Generation of locus coeruleus norepinephrine neurons from human pluripotent stem cells.

Central norepinephrine (NE) neurons, located mainly in the locus coeruleus (LC), are implicated in diverse psychiatric and neurodegenerative diseases and are an emerging target for drug discovery. To facilitate their study, we developed a method to generate 40-60% human LC-NE neurons from human pluripotent stem cells. The approach depends on our identification of ACTIVIN A in regulating LC-NE transcription factors in dorsal rhombomere 1 (r1) progenitors. In vitro generated human LC-NE neurons display extensive axonal arborization; release and uptake NE; and exhibit pacemaker activity, calcium oscillation and chemoreceptor activity in response to CO2. Single-nucleus RNA sequencing (snRNA-seq) analysis at multiple timepoints confirmed NE cell identity and revealed the differentiation trajectory from hindbrain progenitors to NE neurons via an ASCL1-expressing precursor stage. LC-NE neurons engineered with an NE sensor reliably reported extracellular levels of NE. The availability of functional human LC-NE neurons enables investigation of their roles in psychiatric and neurodegenerative diseases and provides a tool for therapeutics development.

Generation of serotonin neurons from human pluripotent stem cells.

Serotonin neurons located in the raphe nucleus of the hindbrain have crucial roles in regulating brain functions and have been implicated in various psychiatric disorders. Yet functional human serotonin neurons are not available for in vitro studies. Through manipulation of the WNT pathway, we demonstrate efficient differentiation of human pluripotent stem cells (hPSCs) to cells resembling central serotonin neurons, primarily those located in the rhombomeric segments 2-3 of the rostral raphe, which participate in high-order brain functions. The serotonin neurons express a series of molecules essential for serotonergic development, including tryptophan hydroxylase 2, exhibit typical electrophysiological properties and release serotonin in an activity-dependent manner. When treated with the FDA-approved drugs tramadol and escitalopram oxalate, they release or uptake serotonin in a dose- and time-dependent manner, suggesting the utility of these cells for the evaluation of drug candidates.

Human-derived neural progenitors functionally replace astrocytes in adult mice.

Astrocytes are integral components of the homeostatic neural network as well as active participants in pathogenesis of and recovery from nearly all neurological conditions. Evolutionarily, compared with lower vertebrates and nonhuman primates, humans have an increased astrocyte-to-neuron ratio; however, a lack of effective models has hindered the study of the complex roles of human astrocytes in intact adult animals. Here, we demonstrated that after transplantation into the cervical spinal cords of adult mice with severe combined immunodeficiency (SCID), human pluripotent stem cell-derived (PSC-derived) neural progenitors migrate a long distance and differentiate to astrocytes that nearly replace their mouse counterparts over a 9-month period. The human PSC-derived astrocytes formed networks through their processes, encircled endogenous neurons, and extended end feet that wrapped around blood vessels without altering locomotion behaviors, suggesting structural, and potentially functional, integration into the adult mouse spinal cord. Furthermore, in SCID mice transplanted with neural progenitors derived from induced PSCs from patients with ALS, astrocytes were generated and distributed to a similar degree as that seen in mice transplanted with healthy progenitors; however, these mice exhibited motor deficit, highlighting functional integration of the human-derived astrocytes. Together, these results indicate that this chimeric animal model has potential for further investigating the roles of human astrocytes in disease pathogenesis and repair.

Modeling ALS with iPSCs reveals that mutant SOD1 misregulates neurofilament balance in motor neurons.

Amyotrophic lateral sclerosis (ALS) presents motoneuron (MN)-selective protein inclusions and axonal degeneration but the underlying mechanisms of such are unknown. Using induced pluripotent cells (iPSCs) from patients with mutation in the Cu/Zn superoxide dismutase (SOD1) gene, we show that spinal MNs, but rarely non-MNs, exhibited neurofilament (NF) aggregation followed by neurite degeneration when glia were not present. These changes were associated with decreased stability of NF-L mRNA and binding of its 3' UTR by mutant SOD1 and thus altered protein proportion of NF subunits. Such MN-selective changes were mimicked by expression of a single copy of the mutant SOD1 in human embryonic stem cells and were prevented by genetic correction of the SOD1 mutation in patient's iPSCs. Importantly, conditional expression of NF-L in the SOD1 iPSC-derived MNs corrected the NF subunit proportion, mitigating NF aggregation and neurite degeneration. Thus, NF misregulation underlies mutant SOD1-mediated NF aggregation and axonal degeneration in ALS MNs.

Medial ganglionic eminence-like cells derived from human embryonic stem cells correct learning and memory deficits.

Dysfunction of basal forebrain cholinergic neurons (BFCNs) and γ-aminobutyric acid (GABA) interneurons, derived from medial ganglionic eminence (MGE), is implicated in disorders of learning and memory. Here we present a method for differentiating human embryonic stem cells (hESCs) to a nearly uniform population of NKX2.1(+) MGE-like progenitor cells. After transplantation into the hippocampus of mice in which BFCNs and some GABA neurons in the medial septum had been destroyed by mu P75-saporin, human MGE-like progenitors, but not ventral spinal progenitors, produced BFCNs that synaptically connected with endogenous neurons, whereas both progenitors generated similar populations of GABA neurons. Mice transplanted with MGE-like but not spinal progenitors showed improvements in learning and memory deficits. These results suggest that progeny of the MGE-like progenitors, particularly BFCNs, contributed to learning and memory. Our findings support the prospect of using human stem cell-derived MGE-like progenitors in developing therapies for neurological disorders of learning and memory.

A new parametric feature descriptor for the classification of epileptic and control EEG records in pediatric population.

This study evaluates the sensitivity, specificity and accuracy in associating scalp EEG to either control or epileptic patients by means of artificial neural networks (ANNs) and support vector machines (SVMs). A confluence of frequency and temporal parameters are extracted from the EEG to serve as input features to well-configured ANN and SVM networks. Through these classification results, we thus can infer the occurrence of high-risk (epileptic) as well as low risk (control) patients for potential follow up procedures.

Sub-patterns of language network reorganization in pediatric localization related epilepsy: a multisite study.

To study the neural networks reorganization in pediatric epilepsy, a consortium of imaging centers was established to collect functional imaging data. Common paradigms and similar acquisition parameters were used. We studied 122 children (64 control and 58 LRE patients) across five sites using EPI BOLD fMRI and an auditory description decision task. After normalization to the MNI atlas, activation maps generated by FSL were separated into three sub-groups using a distance method in the principal component analysis (PCA)-based decisional space. Three activation patterns were identified: (1) the typical distributed network expected for task in left inferior frontal gyrus (Broca's) and along left superior temporal gyrus (Wernicke's) (60 controls, 35 patients); (2) a variant left dominant pattern with greater activation in IFG, mesial left frontal lobe, and right cerebellum (three controls, 15 patients); and (3) activation in the right counterparts of the first pattern in Broca's area (one control, eight patients). Patients were over represented in Groups 2 and 3 (P < 0.0004). There were no scanner (P = 0.4) or site effects (P = 0.6). Our data-driven method for fMRI activation pattern separation is independent of a priori notions and bias inherent in region of interest and visual analyses. In addition to the anticipated atypical right dominant activation pattern, a sub-pattern was identified that involved intensity and extent differences of activation within the distributed left hemisphere language processing network. These findings suggest a different, perhaps less efficient, cognitive strategy for LRE group to perform the task.

Subdural EEG classification into seizure and nonseizure files using neural networks in the gamma frequency band.

This study describes a new method for offline seizure detection using intracranial EEG (iEEG). The proposed method integrated two interrelated steps: (1) establishing a decisional space on the basis of the interelectrode mean of the spectral power in the gamma frequencies after a thorough evaluation of temporal and frequency-based features and (2) constructing an artificial neural network that operated on this decisional space to delineate EEG files that contained seizures from those that did not. The data were obtained from 14 patients who underwent two-stage epilepsy surgery with subdural recordings. Of the total 157 files considered, 35 (21 interictal and 14 ictal) iEEG data files or 22% were selected randomly and used initially in a training phase. The remaining 122 iEEG data files or 78% were then used in the testing phase to assess the merits in selecting gamma power as means to detect a seizure. The results obtained exhibited an accuracy of 95.90%, a sensitivity of 92.59%, and a specificity of 96.84%. Although this method had to contend with the complex nature of iEEG and the inherent heavy computational load, the constructed artificial neural networks together with the chosen decisional space yielded the best possible outcome. The proposed method was based on aggregating the power in the 36 to 44-Hz frequency range and analyzing its behavior in time, looking for patterns indicative of seizure evolution. It was shown that the power measurement in the gamma range contains the information needed to discriminate seizure files from nonseizure files. The algorithm consisted in establishing a decision space most suitable for iEEG data classification by relying on the power spectra in the gamma frequencies and constructing and implementing an artificial neural network that generates the highest classification accuracy possible. It was noted that although only 29% (35/122) of the files were used randomly for training the detector, high measures in sensitivity, specificity, and accuracy were still achieved in the remaining files, which were subsequently used in the testing phase. Seizures are known to occur intermittently and unpredictably, and massive amounts of EEG or iEEG data need to be analyzed offline to detect seizures. This is a challenge that can only be met through reliable and time-efficient seizure-detection paradigms, an affirmation this study attempted to prove.

Pax6 is a human neuroectoderm cell fate determinant.

The transcriptional regulation of neuroectoderm (NE) specification is unknown. Here we show that Pax6 is uniformly expressed in early NE cells of human fetuses and those differentiated from human embryonic stem cells (hESCs). This is in contrast to the later expression of Pax6 in restricted mouse brain regions. Knockdown of Pax6 blocks NE specification from hESCs. Overexpression of either Pax6a or Pax6b, but not Pax6triangle upPD, triggers hESC differentiation. However, only Pax6a converts hESCs to NE. In contrast, neither loss nor gain of function of Pax6 affects mouse NE specification. Both Pax6a and Pax6b bind to pluripotent gene promoters but only Pax6a binds to NE genes during human NE specification. These findings indicate that Pax6 is a transcriptional determinant of the human NE and suggest that Pax6a and Pax6b coordinate with each other in determining the transition from pluripotency to the NE fate in human by differentially targeting pluripotent and NE genes.

A comparative study of intracranial EEG files using nonlinear classification methods.

This study is a comparative evaluation of nonlinear classification methods with a focus on nonlinear decision functions and the standard method of support vector machines for seizure detection. These nonlinear classification methods are used on key features that were extracted on subdural EEG data after a thorough evaluation of all the frequency bands from 1 to 44 Hz. The sensitivity, specificity, and accuracy of seizure detection reveal that the gamma frequencies (36-44 Hz) are most suitable for detecting seizure files using a unique 2D decisional plane. We evaluated 157 intracranial EEG files from 14 patients by calculating the spectral power using nonoverlapping 1-s windows on different frequency bands. A key finding is in establishing a 2D decision plane, where duration of the seizure is used as the first dimension (x coordinate) and the maximum of the gamma frequency components is used as the second dimension (y coordinate). Within this 2D plane, the best results were observed when the nonlinearity degree is three for the proposed nonlinear decision functions, with a sensitivity of 96.3%, a specificity of 96.8%, and accuracy of 96.7%.

Classification of leukemia blood samples using neural networks.

Pattern recognition applied to blood samples for diagnosing leukemia remains an extremely difficult task which frequently leads to misclassification errors due in large part to the inherent problem of data overlap. A novel artificial neural network (ANN) algorithm is proposed for optimizing the classification of multidimensional data, focusing on acute leukemia samples. The programming tool established around the ANN architecture focuses on the classification of normal vs. abnormal blood samples, namely acute lymphocytic leukemia (ALL) and acute myeloid leukemia (AML). There were 220 blood samples considered with 60 abnormal samples and 160 normal samples. The algorithm produced very high sensitivity results that improved up to 96.67% in ALL classification with increased data set size. With this type of accuracy, this programming tool provides information to medical doctors in the form of diagnostic references for the specific disease states that are considered for this study. The results obtained prove that a neural network classifier can perform remarkably well for this type of flow-cytometry data. Even more significant is the fact that experimental evaluations in the testing phase reveal that as the ALL data considered is gradually increased from small to large data sets, the more accurate are the classification results.

Seizure detection: an assessment of time- and frequency-based features in a unified two-dimensional decisional space using nonlinear decision functions.

OBJECTIVE: This study proposes a new approach for offline seizure detection in intracranial (subdural) electroencephalogram recordings using nonlinear decision functions. It implements well-established features that are designed to deal with complex signals, such as brain recordings, and proposes a two-dimensional (2D) domain of analysis that overcomes the dilemma faced with the selection of empirical thresholds often used to delineate epileptic events. This unifying approach makes it possible for researchers in epilepsy to establish other performance evaluation criteria on the basis of the proposed nonlinear decision functions as well as introduce additional dimensions toward multidimensional analysis because the mathematics of these decision functions allows for any number of dimensions and any degree of complexity. Furthermore, because the features considered assume both time and frequency domains, the analysis is performed both temporally and as a function of different frequency ranges to ascertain those measures that are most suitable for seizure detection. In retrospect, by using nonlinear decision functions and by establishing a unified 2D domain of analysis, this study establishes a generalized approach to seizure detection that works across several features and across patients. METHODS: Clinical experiments involved 14 patients with intractable seizures that were evaluated for potential surgical interventions. Of the total 157 files considered, 35 (21 interictal and 14 ictal) intracranial electroencephalogram data files or 22% were used initially in a training phase to ascertain the reliability of the formulated features that were implemented in the seizure detection process. The remaining 122 intracranial electroencephalogram data files or 78% were then used in the testing phase to assess the merits of each feature considered as means to detect a seizure. RESULTS: The testing phase using the remaining 122 intracranial electroencephalogram data files revealed that the gamma power in the frequency domain is the feature that performed best across all patients with a sensitivity of 96.296%, an accuracy of 96.721%, and a specificity of 96.842%. The second best feature in the time domain was the mobility with a sensitivity of 81.481% an accuracy of 90.169%, and a specificity of 92.632%. In the frequency domain, all of the five other spectral bands lesser than 36 Hz revealed mixed results in terms of low sensitivity in some frequency bands and low accuracy in other frequency bands, which is expected given that the dominant frequencies during an ictal state are those higher than 30 Hz. In the time domain, other features, including complexity and correlation sum, revealed mixed success. CONCLUSIONS: All the features that are based on the time domain performed well, with mobility being the optimal feature for seizure detection. In the frequency domain, the gamma power outperformed the other frequency bands. Within this 2D plane, the best results were also observed when the degree of complexity is 3 or 4 in the implementation of the proposed nonlinear decision functions. SIGNIFICANCE: : A singular contribution of this study is in creating a common 2D space for analysis through the use of nonlinear decision functions for delineating data clusters of ictal files from data clusters of interictal files. This is critically important in establishing unifying measures that work across different features as expressed by the weight vector of the decision functions for a standardized assessment. The mathematical foundation is consequently established in support of a generalized seizure detection algorithm that works across patients, and in which all type of features that have been amply tested in the literature could be assessed within the realm of nonlinear decision functions.

A spreadsheet application for processing long-term EEG recordings.

This study develops a Windows application for processing huge tabular text files. The tool has been especially designed for handling EEG files. As a consequence, tables with more than 65,536 rows and 256 columns, which is a limitation found in Microsoft's Excel, can be loaded, visualized and processed with no more restrictions than the ones imposed by the memory of the operating system. Beyond tabular visualization, additional tools are available for chart customization and spreadsheet like cell processing commands. Additionally, commands are included for signal processing, cluster analysis and computationally taxing matrix algebra operations.

Classification of electroencephalographic seizure recordings into ictal and interictal files using correlation sum.

This study provides a performance evaluation of the correlation sum in terms of accuracy, sensitivity, and specificity in its ability to classify seizure files from non-seizure files. The main thrust of the study is whether computable properties ("metrics") of EEG tracings over time allow a seizure to be detected. This study evaluates raw intracranial EEG (iEEG) recordings with the intent to detect a seizure and classify different EEG epoch files. One hundred twenty-six iEEG files from eleven sequential patients are processed and the correlation sum is extracted from non-overlapping scrolling windows of 1-s duration. The novelty of this research is in defining a generalized nonlinear approach to classify EEG seizure segments by introducing nonlinear decision functions with the flexibility in choosing any degree of complexity and with any number of dimensions, lending resiliency to data overlap and opportunity for multidimensional data analysis. A singular contribution of this work is in determining a 2-D decision plane, in this case, where duration is one dimension and window-based minima of the correlation sum is the second dimension. Also, experimental observations clearly indicate that a significant drop in the magnitude of the correlation sum signal actually coincides with the clinical seizure onset more so than the electrographic seizure onset as provided by the medical experts. The method with k-fold cross validation performed with an accuracy of 91.84%, sensitivity of 92.31%, and specificity of 91.67%, which makes this classification method most suitable for offline seizure detection applications.

Cre recombination-mediated cassette exchange for building versatile transgenic human embryonic stem cells lines.

To circumvent the silencing effect of transgene expression in human embryonic stem cells (hESCs), we employed the Cre recombination-mediated cassette exchange strategy to target the silencing-resistant site in the genome. We have identified new loci that sustain transgene expression during stem cell expansion and differentiation to cells representing the three germ layers in vitro and in vivo. The built-in double loxP cassette in the established master hESC lines was specifically replaced by a targeting vector containing the same loxP sites, using the cell-permeable Cre protein transduction method, resulting in successful generation of new hESC lines with constitutive functional gene expression, inducible transgene expression, and lineage-specific reporter gene expression. This strategy and the master cell lines allow for rapid production of transgenic hESC lines in ordinary laboratories.

Human oligodendrocytes from embryonic stem cells: conserved SHH signaling networks and divergent FGF effects.

Human embryonic stem cells (hESCs) offer a platform to bridge what we have learned from animal studies to human biology. Using oligodendrocyte differentiation as a model system, we show that sonic hedgehog (SHH)-dependent sequential activation of the transcription factors OLIG2, NKX2.2 and SOX10 is required for sequential specification of ventral spinal OLIG2-expressing progenitors, pre-oligodendrocyte precursor cells (pre-OPCs) and OPCs from hESC-derived neuroepithelia, indicating that a conserved transcriptional network underlies OPC specification in human as in other vertebrates. However, the transition from pre-OPCs to OPCs is protracted. FGF2, which promotes mouse OPC generation, inhibits the transition of pre-OPCs to OPCs by repressing SHH-dependent co-expression of OLIG2 and NKX2.2. Thus, despite the conservation of a similar transcriptional network across vertebrates, human stem/progenitor cells may respond differently to those of other vertebrates to certain extrinsic factors.

Adaptive eye-gaze tracking using neural-network-based user profiles to assist people with motor disability.

This study developed an adaptive real-time human-computer interface (HCI) that serves as an assistive technology tool for people with severe motor disability. The proposed HCI design uses eye gaze as the primary computer input device. Controlling the mouse cursor with raw eye coordinates results in sporadic motion of the pointer because of the saccadic nature of the eye. Even though eye movements are subtle and completely imperceptible under normal circumstances, they considerably affect the accuracy of an eye-gaze-based HCI. The proposed HCI system is novel because it adapts to each specific user's different and potentially changing jitter characteristics through the configuration and training of an artificial neural network (ANN) that is structured to minimize the mouse jitter. This task is based on feeding the ANN a user's initially recorded eye-gaze behavior through a short training session. The ANN finds the relationship between the gaze coordinates and the mouse cursor position based on the multilayer perceptron model. An embedded graphical interface is used during the training session to generate user profiles that make up these unique ANN configurations. The results with 12 subjects in test 1, which involved following a moving target, showed an average jitter reduction of 35%; the results with 9 subjects in test 2, which involved following the contour of a square object, showed an average jitter reduction of 53%. For both results, the outcomes led to trajectories that were significantly smoother and apt at reaching fixed or moving targets with relative ease and within a 5% error margin or deviation from desired trajectories. The positive effects of such jitter reduction are presented graphically for visual appreciation.

In vitro- and in vivo-induced transgene expression in human embryonic stem cells and derivatives.

The use of human embryonic stem cells (hESCs) as a research and therapeutic tool will be facilitated by conditional gene expression. Here, we report drug-induced transgene expression, both in vitro and in vivo, from a tet-on hESC line with >95% purity. Using green fluorescent protein as an indicator, we demonstrated that the tet-on system allowed a tight control of the gene expression in both undifferentiated hESCs and differentiated cells of the three germ layers. More importantly, after the cells were transplanted into animals, the gene expression remained to be regulated by an orally administered drug. These results provide a technical basis for regulation of gene expression in hESCs and derivatives in vitro and in vivo.

Human embryonic stem cell-derived dopaminergic neurons reverse functional deficit in parkinsonian rats.

We show that human embryonic stem cell-derived dopaminergic neurons survived transplantation to the neurotoxin 6-hydroxydopamine-lesioned rat striatum and, in combination with the cells newly differentiated from their progenitors, contributed to locomotive function recovery at 5 months. The animal behavioral improvement was correlated with the dopamine neurons present in the graft. Although the donor cells contained forebrain and midbrain dopamine neurons, the dopamine neurons present in the graft mainly exhibited a midbrain, or nigra, phenotype, suggesting the importance of midbrain dopamine neurons in functional repair. Furthermore, progenies of grafted cells were neurons and glia with greatly diminished mitotic activity by 5 months. Thus, the in vitro-produced human dopamine neurons can functionally engraft in the brain.

Pattern extraction in interictal EEG recordings towards detection of electrodes leading to seizures.

This study introduces an algorithm for a new application dedicated at discriminating between electrodes leading to a seizure onset and those that do not lead to seizure using interictal subdural EEG data. The significance of this study is in determining among all of these channels, all containing interictal spikes that are asynchronously, independent of region and time, which are selected randomly (these EEG portions may or may not contain spikes), and yet through the developed algorithm, we are able to classify those channels that lead to seizure and those that do not. The main zones of ictal activity are supposed to evolve from the tissue located at the channels that present interictal activity, but sometimes this is no the case. The purpose is to gain a better understanding of the dynamics of the human brain through a study of subdural EEG, with an emphasis on attempting to characterize the common behaviors of interictal EEG channels prior to an ictal activity. The study will try to correlate the clinical features with the EEG findings and to determine whether the patient has a consistent source of ictal activity, which is coming from the location of the group of channels that present interictal activity. If a method was found to detect the electrodes that present interictal activity, with the most potential to lead to an pileptic seizure, then the epilepsy focus could be located with a higher degree of certainty. This analysis allows for the detection of neurological disorders due to epileptic activity in the brain, and rings out how different patients react prior to a seizure.