Human Neural Development Research Articles

LHX2 regulates the neural differentiation of human embryonic stem cells via transcriptional modulation of PAX6 and CER1

The LIM homeobox 2 transcription factor Lhx2 is known to control crucial aspects of neural development in various species. However, its function in human neural development is still elusive. Here, we demonstrate that LHX2 plays a critical role in human neural differentiation, using human embryonic stem cells (hESCs) as a model. In hESC-derived neural progenitors (hESC-NPs), LHX2 was found to be expressed before PAX6, and co-expressed with early neural markers. Conditional ectopic expression of LHX2 promoted neural differentiation, whereas disruption of LHX2 expression in hESCs significantly impaired neural differentiation. Furthermore, we have demonstrated that LHX2 regulates neural differentiation at two levels: first, it promotes expression of PAX6 by binding to its active enhancers, and second, it attenuates BMP and WNT signaling by promoting expression of the BMP and WNT antagonist Cerberus 1 gene (CER1), to inhibit non-neural differentiation. These findings indicate that LHX2 regulates the transcription of downstream intrinsic and extrinsic molecules that are essential for early neural differentiation in human.

Functional Maturation of hPSC-Derived Forebrain Interneurons Requires an Extended Timeline and Mimics Human Neural Development

Directed differentiation from human pluripotent stem cells (hPSCs) has seen significant progress in recent years. However, most differentiated populations exhibit immature properties of an early embryonic stage, raising concerns about their ability to model and treat disease. Here, we report the directed differentiation of hPSCs into medial ganglionic eminence (MGE)-like progenitors and their maturation into forebrain type interneurons. We find that early-stage progenitors progress via a radial glial-like stem cell enriched in the human fetal brain. Both invitro and posttransplantation into the rodent cortex, the MGE-like cells develop into GABAergic interneuron subtypes with mature physiological properties along a prolonged intrinsic timeline of up to 7months, mimicking endogenous human neural development. MGE-derived cortical interneuron deficiencies are implicated in a broad range of neurodevelopmental and degenerative disorders, highlighting the importance of these results for modeling human neural development and disease.

Pluripotent Stem Cell-Derived Somatic Stem Cells as Tool to Study the Role of MicroRNAs in Early Human Neural Development

The in vitro differentiation of human pluripotent stem cells represents a convenient approach to generate large numbers of neural cells for basic and translational research. We recently described the derivation of homogeneous populations of long-term self-renewing neuroepithelial-like stem cells from human pluripotent stem cells (lt-NES® cells). These cells constitute a suitable source of neural stem cells for in vitro modelling of early human neural development. Recent evidence demonstrates that microRNAs are important regulators of stem cells and nervous system development. Studies in several model organisms suggest that microRNAs contribute to different stages of neurogenesis - from progenitor self-renewal to survival and function of differentiated neurons. However, the understanding of the impact of microRNA-based regulation in human neural development is still at its dawn. Here, we give an overview on the current state of microRNA biology in stem cells and neural development and examine the role of the neural-associated miR-124, miR- 125b and miR-9/9* in human lt-NES® cells. We show that overexpression of miR-124, as well as overexpression of miR-125b, impair lt-NES® cell self-renewal and induce differentiation into neurons. Overexpression of the miR-9/9* locus also impairs self-renewal of lt-NES® cells and supports their commitment to neuronal differentiation. A detailed examination revealed that overexpression of miR-9 promotes differentiation, while overexpression of miR-9* affects both proliferation and differentiation of lt-NES® cells. This work provides insights into the regulation of early human neuroepithelial cells by microRNAs and highlights the potential of controlling differentiation of human stem cells by modulating the expression of selected microRNAs.

Quercetin and lithium chloride modulate Wnt signaling in pluripotent embryonal carcinoma NT2/D1 cells

Wnt signaling functions in numerous cellular activities such as cell fate determination, patterning, and migration in embryogenesis, apoptosis, etc. In this study, we used quercetin and lithium chloride to investigate modulations of the Wnt signaling pathway in human pluripotent embryonal carcinoma NT2/D1 cell line. First, we optimized conditions for NT2/D1 cell treatments with quercetin and lithium chloride and assessed their cytotoxic effects on the cells, cell viability and proliferation rate. Our results showed that induction of cell death by quercetin and LiCl is p53-dependent in NT2/D cells. We also examined the degree of Wnt signaling modulations by analyzing the expression of c-myc, a wellknown Wnt signaling target gene. Since the retinoic acid induction of NT2/D1 cells is good in an in vitro model system for human neural differentiation, studying Wnt signaling modulation in NT2/D1 would contribute to a better understanding of the mechanisms involved in neural stem cell maintenance and human neural development.

Modeling Brain Disease in a Dish: Really?

Cellular programming and reprogramming technology (CPART) presents a novel approach for understanding disease progression and mechanism. In addition, CPART provides an innovative opportunity for developing diagnostic tools and novel drug candidates for therapy. In this Forum, we will discuss obstacles and solutions for modeling brain disease using CPART.

Pharmacological Modulation of the Hedgehog Pathway Differentially Affects Dorsal/Ventral Patterning in Mouse and Human Embryonic Stem Cell Models of Telencephalic Development

A complex set of extrinsic and intrinsic signals acts in specific temporal and spatial orders to enable neural differentiation during development. These processes have been extensively studied in animal models, but human neural development remains much less understood. This lack of detailed information about human early neurogenesis is a hindrance for the differentiation of pluripotent stem cell lines into specific neuronal phenotypes. Therefore, it is important to strengthen the interspecies comparative approaches. We describe a novel model system in which in vitro differentiation of human and mouse embryonic stem (ES) cells are temporally aligned to each other and compared with mouse telencephalic neurogenesis in vivo. In this comparative model system, we tested the in vitro role of Hedgehog (Hh) signaling for ES cell-derived telencephalic differentiation. In vivo, Hh signaling mediates dorsal/ventral patterning during early stages of telencephalic development. We monitored the effect of pharmacological modulators of the Hh signaling pathway, purmorphamine-an agonist and cyclopamine-an antagonist of the Smoothened receptor (Smo), on the expression of region-specific transcription factors and signaling molecules relevant for telencephalic development in vivo. Purmorphamine strongly upregulated the expression of telencephalic ventral markers Nkx2.1, Nkx6.2, Lhx6, and Lhx8 in mouse and human cells, thus reflecting the in vivo process of the medial ganglionic eminence patterning and specification. Cyclopamine upregulated the expression of telencephalic dorsal markers, but at lower levels in human compared with mouse cells. Modulation of Smo in vitro differentially affected, in mouse and human cells, the expression of molecules of the Hh pathway, especially the Gli1 and Gli3 effectors, Sonic Hh ligand and Ptch receptors. These results provide evidence for the different default differentiation of mouse and human ES cells and prove the utility of the comparative system for optimizing the directed differentiation of human pluripotent stem cells.

Differentiation of human ES cell-derived neural progenitors to neuronal cells with regional specific identity by co-culturing of notochord and somite

Limitations to the in vivo study of human nervous system development make it necessary to design an in vitro model to evaluate the in vivo effects of surrounding tissues on neurogenesis and regional identity of the human neural plate. Rostral neural progenitors (NPs) were initially generated from adherent human embryonic stem cells (hESCs) in a defined condition and characterized. Then, to find the role of somites (S) and notochords (N) in rostro-caudal (RC) and dorso-ventral (DV) patterning of neuronal cells, NPs were co-cultured with microencapsulated chicken S or N in alginate beads. In this study, N induced more neurogenesis as evaluated by expression of TUJ1 and MAP2-positive cells. Additionally, N induced spinal cord ventral brachiothoracic identity as well as NPs proliferation. We observed a synergic effect on motoneuron induction when S and N were used together. Moreover, S induced hindbrain identity in differentiated neuronal cells from NPs. These results indicate that highly enriched NPs can be generated in an adherent and defined system from hESCs. Moreover, S and N tissues highly influenced neuronal differentiation in point of proliferation, neurogenesis, and RC and DV regional identity. These results indicate a very simple and efficient protocol to mimic in vivo events of human neural development in vitro which is important in the context of developmental neuroscience and cellular replacement therapies.

Neural development in human embryonic stem cells-applications of lentiviral vectors

The derivation of neural lineages from human embryonic stem cells (hESCs) in vitro is based largely on exposure of hESCs to exogenous signals and substrates, designed to mimic conditions in the developing embryo. However, selection of specific lineages and the discovery of gene function in human neural development may be enhanced by the ability to intrinsically regulate gene expression. Recombinant lentiviral vectors provide an efficient method to stably introduce genes into hESC and their differentiating derivatives. Here we review the methods used to derive neural cells from hESCs, transduction of these cells with lentiviral vectors, and improvements that have been made to the vectors to enhance viral integration and transgene expression. Finally, we explore prospects for future uses of lentiviral vectors in hESC research, including their applications in library screening for drug development, zinc finger nucleases for gene editing and optogenetics to interrogate cellular pathways and function.

RE: Infantile and early childhood masturbation: Sex hormones and clinical profile

RE: Infantile and early childhood masturbation: Sex hormones and clinical profile

P.2.023 In vivo microdialysis studies of brain dopamine, glutamate and GABA overflow during cocaine self-administration and its extinction

Epigenetic regulation plays an important role in controlling gene expression during complex processes, such as development of the human brain. Mutations in genes encoding chromatin modifying proteins and in the non-protein coding sequences of the genome can potentially alter transcription factor binding or chromatin accessibility. Such mutations can frequently cause neurodevelopmental disorders, therefore understanding how epigenetic regulation shapes brain development is of particular interest. While epigenetic regulation of neural development has been extensively studied in murine models, significant species-specific differences in both the genome sequence and in brain development necessitate human models. However, access to human fetal material is limited and these tissues cannot be grown or experimentally manipulated ex vivo. Therefore, models that recapitulate particular aspects of human fetal brain development, such as the in vitro differentiation of human pluripotent stem cells (hPSCs), are instrumental for studying the epigenetic regulation of human neural development. Here, we examine recent studies that have defined changes in the epigenomic landscape during fetal brain development. We compare these studies with analogous data derived by in vitro differentiation of hPSCs into specific neuronal cell types or as three-dimensional cerebral organoids. Such comparisons can be informative regarding which aspects of fetal brain development are faithfully recapitulated by in vitro differentiation models and provide a foundation for using experimentally tractable in vitro models of human brain development to study neural gene regulation and the basis of its disruption to cause neurodevelopmental disorders.

Network analysis: applications for the developing brain.

Development of the human brain follows a complex trajectory of age-specific anatomical and physiological changes. The application of network analysis provides an illuminating perspective on the dynamic interregional and global properties of this intricate and complex system. Here, we provide a critical synopsis of methods of network analysis with a focus on developing brain networks. After discussing basic concepts and approaches to network analysis, we explore the primary events of anatomical cortical development from gestation through adolescence. Upon this framework, we describe early work revealing the evolution of age-specific functional brain networks in normal neurodevelopment. Finally, we review how these relationships can be altered in disease and perhaps even rectified with treatment. While this method of description and inquiry remains in early form, there is already substantial evidence that the application of network models and analysis to understanding normal and abnormal human neural development holds tremendous promise for future discovery.

Neural Differentiation from Human Embryonic Stem Cells as a Tool to Study Early Brain Development and the Neuroteratogenic Effects of Ethanol

The in vitro generation of neural cells from human embryonic stem cells is a powerful tool to acquire better knowledge of the cellular and molecular events involved in early human neural and brain development under physiological and pathological conditions. Prenatal alcohol exposure can induce important anomalies in the developing brain, the embryogenesis being an important critical period for the craniofacial defects and mental disabilities associated with fetal alcohol syndrome. Here, we report the generation of neural progenitors (NPs) from human embryonic stem cells. Neuroepithelial progenitors display the morphological and functional characteristics of their embryonic counterparts and the proper timing of neurons and glia cells generation. Immunocytochemical and real time (RT)-polymerase chain reaction analyses reveal that cells appeared as clusters during neuroepithelial cell proliferation and that the genes associated with the neuroectodermal (Pax-6) and the endodermic (α-fetoprotein) lineages decreased in parallel to the upregulation of the genes of NPs (nestin and Tuj1), followed by their differentiation into neurons (MAP-2+, GABA+), oligodendrocytes [galactocerebroside (GalC+)], and astrocytes (GFAP+). We further demonstrate, for the first time, that human NPs express the endocannabinoid receptors (CB1 and CB2) and the enzymes involved in endocannabinoids synthesis (NAPE-PLD) and degradation (FAAH). Using this in vitro culture, we demonstrate that ethanol exposure impairs NPs survival, affects the differentiation of NPs into neurons and astrocytes, disrupts the actin cytoskeleton, and affects the expression of different genes associated with neural differentiation. The results provide new insights into the effects of ethanol on human embryogenesis and neuroprogenitors and offer an opportunity to delineate potential therapeutic strategies to restore early ethanol-induced brain damage.

Real Time Imaging of Human Progenitor Neurogenesis

Human neural progenitors are increasingly being employed in drug screens and emerging cell therapies targeted towards neurological disorders where neurogenesis is thought to play a key role including developmental disorders, Alzheimer’s disease, and depression. Key to the success of these applications is understanding the mechanisms by which neurons arise. Our understanding of development can provide some guidance but since little is known about the specifics of human neural development and the requirement that cultures be expanded in vitro prior to use, it is unclear whether neural progenitors obey the same developmental mechanisms that exist in vivo. In previous studies we have shown that progenitors derived from fetal cortex can be cultured for many weeks in vitro as undifferentiated neurospheres and then induced to undergo neurogenesis by removing mitogens and exposing them to supportive substrates. Here we use live time lapse imaging and immunocytochemical analysis to show that neural progenitors use developmental mechanisms to generate neurons. Cells with morphologies and marker profiles consistent with radial glia and recently described outer radial glia divide asymmetrically and symmetrically to generate multipolar intermediate progenitors, a portion of which express ASCL1. These multipolar intermediate progenitors subsequently divide symmetrically to produce CTIP2+ neurons. This 3-cell neurogenic scheme echoes observations in rodents in vivo and in human fetal slice cultures in vitro, providing evidence that hNPCs represent a renewable and robust in vitro assay system to explore mechanisms of human neurogenesis without the continual need for fresh primary human fetal tissue. Knowledge provided by this and future explorations of human neural progenitor neurogenesis will help maximize the safety and efficacy of new stem cell therapies by providing an understanding of how to generate physiologically-relevant cell types that maintain their identities when placed in diagnostic or transplantation environments.

High-Efficiency Induction of Neural Conversion in Human ESCs and Human Induced Pluripotent Stem Cells with a Single Chemical Inhibitor of Transforming Growth Factor Beta Superfamily Receptors

Chemical compounds have emerged as powerful tools for modulating ESC functions and deriving induced pluripotent stem cells (iPSCs), but documentation of compound-induced efficient directed differentiation in human ESCs (hESCs) and human iPSC (hiPSCs) is limited. By screening a collection of chemical compounds, we identified compound C (also denoted as dorsomorphin), a protein kinase inhibitor, as a potent regulator of hESC and hiPSC fate decisions. Compound C suppresses mesoderm, endoderm, and trophoectoderm differentiation and induces rapid and high-efficiency neural conversion in both hESCs and hiPSCs, 88.7% and 70.4%, respectively. Interestingly, compound C is ineffective in inducing neural conversion in mouse ESCs (mESCs). Large-scale kinase assay revealed that compound C targets at least seven transforming growth factor beta (TGF-β) superfamily receptors, including both type I and type II receptors, and thereby blocks both the Activin and bone morphogenesis protein (BMP) signaling pathways in hESCs. Dual inhibition of Activin and BMP signaling accounts for the effects of compound C on hESC differentiation and neural conversion. We also identified muscle segment homeobox gene 2 (MSX2) as a downstream target gene of compound C and a key signaling intermediate of the BMP pathway in hESCs. Our findings provide a single-step cost-effective method for efficient derivation of neural progenitor cells in adherent culture from human pluripotent stem cells. Therefore, it will be uniquely suitable for the production of neural progenitor cells in large scale and should facilitate the use of stem cells in drug screening and regenerative medicine and study of early human neural development.

Analysis of early human neural crest development

The outstanding migration and differentiation capacities of neural crest cells (NCCs) have fascinated scientists since Wilhelm His described this cell population in 1868. Today, after intense research using vertebrate model organisms, we have gained considerable knowledge regarding the origin, migration and differentiation of NCCs. However, our understanding of NCC development in human embryos remains largely uncharacterized, despite the role the neural crest plays in several human pathologies. Here, we report for the first time the expression of a battery of molecular markers before, during, or following NCC migration in human embryos from Carnegie Stages (CS) 12 to 18. Our work demonstrates the expression of Sox9, Sox10 and Pax3 transcription factors in premigratory NCCs, while actively migrating NCCs display the additional transcription factors Pax7 and AP-2α. Importantly, while HNK-1 labels few migrating NCCs, p75 NTR labels a large proportion of this population. However, the broad expression of p75 NTR – and other markers – beyond the neural crest stresses the need for the identification of additional markers to improve our capacity to investigate human NCC development, and to enable the generation of better diagnostic and therapeutic tools.

Polybrominated Diphenyl Ethers Induce Developmental Neurotoxicity in a Human in Vitro Model: Evidence for Endocrine Disruption

BackgroundPolybrominated diphenyl ethers (PBDEs) are persistent and bioaccumulative flame retardants, which are found in rising concentrations in human tissues. They are of concern for human health because animal studies have shown that they possess the potential to be developmentally neurotoxic.ObjectiveBecause there is little knowledge of the effects of PBDEs on human brain cells, we investigated their toxic potential for human neural development in vitro. Moreover, we studied the involvement of thyroid hormone (TH) disruption in the effects caused by PBDEs.MethodsWe used the two PBDE congeners BDE-47 and BDE-99 (0.1–10 μM), which are most prominent in human tissues. As a model of neural development, we employed primary fetal human neural progenitor cells (hNPCs), which are cultured as neurospheres and mimic basic processes of brain development in vitro: proliferation, migration, and differentiation.ResultsPBDEs do not disturb hNPC proliferation but decrease migration distance of hNPCs. Moreover, they cause a reduction of differentiation into neurons and oligodendrocytes. Simultaneous exposure with the TH receptor (THR) agonist triiodothyronine rescues these effects on migration and differentiation, whereas the THR antagonist NH-3 does not exert an additive effect.ConclusionPBDEs disturb development of hNPCs in vitro via endocrine disruption of cellular TH signaling at concentrations that might be of relevance for human exposure.

Converging molecular pathways in human neural development and degeneration

Animal studies suggest that phosphorylation of microtubule-associated protein tau is a physiological way of destabilizing axons in the developing brain, promoting synaptic plasticity, while in the adult human brain tau phosphorylation is a specific sign of Alzheimer's disease. We here show, for the first time, that newborn human infants have extremely high levels of phosphorylated tau in their cerebrospinal fluid, and that these levels decrease during the first years of life. Tau phosphorylation in Alzheimer's disease may be a physiological response to Alzheimer-associated synaptotoxicity.

Autism: The Ups and Downs of Neuroligin

Autism: The Ups and Downs of Neuroligin

Isolation and Characterization of Neural Crest Stem Cells Derived From In Vitro–Differentiated Human Embryonic Stem Cells

The neural crest is a transient structure of vertebrate embryos that initially generates neural crest stem cells (NCSCs) which then migrate throughout the body to produce a diverse array of mature tissue types. Due to the rarity of adult NCSCs as well as ethical and technical issues surrounding isolation of early embryonic tissues, biologic studies of human NCSCs are extremely challenging. Thus, much of what is known about human neural crest development has been inferred from model organisms. In this study, we report that functional NCSCs can be rapidly generated and isolated from in vitro-differentiated human embryonic stem cells (hESCs). Using the stromal-derived inducing activity (SDIA) of PA6 fibroblast co-culture we have induced hESCs to differentiate into neural crest. Within 1 week, migrating cells that express the early neural crest markers p75 and HNK1 as well as numerous other genes associated with neural crest induction such as SNAIL, SLUG, and SOX10 are detectable. Fluorescence-activated cell sorting (FACS)-based isolation of the p75-positive population enriches for cells with genetic, phenotypic, and functional characteristics of NCSCs. These p75-enriched cells readily form neurospheres in suspension culture, self-renew to form secondary spheres, and give rise under differentiation conditions to multiple neural crest lineages including peripheral nerves, glial, and myofibroblastic cells. Importantly, these cells differentiate into neural crest derivatives when transplanted into developing chick embryos in vivo. Thus, this SDIA protocol can be used to successfully and efficiently isolate early human NCSCs from hESCs in vitro. This renewable source of NCSCs provides an invaluable source of cells for studies of both normal and disordered human neural crest development.

Polybrominated diphenyl ethers disturb human neural development in vitro

Polybrominated diphenyl ethers disturb human neural development in vitro