Implication of thermal signaling in neuronal differentiation revealed by manipulation and measurement of intracellular temperature.

Neuronal differentiation-the development of neurons from neural stem cells-involves neurite outgrowth and is a key process during the development and regeneration of neural functions. In addition to various chemical signaling mechanisms, it has been suggested that thermal stimuli induce neuronal differentiation. However, the function of physiological subcellular thermogenesis during neuronal differentiation remains unknown. Here we create methods to manipulate and observe local intracellular temperature, and investigate the effects of noninvasive temperature changes on neuronal differentiation using neuron-like PC12 cells. Using quantitative heating with an infrared laser, we find an increase in local temperature (especially in the nucleus) facilitates neurite outgrowth. Intracellular thermometry reveals that neuronal differentiation is accompanied by intracellular thermogenesis associated with transcription and translation. Suppression of intracellular temperature increase during neuronal differentiation inhibits neurite outgrowth. Furthermore, spontaneous intracellular temperature elevation is involved in neurite outgrowth of primary mouse cortical neurons. These results offer a model for understanding neuronal differentiation induced by intracellular thermal signaling.

Neutral competition explains the clonal composition of neural organoids.

Neural organoids model the development of the human brain and are an indispensable tool for studying neurodevelopment. Whole-organoid lineage tracing has revealed the number of progenies arising from each initial stem cell to be highly diverse, with lineage sizes ranging from one to more than 20,000 cells. This high variability exceeds what can be explained by existing stochastic models of corticogenesis and indicates the existence of an additional source of stochasticity. To explain this variability, we introduce the SAN model which distinguishes Symmetrically diving, Asymmetrically dividing, and Non-proliferating cells. In the SAN model, the additional source of stochasticity is the survival time of a lineage's pool of symmetrically dividing cells. These survival times result from neutral competition within the sub-population of all symmetrically dividing cells. We demonstrate that our model explains the experimentally observed variability of lineage sizes and derive the quantitative relationship between survival time and lineage size. We also show that our model implies the existence of a regulatory mechanism which keeps the size of the symmetrically dividing cell population constant. Our results provide quantitative insight into the clonal composition of neural organoids and how it arises. This is relevant for many applications of neural organoids, and similar processes may occur in other developing tissues both in vitro and in vivo.

Spatial organization of astrocyte clones: The role of developmental progenitor timing.

Astrocytes represent a diverse and morphologically complex group of glial cells critical for shaping and maintaining nervous system homeostasis, as well as responding to injuries. Understanding the origins of astroglial heterogeneity, originated from a limited number of progenitors, has been the focus of many studies. Most of these investigations have centered on protoplasmic and pial astrocytes, while the clonal relationship of fibrous astrocytes or juxtavascular astrocytes has remained relatively unexplored. In this study, we sought to elucidate the morphological diversity and clonal distribution of astrocytes across adult cortical layers, with particular emphasis on their ontogenetic origins. Using the StarTrack lineage tracing tool, we explored the characteristics of adult astroglial clones derived from single and specific progenitors at various embryonic stages. Our results revealed a heterogeneous spatial distribution of astroglial clones, characterized by variations in location, clonal size, and rostro-caudal dispersion. While a considerable proportion of clones were confined within specific cortical layers, others displayed sibling cells crossing layer boundaries. Notably, we observed a correlation between clone location and developmental stage at earlier embryonic stages, although this relationship diminished in later stages. Fibrous astrocyte clones were exclusively confined to the corpus callosum. In contrast, protoplasmic or juxtavascular clones were located in either the upper or lower cortical layers, with certain clones displayed sibling cells distributed across both regions. Our findings underscore the developmental origins and spatial distribution of astroglial clones within cortical layers, providing new insights into the interplay between their morphology, clonal sizes, and progenitor heterogeneity.

[A temporal mechanism for the generation of neuronal diversity]. / Un mécanisme temporel pour la génération de la diversité neuronale.

One of the greatest challenges in neuroscience is to understand how a complex structure, such as the brain, is built. Spatial and temporal patternings of neuronal progenitors are responsible for the generation of most of the neuronal diversity observed in the brain. This review focuses on the temporal patterning of neuronal progenitors, i.e. the sequential expression of transcription factors that changes the capacity of stem cells to generate different neuronal types, and which is conserved in animals. Recent papers have offered a near complete understanding of the mechanism of temporal patterning in the developing visual system of Drosophila, and of how this contributes to the specification of diverse neuronal identities, which are then maintained by the sustained expression of downstream transcription factors. The insect visual system provides a unique model to study the evolution of neuronal cell types, as well as the evolution of neurodevelopmental mechanisms that generate them.

Title: Un mécanisme temporel pour la génération de la diversité neuronale. Abstract: L'un des plus grands défis des neurosciences est de comprendre comment une structure complexe, telle que le cerveau, se construit. L'encodage spatial et temporel des progéniteurs neuronaux permet la génération de l'essentiel de la diversité neuronale. Cette revue se concentre sur l'expression séquentielle de facteurs de transcription temporels, qui modifie la capacité des cellules souches à générer différents types de neurones et qui est conservée chez plusieurs espèces animales. Des publications récentes ont permis, en particulier, une compréhension fine de ce processus au cours du développement du système visuel de la drosophile, en éclairant la manière dont il contribue à la spécification de diverses identités neuronales. Le système visuel des insectes constitue un modèle unique pour étudier l'évolution des mécanismes neurodéveloppementaux qui génèrent la diversité neuronale.

The Origin and Regulation of Neuromesodermal Progenitors (NMPs) in Embryos.

Neuromesodermal progenitors (NMPs), serving as the common origin of neural and paraxial mesodermal development in a large part of the trunk, have recently gained significant attention because of their critical importance in the understanding of embryonic organogenesis and the design of in vitro models of organogenesis. However, the nature of NMPs at many essential points remains only vaguely understood or even incorrectly assumed. Here, we discuss the nature of NMPs, focusing on their dynamic migratory behavior during embryogenesis and the mechanisms underlying their neural vs. mesodermal fate choice. The discussion points include the following: (1) How the sinus rhomboidals is organized; the tissue where the neural or mesodermal fate choice of NMPs occurs. (2) NMPs originating from the broad posterior epiblast are associated with Sox2 N1 enhancer activity. (3) Tbx6-dependent Sox2 repression occurs during NMP-derived paraxial mesoderm development. (4) The nephric mesenchyme, a component of the intermediate mesoderm, was newly identified as an NMP derivative. (5) The transition of embryonic tissue development from tissue-specific progenitors in the anterior part to that from NMPs occurs at the forelimb bud axial level. (6) The coexpression of Sox2 and Bra in NMPs is conditional and is not a hallmark of NMPs. (7) The ability of the NMP pool to sustain axial embryo growth depends on Wnt3a signaling in the NMP population. Current in vitro models of NMPs are also critically reviewed.

Generation of induced pluripotent stem cells from rat fibroblasts and optimization of its differentiation into mature functional neurons.

BACKGROUND: Induced pluripotent stem cells (iPSCs) derived neural stem cells (NSCs) provide a potential for autologous neural transplantation therapy following neurological insults. Thus far, in preclinical studies the donor iPSCs-NSCs are mostly of human or mouse origin with concerns centering around graft rejection when applied to rat brain injury models. For better survival and integration of transplanted cells in the injured brain in rat models, use of rat-iPSC-NSCs and in combination with biomaterials is of advantageous. Herein, we report a detailed method in generating rat iPSCs with improved reprogramming efficiency and differentiation into neurons. NEW METHOD: Rat fibroblasts were reprogrammed into iPSCs with polybrene and EF1α-STEMCCA-LoxP lentivirus vector. Pluripotency characterization, differentiation into neuronal linage cells were assessed with RT-qPCR, Western blotting, immunostaining and patch-clamp methods. Cells were cultured in a custom-designed integrin array system as well as in a hydrogel-based 3D condition. RESULTS: We describe a thorough method for the generation of rat-iPSC-NSCs, and identify integrin αvß8 as a substrate for the optimal growth of rat-iPSC-NSCs. Furthermore, with hydrogel as the supporting biomaterial in the 3-D culture, when combined with integrin αvß8 binding peptide, it forms a conducive environment for optimal growth and differentiation of iPSC-NSCs into mature neurons. COMPARISON WITH EXISTING METHODS: Published studies about rat-iPSC-NSCs are rare. This study provides a detailed protocol for the generation of rat iPSC-NSCs and optimal growth conditions for neuronal differentiation. Our method is useable for studies to assess the utility of rat iPSC-NSCs for neural transplantation in rat brain injury models.

Neuron and Brain Maturation 2.0.

The mammalian central nervous system (CNS) is built up during embryogenesis by neural stem cells located in the periventricular germinal layers which undergo multiple division cycles [...].

Pregnancy-responsive pools of adult neural stem cells for transient neurogenesis in mothers.

Adult neural stem cells (NSCs) contribute to lifelong brain plasticity. In the adult mouse ventricular-subventricular zone, NSCs are heterogeneous and, depending on their location in the niche, give rise to different subtypes of olfactory bulb (OB) interneurons. Here, we show that multiple regionally distinct NSCs, including domains that are usually quiescent, are recruited on different gestation days during pregnancy. Synchronized activation of these adult NSC pools generates transient waves of short-lived OB interneurons, especially in layers with less neurogenesis under homeostasis. Using spatial transcriptomics, we identified molecular markers of pregnancy-associated interneurons and showed that some subsets are temporarily needed for own pup recognition. Thus, pregnancy triggers transient yet behaviorally relevant neurogenesis, highlighting the physiological relevance of adult stem cell heterogeneity.

Subventricular zone cytogenesis provides trophic support for neural repair in a mouse model of stroke.

Stroke enhances proliferation of neural precursor cells within the subventricular zone (SVZ) and induces ectopic migration of newborn cells towards the site of injury. Here, we characterize the identity of cells arising from the SVZ after stroke and uncover a mechanism through which they facilitate neural repair and functional recovery. With genetic lineage tracing, we show that SVZ-derived cells that migrate towards cortical photothrombotic stroke in mice are predominantly undifferentiated precursors. We find that ablation of neural precursor cells or conditional knockout of VEGF impairs neuronal and vascular reparative responses and worsens recovery. Replacement of VEGF is sufficient to induce neural repair and recovery. We also provide evidence that CXCL12 from peri-infarct vasculature signals to CXCR4-expressing cells arising from the SVZ to direct their ectopic migration. These results support a model in which vasculature surrounding the site of injury attracts cells from the SVZ, and these cells subsequently provide trophic support that drives neural repair and recovery.

Parental folic acid deficiency delays neurobehavioral development in rat offspring by inhibiting the differentiation of neural stem cells into neurons.

Maternal folate status during pregnancy is associated with the neurodevelopment of offspring; however, study results on the association between paternal folate status and offspring neurodevelopment are inconsistent. This study aimed to explore whether parental folic acid deficiency affects the neurobehavioral development of offspring by affecting the differentiation of neural stem cells (NSCs) into neurons. In the present study, the offspring were divided into four groups: parental folic acid deficient group (D-D), maternal folic acid deficient and paternal folic acid normal group (D-N), maternal folic acid normal and paternal folic acid deficient group (N-D), and parental folic acid normal group (N-N). For in vivo study, neurobehavioral indexes, and neuron-specific nuclear protein (NeuN) and glial fibrillary acidic protein (GFAP) expression in the brain hippocampus and cerebral cortex of offspring were measured at different time points. For in vitro study, NSCs were cultured from the hippocampus and striatum, and neuronal and astrocytic differentiation were measured. The results demonstrated that parental folic acid deficiency decreased the brain folate level in offspring, delayed early sensory-motor reflex development, impaired spatial learning and memory ability in adolescence and adulthood, decreased differentiation of NSCs into neurons and increased differentiation of NSCs into astrocytes in vivo and in vitro. These impacts on the neurodevelopment of offspring were most pronounced in D-D group, followed by D-N group and N-D group. In conclusion, parental folic acid deficiency inhibits the neurobehavioral development of offspring, possibly by inhibiting the differentiation of NSCs into neurons.

Molecular programs of regional specification and neural stem cell fate progression in macaque telencephalon.

During early telencephalic development, intricate processes of regional patterning and neural stem cell (NSC) fate specification take place. However, our understanding of these processes in primates, including both conserved and species-specific features, remains limited. Here, we profiled 761,529 single-cell transcriptomes from multiple regions of the prenatal macaque telencephalon. We deciphered the molecular programs of the early organizing centers and their cross-talk with NSCs, revealing primate-biased galanin-like peptide (GALP) signaling in the anteroventral telencephalon. Regional transcriptomic variations were observed along the frontotemporal axis during early stages of neocortical NSC progression and in neurons and astrocytes. Additionally, we found that genes associated with neuropsychiatric disorders and brain cancer risk might play critical roles in the early telencephalic organizers and during NSC progression.

Crosstalk between Adult Hippocampal Neurogenesis and Its Role in Alzheimer's Disease.

The functional and developmental unit of neurogenesis is neural stem cells (NSCs). These NSCs have self-renewal capacity and produce new neurons throughout life in different neurogenic niche. Neurogenesis in adult brain is associated with synaptic plasticity, learning, and memory in dentate gyrus (DG) of hippocampus and olfactory bulb. Remarkably, weakened neurogenesis has been viewed before the onset of different pathological hallmarks of neurological disorders. In this review, we have provided evidence which implicates impaired neurogenesis as a culprit in age associated neurological disorders with greater emphasis on Alzheimer's disease (AD). Moreover, an insight about the molecular and cellular regulation linked with altered neurogenesis in young and aging brain has also been discussed. This review further summarizes the therapeutic strategies for targeting the manipulation of the neural stem cell pool and factors affecting the pool involved in AD.

Regulation of Primary Cilium Length by O-GlcNAc during Neuronal Development in a Human Neuron Model.

The primary cilium plays critical roles in the homeostasis and development of neurons. Recent studies demonstrate that cilium length is regulated by the metabolic state of cells, as dictated by processes such as glucose flux and O-GlcNAcylation (OGN). The study of cilium length regulation during neuron development, however, has been an area left largely unexplored. This project aims to elucidate the roles of O-GlcNAc in neuronal development through its regulation of the primary cilium. Here, we present findings suggesting that OGN levels negatively regulate cilium length on differentiated cortical neurons derived from human-induced pluripotent stem cells. In neurons, cilium length increased significantly during maturation (after day 35), while OGN levels began to drop. Long-term perturbation of OGN via drugs, which inhibit or promote its cycling, during neuron development also have varying effects. Diminishing OGN levels increases cilium length until day 25, when neural stem cells expand and undergo early neurogenesis, before causing cell cycle exit defects and multinucleation. Elevating OGN levels induces greater primary cilia assembly but ultimately results in the development of premature neurons, which have higher insulin sensitivity. These results indicate that OGN levels and primary cilium length are jointly critical in proper neuron development and function. Understanding the interplays between these two nutrient sensors, O-GlcNAc and the primary cilium, during neuron development is important in paving connections between dysfunctional nutrient-sensing and early neurological disorders.

Bioengineered perfused human brain microvascular networks enhance neural progenitor cell survival, neurogenesis, and maturation.

Neural progenitor cells (NPCs) have the capability to self-renew and differentiate into neurons and glial cells. In the adult brain, NPCs are found near brain microvascular networks (BMVNs) in specialized microenvironments called the neurovascular niche (NVN). Although several in vitro NVN models have been previously reported, most do not properly recapitulate the intimate cellular interactions between NPCs and perfused brain microvessels. Here, we developed perfused BMVNs composed of primary human brain endothelial cells, pericytes, and astrocytes within microfluidic devices. When induced pluripotent stem cell-derived NPCs were introduced into BMVNs, we found that NPC survival, neurogenesis, and maturation were enhanced. The application of flow during BMVN coculture was also beneficial for neuron differentiation. Collectively, our work highlighted the important role of BMVNs and flow in NPC self-renewal and neurogenesis, as well as demonstrated our model's potential to study the biological and physical interactions of human NVN in vitro.

Developmental stage of transplanted neural progenitor cells influences anatomical and functional outcomes after spinal cord injury in mice.

Neural progenitor cell (NPC) transplantation is a promising therapeutic strategy for replacing lost neurons following spinal cord injury (SCI). However, how graft cellular composition influences regeneration and synaptogenesis of host axon populations, or recovery of motor and sensory functions after SCI, is poorly understood. We transplanted developmentally-restricted spinal cord NPCs, isolated from E11.5-E13.5 mouse embryos, into sites of adult mouse SCI and analyzed graft axon outgrowth, cellular composition, host axon regeneration, and behavior. Earlier-stage grafts exhibited greater axon outgrowth, enrichment for ventral spinal cord interneurons and Group-Z spinal interneurons, and enhanced host 5-HT+ axon regeneration. Later-stage grafts were enriched for late-born dorsal horn interneuronal subtypes and Group-N spinal interneurons, supported more extensive host CGRP+ axon ingrowth, and exacerbated thermal hypersensitivity. Locomotor function was not affected by any type of NPC graft. These findings showcase the role of spinal cord graft cellular composition in determining anatomical and functional outcomes following SCI.

Expandable Sendai-Virus-Reprogrammed Human iPSC-Neuronal Precursors: In Vivo Post-Grafting Safety Characterization in Rats and Adult Pig.

One of the challenges in clinical translation of cell-replacement therapies is the definition of optimal cell generation and storage/recovery protocols which would permit a rapid preparation of cell-treatment products for patient administration. Besides, the availability of injection devices that are simple to use is critical for potential future dissemination of any spinally targeted cell-replacement therapy into general medical practice. Here, we compared the engraftment properties of established human-induced pluripotent stem cells (hiPSCs)-derived neural precursor cell (NPCs) line once cells were harvested fresh from the cell culture or previously frozen and then grafted into striata or spinal cord of the immunodeficient rat. A newly developed human spinal injection device equipped with a spinal cord pulsation-cancelation magnetic needle was also tested for its safety in an adult immunosuppressed pig. Previously frozen NPCs showed similar post-grafting survival and differentiation profile as was seen for freshly harvested cells. Testing of human injection device showed acceptable safety with no detectable surgical procedure or spinal NPCs injection-related side effects.

The vasculature of neurogenic niches: Properties and function.

In the adult rodent brain, neural stem cells (NSCs) reside in the subventricular zone (SVZ) of the lateral ventricles and the subgranular zone (SGZ) of the hippocampus. In these areas, NSCs and their progeny integrate intrinsic signals and extrinsic cues provided by their microenvironment that control their behavior. The vasculature in the SVZ and SGZ, and the choroid plexus (ChP) in the SVZ, have emerged as critical compartments of the neurogenic niches as they provide a rich repertoire of cues to regulate NSC quiescence, proliferation, self-renewal and differentiation. Physical contact between NSCs and blood vessels is also a feature within the niches and supports different processes such as quiescence, migration and vesicle transport. In this review, we provide a description of the brain and choroid plexus vasculature in both stem cell niches, highlighting the main properties and role of the vasculature in each niche. We also summarize the current understanding of how blood vessel- and ChP-derived signals influence the behavior of NSCs in physiological adulthood, as well as upon aging.

Profiling spatiotemporal gene expression of the developing human spinal cord and implications for ependymoma origin.

The spatiotemporal regulation of cell fate specification in the human developing spinal cord remains largely unknown. In this study, by performing integrated analysis of single-cell and spatial multi-omics data, we used 16 prenatal human samples to create a comprehensive developmental cell atlas of the spinal cord during post-conceptional weeks 5-12. This revealed how the cell fate commitment of neural progenitor cells and their spatial positioning are spatiotemporally regulated by specific gene sets. We identified unique events in human spinal cord development relative to rodents, including earlier quiescence of active neural stem cells, differential regulation of cell differentiation and distinct spatiotemporal genetic regulation of cell fate choices. In addition, by integrating our atlas with pediatric ependymomas data, we identified specific molecular signatures and lineage-specific genes of cancer stem cells during progression. Thus, we delineate spatiotemporal genetic regulation of human spinal cord development and leverage these data to gain disease insight.

Hyperbaric Oxygenation Prevents Loss of Immature Neurons in the Adult Hippocampal Dentate Gyrus Following Brain Injury.

A growing body of evidence suggests that hyperbaric oxygenation (HBO) may affect the activity of adult neural stem cells (NSCs). Since the role of NSCs in recovery from brain injury is still unclear, the purpose of this study was to investigate the effects of sensorimotor cortex ablation (SCA) and HBO treatment (HBOT) on the processes of neurogenesis in the adult dentate gyrus (DG), a region of the hippocampus that is the site of adult neurogenesis. Ten-week-old Wistar rats were divided into groups: Control (C, intact animals), Sham control (S, animals that underwent the surgical procedure without opening the skull), SCA (animals in whom the right sensorimotor cortex was removed via suction ablation), and SCA + HBO (operated animals that passed HBOT). HBOT protocol: pressure applied at 2.5 absolute atmospheres for 60 min, once daily for 10 days. Using immunohistochemistry and double immunofluorescence labeling, we show that SCA causes significant loss of neurons in the DG. Newborn neurons in the subgranular zone (SGZ), inner-third, and partially mid-third of the granule cell layer are predominantly affected by SCA. HBOT decreases the SCA-caused loss of immature neurons, prevents reduction of dendritic arborization, and increases proliferation of progenitor cells. Our results suggest a protective effect of HBO by reducing the vulnerability of immature neurons in the adult DG to SCA injury.

Radial stem astrocytes (aka neural stem cells): Identity, development, physio-pathology, and therapeutic potential.

Adult neurogenesis is a striking example of neuroplasticity, which enables adaptive network remodelling in response to all forms of environmental stimulation in physiological and pathological contexts. Dysregulation or cessation of adult neurogenesis contributes to neuropathology negatively affecting brain functions and hampering regeneration of the nervous tissue while targeting adult neurogenesis may provide the basis for potential therapeutic interventions. Neural stem cells in the adult mammalian brain are at the core and the entry point of adult neurogenesis. By their origin and properties, these cells belong to astroglia, and are represented by stem radial astrocytes (RSA) which exhibit multipotent "stemness". In the neurogenic niches, RSA interact with other cellular components, including protoplasmic astrocytes, which in turn regulate their neurogenic activity. In pathology, RSA become reactive, which affects their neurogenic capabilities, whereas reactive parenchymal astrocytes up-regulate stem cell hallmarks and are able to generate progeny that remain within astrocyte lineage. What makes RSA special is their multipotency, represented by self-renewing capacity capability to generate other cellular types as progeny. A broad understanding of the cellular features of RSA and parenchymal astrocytes provides an insight into the machinery that promotes/suppresses adult neurogenesis, clarifying principles of network remodelling. In this review, we discuss the cellular hallmarks, research tools, and models of RSA and astrocytes of the subventricular zone along the lateral ventricle and dentate gyrus of the hippocampus. We also discuss RSA in ageing, which has a great impact on the proliferative capacity of RSA, as well as the potential of RSA and astrocytes in therapeutic strategies aimed at cell replacement and regeneration.