Multi-omics integration of scRNA-seq time series data predicts new intervention points for Parkinson's disease.

Parkinson's disease (PD) is a complex neurodegenerative disorder without a cure. The onset of PD symptoms corresponds to 50% loss of midbrain dopaminergic (mDA) neurons, limiting early-stage understanding of PD. To shed light on early PD development, we study time series scRNA-seq datasets of mDA neurons obtained from patient-derived induced pluripotent stem cell differentiation. We develop a new data integration method based on Non-negative Matrix Tri-Factorization that integrates these datasets with molecular interaction networks, producing condition-specific "gene embeddings". By mining these embeddings, we predict 193 PD-related genes that are largely supported (49.7%) in the literature and are specific to the investigated PINK1 mutation. Enrichment analysis in Kyoto Encyclopedia of Genes and Genomes pathways highlights 10 PD-related molecular mechanisms perturbed during early PD development. Finally, investigating the top 20 prioritized genes reveals 12 previously unrecognized genes associated with PD that represent interesting drug targets.

Sex differences in neuronal activation in the cortex and midbrain during quinine-adulterated alcohol intake.

AIMS: Continued alcohol consumption despite negative consequences is a core symptom of alcohol use disorder. This is modeled in mice by pairing negative stimuli with alcohol, such as adulterating alcohol solution with quinine. Mice consuming alcohol under these conditions are considered to be engaging in aversion-resistant intake. Previously, we have observed sex differences in this behavior, with females more readily expressing aversion-resistant consumption. We also identified three brain regions that exhibited sex differences in neuronal activation during quinine-alcohol drinking: ventromedial prefrontal cortex (vmPFC), posterior insular cortex (PIC), and ventral tegmental area (VTA). Specifically, male mice showed increased activation in vmPFC and PIC, while females exhibited increased activation in VTA. In this study, we aimed to identify what specific type of neurons are activated in these regions during quinine-alcohol drinking. METHOD: We assessed quinine-adulterated alcohol intake using the two-bottle choice procedure. We also utilized RNAscope in situ hybridization in the three brain regions that previously exhibited a sex difference to examine colocalization of Fos, glutamate, GABA, and dopamine. RESULT: Females showed increased aversion-resistant alcohol consumption compared to males. We also found that males had higher colocalization of glutamate and Fos in vmPFC and PIC, while females had greater dopamine and Fos colocalization in the VTA. CONCLUSIONS: Collectively, these experiments suggest that glutamatergic output from the vmPFC and PIC may have a role in suppressing, and dopaminergic activity in the VTA may promote, aversion-resistant alcohol consumption. Future experiments will examine neuronal circuits that contribute to sex differences in aversion resistant consumption.

Delayed levodopa-responsive parkinsonism following acute midbrain injury.

Acute midbrain injury may cause both hyperkinetic movement disorders and parkinsonism. The temporal interval between the insult and the emergence of hyperkinetic disorders can last years. A delayed appearance of parkinsonism, on the other hand, was rarely described. We present three cases of male patients (50-, 58- and 28-year-old) who developed levodopa-responsive parkinsonism 20, 8 and two years, respectively, after acute brain insult involving the midbrain. Insults included subcortical intracerebral hemorrhage dissecting into the midbrain, embolic basilar occlusion and trauma. A fluorodopa scan, performed in two cases, revealed reduced striatal uptake. All individuals improved on low doses of levodopa and developed motor fluctuations shortly after levodopa was introduced. We conclude that delayed, levodopa-responsive parkinsonism following midbrain injury should be recognized in the relevant clinical setup. Possible mechanisms include age-related loss of dopaminergic neurons superimposed on acute injury and secondary neurodegeneration.

Loss of Midbrain Dopamine Neurons Does Not Alter GABAergic Inhibition Mediated by Parvalbumin-Expressing Interneurons in Mouse Primary Motor Cortex.

The primary motor cortex (M1) integrates sensory and cognitive inputs to generate voluntary movement. Its functional impairments have been implicated in the pathophysiology of motor symptoms in Parkinson's disease (PD). Specifically, dopaminergic degeneration and basal ganglia dysfunction entrain M1 neurons into the abnormally synchronized bursting pattern of activity throughout the cortico-basal ganglia-thalamocortical network. However, how degeneration of the midbrain dopaminergic neurons affects the anatomy, microcircuit connectivity, and function of the M1 network remains poorly understood. The present study examined whether and how the loss of dopamine (DA) affects the morphology, cellular excitability, and synaptic physiology of Layer 5 parvalbumin-expressing (PV+) cells in the M1 of mice of both sexes. Here, we reported that loss of midbrain dopaminergic neurons does not alter the number, morphology, and physiology of Layer 5 PV+ cells in M1. Moreover, we demonstrated that the number of perisomatic PV+ puncta of M1 pyramidal neurons as well as their functional innervation of cortical pyramidal neurons were not altered following the loss of DA. Together, the present study documents an intact GABAergic inhibitory network formed by PV+ cells following the loss of midbrain dopaminergic neurons.

Midbrain organoids for Parkinson's disease (PD) - A powerful tool to understand the disease pathogenesis.

Brain Organiods (BOs) are a promising technique for researching disease progression in the human brain. These organoids, which are produced from human induced pluripotent stem cells (HiPSCs), can construct themselves into structured frameworks. In the context of Parkinson's disease (PD), recent advancements have been made in the development of Midbrain organoids (MBOs) models that consider key pathophysiological mechanisms such as alpha-synuclein (α-Syn), Lewy bodies, dopamine loss, and microglia activation. However, there are limitations to the current use of BOs in disease modelling and drug discovery, such as the lack of vascularization, long-term differentiation, and absence of glial cells. To address these limitations, researchers have proposed the use of spinning bioreactors to improve oxygen and nutrient perfusion. Modelling PD utilising modern experimental in vitro models is a valuable tool for studying disease mechanisms and elucidating previously unknown features of PD. In this paper, we exclusively review the unique methods available for cultivating MBOs using a pumping system that mimics the circulatory system. This mechanism may aid in delivering the required amount of oxygen and nutrients to all areas of the organoids, preventing cell death, and allowing for long-term culture and using co-culturing techniques for developing glial cell in BOs. Furthermore, we emphasise some of the significant discoveries about the BOs and the potential challenges of using BOs will be discussed.

Distributional coding of associative learning in discrete populations of midbrain dopamine neurons.

Midbrain dopamine neurons are thought to play key roles in learning by conveying the difference between expected and actual outcomes. Recent evidence suggests diversity in dopamine signaling, yet it remains poorly understood how heterogeneous signals might be organized to facilitate the role of downstream circuits mediating distinct aspects of behavior. Here, we investigated the organizational logic of dopaminergic signaling by recording and labeling individual midbrain dopamine neurons during associative behavior. Our findings show that reward information and behavioral parameters are not only heterogeneously encoded but also differentially distributed across populations of dopamine neurons. Retrograde tracing and fiber photometry suggest that populations of dopamine neurons projecting to different striatal regions convey distinct signals. These data, supported by computational modeling, indicate that such distributional coding can maximize dynamic range and tailor dopamine signals to facilitate specialized roles of different striatal regions.

Genetic and pharmacological reduction of CDK14 mitigates synucleinopathy.

Parkinson's disease (PD) is a debilitating neurodegenerative disease characterized by the loss of midbrain dopaminergic neurons (DaNs) and the abnormal accumulation of α-Synuclein (α-Syn) protein. Currently, no treatment can slow nor halt the progression of PD. Multiplications and mutations of the α-Syn gene (SNCA) cause PD-associated syndromes and animal models that overexpress α-Syn replicate several features of PD. Decreasing total α-Syn levels, therefore, is an attractive approach to slow down neurodegeneration in patients with synucleinopathy. We previously performed a genetic screen for modifiers of α-Syn levels and identified CDK14, a kinase of largely unknown function as a regulator of α-Syn. To test the potential therapeutic effects of CDK14 reduction in PD, we ablated Cdk14 in the α-Syn preformed fibrils (PFF)-induced PD mouse model. We found that loss of Cdk14 mitigates the grip strength deficit of PFF-treated mice and ameliorates PFF-induced cortical α-Syn pathology, indicated by reduced numbers of pS129 α-Syn-containing cells. In primary neurons, we found that Cdk14 depletion protects against the propagation of toxic α-Syn species. We further validated these findings on pS129 α-Syn levels in PD patient neurons. Finally, we leveraged the recent discovery of a covalent inhibitor of CDK14 to determine whether this target is pharmacologically tractable in vitro and in vivo. We found that CDK14 inhibition decreases total and pathologically aggregated α-Syn in human neurons, in PFF-challenged rat neurons and in the brains of α-Syn-humanized mice. In summary, we suggest that CDK14 represents a novel therapeutic target for PD-associated synucleinopathy.

Label-free, whole-brain in vivo mapping in an adult vertebrate with third harmonic generation microscopy.

Comprehensive understanding of interconnected networks within the brain requires access to high resolution information within large field of views and over time. Currently, methods that enable mapping structural changes of the entire brain in vivo are extremely limited. Third harmonic generation (THG) can resolve myelinated structures, blood vessels, and cell bodies throughout the brain without the need for any exogenous labeling. Together with deep penetration of long wavelengths, this enables in vivo brain-mapping of large fractions of the brain in small animals and over time. Here, we demonstrate that THG microscopy allows non-invasive label-free mapping of the entire brain of an adult vertebrate, Danionella dracula, which is a miniature species of cyprinid fish. We show this capability in multiple brain regions and in particular the identification of major commissural fiber bundles in the midbrain and the hindbrain. These features provide readily discernable landmarks for navigation and identification of regional-specific neuronal groups and even single neurons during in vivo experiments. We further show how this label-free technique can easily be coupled with fluorescence microscopy and used as a comparative tool for studies of other species with similar body features to Danionella, such as zebrafish (Danio rerio) and tetras (Trochilocharax ornatus). This new evidence, building on previous studies, demonstrates how small size and relative transparency, combined with the unique capabilities of THG microscopy, can enable label-free access to the entire adult vertebrate brain.

Decoding Typical Flight States Based on Neural Signals from the Midbrain Motor Nuclei of Pigeons.

BACKGROUND: Exploring the neural encoding mechanism and decoding of motion state switching during flight can advance our knowledge of avian behavior control and contribute to the development of avian robots. However, limited acquisition equipment and neural signal quality have posed challenges, thus we understand little about the neural mechanisms of avian flight. METHODS: We used chronically implanted micro-electrode arrays to record the local field potentials (LFPs) in the formation reticularis medialis mesencephali (FRM) of pigeons during various motion states in their natural outdoor flight. Subsequently, coherence-based functional connectivity networks under different bands were constructed and the topological features were extracted. Finally, we used a support vector machine model to decode different flight states. RESULTS: Our findings indicate that the gamma band (80-150 Hz) in the FRM exhibits significant power for identifying different states in pigeons. Specifically, the avian brain transmitted flight related information more efficiently during the accelerated take-off or decelerated landing states, compared with the uniform flight and baseline states. Finally, we achieved a best average accuracy of 0.86 using the connectivity features in the 80-150 Hz band and 0.89 using the fused features for state decoding. CONCLUSIONS: Our results open up possibilities for further research into the neural mechanism of avian flight and contribute to the understanding of flight behavior control in birds.

Effects of electroacupuncture on Sirt3/NLRP3/GSDMD signaling pathway in the substantia nigra of midbrain of rats with Parkinson's disease. / ç”µé’ˆå¯¹å¸•é‡‘æ£®ç— å¤§é¼ ä¸­è„‘é»‘è´¨Sirt3/NLRP3/GSDMD信号通路的影响.

OBJECTIVES: To observe the effects on tyrosine hydroxylase (TH), α-synaptic nucleoprotein (α-syn), sirtuin 3 (Sirt3), NOD-like receptor 3 (NLRP3) and gasdermin-D (GSDMD) in the substantia nigra of midbrain after electroacupuncture (EA) at "Fengfu"(GV16), "Taichong" (LR3) and "Zusanli" (ST36) in rats of Parkinson's disease (PD), so as to explore the mechanism of EA in treatment of PD. METHODS: SD rats were randomly divided into control, model and EA groups, with 10 rats in each group. The PD model was established by injecting rotenone into the neck and back, lasting 28 days. In the EA group, EA was applied to GV16, LR3 and ST36, 30 min each time, once daily, consecutively for 28 days. The open-field test was adopted to detect the total distance of autonomic movement of rats, and the pole climbing test was used to detect the body coordination ability of rats. In the substania nigra of midbrain, the positive expression of TH was determined using immunohistochemistry, the mRNA expression levels of α - syn, Sirt3, NLRP3 and GSDMD were detected by quantitative real-time fluorescence PCR, and the protein expression levels of NLRP3, apoptosis-associated speck-like protein containing a caspase-recruitment domain (ASC) and cysteinyl aspartate specific proteinase (Caspase)-1 were detected by Western blot. RESULTS: Compared with the control group, the total distance of autonomous movement was decreased (P<0.01) in the model group, and the score of pole climbing experiment was increased (P<0.01)；in the midbrain substantia nigra the positive expression of TH was decreased (P<0.01)；the mRNA expression level of Sirt3 was decreased (P<0.01), and those of α-syn, NLRP3 and GSDMD were increased (P<0.01)；while the protein expression levels of NLRP3, ASC and Caspase-1 were increased (P<0.01). When compared with the model group, the total distance of autonomous movement in open field experiment was increased (P<0.01) in the EA group and the score of pole climbing experiment was lower (P<0.05)；in the midbrain substantia nigra the positive expression of TH was increased (P<0.01)；the mRNA expression level of Sirt3 in the midbrain substantia nigra was increased (P<0.01), and those of α-syn, NLRP3 and GSDMD were reduced (P<0.01)；while the protein expression levels of NLRP3, ASC and Caspase-1 decreased (P<0.01, P<0.05). CONCLUSIONS: EA at "GV16" "LR3" and "ST36" can repair the neuronal injury, clear the abnormal accumulation of α-syn in the substania nigra of midbrain, and ameliorate mitochondrial damage in PD rats, which may be obtained by regulating Sirt3/NLRP3/GSDMD signaling pathway, so as to delay the occurrence and development of Parkinson's disease.

Transcriptomic atlas of midbrain dopamine neurons uncovers differential vulnerability in a Parkinsonism lesion model.

Midbrain dopamine (mDA) neurons comprise diverse cells with unique innervation targets and functions. This is illustrated by the selective sensitivity of mDA neurons of the substantia nigra compacta (SNc) in patients with Parkinson's disease, while those in the ventral tegmental area (VTA) are relatively spared. Here, we used single nuclei RNA sequencing (snRNA-seq) of approximately 70,000 mouse midbrain cells to build a high-resolution atlas of mouse mDA neuron diversity at the molecular level. The results showed that differences between mDA neuron groups could best be understood as a continuum without sharp differences between subtypes. Thus, we assigned mDA neurons to several 'territories' and 'neighborhoods' within a shifting gene expression landscape where boundaries are gradual rather than discrete. Based on the enriched gene expression patterns of these territories and neighborhoods, we were able to localize them in the adult mouse midbrain. Moreover, because the underlying mechanisms for the variable sensitivities of diverse mDA neurons to pathological insults are not well understood, we analyzed surviving neurons after partial 6-hydroxydopamine (6-OHDA) lesions to unravel gene expression patterns that correlate with mDA neuron vulnerability and resilience. Together, this atlas provides a basis for further studies on the neurophysiological role of mDA neurons in health and disease.

Wasp sting-induced bilateral thalamic and midbrain infarction.

A male patient in his early 40s presented to the emergency department with an acute onset of respiratory distress and facial oedema, indicative of anaphylaxis. These symptoms emerged 2 hours subsequent to a wasp sting on the left side of his face. Despite initial stabilisation, the patient's state deteriorated into somnolence and disorientation. Notably, he denied any history of seizures, sensory or motor deficits, or bowel/bladder complications. Physical examination unveiled no focal neurological deficits. Routine laboratory tests and drug screening yielded no significant findings. Subsequent brain MRI with angiography exposed bilateral thalami diffusion restriction, strongly implying an acute infarction within the artery of Percheron territory, an atypical vascular variant. The sequence of events, alongside the absence of other conclusive aetiologies, indicated a wasp sting-induced thalamic infarction driven by vasogenic and thrombogenic effects of inflammatory substances.

Optical Intracranial Self-Stimulation (oICSS): A New Behavioral Model for Studying Drug Reward and Aversion in Rodents.

Brain-stimulation reward, also known as intracranial self-stimulation (ICSS), is a commonly used procedure for studying brain reward function and drug reward. In electrical ICSS (eICSS), an electrode is surgically implanted into the medial forebrain bundle (MFB) in the lateral hypothalamus or the ventral tegmental area (VTA) in the midbrain. Operant lever responding leads to the delivery of electrical pulse stimulation. The alteration in the stimulation frequency-lever response curve is used to evaluate the impact of pharmacological agents on brain reward function. If a test drug induces a leftward or upward shift in the eICSS response curve, it implies a reward-enhancing or abuse-like effect. Conversely, if a drug causes a rightward or downward shift in the functional response curve, it suggests a reward-attenuating or aversive effect. A significant drawback of eICSS is the lack of cellular selectivity in understanding the neural substrates underlying this behavior. Excitingly, recent advancements in optical ICSS (oICSS) have facilitated the development of at least three cell type-specific oICSS models-dopamine-, glutamate-, and GABA-dependent oICSS. In these new models, a comparable stimulation frequency-lever response curve has been established and employed to study the substrate-specific mechanisms underlying brain reward function and a drug's rewarding versus aversive effects. In this review article, we summarize recent progress in this exciting research area. The findings in oICSS have not only increased our understanding of the neural mechanisms underlying drug reward and addiction but have also introduced a novel behavioral model in preclinical medication development for treating substance use disorders.

A novel method of neurophysiological brainstem mapping in neurosurgery.

BACKGROUND: Brainstem mapping with electrical stimulation allows functional identification of neural structures during resection of deep lesions. Single pulses or train of pulses are delivered to map cranial nerves and corticospinal tracts, respectively. NEW METHOD: We introduce a hybrid stimulation technique for mapping the brainstem. The stimulus consists of an electrical single pulse followed by a short train of 3-5 pulses at 500 Hz, at an interval of 60-75 ms. The responses to this stimulation pattern are recorded from appropriate cranial and limb muscles. RESULTS: Both the single pulse and the short train elicit electromyographic responses when motor fibers or motor nuclei of the cranial nerves are stimulated. Responses to the train but not to the preceding single pulse indicate activation of the descending motor tracts, in the mesencephalon and the pons. Conversely, in the medulla, limb responses to stimulation of the corticospinal tracts are elicited by a single pulse. Identification of the extra and intra-axial courses of the trigeminal motor and sensory fibers is possible by recording responses from the masseter and the tongue muscles. COMPARISON WITH EXISTING METHOD(S): To date, either a pulse or a train is delivered during brainstem mapping, switching from one to the other modality according to the expected target structure. This procedure can be time-consuming and may even lead to false negative responses to the stimulation, eventually leading to inaccurate neurosurgical procedures. CONCLUSIONS: The novel hybrid pulse-train technique enhances the advantage of brainstem mapping procedure, minimizing pitfalls and improving patient safety.

The Mouse Inferior Colliculus Responds Preferentially to Non-Ultrasonic Vocalizations.

The inferior colliculus (IC), the midbrain auditory integration center, analyzes information about social vocalizations and provides substrates for higher level processing of vocal signals. We used multichannel recordings to characterize and localize responses to social vocalizations and synthetic stimuli within the IC of female and male mice, both urethane anesthetized and unanesthetized. We compared responses to ultrasonic vocalizations (USVs) with other vocalizations in the mouse repertoire and related vocal responses to frequency tuning, IC subdivisions, and sex. Responses to lower frequency, broadband social vocalizations were widespread in IC, well represented throughout the tonotopic axis, across subdivisions, and in both sexes. Responses to USVs were much more limited. Although we observed some differences in tonal and vocal responses by sex and subdivision, representations of vocal responses by sex and subdivision were largely the same. For most units, responses to vocal signals occurred only when frequency response areas overlapped with spectra of the vocal signals. Since tuning to frequencies contained within the highest frequency USVs is limited (<15% of IC units), responses to these vocalizations are correspondingly limited (<5% of sound-responsive units). These results highlight a paradox of USV processing in some rodents: although USVs are the most abundant social vocalization, their representation and the representation of corresponding frequencies are less than lower frequency social vocalizations. We interpret this paradox in light of observations suggesting that USVs with lower frequency elements (<50 kHz) are associated with increased emotional intensity and engage a larger population of neurons in the mouse auditory system.

Development of Midbrain Dopaminergic Neurons and the Advantage of Using hiPSCs as a Model System to Study Parkinson's Disease.

Midbrain dopaminergic (mDA) neurons are significantly impaired in patients inflicted with Parkinson's disease (PD), subsequently affecting a variety of motor functions. There are four pathways through which dopamine elicits its function, namely, nigrostriatal, mesolimbic, mesocortical and tuberoinfundibular dopamine pathways. SHH and Wnt signalling pathways in association with favourable expression of a variety of genes, promotes the development and differentiation of mDA neurons in the brain. However, there is a knowledge gap regarding the complex signalling pathways involved in development of mDA neurons. hiPSC models have been acclaimed to be effective in generating complex disease phenotypes. These models mimic the microenvironment found in vivo thus ensuring maximum reliability. Further, a variety of therapeutic compounds can be screened using hiPSCs since they can be used to generate neurons that could carry an array of mutations associated with both familial and sporadic PD. Thus, culturing hiPSCs to study gene expression and dysregulation of cellular processes associated with PD can be useful in developing targeted therapies that will be a step towards halting disease progression.

A single-nuclei paired multiomic analysis of the human midbrain reveals age- and Parkinson's disease-associated glial changes.

Age is the primary risk factor for Parkinson's disease (PD), but how aging changes the expression and regulatory landscape of the brain remains unclear. Here we present a single-nuclei multiomic study profiling shared gene expression and chromatin accessibility of young, aged and PD postmortem midbrain samples. Combined multiomic analysis along a pseudopathogenesis trajectory reveals that all glial cell types are affected by age, but microglia and oligodendrocytes are further altered in PD. We present evidence for a disease-associated oligodendrocyte subtype and identify genes lost over the aging and disease process, including CARNS1, that may predispose healthy cells to develop a disease-associated phenotype. Surprisingly, we found that chromatin accessibility changed little over aging or PD within the same cell types. Peak-gene association patterns, however, are substantially altered during aging and PD, identifying cell-type-specific chromosomal loci that contain PD-associated single-nucleotide polymorphisms. Our study suggests a previously undescribed role for oligodendrocytes in aging and PD.

The people behind the papers - Andy Yang and Stephane Angers.

Activation of the Wnt signalling pathway is vital in the anterior-posterior patterning during neural development. In a new study, Stephane Angers and colleagues leverage previously developed selective antibodies against Frizzled receptors of the Wnt pathway to stimulate midbrain progenitor differentiation in human pluripotent stem cells. We caught up with first author Andy Yang and corresponding author Stephane Angers, Professor at the University of Toronto, to learn more about the story behind the paper.

A midbrain GABAergic circuit constrains wakefulness in a mouse model of stress.

Enhancement of wakefulness is a prerequisite for adaptive behaviors to cope with acute stress, but hyperarousal is associated with impaired behavioral performance. Although the neural circuitries promoting wakefulness in acute stress conditions have been extensively identified, less is known about the circuit mechanisms constraining wakefulness to prevent hyperarousal. Here, we found that chemogenetic or optogenetic activation of GAD2-positive GABAergic neurons in the midbrain dorsal raphe nucleus (DRNGAD2) decreased wakefulness, while inhibition or ablation of these neurons produced an increase in wakefulness along with hyperactivity. Surprisingly, DRNGAD2 neurons were paradoxically wakefulness-active and were further activated by acute stress. Bidirectional manipulations revealed that DRNGAD2 neurons constrained the increase of wakefulness and arousal level in a mouse model of stress. Circuit-specific investigations demonstrated that DRNGAD2 neurons constrained wakefulness via inhibition of the wakefulness-promoting paraventricular thalamus. Therefore, the present study identified a wakefulness-constraining role DRNGAD2 neurons in acute stress conditions.

[Rho kinase inhibitor Y27632 promotes survival of human induced pluripotent stem cells during differentiation into functional midbrain dopaminergic progenitor cells in vitro].

OBJECTIVE: To improve the efficiency of induced differentiation of primitive neural epithelial cells derived from human induced pluripotent stem cells (hiPSCs-NECs) into functional midbrain dopaminergic progenitor cells (DAPs). METHODS: HiPSCs were cultured in mTeSRTM medium containing DMH1 (10 µmol/L), SB431542 (10 µmol/L), SHH (200 ng/mL), FGF8 (100 ng/mL), purmorphamine (2 µmol/L), CHIR99021 (3 µmol/L), and N2 (1%) for 12 days to induce their differentiation into primitive neuroepithelial cells (NECs). The hiPSCs-NECs were digested with collagenase Ⅳ and then cultured in neurobasal medium supplemented with 1% N2, 2% B27-A, BDNF (10 ng/mL), GDNF (10 ng/mL), AA, TGF-ß, cAMP, and 1% GlutaMax in the presence of different concentrations of Rho kinase inhibitor Y27632, and the culture medium was changed the next day to remove Y27632. Continuous induction was performed until day 28 to obtain DAPs. RESULTS: Human iPSCs expressed the pluripotency markers OCT4, SOX2, Nanog, and SSEA1 and were positive for alkaline phosphatase staining. The hiPSCs-NECs were obtained on day 13 in the form of neural rosettes expressing neuroepithelial markers SOX2, nestin, and PAX6. In digested hiPSCs-NECs, the addition of 5 µmol/L Y27632 significantly promoted survival of the adherent cells, increased cell viability and the proportion of S-phase cells (P < 0.01), and reduced the rate of apoptotic cells (P < 0.05). On day 28 of induction, the obtained cells highly expressed the specific markers of DAPS (TH, FOXA2, NURR1, and Tuj1). CONCLUSION: Treatment with Y27632 (5 µmol/L) for 24 h significantly promotes the survival of human iPSCs-NECs during their differentiation into DPAs without affecting the cell differentiation, which indirectly enhances the efficiency of cell differentiation.