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Valproic acid (VPA) is an effective and widely used anti-seizure medication but is teratogenic when used during pregnancy, affecting brain and spinal cord development for reasons that remain largely unclear. Here we designed a genetic recombinase-based SOX10 reporter system in human pluripotent stem cells that enables tracking and lineage tracing of Neural Crest cells (NCCs) in a human organoid model of the developing neural tube. We found that VPA induces extensive cellular senescence and promotes mesenchymal differentiation of human NCCs. We next show that the clinically approved drug Rapamycin inhibits senescence and restores aberrant NCC differentiation trajectory after VPA exposure in human organoids and in developing zebrafish, highlighting the therapeutic promise of this approach. Finally, we identify the pioneer factor AP1 as a key element of this process. Collectively our data reveal cellular senescence as a central driver of VPA-associated neurodevelopmental teratogenicity and identifies a new pharmacological strategy for prevention. These results exemplify the power of genetically modified human stem cell-derived organoid models for drug discovery., (© 2024. The Author(s).)

Why individuals with Down syndrome (DS) are more susceptible to SARS-CoV-2-induced neuropathology remains elusive. Choroid plexus (ChP) plays critical roles in barrier function and immune response modulation and expresses the ACE2 receptor and the chromosome 21-encoded TMPRSS2 protease, suggesting its substantial role in establishing SARS-CoV-2 infection in the brain. To explore this, we established brain organoids from DS and isogenic euploid iPSC that consist of a core of functional cortical neurons surrounded by a functional ChP-like epithelium (ChPCOs). DS-ChPCOs recapitulated abnormal DS cortical development and revealed defects in ciliogenesis and epithelial cell polarity in ChP-like epithelium. We then demonstrated that the ChP-like epithelium facilitates infection and replication of SARS-CoV-2 in cortical neurons and that this is increased in DS. Inhibiting TMPRSS2 and furin activity reduced viral replication in DS-ChPCOs to euploid levels. This model enables dissection of the role of ChP in neurotropic virus infection and euploid forebrain development and permits screening of therapeutics for SARS-CoV-2-induced neuropathogenesis.

Stem cells and the cells they produce are unique because they vary from one cell to another. Traditional methods of studying cells often overlook these differences. However, the development of new technologies for studying individual cells has greatly changed biological research in recent years. Among these innovations, single-cell RNA sequencing (scRNA-seq) stands out. This technique allows scientists to examine the activity of genes in each cell, across thousands or even millions of cells. This makes it possible to understand the diversity of cells, identify new types of cells, and see how cells differ across different tissues, individuals, species, times, and conditions. This paper discusses the importance of scRNA-seq and the computational tools and software that are essential for analyzing the vast amounts of data generated by scRNA-seq studies. Our goal is to provide practical advice for bioinformaticians and biologists who are using scRNA-seq to study stem cells. We offer an overview of the scRNA-seq field, including the tools available, how they can be used, and how to present the results of these studies effectively. Our findings include a detailed overview and classification of tools used in scRNA-seq analysis, based on a review of 2,733 scientific publications. This review is complemented by information from the scRNA-tools database, which lists over 1,400 tools for analyzing scRNA-seq data. This database is an invaluable resource for researchers, offering a wide range of options for analyzing their scRNA-seq data.

In response to the scarcity of advanced in vitro models dedicated to human CNS white matter research, we present a protocol to generate neuroectoderm-derived embedding-free human brain organoids enriched with oligodendrocytes. We describe steps for neuroectoderm differentiation, development of neural spheroids, and their transferal to Matrigel. We then detail procedures for the development, maturation, and application of oligodendrocyte-enriched brain organoids. The presence of myelin-producing cells makes these organoids useful for studying human white matter diseases, such as leukodystrophy., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2023 The Author(s). Published by Elsevier Inc. All rights reserved.)

In vertebrates, the entire central nervous system is derived from the neural tube, which is formed through a conserved early developmental morphogenetic process called neurulation. Although the perturbations in neurulation caused by genetic or environmental factors lead to neural tube defects (NTDs), the most common congenital malformation and the precise molecular pathological cascades mediating NTDs are not well understood. Recently, we have developed human spinal cord organoids (hSCOs) that recapitulate some aspects of human neurulation and observed that valproic acid (VPA) could cause neurulation defects in an organoid model. In this study, we identified and verified the significant changes in cell-cell junctional genes/proteins in VPA-treated organoids using transcriptomic and immunostaining analysis. Furthermore, VPA-treated mouse embryos exhibited impaired gene expression and NTD phenotypes, similar to those observed in the hSCO model. Collectively, our data demonstrate that hSCOs provide a valuable biological resource for dissecting the molecular pathways underlying the currently unknown human neurulation process using destructive biological analysis tools.

Brain organoids are three-dimensional models of the developing human brain and provide a compelling, cutting-edge platform for disease modeling and large-scale genomic and drug screening. Due to the self-organizing nature of cells in brain organoids and the growing range of available protocols for their generation, issues with heterogeneity and variability between organoids have been identified. In this protocol paper, we describe a robust and replicable protocol that largely overcomes these issues and generates cortical organoids from neuroectodermal progenitors within 1 month, and that can be maintained for more than 1 year. This highly reproducible protocol can be easily carried out in a standard tissue culture room and results in organoids with a rich diversity of cell types typically found in the developing human cortex. Despite their early developmental make-up, neurons and other human brain cell types will start to exhibit the typical signs of senescence in neuronal cells after prolonged in vitro culture, making them a valuable and useful platform for studying aging-related neuronal processes. This protocol also outlines a method for detecting such senescent cells in cortical brain organoids using senescence-associated beta-galactosidase staining.

Human spinal-cord-like tissues induced from human pluripotent stem cells are typically insufficiently mature and do not mimic the morphological features of neurulation. Here, we report a three-dimensional culture system and protocol for the production of human spinal-cord-like organoids (hSCOs) recapitulating the neurulation-like tube-forming morphogenesis of the early spinal cord. The hSCOs exhibited neurulation-like tube-forming morphogenesis, cellular differentiation into the major types of spinal-cord neurons as well as glial cells, and mature synaptic functional activities, among other features of the development of the spinal cord. We used the hSCOs to screen for antiepileptic drugs that can cause neural-tube defects. hSCOs may also facilitate the study of the development of the human spinal cord and the modelling of diseases associated with neural-tube defects., (© 2022. The Author(s), under exclusive licence to Springer Nature Limited.)

In this paper, we describe the generation and validation of human induced pluripotent stem cell (hiPSC) lines from peripheral blood mononuclear cells (PBMCs) from 6 epilepsy patients using a non-integrative Sendai virus vector. These human cellular models will enable patient-specific drug screening to improve outcomes for individuals with this disorder., (Copyright © 2022 The Author(s). Published by Elsevier B.V. All rights reserved.)

Neural epidermal growth factor-like like 2 (NELL2) is a cytoplasmic and secreted glycosylated protein with six epidermal growth factor-like domains. In animal models, NELL2 is predominantly expressed in neural tissues where it regulates neuronal differentiation, polarization, and axon guidance, but little is known about the role of NELL2 in human brain development. In this study, we show that rostral neural stem cells (rNSC) derived from human-induced pluripotent stem cell (hiPSC) exhibit particularly strong NELL2 expression and that NELL2 protein is enriched at the apical side of neural rosettes in hiPSC-derived brain organoids. Following differentiation of human rostral NSC into neurons, NELL2 remains robustly expressed but changes its subcellular localization from >20 small cytoplasmic foci in NSC to one-five large peri-nuclear puncta per neuron. Unexpectedly, we discovered that in human brain organoids, NELL2 is readily detectable in the oligodendroglia and that the number of NELL2 puncta increases as oligodendrocytes mature. Artificial intelligence-based machine learning further predicts a strong association of NELL2 with multiple human white matter diseases, suggesting that NELL2 may possess yet unexplored roles in regulating oligodendrogenesis and/or myelination during human cortical development and maturation., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Shaker, Kahtan, Prasad, Lee, Pietrogrande, Leeson, Sun, Wolvetang and Slonchak.)

Alzheimer's disease (AD) is a progressive neurodegenerative disorder that leads to dementia and behavioral changes. Extracellular deposition of amyloid plaques (Aβ) and intracellular deposition of neurofibrillary tangles in neurons are the major pathogenicities of AD. However, drugs targeting these therapeutic targets are not effective. Therefore, novel targets for the treatment of AD urgently need to be identified. Expression of the mesoderm-specific transcript (Mest) is regulated by genomic imprinting, where only the paternal allele is active for transcription. We identified hypermethylation on the Mest promoter, which led to a reduction in Mest mRNA levels and activation of Wnt signaling in brain tissues of AD patients. Mest knockout (KO) using the CRIPSR/Cas9 system in mouse embryonic stem cells and P19 embryonic carcinoma cells leads to neuronal differentiation arrest. Depletion of Mest in primary hippocampal neurons via lentivirus expressing shMest or inducible KO system causes neurodegeneration. Notably, depletion of Mest in primary cortical neurons of rats leads to tau phosphorylation at the S199 and T231 sites. Overall, our data suggest that hypermethylation of the Mest promoter may cause or facilitate the progression of AD., (© 2021. The Author(s).)

Epilepsy is a common neurological disorder characterized by seizures. Unfortunately, 30-40% of all epilepsy patients are resistant to at least two or more anti-seizure medications. Attempts to treat these patients and prevent further seizures necessitates multiple drug trials for the patient. Here we describe the generation and validation of induced pluripotent stem cell (iPSC) lines from peripheral blood mononuclear cells (PBMCs) from 3 drug responsive and 3 drug resistant patients, using a non-integrative Sendai virus vector. These lines can be used to generate 2D and 3D patient-specific human cellular models that will enable personalised drug screening and pharmacogenomic studies., (Copyright © 2021 The Authors. Published by Elsevier B.V. All rights reserved.)

Aims: During vertebrate development, the posterior end of the embryo progressively elongates in a head-to-tail direction to form the body plan. Recent lineage tracing experiments revealed that bi-potent progenitors, called neuromesodermal progenitors (NMPs), produce caudal neural and mesodermal tissues during axial elongation. However, their precise location and contribution to spinal cord development remain elusive., Main Methods: Here we used NMP-specific markers (Sox2 and BraT) and a genetic lineage tracing system to localize NMP progeny in vivo., Key Findings: Sox2 and BraT double positive cells were initially located at the tail tip, but were later found in the caudal neural tube, which is a unique feature of mouse development. In the neural tube, they produced neural progenitors (NPCs) and contributed to the spinal cord gradually along the AP axis during axial elongation. Interestingly, NMP-derived NPCs preferentially contributed to the ventral side first and later to the dorsal side at the lumbar spinal cord level, which may be associated with atypical junctional neurulation in mice., Significance: Our current observations detail the contribution of NMP progeny to spinal cord elongation and provide insights into how different species uniquely execute caudal morphogenesis., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)

Ataxia-telangiectasia (A-T) is a genetic disorder caused by the lack of functional ATM kinase. A-T is characterized by chronic inflammation, neurodegeneration and premature ageing features that are associated with increased genome instability, nuclear shape alterations, micronuclei accumulation, neuronal defects and premature entry into cellular senescence. The causal relationship between the detrimental inflammatory signature and the neurological deficiencies of A-T remains elusive. Here, we utilize human pluripotent stem cell-derived cortical brain organoids to study A-T neuropathology. Mechanistically, we show that the cGAS-STING pathway is required for the recognition of micronuclei and induction of a senescence-associated secretory phenotype (SASP) in A-T olfactory neurosphere-derived cells and brain organoids. We further demonstrate that cGAS and STING inhibition effectively suppresses self-DNA-triggered SASP expression in A-T brain organoids, inhibits astrocyte senescence and neurodegeneration, and ameliorates A-T brain organoid neuropathology. Our study thus reveals that increased cGAS and STING activity is an important contributor to chronic inflammation and premature senescence in the central nervous system of A-T and constitutes a novel therapeutic target for treating neuropathology in A-T patients., (© 2021 The Authors. Aging Cell published by Anatomical Society and John Wiley & Sons Ltd.)

Aging is a major risk factor for many neurodegenerative diseases. Klotho (KL) is a glycosylated transmembrane protein that is expressed in the choroid plexus and neurons of the brain. KL exerts potent anti-aging effects on multiple cell types in the body but its role in human brain cells remains largely unclear. Here we show that human cortical neurons, derived from human pluripotent stem cells in 2D cultures or in cortical organoids, develop the typical hallmarks of senescent cells when maintained in vitro for prolonged periods of time, and that moderate upregulation or repression of endogenous KL expression in cortical organoids inhibits and accelerates senescence, respectively. We further demonstrate that KL expression alters the expression of senescence-associated genes including, extracellular matrix genes, and proteoglycans, and can act in a paracrine fashion to inhibit neuronal senescence. In summary, our results establish an important role for KL in the regulation of human neuronal senescence and offer new mechanistic insight into its role in human brain aging., (© 2021. The Author(s).)

Neuromesodermal progenitors (NMPs) constitute a bipotent cell population that generates a wide variety of trunk cell and tissue types during embryonic development. Derivatives of NMPs include both mesodermal lineage cells such as muscles and vertebral bones, and neural lineage cells such as neural crests and central nervous system neurons. Such diverse lineage potential combined with a limited capacity for self-renewal, which persists during axial elongation, demonstrates that NMPs are a major source of trunk tissues. This review describes the identification and characterization of NMPs across multiple species. We also discuss key cellular and molecular steps for generating neural and mesodermal cells for building up the elongating trunk tissue.

Human stem cell derived brain organoids are increasingly gaining attention as an ideal model system for investigating neurological diseases, particularly those that involve myelination defects. However, current protocols for generating brain organoids with sufficiently mature oligodendrocytes that deposit myelin on endogenously produced neurons are lengthy and complicated. Taking advantage of a human pluripotent stem cell line that reports on SOX10 expression, we developed a protocol that involves a 42 day exposure of neuroectoderm-derived organoids to a cocktail of growth factors and small molecules that collectively foster oligodendrocyte specification and survival. Importantly, the resulting day 42 brain organoids contain both myelinating oligodendrocytes, cortical neuronal cells and astrocytes. These oligodendrocyte brain organoids therefore constitute a valuable and tractable platform for functional neurogenomics and drug screening for white matter diseases., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Shaker, Pietrogrande, Martin, Lee, Sun and Wolvetang.)

Mammalian embryos exhibit a transition from head morphogenesis to trunk elongation to meet the demand of axial elongation. The caudal neural tube (NT) is formed with neural progenitors (NPCs) derived from neuromesodermal progenitors localized at the tail tip. However, the molecular and cellular basis of elongating NT morphogenesis is yet elusive. Here, we provide evidence that caudal NPCs exhibit strong adhesion affinity that is gradually decreased along the anteroposterior (AP) axis in mouse embryonic spinal cord and human cellular models. Strong cell-cell adhesion causes collective migration, allowing AP alignment of NPCs depending on their birthdate. We further validated that this axial adhesion gradient is associated with the extracellular matrix and is under the control of graded Wnt signaling emanating from tail buds and antagonistic retinoic acid (RA) signaling. These results suggest that progressive reduction of NPC adhesion along the AP axis is under the control of Wnt-RA molecular networks, which is essential for a proper elongation of the spinal cord., (Copyright © 2020 The Authors. Published by Elsevier Inc. All rights reserved.)

Neural stem cells (NSCs) in the embryonic neocortex have the potential to generate a well-organized laminar architecture of the cerebral cortex through precise regulation of the proliferation, differentiation, and migration of neural cells. NSCs can be isolated in vitro and expanded as cell clusters, called neurospheres, which are primarily related to the proliferation ability of NSCs. Conversely, the tissue-organizing properties of NSCs via regulated differentiation and migration of the cells are not well understood. In this study, we established a three-dimensional (3D) differentiation model of neurospheres, which produce unique neuronal clusters, termed NeuroCore (NC). NC formation was initiated by the aggregation of young neurons. Upon maturation of the neurons and the establishment of radial glia-like structures, the initial organization of the NCs transformed into a glomeruli-like arrangement of cortical neurons. These neurons expressed multiple markers of upper and deep cortical neurons. Taken together, we propose that NSCs in vitro maintain some aspects of their original in vivo tissue-organizing properties, providing an alternative opportunity to explore the fundamental components of brain histogenesis in vitro., Competing Interests: Declaration of Competing Interest The authors declare no conflicts of interest., (Copyright © 2020 The Authors. Published by Elsevier B.V. All rights reserved.)

Decellularization of tissues provides extracellular matrix (ECM) scaffolds for regeneration therapy and an experimental model to understand ECM and cellular interactions. However, decellularization often causes microstructure disintegration and reduction of physical strength, which greatly limits the use of this technique in soft organs or in applications that require maintenance of physical strength. Here, we present a new tissue decellularization procedure, namely CASPER ( C linically and Experimentally A pplicable Acellular Tissue S caffold P roduction for Tissue E ngineering and R egenerative Medicine), which includes infusion and hydrogel polymerization steps prior to robust chemical decellularization treatments. Polymerized hydrogels serve to prevent excessive damage to the ECM while maintaining the sophisticated structures and biological activities of ECM components in various organs, including soft tissues such as brains and embryos. CASPERized tissues were successfully recellularized to stimulate a tissue-regeneration-like process after implantation without signs of pathological inflammation or fibrosis in vivo, suggesting that CASPERized tissues can be used for monitoring cell-ECM interactions and for surrogate organ transplantation.

The inability of neurons to undergo mitosis renders damage to the central or peripheral nervous system. Neural stem cell therapy could provide a path for treating the neurodegenerative diseases. However, reliable and simple tools for the developing and testing neural stem cell therapy are still required. Here, we show the development of a micropillar-based microfluidic device to trap the uniform-sized neurospheres. The neurospheres trapped within micropillar arrays were largely differentiated into neuronal cells, and their neurite networks were observed in the microfluidic device. Compared to conventional cultures on glass slides, the neurite networks generated with this method have a higher reproducibility. Furthermore, we demonstrated the effect of thapsigargin on the neurite networks in the microfluidic device, demonstrating that neural networks exposed to thapsigargin were largely diminished and disconnected from each other. Therefore, this micropillar-based microfluidic device could be a potential tool for screening of neurotoxins., (© 2018 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

We developed the photocrosslinkable hydrogel microwell arrays for uniform-sized neurosphere-mediated motoneuron differentiation. Neural stem cells (NSCs) were obtained from embryonic cerebral cortex and spinal cord. To generate uniform-sized neurospheres in a homogeneous manner, the dissociated cells were cultured in the hydrogel microwell arrays for 3 days. Uniform-sized neurospheres harvested from microwell arrays were replated into laminin-coated substrate. In parallel, uniform-sized neurospheres cultured in microwell arrays were encapsulated by photocrosslinkable gelatin methacrylate hydrogels in a three-dimensional manner. We demonstrated the effect of hydrogel microwell sizes (e.g., 50, 100, 150 μm in diameter) on motoneuron differentiation, showing that the largest uniform-sized neurospheres derived from embryonic spinal cord efficiently differentiated into motoneurons. Therefore, this hydrogel microwell array could be a powerful array to regulate the uniform-sized neurosphere-mediated motoneuron differentiation., (© 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Neural stem cells (NSCs) in the adult subventricular zone (SVZ) are regionally specified and have distinct molecular gene expression signatures. Recently, we identified the subcallosal zone (SCZ) as a novel brain region where adult NSCs maintain and spontaneously produce neuroblasts. In an attempt to isolate genes specifically expressed in the SCZ or SVZ, microarray analyses of their differentially expressing transcripts were done. The comparison between neurospheres generated from SVZ and SCZ revealed differential expression >1.5-fold in two groups in only 83 genes, representing <0.03% of the genes examined, suggesting that these two populations are largely similar. The differential expression patterns SCZ and SVZ genes were confirmed by RT-PCR and Western blots. The selective expressions of two genes ( CRBP1 , HMGA1 ) in SVZ-NSCs were further confirmed by immunohistochemistry. These molecular markers could be useful for further molecular and cellular characterization of NSCs.

Background: Coconut oil is commonly used as herbal medicine worldwide. There is limited information regarding its effects on the developing embryo and infant growth., Methods: We investigated the effect of virgin coconut oil post-natally and until 6 weeks old in mice (age of maturity). Females were fed with either standard, virgin olive oil or virgin coconut oil diets 1 month prior to copulation, during gestation and continued until weaning of pups. Subsequently, groups of pups borne of the respective diets were continuously fed the same diet as its mother from weaning until 6 weeks old. Profiles of the standard and coconut oil diets were analysed by gas chromatography flame ionization detector (GCFID)., Results: Analysis of the mean of the total weight gained/ loss over 6 weeks revealed that in the first 3 weeks, pups whose mothers were fed virgin coconut oil and virgin olive oil have a significantly lower body weight than that of standard diet pups. At 6 weeks of age, only virgin coconut oil fed pups exhibited significantly lower body weight. We report that virgin coconut oil modifies the fatty acid profiles of the standard diet by inducing high levels of medium chain fatty acids with low levels of essential fatty acids. Furthermore, pups borne by females fed with virgin coconut oil developed spiky fur., Conclusion: Our study has demonstrated that virgin coconut oil could affect infant growth and appearance via maternal intake; we suggest the use of virgin coconut oil as herbal medicine to be treated with caution.

Nanoparticle-mediated delivery of functional macromolecules is a promising method for treating a variety of human diseases. Among nanoparticles, cell-derived exosomes have recently been highlighted as a new therapeutic strategy for the in vivo delivery of nucleotides and chemical drugs. Here we describe a new tool for intracellular delivery of target proteins, named 'exosomes for protein loading via optically reversible protein-protein interactions' (EXPLORs). By integrating a reversible protein-protein interaction module controlled by blue light with the endogenous process of exosome biogenesis, we are able to successfully load cargo proteins into newly generated exosomes. Treatment with protein-loaded EXPLORs is shown to significantly increase intracellular levels of cargo proteins and their function in recipient cells in vitro and in vivo. These results clearly indicate the potential of EXPLORs as a mechanism for the efficient intracellular transfer of protein-based therapeutics into recipient cells and tissues.

Neurogenesis occurs spontaneously in the subventricular zone (SVZ) of the lateral ventricle in adult rodent brain, but it has long been debated whether there is sufficient adult neurogenesis in human SVZ. Subcallosal zone (SCZ), a posterior continuum of SVZ closely associated with posterior regions of cortical white matter, has also been reported to contain adult neural stem cells (aNSCs) in both rodents and humans. However, little is known whether SCZ-derived aNSC (SCZ-aNSCs) can produce cortical neurons following brain injury. We found that SCZ-aNSCs exhibited limited neuronal differentiation potential in culture and after transplantation in mice. Neuroblasts derived from SCZ initially migrated toward injured cortex regions following brain injury, but later exhibited apoptosis. Overexpression of anti-apoptotic bcl-xL in the SCZ by retroviral infection rescued neuroblasts from cell death in the injured cortex, but neuronal maturation was still limited, resulting in atrophy. In combination with Bcl-xL, infusion of brain-derived neurotropic factor rescued atrophy, and importantly, a subset of such SCZ-aNSCs differentiated and attained morphological and physiological characteristics of mature, excitatory neurons. These results suggest that the combination of anti-apoptotic and neurotrophic factors might enable the use of aNSCs derived from the SCZ in cortical neurogenesis for neural replacement therapy., (© 2015 AlphaMed Press.)

Mitochondria are important in many essential cellular functions, including energy production, calcium homeostasis, and apoptosis. The organelles are scattered throughout the cytoplasm, but their distribution can be altered in response to local energy demands, such as cell division and neuronal maturation. Mitochondrial distribution is closely associated with mitochondrial fission, and blocking the fission-promoting protein dynamin-related protein 1 (Drp1) activity often results in mitochondrial elongation and clustering. In this study, we observed that mitochondria were preferentially localized at the leading process of migratory adult neural stem cells (aNSCs), whereas neuronal differentiating cells transiently exhibited perinuclear condensation of mitochondria. Inhibiting Drp1 activity altered the typical migratory cell morphology into round shapes while the polarized mitochondrial distribution was maintained. With these changes, aNSCs failed to migrate, and neuronal differentiation was prevented. Because Drp1 blocking also impaired the mitochondrial membrane potential, we tested whether supplementing with L-carnitine, a compound that restores mitochondrial membrane potential and ATP synthesis, could revert the defects induced by Drp1 inhibition. Interestingly, L-carnitine fully restored the aNSC defects, including cell shrinkage, migration, and impaired neuronal differentiation. These results suggest that Drp1 is required for functionally active mitochondria, and supplementing with ATP can restore the defects induced by Drp1 suppression.

Secondary neurulation is an embryonic progress that gives rise to the secondary neural tube, the precursor of the lower spinal cord region. The secondary neural tube is derived from aggregated Sox2-expressing neural cells at the dorsal region of the tail bud, which eventually forms rosette or tube-like structures to give rise to neural tissues in the tail bud. We addressed whether the embryonic tail contains neural stem cells (NSCs), namely secondary NSCs (sNSCs), with the potential for self-renewal in vitro. Using in vitro neurosphere assays, neurospheres readily formed at the rosette and neural-tube levels, but less frequently at the tail bud tip level. Furthermore, we identified that sNSC-generated neurospheres were significantly smaller in size compared with cortical neurospheres. Interestingly, various cell cycle analyses revealed that this difference was not due to a reduction in the proliferation rate of NSCs, but rather the neuronal commitment of sNSCs, as sNSC-derived neurospheres contain more committed neuronal progenitor cells, even in the presence of epidermal growth factor (EGF) and basic fibroblast growth factor (bFGF). These results suggest that the higher tendency for sNSCs to spontaneously differentiate into progenitor cells may explain the limited expansion of the secondary neural tube during embryonic development.

Background: The suburban transmission of malaria in Selangor, Malaysia's most developed and populous state still remains a concern for public health in this region. Despite much successful control efforts directed at its reduction, sporadic cases, mostly brought in by foreigners have continued to occur. In addition, cases of simian malaria caused by Plasmodium knowlesi, some with fatal outcome have caused grave concern to health workers. The aim of this study was to investigate the possibility of local malaria transmission in suburban regions of Selangor, which are adjacent to secondary rainforests., Findings: A malaria survey spanning 7 years (2006 - 2012) was conducted in Selangor. A total of 1623 laboratory confirmed malaria cases were reported from Selangor's nine districts. While 72.6% of these cases (1178/1623) were attributed to imported malaria (cases originating from other countries), 25.5% (414/1623) were local cases and 1.9% (31/1623) were considered as relapse and unclassified cases combined. In this study, the most prevalent infection was P. vivax (1239 cases, prevalence 76.3%) followed by P. falciparum (211, 13.0%), P. knowlesi (75, 4.6%), P. malariae (71, 4.4%) and P. ovale (1, 0.06%). Mixed infections comprising of P. vivax and P. falciparum were confirmed (26, 1.6%). Entomological surveys targeting the residences of malaria patients' showed that the most commonly trapped Anopheles species was An. maculatus. No oocysts or sporozoites were found in the An. maculatus collected. Nevertheless, the possibility of An. maculatus being the malaria vector in the investigated locations was high due to its persistent occurrence in these areas., Conclusions: Malaria cases reported in this study were mostly imported cases. However the co-existence of local cases and potential Plasmodium spp. vectors should be cause for concern. The results of this survey reflect the need of maintaining closely monitored malaria control programs and continuous extensive malaria surveillance in Peninsula Malaysia.

Dielectric spectroscopy is a widely utilized electrophysiological characterization method. The obtained dielectric spectra and derived properties have the potential of providing significant information regarding changes in the physiological state of a biological system. However, since many of the dielectric properties are obtained in vitro from excised tissue far removed from physiological conditions, the value of the information obtained may be diminished. In this paper, we introduce a superfusion system that is designed to produce ex vivo dielectric spectroscopy measurements by providing the living tissue with a continuous and ample supply of nutrients and oxygen while removing metabolites and other waste. This superfusion system provides the convenience of in vitro measurement while concurrently producing results that can be more closely correlated with actual physiological changes in the biological system.

The effects of the synthetic retinoid N-(4-hydroxyphenyl) retinamide (4-HPR) on prostate cancer metastasis in vivo were evaluated in the mouse prostate reconstitution (MPR) model. MPRs were produced by infection of either heterozygous (+/-) or nullizygous (-/-) p53-mutant fetal prostatic epithelial cells with the recombinant retrovirus Zipras/myc 9. Previous studies have documented that loss of p53 function potentiates metastasis in this model system. MPRs were grafted into homozygous (+/+) p53 male mice, fed a 4-HPR containing diet or a control diet and maintained until the status of tumor progression dictated sacrifice. Under these experimental conditions, treatment with 4-HPR did not have a significant effect on primary tumor wet weight for either p53 +/- or p53 -/- MPRs. For, p53 +/- MPRs the animals fed the 4-HPR diet had a slight improvement in survival and a significant reduction in the number of mesenteric metastases (P = 0.0477, t-test). Notably, in p53 +/- MPRs the incidence of metastasis to lumbar spine and sternum was 92% in the control animals compared to 54% in the 4-HPR treated animals (P = 0.035, chi2-test). In p53 -/- MPRs there was a trend toward a reduction in the number of soft tissue metastases to lung and liver in the 4-HPR group relative to the control diet group and a statistically significant reduction in the incidence of metastasis to bone was demonstrated in that 50% of control animals versus 30% of 4-HPR treated p53 -/- animals harbored bone metastases (P = 0 < 0.05, chi2-test). Cell lines were established from portions of the primary tumor and from selected metastatic deposits in each experimental group. Clonal analysis, by retroviral integration pattern, indicated increased clonal diversity in both the primary tumors and metastasis-derived cell lines from 4-HPR treated animals relative to the control animals. In vitro treatment with 4-HPR did not reveal discriminating differences between cell lines derived from primary tumors and bone metastases or control and treatment groups in regard to growth arrest or apoptotic responses. Overall these studies indicate limited anti-tumor and anti-metastatic activity in this highly aggressive in vivo mouse model of prostate cancer, yet 4-HPR treatment significantly suppressed the development of bone metastases in p53 +/- and p53 -/- MPRs revealing a novel and potentially clinically useful activity of this retinoid.

We targeted a mutant p53 gene (val135), previously shown to cause tumors in transgenic mice, to the kidney and eye using a gamma-glutamyltranspeptidase promoter. Although transgene RNA was expressed in both tissues, and mutant protein could be detected at high levels in the kidney and was appropriately localized to the nuclei of proximal tubules, no gross or microscopic lesions developed, even when mice were held as long as 75 weeks. When these mice were crossed with transgenic mice carrying HrasT24 (containing a codon 12 mutation) driven by the same promoter, the p53val135 transgene partially suppressed the mutant ras phenotype (proximal tubular hyperplasia and adenomas and carcinomas of the ciliary body and retinal pigment epithelium). The kidneys of double transgenic mice younger than 25 weeks showed less tubular hyperplasia and cystic change than littermates carrying gamma-glutamyltranspeptidase(I)rasT24 alone. By 33 weeks, there was no difference in the severity of the kidney lesions. The eye lesions were less aggressive, and no malignant lesions were identified. Our findings are consistent with the work of others, indicating that p53val135 is not tumorigenic under all conditions; in fact, in some circumstances, it retains some of the suppressing activity of wild-type p53.

Background: Although prostate cancer is one of the most prevalent tumors in men, knowledge of its biology has been hindered by lack of animal models. We have attempted to develop a prostate cancer model utilizing transgenic mouse technology., Experimental Design: Two lines of transgenic mice were derived from one cell stage embryos injected with a fusion gene consisting of a mutated (codon 12) ras gene driven by the human prostate specific antigen (PSA) promoter in an attempt to target the oncogene specifically to the mouse prostate gland. Nontransgenic FVB/N mice were used as controls. The animals were sacrificed for study between 4 and 55 weeks of age., Results: All organs were normal except the salivary glands and gastrointestinal tracts, both of which developed carcinomas in animals older than 44 weeks. The salivary gland tumors were of ductal origin, exhibited a variable degree of differentiation, and were shown to contain abundant PSAras mRNA by in situ hybridization. The gastrointestinal tract tumors were undifferentiated but appeared to be of stromal origin. Both salivary gland and gastrointestinal tumors occasionally metastasized. No transgene expression could be demonstrated in the prostate gland by either reverse transcription-polymerase chain reaction or in situ hybridization., Conclusions: Lack of transgene expression by the prostate can be explained on the basis of the apparent species specificity previously observed for PSA. Expression in salivary gland is best attributed to identity between the nucleotide sequences of the PSA promoter and of a mouse glandular kallikrein normally secreted by the salivary gland.

Harrison (1918: J. Exp. Zool. 25: 413-461) described a developmental field as an "equipotential self-differentiating system." The present study was undertaken to address the question: To what extent can be pre-limb territory of a chick embryo be considered a developmental field? To what extent is the chick pre-limb territory an equipotential self-differentiating system? Two sets of experiments were undertaken to address these questions: (1) Whole and half limb territories were explanted to the celoma of host embryos, and (2) portions of the wing territories were extirpated. The wing exhibited the quality of self-differentiation after stage 12, in that the isolated wing territory, grafted to a host celom, could form limbs beginning at stage 12 (however, complete wings formed only from wing territories of stage 16 and older). On the other hand, the chick wing territory did not appear to exhibit equipotentiality. No posterior half limb graft formed normal limbs, and only in two exceptional cases did anterior half limb grafts form limbs. If part or all of the wing territory was removed from chick embryos, normal limbs formed in less than 15% of the cases after stage 15, in about 30% of the cases at stages 13 and 14, but in over half the cases at stages 10-12. Wound healing and reinitiation of limb potential may be responsible for the higher incidence of limb formation at the younger ages.

Previous studies by Stephens and McNulty and Strecker and Stephens have demonstrated that foil barriers placed between the mesonephros and lateral plate at stages 12 to 15 inhibited limb development, but foil barriers placed between the neural tube and somites at stages 11 to 12 resulted in limbs with normal skeletal patterns. It was concluded that some influence present in the paraxial region of the embryo at stages 11 to 15 is necessary for normal limb development. The present study was undertaken to localize that influence more precisely. Foil barriers were placed in the lateral edge of the somites or segmental plate of stage 10 to 15 chick embryos. Barriers placed into stage 13 to 15 embryos resulted in chicks with normal limbs, but barriers placed into stage 10 to 11 embryos resulted in chicks with defective limbs. Barriers inserted just lateral to Hensen's node at stages 6 to 8 resulted in embryos with defective or absent wings. We also grafted stage 4 to 9 presumptive limb territories with and without Hensen's node. Explants without Hensen's node formed limb-like structures in 1% of the cases. Explants with Hensen's node formed limb-like structures in 27% of the cases. When barriers were implanted and a node was placed on the lateral side of the barrier, limbs formed in 40% of the cases. These data suggest a medial to lateral progression of some as yet unknown morphogenetic influence necessary for normal limb development and we hypothesized that the influence may initially emanate from Hensen's node., (Copyright © 1991 Wiley-Liss, Inc.)