Stem cell models of Angelman syndrome.

Angelman syndrome (AS) is an imprinted neurodevelopmental disorder that lacks a cure, characterized by developmental delay, intellectual impairment, seizures, ataxia, and paroxysmal laughter. The condition arises due to the loss of the maternally inherited copy of the UBE3A gene in neurons. The paternally inherited UBE3A allele is unable to compensate because it is silenced by the expression of an antisense transcript (UBE3A-ATS) on the paternal chromosome. UBE3A, encoding enigmatic E3 ubiquitin ligase variants, regulates target proteins by either modifying their properties/functions or leading them to degradation through the proteasome. Over time, animal models, particularly the Ube3a mat-/pat+ Knock-Out (KO) mice, have significantly contributed to our understanding of the molecular mechanisms underlying AS. However, a shift toward human pluripotent stem cell models (PSCs), such as human embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs), has gained momentum. These stem cell models accurately capture human genetic and cellular characteristics, offering an alternative or a complement to animal experimentation. Human stem cells possess the remarkable ability to recapitulate neurogenesis and generate "brain-in-a-dish" models, making them valuable tools for studying neurodevelopmental disorders like AS. In this review, we provide an overview of the current state-of-the-art human stem cell models of AS and explore their potential to become the preclinical models of choice for drug screening and development, thus propelling AS therapeutic advancements and improving the lives of affected individuals.

Generation and characterization of induced pluripotent stem cell line (IBBISTi004-A) from an Angelman syndrome patient carrying a class II deletion of the maternal chromosome 15q11.2-q13.

Angelman Syndrome is a rare neurodevelopmental disorder caused by several (epi)genetic alterations. The patients present strong neurological impairment due to the absence of a functional maternal UBE3A gene in neurons. Here, we generated and characterized a new induced pluripotent stem cell (iPSC) line from a female child with Angelman syndrome harbouring a class II deletion. iPSCs were reprogrammed from fibroblasts using Sendai viruses. The new iPSCs express pluripotency markers, are capable of trilineage in vitro differentiation and have the expected imprinting status of Angelman syndrome. These iPSCs are a valuable tool to elucidate the pathophysiological mechanisms associated with this disease.

Transcriptome profiling of human pluripotent stem cell-derived cerebellar organoids reveals faster commitment under dynamic conditions.

Human-induced pluripotent stem cells (iPSCs) have great potential for disease modeling. However, generating iPSC-derived models to study brain diseases remains a challenge. In particular, the ability to recapitulate cerebellar development in vitro is still limited. We presented a reproducible and scalable production of cerebellar organoids by using the novel single-use Vertical-Wheel bioreactors, in which functional cerebellar neurons were obtained. Here, we evaluate the global gene expression profiles by RNA sequencing (RNA-seq) across cerebellar differentiation, demonstrating a faster cerebellar commitment in this novel dynamic differentiation protocol. Furthermore, transcriptomic profiles suggest a significant enrichment of extracellular matrix (ECM) in dynamic-derived cerebellar organoids, which can better mimic the neural microenvironment and support a consistent neuronal network. Thus, an efficient generation of organoids with cerebellar identity was achieved for the first time in a continuous process using a dynamic system without the need of organoids encapsulation in ECM-based hydrogels, allowing the possibility of large-scale production and application in high-throughput processes. The presence of factors that favors angiogenesis onset was also detected in dynamic conditions, which can enhance functional maturation of cerebellar organoids. We anticipate that large-scale production of cerebellar organoids may help developing models for drug screening, toxicological tests, and studying pathological pathways involved in cerebellar degeneration.

Modeling Rett Syndrome With Human Patient-Specific Forebrain Organoids.

Engineering brain organoids from human induced pluripotent stem cells (hiPSCs) is a powerful tool for modeling brain development and neurological disorders. Rett syndrome (RTT), a rare neurodevelopmental disorder, can greatly benefit from this technology, since it affects multiple neuronal subtypes in forebrain sub-regions. We have established dorsal and ventral forebrain organoids from control and RTT patient-specific hiPSCs recapitulating 3D organization and functional network complexity. Our data revealed a premature development of the deep-cortical layer, associated to the formation of TBR1 and CTIP2 neurons, and a lower expression of neural progenitor/proliferative cells in female RTT dorsal organoids. Moreover, calcium imaging and electrophysiology analysis demonstrated functional defects of RTT neurons. Additionally, assembly of RTT dorsal and ventral organoids revealed impairments of interneuron's migration. Overall, our models provide a better understanding of RTT during early stages of neural development, demonstrating a great potential for personalized diagnosis and drug screening.

hiPSC-Based Model of Prenatal Exposure to Cannabinoids: Effect on Neuronal Differentiation.

Phytocannabinoids are psychotropic substances ofcannabis with the ability to bind endocannabinoid (eCB) receptors that regulate synaptic activity in the central nervous system (CNS). Synthetic cannabinoids (SCs) are synthetic analogs of Δ9-tetrahydrocannabinol (Δ9-THC), the psychotropic compound of cannabis, acting as agonists of eCB receptor CB1. SC is an easily available and popular alternative to cannabis, and their molecular structure is always changing, increasing the hazard for the general population. The popularity of cannabis and its derivatives may lead, and often does, to a child's exposure to cannabis both in utero and through breastfeeding by a drug-consuming mother. Prenatal exposure to cannabis has been associated with an altered rate of mental development and significant changes in nervous system functioning. However, the understanding of mechanisms of its action on developing the human CNS is still lacking. We investigated the effect of continuous exposure to cannabinoids on developing human neurons, mimicking the prenatal exposure by drug-consuming mother. Two human induced pluripotent stem cells (hiPSC) lines were induced to differentiate into neuronal cells and exposed for 37 days to cannabidiol (CBD), Δ9-THC, and two SCs, THJ-018 and EG-018. Both Δ9-THC and SC, at 10 µM, promote precocious neuronal and glial differentiation, while CBD at the same concentration is neurotoxic. Neurons exposed to Δ9-THC and SC show abnormal functioning of voltage-gated calcium channels when stimulated by extracellular potassium. In sum, all studied substances have a profound impact on the developing neurons, highlighting the importance of thorough research on the impact of prenatal exposure to natural and SC.

Scalable Generation of Mature Cerebellar Organoids from Human Pluripotent Stem Cells and Characterization by Immunostaining.

The cerebellum plays a critical role in the maintenance of balance and motor coordination, and a functional defect in different cerebellar neurons can trigger cerebellar dysfunction. Most of the current knowledge about disease-related neuronal phenotypes is based on postmortem tissues, which makes understanding of disease progression and development difficult. Animal models and immortalized cell lines have also been used as models for neurodegenerative disorders. However, they do not fully recapitulate human disease. Human induced pluripotent stem cells (iPSCs) have great potential for disease modeling and provide a valuable source for regenerative approaches. In recent years, the generation of cerebral organoids from patient-derived iPSCs improved the prospects for neurodegenerative disease modeling. However, protocols that produce large numbers of organoids and a high yield of mature neurons in 3D culture systems are lacking. The protocol presented is a new approach for reproducible and scalable generation of human iPSC-derived organoids under chemically-defined conditions using scalable single-use bioreactors, in which organoids acquire cerebellar identity. The generated organoids are characterized by the expression of specific markers at both mRNA and protein level. The analysis of specific groups of proteins allows the detection of different cerebellar cell populations, whose localization is important for the evaluation of organoid structure. Organoid cryosectioning and further immunostaining of organoid slices are used to evaluate the presence of specific cerebellar cell populations and their spatial organization.

Maturation of Human Pluripotent Stem Cell-Derived Cerebellar Neurons in the Absence of Co-culture.

The cerebellum plays a critical role in all vertebrates, and many neurological disorders are associated with cerebellum dysfunction. A major limitation in cerebellar research has been the lack of adequate disease models. As an alternative to animal models, cerebellar neurons differentiated from pluripotent stem cells have been used. However, previous studies only produced limited amounts of Purkinje cells. Moreover, in vitro generation of Purkinje cells required co-culture systems, which may introduce unknown components to the system. Here we describe a novel differentiation strategy that uses defined medium to generate Purkinje cells, granule cells, interneurons, and deep cerebellar nuclei projection neurons, that self-formed and differentiated into electrically active cells. Using a defined basal medium optimized for neuronal cell culture, we successfully promoted the differentiation of cerebellar precursors without the need for co-culturing. We anticipate that our findings may help developing better models for the study of cerebellar dysfunctions, while providing an advance toward the development of autologous replacement strategies for treating cerebellar degenerative diseases.

Angelman syndrome: a journey through the brain.

Angelman syndrome (AS) is an incurable neurodevelopmental disease caused by loss of function of the maternally inherited UBE3A gene. AS is characterized by a defined set of symptoms, namely severe developmental delay, speech impairment, uncontrolled laughter, and ataxia. Current understanding of the pathophysiology of AS relies mostly on studies using the murine model of the disease, although alternative models based on patient-derived stem cells are now emerging. Here, we summarize the literature of the last decade concerning the three major brain areas that have been the subject of study in the context of AS: hippocampus, cortex, and the cerebellum. Our comprehensive analysis highlights the major phenotypes ascribed to the different brain areas. Moreover, we also discuss the major drawbacks of current models and point out future directions for research in the context of AS, which will hopefully lead us to an effective treatment of this condition in humans.

In vitro and in silico evaluations of resin-based dental restorative material toxicity.

OBJECTIVES: This study aims to evaluate the cytocompatibility of three provisional restoration materials and predict neurotoxic potential of their monomers. These materials are Tab 2000® (methyl methacrylate based), ProTemp 4™ (bis-acrylic based) and Structur 3® (urethane dimethacrylate based). MATERIALS AND METHODS: Resin samples were incubated in a cell culture medium and the cytotoxic effects of these extracts were studied in 3T3 fibroblast cells through MTT and crystal violet assays as well as ROS assessment. The presence of relevant leached monomers was determined by HPLC. Additionally, the blood-brain barrier (BBB) permeability to these resin-based monomers was predicted using ACD/Labs algorithms model. RESULTS: Cell survival rates were compared with the resin extracts, and Structur 3® was statistically significant different from the others (p < 0.001) at all-time incubation periods. All materials induced a dose-dependent loss of cell viability; however, only Structur 3 extracts were cytotoxic against 3T3 fibroblasts. The highest cytotoxic effect (77%, p < 0.001) was observed at 24 h incubation period, which may be associated with the presence of urethane dimethacrylate (UDMA) leached monomers. Furthermore, the computational model showed that most monomers under study are expectedly capable of crossing the BBB. CONCLUSIONS: Our results showed that Structur 3® is not cytocompatible with our cell model and UDMA is a potential neurotoxic compound. CLINICAL RELEVANCE: These results indicate that only ProTemp 4™ and Tab 2000® are safe for provisional restorations.

Design Principles for Pluripotent Stem Cell-Derived Organoid Engineering.

Human morphogenesis is a complex process involving distinct microenvironmental and physical signals that are manipulated in space and time to give rise to complex tissues and organs. Advances in pluripotent stem cell (PSC) technology have promoted the in vitro recreation of processes involved in human morphogenesis. The development of organoids from human PSCs represents one reliable source for modeling a large spectrum of human disorders, as well as a promising approach for drug screening and toxicological tests. Based on the "self-organization" capacity of stem cells, different PSC-derived organoids have been created; however, considerable differences between in vitro-generated PSC-derived organoids and their in vivo counterparts have been reported. Advances in the bioengineering field have allowed the manipulation of different components, including cellular and noncellular factors, to better mimic the in vivo microenvironment. In this review, we focus on different examples of bioengineering approaches used to promote the self-organization of stem cells, including assembly, patterning, and morphogenesis in vitro, contributing to tissue-like structure formation.

Quantification of cell cycle kinetics by EdU (5-ethynyl-2'-deoxyuridine)-coupled-fluorescence-intensity analysis.

We propose a novel single-deoxynucleoside-based assay that is easy to perform and provides accurate values for the absolute length (in units of time) of each of the cell cycle stages (G1, S and G2/M). This flow-cytometric assay takes advantage of the excellent stoichiometric properties of azide-fluorochrome detection of DNA substituted with 5-ethynyl-2'-deoxyuridine (EdU). We show that by pulsing cells with EdU for incremental periods of time maximal EdU-coupled fluorescence is reached when pulsing times match the length of S phase. These pulsing times, allowing labelling for a full S phase of a fraction of cells in asynchronous populations, provide accurate values for the absolute length of S phase. We characterized additional, lower intensity signals that allowed quantification of the absolute durations of G1 and G2 phases.Importantly, using this novel assay data on the lengths of G1, S and G2/M phases are obtained in parallel. Therefore, these parameters can be estimated within a time frame that is shorter than a full cell cycle. This method, which we designate as EdU-Coupled Fluorescence Intensity (E-CFI) analysis, was successfully applied to cell types with distinctive cell cycle features and shows excellent agreement with established methodologies for analysis of cell cycle kinetics.

Generation and characterization of a novel mouse embryonic stem cell line with a dynamic reporter of Nanog expression.

BACKGROUND: The pluripotent state in embryonic stem (ES) cells is controlled by a core network of transcription factors that includes Nanog, Oct4 and Sox2. Nanog is required to reach pluripotency during somatic reprogramming and is the only core factor whose overexpression is able to oppose differentiation-promoting signals. Additionally, Nanog expression is known to fluctuate in ES cells, and different levels of Nanog seem to correlate with ES cells' ability to respond to differentiation promoting signals. Elucidating how dynamic Nanog levels are regulated in pluripotent cells and modulate their potential is therefore critical to develop a better understanding of the pluripotent state. METHODOLOGY/PRINCIPAL FINDINGS: We describe the generation and validation of a mouse ES cell line with a novel Nanog reporter (Nd, from Nanog dynamics), containing a BAC transgene where the short-lived fluorescent protein VNP is placed under Nanog regulation. We show that Nanog and VNP have similar half-lives, and that Nd cells provide an accurate and measurable read-out for the dynamic levels of Nanog. Using this reporter, we could show that ES cells with low Nanog levels indeed have higher degree of priming to differentiation, when compared with high-Nanog cells. However, low-Nanog ES cells maintain high levels of Oct4 and Sox2 and can revert to a state of high-Nanog expression, indicating that they are still within the window of pluripotency. We further show that the observed changes in Nanog levels correlate with ES cell morphology and that Nanog dynamic expression is modulated by the cellular environment. CONCLUSIONS/SIGNIFICANCE: The novel reporter ES cell line here described allows an accurate monitoring of Nanog's dynamic expression in the pluripotent state. This reporter will thus be a valuable tool to obtain quantitative measurements of global gene expression in pluripotent ES cells in different states, allowing a detailed molecular mapping of the pluripotency landscape.

Neural differentiation of embryonic stem cells in vitro: a road map to neurogenesis in the embryo.

BACKGROUND: The in vitro generation of neurons from embryonic stem (ES) cells is a promising approach to produce cells suitable for neural tissue repair and cell-based replacement therapies of the nervous system. Available methods to promote ES cell differentiation towards neural lineages attempt to replicate, in different ways, the multistep process of embryonic neural development. However, to achieve this aim in an efficient and reproducible way, a better knowledge of the cellular and molecular events that are involved in the process, from the initial specification of neuroepithelial progenitors to their terminal differentiation into neurons and glial cells, is required. METHODOLOGY/PRINCIPAL FINDINGS: In this work, we characterize the main stages and transitions that occur when ES cells are driven into a neural fate, using an adherent monolayer culture system. We established improved conditions to routinely produce highly homogeneous cultures of neuroepithelial progenitors, which organize into neural tube-like rosettes when they acquire competence for neuronal production. Within rosettes, neuroepithelial progenitors display morphological and functional characteristics of their embryonic counterparts, namely, apico-basal polarity, active Notch signalling, and proper timing of production of neurons and glia. In order to characterize the global gene activity correlated with each particular stage of neural development, the full transcriptome of different cell populations that arise during the in vitro differentiation protocol was determined by microarray analysis. By using embryo-oriented criteria to cluster the differentially expressed genes, we define five gene expression signatures that correlate with successive stages in the path from ES cells to neurons. These include a gene signature for a primitive ectoderm-like stage that appears after ES cells enter differentiation, and three gene signatures for subsequent stages of neural progenitor development, from an early stage that follows neural induction to a final stage preceding terminal differentiation. CONCLUSIONS/SIGNIFICANCE: Overall, our work confirms and extends the cellular and molecular parallels between monolayer ES cell neural differentiation and embryonic neural development, revealing in addition novel aspects of the genetic network underlying the multistep process that leads from uncommitted cells to differentiated neurons.

Bilirubin as a determinant for altered neurogenesis, neuritogenesis, and synaptogenesis.

Elevated levels of serum unconjugated bilirubin (UCB) in the first weeks of life may lead to long-term neurologic impairment. We previously reported that an early exposure of developing neurons to UCB, in conditions mimicking moderate to severe neonatal jaundice, leads to neuritic atrophy and cell death. Here, we have further analyzed the effect of UCB on nerve cell differentiation and neuronal development, addressing how UCB may affect the viability of undifferentiated neural precursor cells and their fate decisions, as well as the development of hippocampal neurons in terms of dendritic and axonal elongation and branching, the axonal growth cone morphology, and the establishment of dendritic spines and synapses. Our results indicate that UCB reduces the viability of proliferating neural precursors, decreases neurogenesis without affecting astrogliogenesis, and increases cellular dysfunction in differentiating cells. In addition, an early exposure of neurons to UCB decreases the number of dendritic and axonal branches at 3 and 9 days in vitro (DIV), and a higher number of neurons showed a smaller growth cone area. UCB-treated neurons also reveal a decreased density of dendritic spines and synapses at 21 DIV. Such deleterious role of UCB in neuronal differentiation, development, and plasticity may compromise the performance of the brain in later life.

Expansion of mouse embryonic stem cells on microcarriers.

Embryonic stem (ES) cells have been shown to differentiate in vitro into a wide variety of cell types having significant potential for tissue regeneration. Therefore, the operational conditions for the ex vivo expansion and differentiation should be optimized for large-scale cultures. The expansion of mouse ES cells has been evaluated in static culture. However, in this system, culture parameters are difficult to monitor and scaling-up becomes time consuming. The use of stirred bioreactors facilitates the expansion of cells under controlled conditions but, for anchorage-dependent cells, a proper support is necessary. Cytodex-3, a microporous microcarrier made up of a dextran matrix with a collagen layer at the surface, was tested for its ability to support the expansion of the mouse S25 ES cell line in spinner flasks. The effect of inocula and microcarrier concentration on cell growth and metabolism were analyzed. Typically, after seeding, the cells exhibited a growth curve consisting of a short death or lag phase followed by an exponential phase leading to the maximum cell density of 2.5-3.9 x 10(6) cells/mL. Improved expansion was achieved using an inoculum of 5 x 10(4) cells/mL and a microcarrier concentration of 0.5 mg/mL. Medium replacement allowed the supply of the nutrients and the removal of waste products inhibiting cell growth, leading to the maintenance of the cultures in steady state for several days. These conditions favored the preservation of the S25 cells pluripotent state, as assessed by quantitative real-time PCR and immunostaining analysis.

Dosage-sensitive requirement for mouse Dll4 in artery development.

Involvement of the Notch signaling pathway in vascular development has been demonstrated by both gain- and loss-of-function mutations in humans, mice, and zebrafish. In zebrafish, Notch signaling is required for arterial identity by suppressing the venous fate in developing artery cells. In mice, the Notch4 receptor and the Delta-like 4 (Dll4) ligand are specifically expressed in arterial endothelial cells, suggesting a similar role. Here we show that the Dll4 ligand alone is required in a dosage-sensitive manner for normal arterial patterning in development. This implicates Dll4 as the specific mammalian endothelial ligand for autocrine endothelial Notch signaling, and suggests that Dll4 may be a suitable target for intervention in arterial angiogenesis.

Expansion and neural differentiation of embryonic stem cells in adherent and suspension cultures.

The embryonic stem cell line, S25, is a genetically modified line that allows lineage selection of neural cells (M. Li, L. Lovell-Badge, A. Smith (1998) Current Biology 8: 971-974). Here, the growth parameters of this cell line were analysed. Serial passaging in adherent conditions enabled these cells to grow rapidly (average specific growth rates of 0.035 h-1) and generate high viable cell densities (above 90%). The aggregation of the S25 cells into embryoid bodies (EBs) was also studied, indicating limited cell growth (maximum cell densities of 2.7 x 10(5) cells ml-1) and a high variability of aggregate size (70-400 microns after 8 d). Enzymatic dissociation of EBs with 1% (v/v) trypsin gave highest cell viability (91%) and density (1.4 x 10(4) cells ml-1) and the cells thus obtained are able to differentiate into neurons.

Embryonic expression of three mouse genes with homology to the Drosophila melanogaster prickle gene.

The Drosophila melanogaster gene prickle-spiny-legs (pk) functions in an intercellular feedback loop that is central to the establishment of planar cell polarity in the eye and epidermis of the fly, by modulating Frizzled-Disheveled signalling. Here we identify three mouse prickle-related genes (dyxin, testin and prickle) and describe their expression pattern during murine embryogenesis (E7.5-E15.5). We report that the three genes are expressed in restricted areas of the developing mouse brain: dyxin in the most ventral region of the neural tube and in some localized regions of the ventricular layer of the mesencephalon and rhombencephalon, prickle in the pons region, ventrolateral part of rhombencephalon and motoneurons in the spinal cord, and testin in differentiating neurons of the spinal cord and retina. At the stages analyzed, the main site of expression of testin is the migrating cranial neural crest, while the expression of dyxin is noticeable in myotomal cells and its derivatives, with prickle expression being reciprocally localized to some sclerotomal derivatives, like bone primordia. prickle is also expressed in the apical ectodermal ridge and the most distal mesenchyme of the forming limb buds.

Embryonic expression of three mouse genes with homology to the Drosophila melanogaster prickle gene.

The Drosophila melanogaster gene prickle-spiny-legs (pk) functions in an intercellular feedback loop that is central to the establishment of planar cell polarity in the eye and epidermis of the fly, by modulating Frizzled-Disheveled signalling. Here we identify three mouse prickle-related genes (dyxin, testin and prickle) and describe their expression pattern during murine embryogenesis (E7.5-E15.5). We report that the three genes are expressed in restricted areas of the developing mouse brain: dyxin in the most ventral region of the neural tube and in some localized regions of the ventricular layer of the mesencephalon and rhombencephalon, prickle in the pons region, ventrolateral part of rhombencephalon and motoneurons in the spinal cord, and testin in differentiating neurons of the spinal cord and retina. At the stages analyzed, the main site of expression of testin is the migrating cranial neural crest, while the expression of dyxin is noticeable in myotomal cells and its derivatives, with prickle expression being reciprocally localized to some sclerotomal derivatives, like bone primordia. prickle is also expressed in the apical ectodermal ridge and the most distal mesenchyme of the forming limb buds.