Pioneer statoacoustic neurons guide neuroblast behaviour during otic ganglion assembly.

Cranial ganglia are aggregates of sensory neurons that mediate distinct types of sensation. The statoacoustic ganglion (SAG) develops into several lobes that are spatially arranged to connect appropriately with hair cells of the inner ear. To investigate the cellular behaviours involved in the 3D organization of the SAG, we use high-resolution confocal imaging of single-cell, labelled zebrafish neuroblasts (NBs), photoconversion, photoablation, and genetic perturbations. We show that otic NBs delaminate out of the otic epithelium in an epithelial-mesenchymal transition-like manner, rearranging apical polarity and primary cilia proteins. We also show that, once delaminated, NBs require RhoGTPases in order to perform active migration. Furthermore, tracking of recently delaminated NBs revealed their directed migration and coalescence around a small population of pioneer SAG neurons. These pioneer SAG neurons, not from otic placode origin, populate the coalescence region before otic neurogenesis begins and their ablation disrupts delaminated NB migratory pathways, consequentially affecting SAG shape. Altogether, this work shows for the first time the role of pioneer SAG neurons in orchestrating SAG development.

Human pluripotent stem cell-derived inner ear organoids recapitulate otic development in vitro.

Our molecular understanding of the early stages of human inner ear development has been limited by the difficulty in accessing fetal samples at early gestational stages. As an alternative, previous studies have shown that inner ear morphogenesis can be partially recapitulated using induced pluripotent stem cells directed to differentiate into inner ear organoids (IEOs). Once validated and benchmarked, these systems could represent unique tools to complement and refine our understanding of human otic differentiation and model developmental defects. Here, we provide the first direct comparisons of the early human embryonic otocyst and fetal sensory organs with human IEOs. We use multiplexed immunostaining and single-cell RNA-sequencing to characterize IEOs at three key developmental steps, providing a new and unique signature of in vitro-derived otic placode, epithelium, neuroblasts and sensory epithelia. In parallel, we evaluate the expression and localization of crucial markers at these equivalent stages in human embryos. Together, our data indicate that the current state-of-the-art protocol enables the specification of bona fide otic tissue, supporting the further application of IEOs to inform inner ear biology and disease.

[Structural changes in human brain tissue during prenatal alcoholization at different stages of intrauterine development]. / Strukturnye izmeneniya v tkani golovnogo mozga cheloveka pri prenatal'noi alkogolizatsii na raznykh srokakh vnutriutrobnogo razvitiya.

OBJECTIVE: To assess the degree of influence of intrauterine alcoholization on the formation of various structural components of the brain of human embryos. MATERIAL AND METHODS: Twenty-six samples of embryonic material from 8 to 11 weeks of intrauterine development were studied. The material was divided into four subgroups in accordance with the gestational age (Control 1 - 8-9 weeks of gestation and Control 2 - 10-11 weeks of gestation) and the history of the mother (presence or absence of the diagnosis «Alcoholism stage I-II¼ in the anamnesis). Morphometry was subjected to semi-thin sections stained by Nissl. The diameter and area of each individual tissue element (neuroblasts, glioblasts, vessels of the microvasculature, as well as the determination of the specific area (the ratio of the total area of the studied structure to the area of the entire section) and the calculation of the average number of these structures per unit area of the section, were determined. The AxioVision 4.8 program (Carl Zeiss, Germany) was used for analysis, and the Mann-Whitney test was used for statistical analysis of differences between the samples (significant differences, p<0.05). RESULTS: An insufficient increase in the area of vessels of the microvasculature was revealed in combination with a compensatory increase in their number per unit area of the section in the Alcohol groups compared with intact groups (48.5 µm2 vs 83.3 µm2, p<0.05). When comparing the sizes of glioblasts in the Control and Alcohol subgroups at different stages of development, a lag in the sizes of cellular structures in the Alcohol groups at the initial stages was revealed (average area 21.3 µm2 vs 32.1 µm2; 12.9 µm2 vs 13.3 µm2). When comparing data on later periods, no significant differences were found, only an increase in the specific number of cells in subgroup Alcohol 2 was noted (p<0.05). In neuroblasts, there was also a decrease in cell size with an increase in gestational age both among the Control and among the Alcohol subgroups. However, the cell sizes in Alcohol 2 exceeded those in Control 2 and their number was smaller (p<0.05). CONCLUSION: Alcohol leads to changes in the size and number of neuroblasts, glioblasts and vessels of the microvasculature and, as a result, to a disproportionate development of the entire brain tissue. The changes progress with an increase in the development period.

The Adult Carotid Body: A Germinal Niche at the Service of Physiology.

The carotid body is the most relevant oxygen sensor in mammalian organisms. This organ helps to detect acute changes in PO2, but it is also crucial for the organismal adaptation to a maintained hypoxemia. Profound angiogenic and neurogenic processes take place in the carotid body to facilitate this adaptation process. We have described a plethora of multipotent stem cells and restricted progenitors, from both vascular and neuronal lineages, existing in the quiescent normoxic carotid body, ready to contribute to organ growth and adaptation upon the arrival of the hypoxic stimulus. Our deep understanding of the functioning of this stunning germinal niche will very likely facilitate the management and treatment of an important group of diseases that course with carotid body over-activation and malfunction.

Atypical Neurogenesis, Astrogliosis, and Excessive Hilar Interneuron Loss Are Associated with the Development of Post-Traumatic Epilepsy.

BACKGROUND: Traumatic brain injury (TBI) remains a significant risk factor for post-traumatic epilepsy (PTE). The pathophysiological mechanisms underlying the injury-induced epileptogenesis are under investigation. The dentate gyrus-a structure that is highly susceptible to injury-has been implicated in the evolution of seizure development. METHODS: Utilizing the murine unilateral focal control cortical impact (CCI) injury, we evaluated seizure onset using 24/7 EEG video analysis at 2-4 months post-injury. Cellular changes in the dentate gyrus and hilus of the hippocampus were quantified by unbiased stereology and Imaris image analysis to evaluate Prox1-positive cell migration, astrocyte branching, and morphology, as well as neuronal loss at four months post-injury. Isolation of region-specific astrocytes and RNA-Seq were performed to determine differential gene expression in animals that developed post-traumatic epilepsy (PTE+) vs. those animals that did not (PTE-), which may be associated with epileptogenesis. RESULTS: CCI injury resulted in 37% PTE incidence, which increased with injury severity and hippocampal damage. Histological assessments uncovered a significant loss of hilar interneurons that coincided with aberrant migration of Prox1-positive granule cells and reduced astroglial branching in PTE+ compared to PTE- mice. We uniquely identified Cst3 as a PTE+-specific gene signature in astrocytes across all brain regions, which showed increased astroglial expression in the PTE+ hilus. CONCLUSIONS: These findings suggest that epileptogenesis may emerge following TBI due to distinct aberrant cellular remodeling events and key molecular changes in the dentate gyrus of the hippocampus.

Transcriptional dynamics of delaminating neuroblasts in the mouse otic vesicle.

An abundance of research has recently highlighted the susceptibility of cochleovestibular ganglion (CVG) neurons to noise damage and aging in the adult cochlea, resulting in hearing deficits. Furthering our understanding of the transcriptional cascades that contribute to CVG development may provide insight into how these cells can be regenerated to treat inner ear dysfunction. Here we perform a high-depth single-cell RNA sequencing analysis of the E10.5 otic vesicle and its surrounding tissues, including CVG precursor neuroblasts and emerging CVG neurons. Clustering and trajectory analysis of otic-lineage cells reveals otic markers and the changes in gene expression that occur from neuroblast delamination toward the development of the CVG. This dataset provides a valuable resource for further identifying the mechanisms associated with CVG development from neurosensory competent cells within the otic vesicle.

Epiretinal Amniotic Membrane in Complicated Retinal Detachment: a Clinical and In Vitro Safety Assessment.

INTRODUCTION: Amniotic membrane (AM) is a popular treatment for external ocular diseases. First intraocular implantations in other diseases reported promising results. Here, we review three cases of intravitreal epiretinal human AM (iehAM) transplantation as an adjunct treatment for complicated retinal detachment and analyze clinical safety. Possible cellular rejection reactions against the explanted iehAM were evaluated and its influence was assessed on three retinal cell lines in vitro. METHODS: Three patients with complicated retinal detachment and implanted iehAM during pars plana vitrectomy are retrospectively presented. After removal of the iehAM at subsequent surgery, tissue-specific cellular responses were studied by light microscopy and immunohistochemical staining. We investigated the influence of AM in vitro on retinal pigment epithelial cells (ARPE-19), Müller cells (Mio-M1), and differentiated retinal neuroblasts (661W) . An anti-histone DNA ELISA for cell apoptosis, a BrdU ELISA for cell proliferation, a WST-1 assay for cell viability, and a live/dead assay for cell death were performed. RESULTS: Despite the severity of the retinal detachment, stable clinical outcomes were obtained in all three cases. Immunostaining of the explanted iehAM showed no evidence of cellular immunological rejection. In vitro, there was no statistical significant change in cell death or cell viability nor were proliferative effects detected on ARPE-19, Müller cells, and retinal neuroblasts exposed to AM. CONCLUSION: iehAM was a viable adjuvant with many potential benefits for treatment of complicated retinal detachment. Our investigations could not detect any signs of rejection reactions or toxicity. Further studies are needed to evaluate this potential in more detail.

Origin of Neuroblasts in the Avian Otic Placode and Their Distributions in the Acoustic and Vestibular Ganglia.

The inner ear is a complex three-dimensional sensorial structure with auditory and vestibular functions. This intricate sensory organ originates from the otic placode, which generates the sensory elements of the membranous labyrinth, as well as all the ganglionic neuronal precursors. How auditory and vestibular neurons establish their fate identities remains to be determined. Their topological origin in the incipient otic placode could provide positional information before they migrate, to later segregate in specific portions of the acoustic and vestibular ganglia. To address this question, transplants of small portions of the avian otic placode were performed according to our previous fate map study, using the quail/chick chimeric graft model. All grafts taking small areas of the neurogenic placodal domain contributed neuroblasts to both acoustic and vestibular ganglia. A differential distribution of otic neurons in the anterior and posterior lobes of the vestibular ganglion, as well as in the proximal, intermediate, and distal portions of the acoustic ganglion, was found. Our results clearly show that, in birds, there does not seem to be a strict segregation of acoustic and vestibular neurons in the incipient otic placode.

Rearrangement of cell types in the rat carotid body neurogenic niche induced by chronic intermittent hypoxia.

The carotid body (CB) is a prototypical acute oxygen (O2 )-sensing organ that mediates reflex hyperventilation and increased cardiac output in response to hypoxaemia. CB overactivation, secondary to the repeated stimulation produced by the recurrent episodes of intermittent hypoxia, is believed to contribute to the pathogenesis of sympathetic hyperactivity present in sleep apnoea patients. Although CB functional plasticity induced by chronic intermittent hypoxia (CIH) has been demonstrated, the underlying mechanisms are not fully elucidated. Here, we show that CIH induces a small increase in CB volume and rearrangement of cell types in the CB, characterized by a mobilization of immature quiescent neuroblasts, which enter a process of differentiation into mature, O2 -sensing and neuron-like, chemoreceptor glomus cells. Prospective isolation of individual cell classes has allowed us to show that maturation of CB neuroblasts is paralleled by an upregulation in the expression of specific glomus cell genes involved in acute O2 -sensing. CIH enhances mitochondrial responsiveness to hypoxia in maturing neuroblasts as well as in glomus cells. These data provide novel perspectives on the pathogenesis of CB-mediated sympathetic overflow that may lead to the development of new pharmacological strategies of potential applicability in sleep apnoea patients. KEY POINTS: Obstructive sleep apnoea is a frequent condition in the human population that predisposes to severe cardiovascular and metabolic alterations. Activation of the carotid body, the main arterial oxygen-sensing chemoreceptor, by repeated episodes of hypoxaemia induces exacerbation of the carotid body-mediated chemoreflex and contributes to sympathetic overflow characteristic of sleep apnoea patients. In rats, chronic intermittent hypoxaemia induces fast neurogenesis in the carotid body with rapid activation of neuroblasts, which enter a process of proliferation and maturation into O2 -sensing chemoreceptor glomus cells. Maturing carotid body neuroblasts and glomus cells exposed to chronic intermittent hypoxia upregulate genes involved in acute O2 sensing and enhance mitochondrial responsiveness to hypoxia. These findings provide novel perspectives on the pathogenesis of carotid body-mediated sympathetic hyperactivation. Pharmacological modulation of carotid body fast neurogenesis could help to ameliorate the deleterious effects of chronic intermittent hypoxaemia in sleep apnoea patients.

The rates of adult neurogenesis and oligodendrogenesis are linked to cell cycle regulation through p27-dependent gene repression of SOX2.

Cell differentiation involves profound changes in global gene expression that often has to occur in coordination with cell cycle exit. Because cyclin-dependent kinase inhibitor p27 reportedly regulates proliferation of neural progenitor cells in the subependymal neurogenic niche of the adult mouse brain, but can also have effects on gene expression, we decided to molecularly analyze its role in adult neurogenesis and oligodendrogenesis. At the cell level, we show that p27 restricts residual cyclin-dependent kinase activity after mitogen withdrawal to antagonize cycling, but it is not essential for cell cycle exit. By integrating genome-wide gene expression and chromatin accessibility data, we find that p27 is coincidentally necessary to repress many genes involved in the transit from multipotentiality to differentiation, including those coding for neural progenitor transcription factors SOX2, OLIG2 and ASCL1. Our data reveal both a direct association of p27 with regulatory sequences in the three genes and an additional hierarchical relationship where p27 repression of Sox2 leads to reduced levels of its downstream targets Olig2 and Ascl1. In vivo, p27 is also required for the regulation of the proper level of SOX2 necessary for neuroblasts and oligodendroglial progenitor cells to timely exit cell cycle in a lineage-dependent manner.

Combined cell-based therapy strategies for the treatment of Parkinson's disease: focus on mesenchymal stromal cells.

Parkinson's disease is a neurodegenerative condition characterized by motor impairments caused by the selective loss of dopaminergic neurons in the substantia nigra. Levodopa is an effective and well-tolerated dopamine replacement agent. However, levodopa provides only symptomatic improvements, without affecting the underlying pathology, and is associated with side effects after long-term use. Cell-based replacement is a promising strategy that offers the possibility to replace lost neurons in Parkinson's disease treatment. Clinical studies of transplantation of human fetal ventral mesencephalic tissue have provided evidence that the grafted dopaminergic neurons can reinnervate the striatum, release dopamine, integrate into the host neural circuits, and improve motor functions. One of the limiting factors for cell therapy in Parkinson's disease is the low survival rate of grafted dopaminergic cells. Different factors could cause cell death of dopaminergic neurons after grafting such as mechanical trauma, growth factor deprivation, hypoxia, and neuroinflammation. Neurotrophic factors play an essential role in the survival of grafted cells. However, direct, timely, and controllable delivery of neurotrophic factors into the brain faces important limitations. Different types of cells secrete neurotrophic factors constitutively and co-transplantation of these cells with dopaminergic neurons represents a feasible strategy to increase neuronal survival. In this review, we provide a general overview of the pioneering studies on cell transplantation developed in patients and animal models of Parkinson's disease, with a focus on neurotrophic factor-secreting cells, with a particular interest in mesenchymal stromal cells; that co-implanted with dopaminergic neurons would serve as a strategy to increase cell survival and improve graft outcomes.

A Notch-dependent transcriptional mechanism controls expression of temporal patterning factors in Drosophila medulla.

Temporal patterning is an important mechanism for generating a great diversity of neuron subtypes from a seemingly homogenous progenitor pool in both vertebrates and invertebrates. Drosophila neuroblasts are temporally patterned by sequentially expressed Temporal Transcription Factors (TTFs). These TTFs are proposed to form a transcriptional cascade based on mutant phenotypes, although direct transcriptional regulation between TTFs has not been verified in most cases. Furthermore, it is not known how the temporal transitions are coupled with the generation of the appropriate number of neurons at each stage. We use neuroblasts of the Drosophila optic lobe medulla to address these questions and show that the expression of TTFs Sloppy-paired 1/2 (Slp1/2) is directly regulated at the transcriptional level by two other TTFs and the cell-cycle dependent Notch signaling through two cis-regulatory elements. We also show that supplying constitutively active Notch can rescue the delayed transition into the Slp stage in cell cycle arrested neuroblasts. Our findings reveal a novel Notch-pathway dependent mechanism through which the cell cycle progression regulates the timing of a temporal transition within a TTF transcriptional cascade.

Deciphering heterogeneous populations of migrating cells based on the computational assessment of their dynamic properties.

Neuronal migration is a highly dynamic process, and multiple cell movement metrics can be extracted from time-lapse imaging datasets. However, these parameters alone are often insufficient to evaluate the heterogeneity of neuroblast populations. We developed an analytical pipeline based on reducing the dimensions of the dataset by principal component analysis (PCA) and determining sub-populations using k-means, supported by the elbow criterion method and validated by a decision tree algorithm. We showed that neuroblasts derived from the same adult neural stem cell (NSC) lineage as well as across different lineages are heterogeneous and can be sub-divided into different clusters based on their dynamic properties. Interestingly, we also observed overlapping clusters for neuroblasts derived from different NSC lineages. We further showed that genetic perturbations or environmental stimuli affect the migratory properties of neuroblasts in a sub-cluster-specific manner. Our data thus provide a framework for assessing the heterogeneity of migrating neuroblasts.

Mutually exclusive expression of sex-specific and non-sex-specific fruitless gene products in the Drosophila central nervous system.

The fruitless gene of Drosophila produces multiple protein isoforms, which are classified into two major classes, sex-specific Fru proteins (FruM) and non-sex specific proteins (FruCOM). Whereas FruM proteins are expressed in ∼2000 neurons to masculinize their structure and function, little is known about FruCOM's roles. As an attempt to obtain clues to the roles of FruCOM, we compared expression patterns of FruCOM and FruM in the central nervous system at the late larval stage. We found that nearly all neuroblasts express FruCOM but not FruM, whereas a subset of ganglion mother cells and differentiated neurons express FruM but not FruCOM. It is inferred that FruCOM proteins support fundamental stem cell functions, contrasting to FruM proteins, which play major roles in sex-specific differentiation of neurons.

Expression of Doublecortin, Glial Fibrillar Acidic Protein, and Vimentin in the Intact Subpallium and after Traumatic Injury to the Pallium in Juvenile Salmon, Oncorhynchus masou.

Fetalization associated with a delay in development and the preservation of the features of the embryonic structure of the brain dominates the ontogeny of salmonids. The aim of the present study was to comparatively analyze the distribution of the glial-type aNSC markers such as vimentin and glial fibrillar acidic protein (GFAP) and the migratory neuronal precursors such as doublecortin in the telencephalon subpallium of juvenile masu salmon, Oncorhynchus masou, in normal conditions and at 1 week after an injury to the dorsal pallium. Immunohistochemical labeling of vimentin, GFAP, and doublecortin in the pallium of intact juvenile masu salmon revealed single cells with similar morphologies corresponding to a persistent pool of neuronal and/or glial progenitors. The study of the posttraumatic process showed the presence of intensely GFAP-labeled cells of the neuroepithelial type that form reactive neurogenic zones in all areas of the subpallial zone of juvenile masu salmon. A comparative analysis of the distribution of radial glia in the dorsal, ventral, and lateral zones of the subpallium showed a maximum concentration of cells in the dorsal part of subpallium (VD) and a minimum concentration in the lateral part of subpallium VL. An essential feature of posttraumatic immunolabeling in the masu salmon subpallium is the GFAP distribution patterns that are granular intracellular in the apical periventricular zone (PVZ) and fibrillar extracellular in the subventricular (SVZ) and parenchymal zones (PZ). In contrast to those in intact animals, most of the GFAP+ granules and constitutive neurogenic niches in injured fish were localized in the basal part of the PVZ. With the traumatic injury to the subpallium, the number of Vim+ cells in the lateral and ventral regions significantly increased. At 1 week post-injury, the total immunolabeling of vimentin cells in the PVZ was replaced by the granular pattern of Vim immunodistribution spreading from the PVZ to the SVZ and deeper parenchymal layers of the brain in all areas of the subpallium. A significant increase in the number of DC+ cells was observed also in all areas of the subpallium. The number of cells increased both in the PVZ and in the SVZ, as well as in the deeper PZ. Thus, at 1 week after the injury to the dorsal pallium, the number of DC, Vim, and GFAP expressing cells of the neuroepithelial type in the subpallium of juvenile masu salmon increased, and additionally GFAP+ radial glia appeared in VD, which was absent from intact animals.

The potential mechanisms of neuroblastoma in children based on bioinformatics big data.

Background: In recent years, miRNAs have become a research hotspot, which is related to the occurrence and development of a variety of malignant tumors, but there are few studies in neuroblastoma. In this study, the differentially expressed microRNAs (miRNAs) in neuroblastoma were identified and analyzed using bioinformatics, and their biological functions and related signaling pathways were examined. Methods: The neuroblastoma miRNA chip GSE121513 was obtained from the Gene Expression Omnibus (GEO) database and the data of 95 neuroblastoma samples and normal fetal adrenal neuroblastoma samples were analyzed to screen the differential miRNAs. The target genes of the differentially expressed miRNAs were predicted using |log fold change (FC)| ≥4. Kyoto Encyclopedia of Genes and Genomes (KEGG) and Gene Ontology (GO) analyses were performed to construct a protein-protein interaction network and identify the core target genes. Results: A total of 91 differentially expressed miRNAs were identified (P<0.05, |logFC| ≥1), including 52 upregulated and 39 downregulated miRNAs. The target genes of the differential miRNAs (P<0.05, |logFC| ≥4) were pretested, and 602 target genes were obtained. Functional analysis showed that these genes were mainly located in the extracellular matrix region of proteins, and were involved in the negative regulation of cytoplasmic translation, mRNA 3'-untranslated region (UTR) binding, and binding to nucleic acid to inhibit the activity of translation factors. They were also involved in RNA degradation, adhesion pathways, and the phosphatidylinositol-3-kinase (PI3K)-Akt signaling pathway. Ten key target genes were identified via protein interaction network screening. Conclusions: The differential miRNAs may be related to the occurrence of neuroblastoma were screened.

Mathematical modeling of Notch dynamics in Drosophila neural development.

Notch signalling is a well-conserved signalling pathway that regulates cell fate through cell-cell communication. A typical feature of Notch signalling is 'lateral inhibition', whereby two neighbouring cells of equivalent state of differentiation acquire different cell fates. Recently, mathematical and computational approaches have addressed the Notch dynamics in Drosophila neural development. Typical examples of lateral inhibition are observed in the specification of neural stem cells in the embryo and sensory organ precursors in the thorax. In eye disc development, Notch signalling cooperates with other signalling pathways to define the evenly spaced positioning of the photoreceptor cells. The interplay between Notch and epidermal growth factor receptor signalling regulates the timing of neural stem cell differentiation in the optic lobe. In this review, we summarize the theoretical studies that have been conducted to elucidate the Notch dynamics in these systems and discuss the advantages of combining mathematical models with biological experiments.

Regulation of Neural Stem Cell Competency and Commitment during Indirect Neurogenesis.

Indirect neurogenesis, during which neural stem cells generate neurons through intermediate progenitors, drives the evolution of lissencephalic brains to gyrencephalic brains. The mechanisms that specify intermediate progenitor identity and that regulate stem cell competency to generate intermediate progenitors remain poorly understood despite their roles in indirect neurogenesis. Well-characterized lineage hierarchy and available powerful genetic tools for manipulating gene functions make fruit fly neural stem cell (neuroblast) lineages an excellent in vivo paradigm for investigating the mechanisms that regulate neurogenesis. Type II neuroblasts in fly larval brains repeatedly undergo asymmetric divisions to generate intermediate neural progenitors (INPs) that undergo limited proliferation to increase the number of neurons generated per stem cell division. Here, we review key regulatory genes and the mechanisms by which they promote the specification and generation of INPs, safeguarding the indirect generation of neurons during fly larval brain neurogenesis. Homologs of these regulators of INPs have been shown to play important roles in regulating brain development in vertebrates. Insight into the precise regulation of intermediate progenitors will likely improve our understanding of the control of indirect neurogenesis during brain development and brain evolution.

Direct differentiation of tonsillar biopsy-derived stem cells to the neuronal lineage.

BACKGROUND: Neurological disorders are considered one of the greatest burdens to global public health and a leading cause of death. Stem cell therapies hold great promise for the cure of neurological disorders, as stem cells can serve as cell replacement, while also secreting factors to enhance endogenous tissue regeneration. Adult human multipotent stem cells (MSCs) reside on blood vessels, and therefore can be found in many tissues throughout the body, including palatine tonsils. Several studies have reported the capacity of MSCs to differentiate into, among other cell types, the neuronal lineage. However, unlike the case with embryonic stem cells, it is unclear whether MSCs can develop into mature neurons. METHODS: Human tonsillar MSCs (T-MSCs) were isolated from a small, 0.6-g sample, of tonsillar biopsies with high viability and yield as we recently reported. Then, these cells were differentiated by a rapid, multi-stage procedure, into committed, post-mitotic, neuron-like cells using defined conditions. RESULTS: Here we describe for the first time the derivation and differentiation of tonsillar biopsy-derived MSCs (T-MSCs), by a rapid, multi-step protocol, into post-mitotic, neuron-like cells using defined conditions without genetic manipulation. We characterized our T-MSC-derived neuronal cells and demonstrate their robust differentiation in vitro. CONCLUSIONS: Our procedure leads to a rapid neuronal lineage commitment and loss of stemness markers, as early as three days following neurogenic differentiation. Our studies identify biopsy-derived T-MSCs as a potential source for generating neuron-like cells which may have potential use for in vitro modeling of neurodegenerative diseases or cell replacement therapies.

Drosophila to Explore Nucleolar Stress.

Nucleolar stress occurs when ribosome production or function declines. Nucleolar stress in stem cells or progenitor cells often leads to disease states called ribosomopathies. Drosophila offers a robust system to explore how nucleolar stress causes cell cycle arrest, apoptosis, or autophagy depending on the cell type. We provide an overview of nucleolar stress in Drosophila by depleting nucleolar phosphoprotein of 140 kDa (Nopp140), a ribosome biogenesis factor (RBF) in nucleoli and Cajal bodies (CBs). The depletion of Nopp140 in eye imaginal disc cells generates eye deformities reminiscent of craniofacial deformities associated with the Treacher Collins syndrome (TCS), a human ribosomopathy. We show the activation of c-Jun N-terminal Kinase (JNK) in Drosophila larvae homozygous for a Nopp140 gene deletion. JNK is known to induce the expression of the pro-apoptotic Hid protein and autophagy factors Atg1, Atg18.1, and Atg8a; thus, JNK is a central regulator in Drosophila nucleolar stress. Ribosome abundance declines upon Nopp140 loss, but unusual cytoplasmic granules accumulate that resemble Processing (P) bodies based on marker proteins, Decapping Protein 1 (DCP1) and Maternal expression at 31B (Me31B). Wild type brain neuroblasts (NBs) express copious amounts of endogenous coilin, but coilin levels decline upon nucleolar stress in most NB types relative to the Mushroom body (MB) NBs. MB NBs exhibit resilience against nucleolar stress as they maintain normal coilin, Deadpan, and EdU labeling levels.