Stem Cells In Brain Research Articles

Quantification of Global DNA Hydroxymethylation Level Using UHRF2 SRA-Luciferase Based on Bioluminescence Resonance Energy Transfer.

5-Methylcytosine (5mC) plays an important role in the regulation of gene expression. Ten-eleven translocation (TET) continuously oxidizes 5mC to 5-hydroxymethylcytosine (5hmC), 5-formylcytosine (5fC), and 5-carboxylcytosine (5caC). High levels of 5hmC are found in the brain and embryonic stem cells, while global hydroxymethylation levels are reduced in several cancer cells. Moreover, alterations in hydroxymethylation levels occur in neurological diseases, such as Alzheimer's disease and Parkinson's disease. In this study, a convenient sensing method for the determination of global hydroxymethylation levels was developed. A bioluminescence resonance energy transfer (BRET) assay for global methylation level determination has been previously reported. In the assay, BOBO-3 DNA intercalating dye is excited by the bioluminescence of methyl-CpG-binding domain-fused firefly luciferase (MBD-Fluc); that is, the BRET signal depends on the content of methylated CpG on genomic DNA. To develop a hydroxymethylation level sensing method, SET- and RING-associated (SRA) domain of ubiquitin-like with PHD and RING finger domains 2 (UHRF2)-fused Fluc (UHRF2 SRA-Fluc) was prepared. UHRF2 SRA is known to bind to both hydroxymethylated and methylated CpG sites; thus, MBD was utilized to mask the methylated CpG on genomic DNA. We demonstrated that the BRET signal between UHRF2 SRA-Fluc and BOBO-3 depends on the global hydroxymethylation level in the presence of MBD (R2 = 0.99, and relative standard deviation < 2.3%). The limit of detection for hydroxymethylated genomic DNA was 0.75 ng μL-1. In this assay, the global hydroxymethylation level was quantified within 40 min in a single tube, indicating that the assay would be utilized not only for clinical diagnostics but also for the elucidation of 5hmC functions.

Hematopoietic Stem Cell Transplantation for Neurological Disorders: A Focus on Inborn Errors of Metabolism.

Hematopoietic stem cells have been investigated and applied for the treatment of certain neurological disorders for a long time. Currently, their therapeutic potential is harnessed in autologous and allogeneic hematopoietic stem cell transplantation (HSCT). Autologous HSCT is helpful in immune-mediated neurological diseases such as Multiple Sclerosis. However, clinical benefits derive more from the immunosuppressive conditioning regimen than the interaction between stem cells and the nervous system. Mainly used for hematologic malignancies, allogeneic HSCT explores the therapeutic potential of donor-derived hematopoietic stem cells. In the neurological setting, it has proven to be most valuable in Inborn Errors of Metabolism, a large spectrum of multisystem disorders characterized by congenital deficiencies in enzymes involved in metabolic pathways. Inborn Errors of Metabolism such as X-linked Adrenoleukodystrophy present with brain accumulation of enzymatic substrates that result in progressive inflammatory demyelination. Allogeneic HSCT can halt ongoing inflammatory neural destruction by replacing hematopoietic-originated microglia with donor-derived myeloid precursors. Microglia, the only neural cells successfully transplanted thus far, are the most valuable source of central nervous system metabolic correction and play a significant role in the crosstalk between the brain and hematopoietic stem cells. After transplantation, engrafted donor-derived myeloid cells modulate the neural microenvironment by recapitulating microglial functions and enhancing repair mechanisms such as remyelination. In some disorders, additional benefits result from the donor hematopoietic stem cell secretome that cross-corrects neighboring neural cells via mannose-6-phosphatase paracrine pathways. The limitations of allogeneic HSCT in this setting relate to the slow turnover of microglia and complications such as graft-vs.-host disease. These restraints have accelerated the development of hematopoietic stem cell gene therapy, where autologous hematopoietic stem cells are collected, manipulated ex vivo to overexpress the missing enzyme, and infused back into the patient. With this cellular drug vehicle strategy, the brain is populated by improved cells and exposed to supraphysiological levels of the flawed protein, resulting in metabolic correction. This review focuses on the mechanisms of brain repair resulting from HSCT and gene therapy in Inborn Errors of Metabolism. A brief mention will also be made on immune-mediated nervous system diseases that are treated with this approach.

Divergence between Neuronal and Oligodendroglial Cell Fate, in Postnatal Brain Neural Stem Cells, Leads to Divergent Properties in Polymorphic In Vitro Assays.

Two main stem cell pools exist in the postnatal mammalian brain that, although they share some “stemness” properties, also exhibit significant differences. Multipotent neural stem cells survive within specialized microenvironments, called niches, and they are vulnerable to ageing. Oligodendroglial lineage-restricted progenitor cells are widely distributed in the brain parenchyma and are more resistant to the effects of ageing. Here, we create polymorphic neural stem cell cultures and allow cells to progress towards the neuronal and the oligodendroglial lineage. We show that the divergence of cell fate is accompanied by a divergence in the properties of progenitors, which reflects their adaptation to life in the niche or the parenchyma. Neurogenesis shows significant spatial restrictions and a dependence on laminin, a major niche component, while oligodendrogenesis shows none of these constraints. Furthermore, the blocking of integrin-β1 leads to opposing effects, reducing neurogenesis and enhancing oligodendrogenesis. Therefore, polymorphic neural stem cell assays can be used to investigate the divergence of postnatal brain stem cells and also to predict the in vivo effects of potential therapeutic molecules targeting stem and progenitor cells, as we do for the microneurotrophin BNN-20.

Lineage Tracing of Inducible Fluorescently-Labeled Stem Cells in the Adult Mouse Brain.

A telomerase reverse transcriptase (Tert) lineage-tracing mouse line was developed to investigate the behavior and fate of adult tissue stem cells, by crossing the 'Tet-On' system oTet-Cre mouse with a novel reverse tetracycline transactivator (rtTA) transgene linked to the Tert promoter, which we have demonstrated marks a novel population of adult brain stem cells. Here, administration of the tetracycline derivative doxycycline to mTert-rtTA::oTet-Cre mice will indelibly mark a population of cells that express a 4.4 kb fragment of the promoter region of the gene Tert. When combined the Rosa-mTmG reporter, mTert-rtTA::oTet-Cre::Rosa-mTmG mice will express membrane tdTomato (mTomato) until doxycycline treatment induces the replacement of mTomato expression with membrane EGFP (mGFP) in cells that also express Tert. Therefore, when these triple-transgenic lineage tracing mice receive doxycycline (the "pulse" period during which TERT expressing cells are marked), these cells will become indelibly marked mGFP+ cells, which can be tracked for any desirable amount of time after doxycycline removal (the "chase" period), even if Tert expression is subsequently lost. Brains are then perfusion-fixed and processed for immunofluorescence and other downstream applications in order to interpret changes to stem cell activation, proliferation, lineage commitment, migration to various brain niches, and differentiation to mature cell types. Using this system, any rtTA mouse can be mated to oTet-Cre and a Rosa reporter to conduct doxycycline-inducible "pulse-chase" lineagetracing experiments using markers of stem cells.

Lineage Tracing of Inducible Fluorescently-Labeled Stem Cells in the Adult Mouse Brain

Lineage Tracing of Inducible Fluorescently-Labeled Stem Cells in the Adult Mouse Brain

Bilirubin Encephalopathy.

Hyperbilirubinemia is commonly seen in neonates. Though hyperbilirubinemia is typically asymptomatic, severe elevation of bilirubin levels can lead to acute bilirubin encephalopathy and progress to kernicterus spectrum disorder, a chronic condition characterized by hearing loss, extrapyramidal dysfunction, ophthalmoplegia, and enamel hypoplasia. Epidemiological data show that the implementation of universal pre-discharge bilirubin screening programs has reduced the rates of hyperbilirubinemia-associated complications. However, acute bilirubin encephalopathy and kernicterus spectrum disorder are still particularly common in low- and middle-income countries. The understanding of the genetic and biochemical processes that increase the susceptibility of defined anatomical areas of the central nervous system to the deleterious effects of bilirubin may facilitate the development of effective treatments for acute bilirubin encephalopathy and kernicterus spectrum disorder. Scoring systems are available for the diagnosis and severity grading of these conditions. The treatment of hyperbilirubinemia in newborns relies on the use of phototherapy and exchange transfusion. However, novel therapeutic options including deep brain stimulation, brain-computer interface, and stem cell transplantation may alleviate the heavy disease burden associated with kernicterus spectrum disorder. Despite improved screening and treatment options, the prevalence of acute bilirubin encephalopathy and kernicterus spectrum disorder remains elevated in low- and middle-income countries. The continued presence and associated long-term disability of these conditions warrant further research to improve their prevention and management.

NanoDam identifies Homeobrain (ARX) and Scarecrow (NKX2.1) as conserved temporal factors in the Drosophila central brain and visual system.

Temporal patterning of neural progenitors is an evolutionarily conserved strategy for generating neuronal diversity. Type II neural stem cells in the Drosophila central brain produce transit-amplifying intermediate neural progenitors (INPs) that exhibit temporal patterning. However, the known temporal factors cannot account for the neuronal diversity in the adult brain. To search for missing factors, we developed NanoDam, which enables rapid genome-wide profiling of endogenously tagged proteins invivo with a single genetic cross. Mapping the targets of known temporal transcription factors with NanoDam revealed that Homeobrain and Scarecrow (ARX and NKX2.1 orthologs) are also temporal factors. We show that Homeobrain and Scarecrow define middle-aged and late INP temporal windows and play a role in cellular longevity. Strikingly, Homeobrain and Scarecrow have conserved functions as temporal factors in the developing visual system. NanoDam enables rapid cell-type-specific genome-wide profiling with temporal resolution and is easily adapted for use in higher organisms.

Bioorthogonally Cross-Linked Hyaluronan-Laminin Hydrogels for 3D Neuronal Cell Culture and Biofabrication.

Laminins (LNs) are key components in the extracellular matrix of neuronal tissues in the developing brain and neural stem cell niches. LN-presenting hydrogels can provide a biologically relevant matrix for the 3D culture of neurons toward development of advanced tissue models and cell-based therapies for the treatment of neurological disorders. Biologically derived hydrogels are rich in fragmented LN and are poorly defined concerning composition, which hampers clinical translation. Engineered hydrogels require elaborate and often cytotoxic chemistries for cross-linking and LN conjugation and provide limited possibilities to tailor the properties of the materials. Here a modular hydrogel system for neural 3D cell cultures, based on hyaluronan and poly(ethylene glycol), that is cross-linked and functionalized with human recombinant LN-521 using bioorthogonal copper-free click chemistry, is shown. Encapsulated human neuroblastoma cells demonstrate high viability and grow into spheroids. Long-term neuroepithelial stem cells (lt-NES) cultured in the hydrogels can undergo spontaneous differentiation to neural fate and demonstrate significantly higher viability than cells cultured without LN. The hydrogels further support the structural integrity of 3D bioprinted structures and maintain high viability of bioprinted and syringe extruded lt-NES, which can facilitate biofabrication and development of cell-based therapies.

Developmental thyroid disruption permanently affects the neuroglial output in the murine subventricular zone.

SummaryNeural stem cells (NSCs) in the adult brain are a source of neural cells for brain injury repair. We investigated whether their capacity to generate new neurons and glia is determined by thyroid hormone (TH) during development because serum levels peak during postnatal reorganization of the main NSC niche, the subventricular zone (SVZ). Re-analysis of mouse transcriptome data revealed increased expression of TH transporters and deiodinases in postnatal SVZ NSCs, promoting local TH action, concomitant with a burst in neurogenesis. Inducing developmental hypothyroidism reduced NSC proliferation, disrupted expression of genes implicated in NSC determination and TH signaling, and altered the neuron/glia output in newborns. Three-month-old adult mice recovering from developmental hypothyroidism had fewer olfactory interneurons and underperformed on short-memory odor tests, dependent on SVZ neurogenesis. Our data provide readouts permitting comparison with adverse long-term events following thyroid disruptor exposure and ideas regarding the etiology of prevalent neurodegenerative diseases in industrialized countries.

Impaired KDM2B-mediated PRC1 recruitment to chromatin causes defective neural stem cell self-renewal and ASD/ID-like behaviors

SummaryRecent clinical studies report that chromosomal 12q24.31 microdeletions are associated with autism spectrum disorder (ASD) and intellectual disability (ID). However, the causality and underlying mechanisms linking 12q24.31 microdeletions to ASD/ID remain undetermined. Here we show Kdm2b, one gene located in chromosomal 12q24.31, plays a critical role in maintaining neural stem cells (NSCs) in the mouse brain. Loss of the CxxC-ZF domain of KDM2B impairs its function in recruiting Polycomb repressive complex 1 (PRC1) to chromatin, resulting in de-repression of genes involved in cell apoptosis, cell-cycle arrest, NSC senescence, and loss of NSC populations in the brain. Of importance, the Kdm2b mutation is sufficient to induce ASD/ID-like behavioral and memory deficits. Thus, our study reveals a critical role of KDM2B in normal brain development, a causality between the Kdm2b mutation and ASD/ID-like phenotypes in mice, and potential molecular mechanisms linking the function of KDM2B-PRC1 in transcriptional regulation to the 12q24.31 microdeletion-associated ASD/ID.

Heterogeneity of quiescent and active neural stem cells in the postnatal brain.

In the postnatal mammalian brain, neurogenic activity is retained in anatomically restricted areas, driven by pools of Neural Stem Cells (NSCs). These cells and their progeny have been studied intensively as potential targets for regenerative treatments, aiming at either their in situ manipulation or their use as sources of cells for transplantation-based strategies. Although their full identity, heterogeneity and differentiation potential remain elusive, due to the absence of specific cell-type markers, our knowledge of their properties is constantly expanding. Here, we focus on the NSC niche that is located at the Subependymal Zone (SEZ/ also known as Subventricular Zone) of the lateral ventricles of the brain. We review, summarize and explain the different faces of the NSC, as they have been described, using a wide range of experimental approaches, over a time-frame of three decades: the primitive, definitive, quiescent or activated NSC. We also review the growing evidence of the existence of latent NSCs outside of niches, in the brain parenchyma, that constitute promising new therapeutic targets, complemented by the novel technologies of in vivo cell reprogramming.

Toll-Like Receptor 4 Gene Knockout Increases the Function of Exogenous Bone Marrow Stem Cells in Ischemic Brain

The present study was to investigate the effects of toll-like receptor 4 on transplanted bone marrow stem cells in the ischemic brain. Focal cerebral ischemia was induced by permanent occlusion of middle cerebral artery in toll-like receptor 4 gene knockout mice and wild type control mice. Bone marrow stem cells were injected into lateral cerebral ventricle. The cylinder tests, in vivo image, polymerase chain reaction array, etc. were applied to evaluate the neurological function and the underlying mechanisms. On the 10th d after permanent occlusion of middle cerebral artery, the ratio of right limb movement in group 1 was significantly higher than that in wild type and toll-like receptor 4 gene knockout mice without injection of bone marrow stem cells (p<0.05). In vivo imaging showed that on the 3rd d after injection of bone marrow stem cells, the fluorescence intensities in group 3 and group 1 were significantly enhanced compared with that on the 1st d and 7th d. There were more green fluorescent protein positive cells in group 3 and group 1 compared with sham controls (p<0.05). Amount of green fluorescent protein positive cells in group 1 was significantly higher compared with group 3. In toll-like receptor 4 gene knockout mice, bone morphogenetic protein 2 messenger ribonucleic acid and fibroblast growth factor 9 messenger ribonucleic acid were up-regulated and interleukin-1-beta protein was decreased, compared with wild type mice. The present study firstly demonstrated that toll-like receptor 4 deficiency further increased the amount of intracerebroventricularly injected bone marrow stem cells in ischemic brain areas, thereby improved neurological function after focal cerebral infarction. The increased expression of bone morphogenetic protein 2 and fibroblast growth factor 9 in the environment of toll-like receptor 4 deficiency might be one of the molecular mechanisms.

Single-Molecule Studies on the Motion and Force Generation of the Kinesin-3 Motor KIF1A.

KIF1A is a neuron-specific member of the kinesin-3 family of microtubule (MT) plus-end-directed motor proteins. It powers the migration of nuclei in differentiating brain stem cells and the transport of synaptic precursors and dense core vesicles in axons. Its dysfunction causes severe neurodevelopmental and neurodegenerative diseases termed KIF1A-associated neurological disorders (KAND). KAND mutations span the entirety of the KIF1A protein sequence, of which the majority are located within the motor domain and are thus predicted to affect the motor's motility and force-generating properties. Unfortunately, the molecular etiologies of KAND remain poorly understood, in part because KIF1A's molecular mechanism remains unclear. Here, we describe detailed methods for how to express a tail-truncated dimeric KIF1A in E. coli cells and provide step-by-step protocols for performing single-molecule studies with total internal reflection fluorescence microscopy and optical tweezers assays, which, when combined with structure-function studies, help to decipher KIF1A's molecular mechanism.

Epigenetics of Mitochondria-Associated Genes in Striated Muscle.

Striated muscle has especially large energy demands. We identified 97 genes preferentially expressed in skeletal muscle and heart, but not in aorta, and found significant enrichment for mitochondrial associations among them. We compared the epigenomic and transcriptomic profiles of the 27 genes associated with striated muscle and mitochondria. Many showed strong correlations between their tissue-specific transcription levels, and their tissue-specific promoter, enhancer, or open chromatin as well as their DNA hypomethylation. Their striated muscle-specific enhancer chromatin was inside, upstream, or downstream of the gene, throughout much of the gene as a super-enhancer (CKMT2, SLC25A4, and ACO2), or even overlapping a neighboring gene (COX6A2, COX7A1, and COQ10A). Surprisingly, the 3′ end of the 1.38 Mb PRKN (PARK2) gene (involved in mitophagy and linked to juvenile Parkinson’s disease) displayed skeletal muscle/myoblast-specific enhancer chromatin, a myoblast-specific antisense RNA, as well as brain-specific enhancer chromatin. We also found novel tissue-specific RNAs in brain and embryonic stem cells within PPARGC1A (PGC-1α), which encodes a master transcriptional coregulator for mitochondrial formation and metabolism. The tissue specificity of this gene’s four alternative promoters, including a muscle-associated promoter, correlated with nearby enhancer chromatin and open chromatin. Our in-depth epigenetic examination of these genes revealed previously undescribed tissue-specific enhancer chromatin, intragenic promoters, regions of DNA hypomethylation, and intragenic noncoding RNAs that give new insights into transcription control for this medically important set of genes.

IL2 Neural stem cell regulation and brain development

Quiescent neural stem cells (NSCs) in the adult mouse brain are the source of neurogenesis that regulates innate and adaptive behaviors. Adult NSCs in the subventricular zone (SVZ) are derived from a subpopulation of embryonic neural stem-progenitor cells (NPCs) that is characterized by a slower cell cycle relative to the more abundant rapid cycling NPCs that build the brain. We have previously shown that slow cell cycle can cause the establishment of adult NSCs at the SVZ, although the underlying mechanism remains unknown. We found that Notch and an effector Hey1 form a module that is upregulated by cell cycle arrest in slowly dividing NPCs. In contrast to the oscillatory expression of the Notch effectors Hes1 and Hes5 in fast cycling progenitors, Hey1 displays a non-oscillatory stationary expression pattern and contributes to the long-term maintenance of NSCs. These findings reveal a novel division of labor in Notch effectors where cell cycle rate biases effector selection and cell fate. I will also discuss the heterogeneity of slowly dividing embryonic NPCs and the lineage relationship between adult NSCs and ependymal cells, which together form the niche for adult neurogenesis at the SVZ.

Blood-Brain Barrier Overview: Structural and Functional Correlation.

The blood-brain barrier (BBB) is a semipermeable and extremely selective system in the central nervous system of most vertebrates, that separates blood from the brain's extracellular fluid. It plays a vital role in regulating the transport of necessary materials for brain function, furthermore, protecting it from foreign substances in the blood that could damage it. In this review, we searched in Google Scholar, Pubmed, Web of Science, and Saudi Digital Library for the various cells and components that support the development and function of this barrier, as well as the different pathways to transport the various molecules between blood and the brain. We also discussed the aspects that lead to BBB dysfunction and its neuropathological consequences, with the identification of some of the most important biomarkers that might be used as a biomarker to predict the BBB disturbances. This comprehensive overview of BBB will pave the way for future studies to focus on developing more specific targeting systems in material delivery as a future approach that assists in combinatorial therapy or nanotherapy to destroy or modify this barrier in pathological conditions such as brain tumors and brain stem cell carcinomas.

Cell-type-specific chromatin occupancy by the pioneer factor Zelda drives key developmental transitions in Drosophila

During Drosophila embryogenesis, the essential pioneer factor Zelda defines hundreds of cis-regulatory regions and in doing so reprograms the zygotic transcriptome. While Zelda is essential later in development, it is unclear how the ability of Zelda to define cis-regulatory regions is shaped by cell-type-specific chromatin architecture. Asymmetric division of neural stem cells (neuroblasts) in the fly brain provide an excellent paradigm for investigating the cell-type-specific functions of this pioneer factor. We show that Zelda synergistically functions with Notch to maintain neuroblasts in an undifferentiated state. Zelda misexpression reprograms progenitor cells to neuroblasts, but this capacity is limited by transcriptional repressors critical for progenitor commitment. Zelda genomic occupancy in neuroblasts is reorganized as compared to the embryo, and this reorganization is correlated with differences in chromatin accessibility and cofactor availability. We propose that Zelda regulates essential transitions in the neuroblasts and embryo through a shared gene-regulatory network driven by cell-type-specific enhancers.

TMOD-25. LATENT SOX9-POSITIVE CELLS BEHIND MYC-DRIVEN MEDULLOBLASTOMA RELAPSE

Abstract Tumor recurrence developing from therapy resistance, immune escape and metastasis is the leading cause of death in medulloblastoma, the most frequent malignant pediatric brain tumor. Amplification of MYC genes is the most common genetic alteration in Group 3 and Group 4 subgroups that constitute two thirds of medulloblastoma. SOX9 is a transcription factor present in stem cells in the normal brain but is limited to rare, quiescent cells in medulloblastoma patients with MYC gene amplifications. By studying paired primary-recurrent patient samples and patient-derived xenografts we here identified significant accumulation of SOX9-positive cells in Group 3 and Group 4 relapses. To follow relapse at the single cell level we developed an inducible dual Tet model of MYC-driven MB, where MYC was re-directed from the treatment-sensitive bulk cells to resistant, dormant SOX9-positive cells by doxycycline. In this model, distant recurrent tumors and spinal metastases developed. SOX9 promoted immune escape, DNA repair suppression and was essential for recurrence. Tumor cell dormancy was non-hierarchical, migratory and depended on MYC suppression by SOX9 to promote relapse. By using computational modeling and treatment we also showed how doxorubicin and MGMT inhibitors were specifically targeting recurrent cells that could be of potential use in future treatments for patients affected by these fatal relapses.

Cell cycle arrest determines adult neural stem cell ontogeny by an embryonic Notch-nonoscillatory Hey1 module

Quiescent neural stem cells (NSCs) in the adult mouse brain are the source of neurogenesis that regulates innate and adaptive behaviors. Adult NSCs in the subventricular zone are derived from a subpopulation of embryonic neural stem-progenitor cells (NPCs) that is characterized by a slower cell cycle relative to the more abundant rapid cycling NPCs that build the brain. Yet, how slow cell cycle can cause the establishment of adult NSCs remains largely unknown. Here, we demonstrate that Notch and an effector Hey1 form a module that is upregulated by cell cycle arrest in slowly dividing NPCs. In contrast to the oscillatory expression of the Notch effectors Hes1 and Hes5 in fast cycling progenitors, Hey1 displays a non-oscillatory stationary expression pattern and contributes to the long-term maintenance of NSCs. These findings reveal a novel division of labor in Notch effectors where cell cycle rate biases effector selection and cell fate.

Role of serotonin in modulating the development and function of adult-born neurons in the olfactory bulb.

Role of serotonin in modulating the development and function of adult-born neurons in the olfactory bulb.