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Project description:Human pluripotent stem cells show considerable promise for applications in regenerative medicine, including the development of cell replacement paradigms for the treatment of Parkinson's disease. Protocols have been developed to generate authentic midbrain dopamine (mDA) neurons capable of reversing dopamine-related deficits in animal models of Parkinson's disease. However, the generation of mDA neurons at clinical scale suitable for human application remains an important challenge. Here, we present an mDA neuron derivation protocol based on a two-step WNT signaling activation strategy that improves expression of midbrain markers, such as Engrailed-1 (EN1), while minimizing expression of contaminating posterior (hindbrain) and anterior (diencephalic) lineage markers. The resulting neurons exhibit molecular, biochemical, and electrophysiological properties of mDA neurons. Cryopreserved mDA neuron precursors can be successfully transplanted into 6-hydroxydopamine (6OHDA) lesioned rats to induce recovery of amphetamine-induced rotation behavior. The protocol presented here is the basis for clinical-grade mDA neuron production and preclinical safety and efficacy studies.
| S-EPMC8006469 | biostudies-literature
Project description:We report a novel midbrain dopamine neuron derivation protocol from human pluripotent stem cells (hPSC)s based on a two-step WNT signaling activation strategy. By comparing next-generation sequencing (NGS) derived transcriptome profiling (RNA-seq) of dopamine neuron differentiated cells derived from hPSC with different concentration of Chir-boosting (day4 – day9) measured at day 11 including hPSC, we find that CHIR-boosting improves expression of midbrain markers such as EN1 while minimizing expression of contaminating posterior (hindbrain) and anterior (diencephalic) lineage markers. Furthermore, gene expression analysis from RNA-seq study confirmed the progressive differentiation and maturation of Boost CHIR treated precursors into mDA neurons by day 30 of differentiation. The protocol presented here is the basis for clinical grade dopamine neuron production and preclinical safety and efficacy studies.
2020-12-18 | GSE162883 | GEO
Project description:We report a novel midbrain dopamine neuron derivation protocol from human pluripotent stem cells (hPSC)s based on a two-step WNT signaling activation strategy. We perform genome-wide chromatin immunoprecipitation (ChIP-seq) for histone modifications including H3K4me3 and H3K27me3 with midbrain dopamine neuron differentiated cells from hPSC in Low-Chir and Boost-Chir (7.5 uM) conditions at day 11. Those results indicate that CHIR-boost promotes a more open chromatin structure at midbrain regions such as an EN1 locus based on increased H3K4me3 and decreased H3K27me3 levels. In contrast, analysis of the contaminating posterior (hindbrain) and anterior (diencephalic) lineage including NKX2-2 and PAX6 loci showed the opposite pattern with decreased H3K4me3 and increased H3K27me3 levels under CHIR-boost conditions. The protocol presented here is the basis for clinical grade dopamine neuron production and preclinical safety and efficacy studies.
2020-12-18 | GSE162882 | GEO
Project description:Biphasic activation of WNT signaling enhances the derivation of midbrain dopamine neurons for translational use.
| PRJNA683629 | ENA
Project description:Biphasic activation of WNT signaling enhances the derivation of midbrain dopamine neurons for translational use [ChIP-seq]
| PRJNA683632 | ENA
Project description:Biphasic activation of WNT signaling enhances the derivation of midbrain dopamine neurons for translational use [RNA-seq]
| PRJNA683633 | ENA
Project description:Human embryonic stem (hES) cells are defined by their extensive self-renewal capacity and their potential to differentiate into any cell type of the human body. The challenge in using hES cells for developmental biology and regenerative medicine has been to direct the wide differentiation potential toward the derivation of a specific cell fate. Within the nervous system, hES cells have been shown to differentiate in vitro into neural progenitor cells, neurons, and astrocytes. However, to our knowledge, the selective derivation of any given neuron subtype has not yet been demonstrated. Here, we describe conditions to direct hES cells into neurons of midbrain dopaminergic identity. Neuroectodermal differentiation was triggered on stromal feeder cells followed by regional specification by means of the sequential application of defined patterning molecules that direct in vivo midbrain development. Progression toward a midbrain dopamine (DA) neuron fate was monitored by the sequential expression of key transcription factors, including Pax2, Pax5, and engrailed-1 (En1), measurements of DA release, the presence of tetrodotoxin-sensitive action potentials, and the electron-microscopic visualization of tyrosinehydroxylase-positive synaptic terminals. High-yield DA neuron derivation was confirmed from three independent hES and two monkey embryonic stem cell lines. The availability of unlimited numbers of midbrain DA neurons is a first step toward exploring the potential of hES cells in preclinical models of Parkinson's disease. This experimental system also provides a powerful tool to probe the molecular mechanisms that control the development and function of human midbrain DA neurons.
| S-EPMC515094 | biostudies-literature
Project description:Inflammation is a characteristic feature of Parkinson's disease (PD). We examined the role of TLR9 and its regulation by glucocorticoid receptors (GRs) in degeneration of substantia nigra dopamine neurons (DNs). TLR9 agonist, CpG-ODN, induced DN degeneration in mice lacking GR in microglia but not in controls. TLR9 deletion reduced DN loss in neurotoxin, 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) mouse model of PD. GR regulates TLR9 activation during MPTP neurotoxicity as TLR9 antagonist suppressed increased DN loss in microglia/macrophage GR mutant mice. GR absence in microglia enhanced TLR9 translocation to endolysosomes and facilitated its cleavage leading to pro-inflammatory gene expression. GR-dependent TLR9 activation also triggered DN loss following intranigral injection of mitochondrial DNA. Finally, microglial GR sensitivity to A53T-alpha-synuclein induced DN degeneration as well as decreased microglial GR expression observed in SN of PD brain samples, all suggest that reduced microglial GR activity in SN can stimulate TLR9 activation and DN loss in PD pathology.
| S-EPMC6015079 | biostudies-literature
Project description:The neurobiological understanding of obsessive-compulsive disorder (OCD) includes dysregulated frontostriatal circuitry and altered monoamine transmission. Repetitive stereotyped behavior (e.g., grooming), a featured symptom in OCD, has been proposed to be associated with perturbed dopamine (DA) signaling. However, the precise brain circuits participating in DA's control over this behavioral phenotype remain elusive. Here, we identified that DA neurons in substantia nigra pars compacta (SNc) orchestrate ventromedial striatum (VMS) microcircuits as well as lateral orbitofrontal cortex (lOFC) during self-grooming behavior. SNc-VMS and SNc-lOFC dopaminergic projections modulate grooming behaviors and striatal microcircuit function differentially. Specifically, the activity of the SNc-VMS pathway promotes grooming via D1 receptors, whereas the activity of the SNc-lOFC pathway suppresses grooming via D2 receptors. SNc DA neuron activity thus controls the OCD-like behaviors via both striatal and cortical projections as dual gating. These results support both pharmacological and brain-stimulation treatments for OCD.
| S-EPMC9674233 | biostudies-literature
Project description:Midbrain dopamine neurons are known to encode reward prediction errors (RPE) used to update value predictions. Here, we examine whether RPE signals coded by midbrain dopamine neurons are modulated by the cost paid to obtain rewards, by recording from dopamine neurons in awake behaving monkeys during performance of an effortful saccade task. Dopamine neuron responses to cues predicting reward and to the delivery of rewards were increased after the performance of a costly action compared to a less costly action, suggesting that RPEs are enhanced following the performance of a costly action. At the behavioral level, stimulus-reward associations are learned faster after performing a costly action compared to a less costly action. Thus, information about action cost is processed in the dopamine reward system in a manner that amplifies the following dopamine RPE signal, which in turn promotes more rapid learning under situations of high cost.
| S-EPMC6695452 | biostudies-literature