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.
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.
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.
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.
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.
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
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.
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.
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.
Display omitted
•NPCs along the spinal cord exhibit a gradient of cell adhesion (anterior < posterior)•Cell adhesion changes depending on the position and developmental stage of NPCs•Wnt-RA signaling acts as an upstream effector of NPCs•Microarray analysis revealed enrichment of ECM among posterior NPCs
In this article, Shaker and colleagues show that cell-cell adhesion and cell-migration properties of the NPCs in the elongating embryonic spinal cord were different depending on their anteroposterior position (anterior < posterior). Transcriptomic analysis revealed ECM enrichment in posterior NPCs, which is dependent on anteroposterior gradient of Wnt-RA signaling.
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.
Brain organoids with ChP-like epithelium show that Down syndrome ChP defects promote SARS-CoV-2 neuronal infection.
PDF
