differentiation from Other Cell Types

Other than iPSCs, dopaminergic neurons can also be differentiated and generated from other cell types.

Neural stem cells

NSCs are self-renewing, multipotent cells that can generate both neuronal and glial cell lines during embryogenesis as well as in the adult central nervous system (Llorente et al., 2022). The functions of NSCs account for continuously maintained neurogenesis throughout adult life in adult mammalian brains. In a recent study by Liu et al. (2024), NSCs with necessary growth factors have been transplanted into the cortex (visual and sensory cortices) of rhesus monkeys after superficial traumatic brain injury. After 1 year of transplantation, the grafted NSCs were found to be differentiated into dopaminergic neurons with higher survivability and functionality (Liu et al., 2024).

A previous study has suggested that NSC transplantation could be considered an efficient therapeutic approach for PD (Marei et al., 2015). It was reported that in the striatum of 6-OHDA lesioned rats, the grafted undifferentiated NSCs (stable clonal cell line C17.2) could be differentiated into dopaminergic neurons (Yang et al., 2002). In the PD rat models, the grafted human olfactory bulb NSCs (genetically modified to express necessary growth factors) have been found to ameliorate cognitive deficits in PD rat models (Marei et al., 2015). These transplanted olfactory bulb NSCs exhibited higher survival and differentiation rates and could migrate to the damaged areas to restore striatal histoarchitecture (Marei et al., 2015). Moreover, transplanted human-brain-derived NSCs have been found to promote the differentiation of dopaminergic neurons in the striatum of 6-OHDA-treated rats, where these differentiated dopaminergic neurons significantly improved motor dysfunction in 6-OHDA-treated rats (Shin et al., 2014). Furthermore, NSCs reprogrammed from human peripheral blood mononuclear cells were able to be differentiated into dopaminergic neurons with potential therapeutic significance (Yuan et al., 2018). These peripheral blood mononuclear cell-derived iNSCs have similar characteristics to fetal NSCs and were able to be differentiated into dopaminergic neurons with high efficiency (Yuan et al., 2018). The NSC-derived dopaminergic neurons showed high survival rates in mice striatum without any graft overgrowths or tumor formation (Yuan et al., 2018). The NSC-derived dopaminergic neurons from peripheral blood mononuclear cell induction can be transplanted into mouse PD models to significantly improve mice motor functions two weeks post-transplantation (Yuan et al., 2018).

NSCs derived from iPSCs can be differentiated into dopaminergic neurons with the help of dopamine-inducing factors and FGF-2 (Daadi, 2019). These two factors enhanced the expression levels of TH and neuron-specific class III beta-tubulin, a neuronal marker, to induce the midbrain dopaminergic neuronal phenotype in NSCs (Daadi, 2019). Microglia are crucial in brain development as they can provide a proper microenvironment for embryonic neurogenesis (Elmore et al., 2014). A recent study has reported that microglia-secreted factors, such as interleukin-1β, insulin-like growth factor 1, and tumor necrosis factor-α, can promote dopaminergic differentiation of somatic and iPSC-derived human NSCs (Schmidt et al., 2021).

Similar to iPSCs, small molecules can also function to promote the differentiation of NSCs into dopaminergic neurons (Ye et al., 2020; Schmidt et al., 2021). (+)4-Cholesten-3-one is a small molecule related to dopaminergic neuron differentiation (Ye et al., 2020). A previous study demonstrated that (+)4-cholesten-3-one can promote the differentiation of rat NSCs into dopaminergic neurons and elevate the TH expression levels in neuron cells (Ye et al., 2020). Further mechanistic studies indicated that (+)4-cholesten-3-one promoted the conversion of NSCs into dopaminergic neurons by up-regulating levels of ten-eleven translocation 1 (an enzyme related to neuronal development) and fork-head box a2 (a nuclear transcription factor activates the promoter of TH) (Ye et al., 2020).

The neuroprotective effects of safranal (SAF), the main component of the Crocus sativus L. (Iridaceae), have been proven previously (Farshid and Tamaddonfard, 2015). Zhao and Xi (2018) verified the role of SAF in promoting dopaminergic neuron growth. SAF has been found to promote the expression of TH and enhance the secretion of DA in an in vitro study of rat NSCs (Zhao and Xi, 2018). A further study has demonstrated that transplantation of SAF-treated NSCs could ameliorate motor deficits and enhance lifespan in 6-OHDA-treated rats (Zhao and Xi, 2018).

These findings highlight the potential of the application of NSCs in cell-based therapies in PD and potentially other neurological disorders. Optimized and efficient differentiation protocols to yield large amounts of pure populations of functional dopaminergic neurons from NSCs need to be further established for PD cell replacement therapy.

Mesenchymal stem cells

MSCs are non-hematopoietic multipotent stem cells found in various adult tissues such as bone marrow, adipose tissue, and umbilical cord tissue (Costela-Ruiz et al., 2022). MSCs can be differentiated into dopaminergic neurons under specific conditions by the application of appropriate growth factors and signaling molecules, and thus, MSCs hold the premise for potential cell replacement therapy for PD (Gaggi et al., 2020). In PD rat models, transplantation of neurotrophic tyrosine receptor kinase 1 overexpressed peripheral blood MSCs into substantia nigra can improve motor deficits and repair dopaminergic neuron damage (Liu et al., 2018). A recent study indicated that after being treated with a dopaminergic differentiation protocol, multiple transcription of dopaminergic markers, including LMX1b, Nurr1, PITX3, and DA active transporter, were gradually enhanced in MSCs and MSCs ultimately obtained a homogenous cell population, resembling dopaminergic neurons with higher expression levels of functional dopaminergic markers, such as TH, DA active transporter and Nurr1 (Gaggi et al., 2020). It was reported that MSCs derived from adipose tissues could be differentiated into dopaminergic neurons by treating with a combination of differentiating factors (including SHH, FGF-2, and FGF-8), evidenced by the demonstration of a surface-specific cluster of differentiation markers and functional dopaminergic markers (Khademizadeh et al., 2019).

Human fetal membrane-mesenchymal stromal cells (hFM-MSCs) are stem cells isolated from the amniochorionic membrane (Gaggi et al., 2020). As a promising candidate in regenerative medicine, hFM-MSCs are not tumorigenic and have low immunogenicity (Gaggi et al., 2019). The epigenetic profile of hFM-MSCs is quite similar to human iPSCs. hFM-MSCs can be differentiated into dopaminergic neurons by sequential exposure to different small molecules, thereby mimicking the steps of dopaminergic differentiation in vivo. It is observed that floor plate cells are first generated by the activation of the SHH pathway and the inhibition of SMAD signaling. Subsequently, midbrain floor plate cells are induced by Wnt signaling, and finally, dopaminergic neurons are obtained after treatment of cells with trophic factors and N-[N-(3,5-difluorophenacetyl-L-alanyl)]-S-phenylglycine tertial butyl ester (DAPT), a gamma-secretase inhibitor to block Notch signaling (Gaggi et al., 2020). The differentiation process correlates with a decline in OCT4 level in line with pluripotency loss and emergence of dopaminergic neuron phenotype (Gaggi et al., 2020).

Bone marrow–derived MSCs were able to be reprogrammed towards a dopaminergic differentiation by transducing the LMX1a gene into the cells via a lentiviral vector transfection (Barzilay et al., 2009). In transduced cells, the LMX1 protein was localized in the nuclei, while the protein expression levels of functional dopaminergic markers, such as TH and vesicular monoamine transporter 2, were elevated and correlated with an increased DA level (Barzilay et al., 2009).

In addition to bone marrow–derived MSCs, human umbilical mesenchymal stem cells were also able to be efficiently differentiated into dopaminergic neurons in the hemiparkinsonian rat (Ko et al., 2015). Human umbilical mesenchymal stem cells were processed through a three-step differentiation protocol involving the treatment of cells with nucampholin, FGF8, and SHH. Ultimately, the expression of TH and the secretion of DA were elevated in differentiated dopaminergic neurons (Ko et al., 2015). Meanwhile, transplantation of human umbilical mesenchymal stem cells into the striatum of hemiparkinsonian rats causes an improvement in their behavioral deficits in rats (Ko et al., 2015).

Other somatic cells

It was reported that some somatic cells can be reprogrammed directly into dopaminergic neurons through genetic manipulation or exposure to specific factors to promote neuronal identity (Aversano et al., 2022). These induced dopaminergic neurons have been shown to integrate into the mouse striatum successfully and alleviate PD motor symptoms in mice (Aversano et al., 2022).

Most of the approaches to direct differentiation of somatic cells into dopaminergic neurons are based on the viral-mediated expression of transcription factors that are crucial for the development of dopaminergic neurons. It is reported that human embryonic fibroblasts can be converted into dopaminergic neurons by combining overexpression of three conversion factors (achaete-scute homolog 1, POU domain class 3 transcription factor 2, and myelin transcription factor 1 like) as well as two genes involved in the generation of dopaminergic neurons (LMX1A and FOXA2) (Pfisterer et al., 2011). Human adipose tissue–derived stem cells (hADSCs) have also been differentiated into dopaminergic neurons (Soheilifar et al., 2018). The hADSCs isolated from subcutaneous abdominal adipose tissue were exposed to a combination of SHH, FGF8, and BDNF. After the differentiation period, a total of 27.9% of cells tested positive for TH with a significant amount of DA released in response to potassium chloride (KCl)–induced depolarisation, indicating positive dopaminergic neuron differentiation from hADSCs (Soheilifar et al., 2018). The direct somatic cell differentiation protocols have also been improved by the use of small molecules and non-coding RNAs (Aversano et al., 2022).

The direct reprogramming of somatic cells into dopaminergic neurons has emerged as a novel strategy to obtain mature and functional dopaminergic neurons, providing a sustainable source of dopaminergic neurons with potential research and clinical application significance.

Challenges and Future Directions

The recent advancement of stem cell techniques has contributed significantly to PD research and provided new hope to PD patients. However, multiple challenges exist for the differentiation and generation of dopaminergic neurons via stem cell techniques. The generated iPSCs may exhibit immature functional characteristics and contain heterogeneous mixtures of different phenotypic cell subtypes (Chun et al., 2015), making it difficult to generate consistent and accurate neurodegenerative disease models (Figure 2). The iPSC differentiation protocols often suffer from variability in terms of efficiency and reproducibility. The variability in protocols may lead to inconsistencies in generated cell populations. Ensuring robust and consistent differentiation across different iPSC lines and batches is a significant challenge to researchers. The future establishment of standardized effective protocols for iPSC differentiation into dopaminergic neurons is essential in ensuring the reducibility in different laboratories across the world.

Figure 2.

Limitations of current approaches and potential strategies to improve dopaminergic neuronal differentiation efficiency from iPSCs.

There are challenges and limitations to current approaches to generating dopaminergic neurons from iPSCs. The current differentiation approaches have difficulty to generate highly purified dopaminergic neurons. The current protocols will produce heterogeneous mixtures of cells, including immature or undifferentiated cells, cells with tumorigenicity, and various subtypes of neuronal and glial cells. Gene editing and single-cell technologies can reduce the potential of tumorigenicity, inhibit the heterogeneity of differentiated cells, and increase the purity of differentiated dopaminergic neurons. The 3D cell culture techniques, organoid/spheroid cultures, allow cells to grow in a 3D environment with an extracellular framework suitable for cell morphology, proliferation, migration, and differentiation. These miniaturized and simplified versions of human brains provide a powerful platform for studying disease mechanisms and potential treatments, including drug screening studies to test the efficacy and toxicity of new drugs compared to traditional 2D cell cultures or animal models. Created with Science Slides 2016 and Photoshop 6.0. iPSCs: Inducible pluripotent stem cells; NPCs: neural progenitor cells.

Gene editing technologies such as CRISPR/Cas9 protocols can be used to enable precise manipulation of the genome, offering unprecedented opportunities for studying disease mechanisms and developing targeted therapies (Figure 2). Using these tools, we can introduce disease-relevant mutations, correct genetic defects, or engineer reporter genes into iPSCs and their derivatives, including dopaminergic neurons, to model neurodegenerative disorders more accurately and develop personalized treatments.

The lack of reliable live-cell lineage identification methods is also a prominent obstacle in confirming the precise lineage of cells during differentiation (Moon et al., 2023). Future implementation of rigorous quality control measures throughout differentiation processes, including assessment of pluripotency, lineage specificity, and functional characterization of iPSC-derived cells can ensure the production of high-quality cell populations suitable for therapeutic applications. The single-cell technologies, such as single-cell RNA sequencing and mass cytometry, are revolutionizing our understanding of cellular heterogeneity and developmental trajectories. Applying these techniques to iPSC-derived dopaminergic neuron differentiation will allow us to dissect complex cell populations, identify rare cell subtypes, and characterize transcriptional dynamics during differentiation and maturation (Figure 2).

The scaling up of the production of dopaminergic neurons from iPSCs is also a crucial factor for both research and clinical applications. Currently, maintaining the consistency and quality of generated dopaminergic neurons across a large-scale production can be challenging. The maturation of differentiated cells seems to follow their trajectories, leading to variations in degrees of maturation after differentiation (Doss and Sachinidis, 2019). It is still challenging to produce considerable amounts and homogeneous populations of dopaminergic neurons for research and therapy, which warrants future investigations. Furthermore, for therapeutic applications, the generated dopaminergic neurons must be able to integrate into existing neural circuits upon transplantation functionally. Ensuring proper connectivity and functionality within host brains is a significant challenge that needs to be overcome. Another concern surrounding the application of iPSC-derived differentiated neurons is tumorigenicity potential due to residual undifferentiated iPSCs or improper differentiation. Robust and optimized differentiation protocols and stringent quality control measures are necessary to minimize this risk of tumorigenicity. Therefore, continual refinement and optimization of differentiation protocols are essential to enhance the efficiency and reproducibility of iPSC differentiation.

The survival and healthy status of the transplanted cells in the brain after long-term transplantation is one of the major issues for the clinical application of stem cell therapies for PD. It was previously reported that the grafted dopaminergic neurons can survive in PD patients long-term (14 years) after transplantation (Mendez et al., 2008). Meanwhile, numerous studies have proven that patients grafted with dopaminergic neurons improved their PD symptoms for a decade year, even the Lewy body (one of the pathological hallmarks of PD) can be formed in grafted dopaminergic neurons in PD brain after long-term transplantation (Chu and Kordower, 2010). Future studies are warranted to enhance the health status and functions of the grafted neurons in PD brains for cell transplantation therapy.

The autologous iPSCs from patient cells can be the ideal source for cell transplantation therapy. However, the autologous iPSC approaches can be expensive and time-consuming, restricting their broad clinical applications (Madrid et al., 2021). A recent study demonstrates that allogeneic hypoimmunogenic iPSC can be an alternative strategy (Hu et al., 2023). The hypoimmunogenic iPSCs are genetically modified to promote transplanted cell survival via reduction of their immunogenicity in the absence of immune suppressants (Deuse et al., 2019). Recently, hypoimmune induced pluripotent stem cells have been reported to survive for a long time in fully immunocompetent, allogeneic rhesus macaques (Hu et al., 2023). Furthermore, the hypoimmunogenic iPSCs with a low immunogenic profile can be induced to differentiate into various cell types successfully, supporting the potential application of hypoimmunogenic iPSCs for human disorders (Andrade da Silva et al., 2023).

The hypoimmunogenic iPSCs can be generated via the removal or alterations of some specific molecules from the iPSC surface (Deuse et al., 2019). Usually, HLA class I and II molecules can be knocked out to suppress immune recognition (Deuse et al., 2019). However, cells with HLA class I molecules deletion can be killed by natural killer cells, which can be abrogated by ligands deletion of active natural killer receptors or increased expression of CD47, PD-L1, PD-L2, HLA-E, or HLA-G, respectively (Deuse et al., 2021; Harding et al., 2024; Tsuneyoshi et al., 2024). However, the improved escape of hypoimmunogenic cells from the host immune response can enhance the risk of potential tumorigenicity of transplanted iPSCs (Liang et al., 2018). So far, some safety switches have been developed, including herpes simplex virus thymidine kinase and Rapamycin-activated Caspase-9, to prevent tumorigenicity of transplanted hypoimmunogenic iPSCs (Liang et al., 2018). Besides, the safety issues of hypoimmunogenic iPSC approaches, other challenges of hypoimmunogenic iPSC techniques, including stability, infection risks, functional integration, and cost issues, should be further investigated in the future.

While using iPSC-differentiated neurons to cultivate neurons for research has provided a very accurate representative model, steps can be taken to further enhance in vitro cellular models for research (Laplaca et al., 2010). The current two-dimensional (2D) cell culture model has been a staple for most of the current researches. These 2D models offer numerous benefits due to their nature of being easier to be cultivated, modified and handled (Xu et al., 2015). However, the 2D models are unable to simulate the actual three-dimensional (3D) structure and environment between cells (Xu et al., 2023). The results obtained from 2D cell culture models can be muddled by the lack of an accurate tissue environment with complicated cell–cell interactions. As a result, even 2D models with accurate cell types can provide results that vary from the in vivo situations (Duval et al., 2017).

The 3D models for cell culture, known as organoid/spheroid cultures, have since been introduced where cells are grown in a 3D environment with the extracellular framework, allowing cells to grow in all directions and grow into a self-assembled cluster or sphere. The 3D stem cell models offer substantial advantages over 2D models to better replicate the in vivo environment and improved cell differentiation, survival, and functionality, which can be more useful than 2D models in cell therapy and tissue transplantation as well as disease modeling (Breslin and O’Driscoll, 2013). The 3D cultures have extracellular matrices similar to the natural structure and thus can simulate the endogenous microenvironment suitable for cell morphology, proliferation, migration, and differentiation (Mimeault et al., 2007). In contrast, 2D models grow cells on flat surfaces, which will limit cell-to-cell and cell-extracellular matrix interactions. This can induce alterations of cell morphology, differentiation potential, and response to signals, contributing to inaccurate predictions of cell behavior in vivo. The resulting 3D model can allow cells to more readily differentiate into an ideal, in vivo-like cell fate, as well as better represent various stages of the cell cycle, including proliferation, differentiation, and cell–cell interactions (Duval et al., 2017). However, 2D models, on the other hand, often lead to incomplete or aberrant differentiation due to the unnatural, planar cell growth in 2D culture. The iPSC-derived organoids can also self-renew and recapitulate the physiologies of various organs (Xu et al., 2023). In 3D cultures, cells are better protected from stresses such as shear forces and unnatural pressures that can occur in 2D cultures. Therefore 3D cultures are suitable for long-term studies or therapies, due to enhanced cell survival and prolonged functionality in 3D culture. Furthermore, in 3D models, cells are seeded and grown onto 3D scaffolds to generate functional tissues, making the engineered tissue more viable for transplantation. However, 2D models lack spatial complexity, restricting their applications in cell therapy and tissue transplantation. By culturing dopaminergic neurons within 3D organoid models, we can study their interactions with other cell types, model complex neural circuits, and investigate disease mechanisms in a more holistic context (Figure 2).

While 3D models seem to be strictly better than current 2D models, there are still limitations to the use of 3D cell culture models. Due to the nature of 3D models, it is more difficult to supplement cells in the center of the organoid model with oxygen and nutrition, and limited interaction of cells inside organoid with cell culture media can result in the build-up and accumulation of toxic waste within the organoid. This can lead to impaired cell viability and degeneration of cultured cells in the middle of organoids, contributing to differences in phenotype and functions between cells on the surface and in the middle of organoids. In addition, while there are varying types of 3D scaffolds available (Murphy et al., 2020; Ranjan et al., 2020; Yan et al., 2023), these models can be cell-line dependent and have varying degrees of success depending on the cells used, and may need to be modified to achieve optimized results.

Actually, 2D culture models still have their place – many 2D models can accurately emulate in vivo environments, and their ease of use and easier modification, make them more attractive for experimentation and research (Duval et al., 2017), as well as being less stressful on cells, with lower rates of cell death, which can be important when working with cells that are scarce or limited (Laplaca et al., 2010). 2D cultures also offer a more standardized environment as compared to 3D models, which can be advantageous in terms of reproducibility and comparison between different studies. The manipulation of various parameters such as growth factors, nutrients, and substrate properties in 2D cultures is also much easier in 3D models, allowing for precise control over the cellular microenvironment.

Conclusions

The discovery of current stem cell techniques for deriving iPSCs from differentiated cells and further differentiation into distinct lineages of cells has opened new avenues of research, including in the field of neurobiology. Being able to derive neurons from patient cells to allow better disease model representation without limitations and ethically challenging as from embryos, the iPSCs techniques are more readily available for research, and allow researchers to generate dopaminergic neurons from PD patient cells. With many distinct protocols to induce the differentiation of iPSCs into dopaminergic neurons, a dopaminergic neuronal cell fate can be selected and verified for research and therapy. With patient-specific cell lines, the research for personalized medicine is also plausible, allowing the development of treatment to meet specific patient needs. Furthermore, besides hESCs and iPSC, other cell types, such as NSCs, MSCs, hADSCs, and human embryonic fibroblasts can also be differentiated into dopaminergic neurons.

Despite various limitations and challenges behind current iPSC techniques, the approach to generate dopaminergic neurons from iPSC holds immense promise, enabling research into previously limited cell lines and potential in PD research and clinical applications. Successful integrations and applications of the iPSC approach and organoid techniques may give us new insights to improve our understanding of PD pathogenesis and add to anti-PD therapy in the future. Overcoming challenges related to safety, efficacy, and immune compatibility will be vital to future clinical application of iPSC approaches for PD patients. With further advancements in culture methods and differentiation protocols in the future, iPSC techniques will likely play an even greater role in both research and clinical applications for PD.

Funding Statement

Funding: This work was supported by Singapore National Medical Research Council (NMRC) grants, including CS-IRG, HLCA2022 (to ZDZ), STaR, OF LCG 000207 (to EKT), a Clinical Translational Research Programme in Parkinson’s Disease, as well as Duke-Duke-NUS collaboration pilot grant (to ZDZ).

Data availability statement:

Not applicable.