Differentiation Of Dopaminergic Neurons Research Articles

Genome-Wide Association Study Reveals Genetic Architecture of Common Epilepsies.

Epilepsy, affecting approximately 50 million individuals worldwide, exhibits a genetic heritability of 32%. While several genes/loci associated with epilepsy have been identified through candidate and genome-wide association studies (GWAS), exploration of population-specific markers remains underexplored. We conducted the first GWAS in north Indian population (~1500 samples) to identify genetic variants/loci associated with epilepsy risk, validated using targeted next-generation sequencing (NGS). Our GWAS revealed 30 variants across seven loci associated with epilepsy risk, including six novel loci. Subtype analysis based on etiology and seizure types, identified 57 variants across 11 loci, 10 of which are novel. Gene-set analysis unveiled enrichment in genes associated with glutathione synthesis and recycling and regulation of dopaminergic neuron differentiation pivotal in epilepsy pathophysiology. Furthermore, PRS analysis revealed a significant genetic contribution to the epilepsy with an R2 of 0.00573. Additionally, targeted NGS showed ~95% concordance with GSA genotypes. Our study highlights six novel loci rs17031055/4q31.3(DCHS2), rs73182224/3q27.2(DGKG), rs9322462/6q25.2(CNKSR3), rs75328617/8q24.23(RNU1-35P), rs2938010/10q26.13(CTBP2) and rs11652575/17p11.2(SLC5A10) associated with epilepsy risk. These findings offer valuable insights into the genetic landscape of epilepsy in the north Indian population, providing foundation for future exploratory studies.

Establishment of a novel method for differentiating into dopaminergic neurons using charged hydrogels.

Parkinson's disease (PD) is a neurodegenerative disease primarily affecting the central nervous system and impacting both the motor system and non-motor systems. Although administration of L-DOPA is effective, it is not a fundamental treatment and has side effects such as diurnal fluctuation and dyskinesia, highlighting the need for new treatment methods. There is a growing interest in dopaminergic neuron transplantation as a potential treatment. Dopaminergic neurons derived from pluripotent stem (iPS) cells provide a valuable source for transplantation therapies. Developing an efficient method to differentiate iPS cells into dopaminergic cells is essential for cell transplantation therapy. While Cell differentiation is typically controlled by the addition of specific reagents, the physical characteristics of culture substrate, especially in the charge and stiffness, are also crucial factors in regulating differentiation. In this research, we show that two newly developed electrically charged polymeric hydrogels composed of cationic (C) and anionic (A) monomers inratio of 1-9 and 2 to 8 can significantly promote Dopaminergic neuron differentiation. Our findings emphasize the importance of culture substrates in effective dopaminergic cell differentiation.

Nanoelectrode-Mediated Extracellular Electrical Stimulation Directing Dopaminergic Neuronal Differentiation of Stem Cells for Improved Parkinson's Disease Therapy.

Parkinson's disease (PD) is a neurodegenerative disease caused by the dysfunction and death of dopaminergic neurons. Neural-stem-cell (NSC)-based therapy is a promising approach for the treatment of PD but its therapeutic performance is limited by low efficiency of differentiation of NSCs to dopaminergic neurons. Although electrical stimulation can promote neuronal differentiation, it is not verified whether it can induce the NSCs to specifically differentiate into dopaminergic neurons. Meanwhile, it is a great challenge to precisely apply electrical stimulation to dynamically migrating NSCs after transplantation. Here, electrochemically exfoliated graphene nanosheets are designed to anchor to the membrane of NSCs to serve as wireless nanoelectrodes. After anchoring to the cell membrane, these nanoelectrodes are able to migrate together with the cells and precisely apply extracellular electrical stimulation to the receptors or ion transport channels on the membrane of transplanted cells under alternating magnetic field. The nanoelectrode-mediated electrical stimulation induces 38.46% of the NSCs to specifically differentiate into dopaminergic neurons, while the percentage is only 5.82% for NSCs without the nanoelectrode stimulation. Transplantation of NSCs anchored with the nanoelectrodes effectively improves the recovery of the motor and memory ability of PD mice under alternating magnetic field within 2 weeks.

A review article on the development of dopaminergic neurons and establishment of dopaminergic neuron-based in vitro models by using immortal cell lines or stem cells to study and treat Parkinson's disease.

The primary pathological hallmark of Parkinson's disease (PD) is the degeneration of dopaminergic (DA) neurons in the substantia nigra pars compacta, a critical midbrain region. In vitro models based on DA neurons provide a powerful platform for investigating the cellular and molecular mechanisms of PD and testing novel therapeutic strategies. A deep understanding of DA neuron development, including the signalling pathways and transcription factors involved, is essential for advancing PD research. This article first explores the differentiation and maturation processes of DA neurons in the midbrain, detailing the relevant signalling pathways. It then compares various in vitro models, including primary cells, immortalized cell lines, and stem cell-based models, focusing on the advantages and limitations of each. Special attention is given to the role of immortalized and stem cell models in PD research. This review aims to guide researchers in selecting the most appropriate model for their specific research goals. Ethical considerations and clinical implications of using stem cells in PD research are also discussed.

Proteome Dynamics in iPSC-Derived Human Dopaminergic Neurons

Dopaminergic neurons participate in fundamental physiological processes and are the cell type primarily affected in Parkinson's disease. Their analysis is challenging due to the intricate nature of their function, involvement in diverse neurological processes, and heterogeneity and localization in deep brain regions. Consequently, most of the research on the protein dynamics of dopaminergic neurons has been performed in animal cells exvivo. Here we use iPSC-derived human mid-brain-specific dopaminergic neurons to study general features of their proteome biology and provide datasets for protein turnover and dynamics, including a human axonal translatome. We cover the proteome to a depth of 9409 proteins and use dynamic SILAC to measure the half-life of more than 4300 proteins. We report uniform turnover rates of conserved cytosolic protein complexes such as the proteasome and map the variable rates of turnover of the respiratory chain complexes in these cells. We use differential dynamic SILAC labeling in combination with microfluidic devices to analyze local protein synthesis and transport between axons and soma. We report 105 potentially novel axonal markers and detect translocation of 269 proteins between axons and the soma in the time frame of our analysis (120h). Importantly, we provide evidence for local synthesis of 154 proteins in the axon and their retrograde transport to the soma, among them several proteins involved in RNA editing such as ADAR1 and the RNA helicase DHX30, involved in the assembly of mitochondrial ribosomes. Our study provides a workflow and resource for the future applications of quantitative proteomics in iPSC-derived human neurons.

State of the Art in Sub-Phenotyping Midbrain Dopamine Neurons.

Dopaminergic neurons in the ventral tegmental area (VTA) and the substantia nigra pars compacta (SNpc) comprise around 75% of all dopaminergic neurons in the human brain. While both groups of dopaminergic neurons are in close proximity in the midbrain and partially overlap, development, function, and impairments in these two classes of neurons are highly diverse. The molecular and cellular mechanisms underlying these differences are not yet fully understood, but research over the past decade has highlighted the need to differentiate between these two classes of dopaminergic neurons during their development and in the mature brain. This differentiation is crucial not only for understanding fundamental circuitry formation in the brain but also for developing therapies targeted to specific dopaminergic neuron classes without affecting others. In this review, we summarize the state of the art in our understanding of the differences between the dopaminergic neurons of the VTA and the SNpc, such as anatomy, structure, morphology, output and input, electrophysiology, development, and disorders, and discuss the current technologies and methods available for studying these two classes of dopaminergic neurons, highlighting their advantages, limitations, and the necessary improvements required to achieve more-precise therapeutic interventions.

Fibrin-Polycaprolactone Scaffolds for the Differentiation of Human Neural Progenitor Cells into Dopaminergic Neurons.

Parkinson's disease (PD), a progressive central nervous system disorder marked by involuntary movements, poses a significant challenge in neurodegenerative research due to the gradual degeneration of dopaminergic (DA) neurons. Early diagnosis and understanding of PD's pathogenesis could slow disease progression and improve patient management. In vitro modeling with DA neurons derived from human-induced pluripotent stem cell-derived neural progenitor cells (NPCs) offers a promising approach. These neurons can be cultured on electrospun (ES) nanofibrous polycaprolactone (PCL) scaffolds, but PCL's hydrophobic nature limits cell adhesion. We investigated the ability of ES PCL scaffolds coated with hydrophilic extracellular matrix-based biomaterials, including cell basement membrane proteins, Matrigel, and Fibrin, to enhance NPC differentiation into DA neurons. We hypothesized that fibrin-coated scaffolds would maximize differentiation based on fibrin's known benefits in neuronal tissue engineering. The scaffolds both coated and uncoated were characterized using scanning electron microscopy (SEM), transmission electron microscopy, Fourier transform infrared spectroscopy-attenuated total reflectance, and dynamic mechanical analysis to assess their properties. NPCs were seeded on the coated scaffolds, differentiated, and matured into DA neurons. Immunocytochemistry targeting tyrosine hydroxylase (TH) and SEM confirmed DA neuronal differentiation and morphological changes. Electrophysiology via microelectrode array recorded their neuronal firing. Results showed enhanced neurite extension, increased TH expression, and active electrical activity in cells on fibrin-coated scaffolds. Diluted fibrin coatings particularly promoted more pronounced neuronal differentiation and maturation. This study introduces a novel tissue-on-a-chip platform for neurodegenerative disease research using DA neurons.

Identification of Potential Drug Targets for Myopia Through Mendelian Randomization.

The purpose of this study was to identify potential drug targets for myopia and explore underlying mechanisms. Mendelian randomization (MR) was implemented to assess the effect of 2684 pharmacologically targetable genes in the blood and retina on the risk of myopia from a genomewide association study (GWAS) for age-at-onset of spectacle wearing-inferred mean spherical equivalent (MSE; discovery cohort, N = 287,448, European), which was further validated in a GWAS for autorefraction-measured MSE (replication cohort, N = 95,619, European). The reliability of the identified significant potential targets was strengthened by colocalization analysis. Additionally, enrichment analysis, protein-protein interaction network, and molecular docking were performed to explore the functional roles and the druggability of these targets. This systematic drug target identification has unveiled 6 putative genetically causal targets for myopia-CD34, CD55, Wnt3, LCAT, BTN3A1, and TSSK6-each backed by colocalization evidence in adult blood eQTL datasets. Functional analysis found that dopaminergic neuron differentiation, cell adhesion, Wnt signaling pathway, and plasma lipoprotein-associated pathways may be involved in myopia pathogenesis. Finally, drug prediction and molecular docking corroborated the pharmacological value of these targets with LCAT demonstrating the strongest binding affinity. Our study not only opens new avenues for the development of therapeutic interventions for myopia but may also help to understand the underlying mechanisms of myopia.

Developmental origins of Parkinson's disease risk: perinatal exposure to the organochlorine pesticide dieldrin leads to sex-specific DNA modifications in critical neurodevelopmental pathways in the mouse midbrain.

Epidemiological studies show that exposure to the organochlorine pesticide dieldrin is associated with an increased risk of Parkinson's disease (PD). Animal studies support a link between developmental dieldrin exposure and increased neuronal susceptibility in the α-synuclein preformed fibril and MPTP models in adult male C57BL/6 mice. In a previous study, we showed that developmental dieldrin exposure was associated with sex-specific changes in DNA modifications within genes related to dopaminergic neuron development and maintenance at 12 wk of age. Here, we used capture hybridization-sequencing with custom baits to interrogate DNA modifications across the entire genetic loci of the previously identified genes at multiple time points-birth, 6, 12, and 36 wk old. We identified largely sex-specific dieldrin-induced changes in DNA modifications at each time point that annotated to pathways important for neurodevelopment, potentially related to critical steps in early neurodevelopment, dopaminergic neuron differentiation, synaptogenesis, synaptic plasticity, and glial-neuron interactions. Despite large numbers of age-specific DNA modifications, longitudinal analysis identified a small number of differential modification of cytosines with dieldrin-induced deflection of epigenetic aging. The sex-specificity of these results adds to evidence that sex-specific responses to PD-related exposures may underly sex-specific differences in disease. Overall, these data support the idea that developmental dieldrin exposure leads to changes in epigenetic patterns that persist after the exposure period and disrupt critical neurodevelopmental pathways, thereby impacting risk of late-life diseases, including PD.

Human platelet lysate supports SH-SY5Y neuroblastoma cell proliferation and differentiation into a dopaminergic-like neuronal phenotype under xenogeneic-free culture conditions.

SH-SY5Y is a human neuroblastoma cell line that can be differentiated into several neuronal phenotypes, depending on culture conditions. For this reason, this cell line has been widely used as an in vitro model of neurodegenerative conditions, such as Parkinson's disease (PD). However, most studies published to date used fetal bovine serum (FBS) as culture medium supplement for SH-SY5Y cell differentiation. We report on the testing of human platelet lysate (hPL) as a culture medium supplement to support SH-SY5Y cell culture. Both standard hPL and a fibrinogen-depleted hPL (FD-hPL) formulation, which does not require the addition of anticoagulants to culture media, promoted an increase in SH-SY5Y cell proliferation in comparison to FBS, without compromising metabolic activity. SH-SY5Y cells cultured in hPL or FD-hPL also displayed a higher number of neurite extensions and stained positive for MAP2 and synaptophysin, in the absence of differentiation stimuli; reducing hPL or FD-hPL concentration to 1% v/v did not affect cell proliferation or metabolic activity. Furthermore, following treatment with retinoic acid (RA) and further stimulation with brain-derived neurotrophic factor (BDNF) and nerve growth factor beta (NGF-β), the percentage of SH-SY5Y cells stained positive for dopaminergic neuronal differentiation markers (tyrosine hydroxylase [TH] and Dopamine Transporter [DAT]) was higher in hPL or FD-hPL than in FBS, and gene expression of dopaminergic markers TH, DAT, and DR2 was also detected. Overall, the data herein presented supports the use of hPL to differentiate SH-SY5Y cells into a neuronal phenotype with dopaminergic features, and the adoption of FD-hPL as a fully xenogeneic free alternative to FBS to support the use of SH-SY5Y cells as a neurodegeneration model.

Chronic ultraviolet irradiation induces memory deficits via dysregulation of the dopamine pathway

The effects of ultraviolet (UV) radiation on brain function have previously been investigated; however, the specific neurotransmitter-mediated mechanisms responsible for UV radiation-induced neurobehavioral changes remain elusive. In this study, we aimed to explore the mechanisms underlying UV radiation-induced neurobehavioral changes. In a mouse model, we observed that UV irradiation of the skin induces deficits in hippocampal memory, synaptic plasticity, and adult neurogenesis, as well as increased dopamine levels in the skin, adrenal glands, and brain. Chronic UV exposure altered the expression of genes involved in dopaminergic neuron differentiation. Furthermore, chronic peripheral dopamine treatments resulted in memory deficits. Systemic administration of a dopamine D1/D5 receptor antagonist reversed changes in memory, synaptic plasticity, adult neurogenesis, and gene expression in UV-irradiated mice. Our findings provide converging evidence that chronic UV exposure alters dopamine levels in the central nervous system and peripheral organs, including the skin, which may underlie the observed neurobehavioral shifts, such as hippocampal memory deficits and impaired neurogenesis. This study underscores the importance of protection from UV exposure and introduces the potential of pharmacological approaches targeting dopamine receptors to counteract the adverse neurological impacts of UV exposure.

The caloric value of food intake structurally adjusts a neuronal mushroom body circuit mediating olfactory learning in Drosophila.

Associative learning enables the adaptive adjustment of behavioral decisions based on acquired, predicted outcomes. The valence of what is learned is influenced not only by the learned stimuli and their temporal relations, but also by prior experiences and internal states. In this study, we used the fruit fly Drosophila melanogaster to demonstrate that neuronal circuits involved in associative olfactory learning undergo restructuring during extended periods of low-caloric food intake. Specifically, we observed a decrease in the connections between specific dopaminergic neurons (DANs) and Kenyon cells at distinct compartments of the mushroom body. This structural synaptic plasticity was contingent upon the presence of allatostatin A receptors in specific DANs and could be mimicked optogenetically by expressing a light-activated adenylate cyclase in exactly these DANs. Importantly, we found that this rearrangement in synaptic connections influenced aversive, punishment-induced olfactory learning but did not impact appetitive, reward-based learning. Whether induced by prolonged low-caloric conditions or optogenetic manipulation of cAMP levels, this synaptic rearrangement resulted in a reduction of aversive associative learning. Consequently, the balance between positive and negative reinforcing signals shifted, diminishing the ability to learn to avoid odor cues signaling negative outcomes. These results exemplify how a neuronal circuit required for learning and memory undergoes structural plasticity dependent on prior experiences of the nutritional value of food.

MicroRNA-Mediated Suppression of Glial Cell Line-Derived Neurotrophic Factor Expression Is Modulated by a Schizophrenia-Associated Non-Coding Polymorphism.

Plasma levels of glial cell line-derived neurotrophic factor (GDNF), a pivotal regulator of differentiation and survival of dopaminergic neurons, are reportedly decreased in schizophrenia. To explore the involvement of GDNF in the pathogenesis of the disease, a case-control association analysis was performed between five non-coding single nucleotide polymorphisms (SNP) across the GDNF gene and schizophrenia. Of them, the 'G' allele of the rs11111 SNP located in the 3' untranslated region (3'-UTR) of the gene was found to associate with schizophrenia. In silico analysis revealed that the rs11111 'G' allele might create binding sites for three microRNA (miRNA) species. To explore the significance of this polymorphism, transient co-transfection assays were performed in human embryonic kidney 293T (HEK293T) cells with a luciferase reporter construct harboring either the 'A' or 'G' allele of the 3'-UTR of GDNF in combination with the hsa-miR-1185-1-3p pre-miRNA. It was demonstrated that in the presence of the rs11111 'G' (but not the 'A') allele, hsa-miR-1185-2-3p repressed luciferase activity in a dose-dependent manner. Deletion of the miRNA binding site or its substitution with the complementary sequence abrogated the modulatory effect. Our results imply that the rs11111 'G' allele occurring more frequently in patients with schizophrenia might downregulate GDNF expression in a miRNA-dependent fashion.

Cathepsin D inhibition during neuronal differentiation selectively affects individual proteins instead of overall protein turnover

Cathepsin D (CTSD) is a lysosomal aspartic protease and its inherited deficiency causes a severe pediatric neurodegenerative disease called neuronal ceroid lipofuscinosis (NCL) type 10. The lysosomal dysfunction in the affected patients leads to accumulation of undigested lysosomal cargo especially in none-dividing cells, such as neurons, resulting in death shortly after birth. To explore which proteins are mainly affected by the lysosomal dysfunction due to CTSD deficiency, Lund human mesencephalic (LUHMES) cells, capable of inducible dopaminergic neuronal differentiation, were treated with Pepstatin A. This inhibitor of “acidic” aspartic proteases caused accumulation of acidic intracellular vesicles in differentiating LUHMES cells. Pulse-chase experiments involving stable isotope labelling with amino acids in cell culture (SILAC) with subsequent mass-spectrometric protein identification and quantification were performed. By this approach, we studied the degradation and synthesis rates of 695 and 680 proteins during early and late neuronal LUHMES differentiation, respectively. Interestingly, lysosomal bulk proteolysis was not altered upon Pepstatin A treatment. Instead, the protease inhibitor selectively changed the turnover of individual proteins. Especially proteins belonging to the mitochondrial energy supply system were differentially degraded during early and late neuronal differentiation indicating a high energy demand as well as stress level in LUHMES cells treated with Pepstatin A.

Effects of Gestational Exercise on Nociception, BDNF, and Irisin Levels in an Animal Model of ADHD

Abnormal cognitive and sensorial properties have been reported in patients with psychiatric and neurodevelopmental conditions, such as attention deficit hyperactivity disorder (ADHD). ADHD patients exhibit impaired dopaminergic signaling and plasticity in brain areas related to cognitive and sensory processing. The spontaneous hypertensive rat (SHR), in comparison to the Wistar Kyoto rat (WKY), is the most used genetic animal model to study ADHD. Brain neurotrophic factor (BDNF), critical for midbrain and hippocampal dopaminergic neuron survival and differentiation, is reduced in both ADHD subjects and SHR. Physical exercise (e.g. swimming) promotes neuroplasticity and improves cognition by increasing BDNF and irisin. Here we investigate the effects of gestational swimming on sensorial and behavioral phenotypes, striatal dopaminergic parameters, and hippocampal FNDC5/irisin and BDNF levels observed in WKY and SHR. Gestational swimming improved nociception in SHR rats (p = 0.006) and increased hippocampal BDNF levels (p = 0.02) in a sex-dependent manner in adolescent offspring. Sex differences were observed in hippocampal FNDC5/irisin levels (p = 0.002), with females presenting lower levels than males. Our results contribute to the notion that swimming during pregnancy is a promising alternative to improve ADHD phenotypes in the offspring.

Multiomics analysis identifies novel facilitators of human dopaminergic neuron differentiation

Midbrain dopaminergic neurons (mDANs) control voluntary movement, cognition, and reward behavior under physiological conditions and are implicated in human diseases such as Parkinson’s disease (PD). Many transcription factors (TFs) controlling human mDAN differentiation during development have been described, but much of the regulatory landscape remains undefined. Using a tyrosine hydroxylase (TH) human iPSC reporter line, we here generate time series transcriptomic and epigenomic profiles of purified mDANs during differentiation. Integrative analysis predicts novel regulators of mDAN differentiation and super-enhancers are used to identify key TFs. We find LBX1, NHLH1 and NR2F1/2 to promote mDAN differentiation and show that overexpression of either LBX1 or NHLH1 can also improve mDAN specification. A more detailed investigation of TF targets reveals that NHLH1 promotes the induction of neuronal miR-124, LBX1 regulates cholesterol biosynthesis, and NR2F1/2 controls neuronal activity.

Neurog1 and Olig2 integrate patterning and neurogenesis signals in development of zebrafish dopaminergic and glutamatergic dual transmitter neurons

Dopaminergic neurons develop in distinct neural domains by integrating local patterning and neurogenesis signals. While the proneural proteins Neurog1 and Olig2 have been previously linked to development of dopaminergic neurons, their dependence on local prepatterning and specific contributions to dopaminergic neurogenesis are not well understood. Here, we show that both transcription factors are differentially required for the development of defined dopaminergic glutamatergic subpopulations in the zebrafish posterior tuberculum, which are homologous to A11 dopaminergic neurons in mammals. Both Olig2 and Neurog1 are expressed in otpa expressing progenitor cells and appear to act upstream of Otpa during dopaminergic neurogenesis. Our epistasis analysis confirmed that Neurog1 acts downstream of Notch signaling, while Olig2 acts downstream of Shh, but upstream and/or in parallel to Notch signaling during neurogenesis of A11-type dopaminergic clusters. Furthermore, we identified Olig2 to be an upstream regulator of neurog1 in dopaminergic neurogenesis. This regulation occurs through Olig2-dependent repression of the proneural repressor and Notch target gene her2. Our study reveals how Neurog1 and Olig2 integrate local patterning signals, including Shh, with Notch neurogenic selection signaling, to specify the progenitor population and initiate neurogenesis and differentiation of A11-type dopaminergic neurons.

The proneural factors Ascl1a and Ascl1b contribute to the terminal differentiation of dopaminergic GABAergic dual transmitter neurons in zebrafish

The proneural factor Ascl1 is involved in several steps of neurogenesis, from neural progenitor maintenance to initiation of terminal differentiation and neuronal subtype specification. In neural progenitor cells, Ascl1 initiates the cell-cycle exit of progenitors, and contributes to their differentiation into mainly GABAergic neurons. Several catecholaminergic neuron groups in the forebrain of zebrafish use GABA as co-transmitter, but a potential role of the two paralogues Ascl1a and Ascl1b in their neurogenesis is not understood. Here, we show that ascl1a, ascl1b double mutant embryos develop a significantly reduced number of neurons in all GABAergic and catecholaminergic dual transmitter neuron anatomical clusters in the fore- and hindbrain, while glutamatergic catecholaminergic clusters develop normally. However, none of the affected catecholaminergic cell clusters are lost completely, suggesting an impairment in progenitor pools, or a requirement of Ascl1a/b for differentiation of a subset of neurons in each cluster. Early progenitors which are dlx2a+, fezf2 + or emx2 + are not reduced whereas late progenitors and differentiating neurons marked by the expression of dlx5a, isl1 and arxa are severely reduced in ascl1a, ascl1b double mutant embryos. This suggests that Ascl1a and Ascl1b play only a minor or no role in the maintenance of their progenitor pools, but rather contribute to the initiation of terminal differentiation of GABAergic catecholaminergic neurons.

Expression of Major Histocompatibility Complex during Neuronal Differentiation of Somatic Cell Nuclear Transfer-Human Embryonic Stem Cells.

Human pluripotent stem cells (hPSCs) such as human embryonic stem cells (hESCs), induced pluripotent stem cells, and somatic cell nuclear transfer (SCNT)-hESCs can permanently self-renew while maintaining their capacity to differentiate into any type of somatic cells, thereby serving as an important cell source for cell therapy. However, there are persistent challenges in the application of hPSCs in clinical trials, where one of the most significant is graft rejection by the patient immune system in response to human leukocyte antigen (HLA) mismatch when transplants are obtained from an allogeneic (non-self) cell source. Homozygous SCNT-hESCs (homo-SCNT-hESCs) were used to simplify the clinical application and to reduce HLA mismatch. Here, we present a xeno-free protocol that confirms the efficient generation of neural precursor cells in hPSCs and also the differentiation of dopaminergic neurons. Additionally, there was no difference when comparing the HLA expression patterns of hESC, homo-SCNT-hESCs and hetero-SCNT-hESCs. We propose that there are no differences in the differentiation capacity and HLA expression among hPSCs that can be cultured in vitro. Thus, it is expected that homo-SCNT-hESCs will possess a wider range of applications when transplanted with neural precursor cells in the context of clinical trials.

A Modified Differentiation Protocol In Vitro to Generate Dopaminergic Neurons from Pluripotent Stem Cells

Cell transplantation is considered a promising therapeutic strategy for the treatment of Parkinson's disease. Because of their strong differentiation potential, pluripotent stem cells may become a source of dopaminergic neurons for cell transplantation. Although published protocols have revealed that pluripotent stem cells can be successfully induced into dopaminergic neurons, unwanted cell types still exist in PSC-derived cultures. Therefore, signaling parameters for dopaminergic neuron patterning in differentiation protocols need to be further identified and optimized. In this study, we explored an In Vitro modified differentiation protocol for efficiently inducing dopaminergic neurons from pluripotent stem cells. Briefly, pluripotent stem cells were incubated in N2B27 medium for a 4-day culture, and then bFGF, SHH-C24II, purmorphamine, FGF8a and laminin were added to the medium. After a 6-day culture, the medium was replaced with N2B27 medium containing L-ascorbic acid, glial cell line-derived neurotrophic factor, cyclic adenosine monophosphate, laminin, and brain-derived neurotrophic factor for an additional 10 days. We confirmed that combined treatment with bFGF, SHH-C24II, purmorphamine, FGF8a and laminin significantly promoted the differentiation of pluripotent stem cells into dopaminergic neurons. Additionally, we determined a reasonable time window for the use of these factors. Our study provides new insights into the role of cell factors in dopaminergic neuron differentiation of pluripotent stem cells.