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Transcriptome profiling of human pluripotent stem cell-derived cerebellar organoids reveals faster commitment under dynamic conditions.
Human-induced pluripotent stem cells (iPSCs) have great potential for disease modeling. However, generating iPSC-derived models to study brain diseases remains a challenge. In particular, the ability to recapitulate cerebellar development in vitro is still limited. We presented a reproducible and scalable production of cerebellar organoids by using the novel single-use Vertical-Wheel bioreactors, in which functional cerebellar neurons were obtained. Here, we evaluate the global gene expression profiles by RNA sequencing (RNA-seq) across cerebellar differentiation, demonstrating a faster cerebellar commitment in this novel dynamic differentiation protocol. Furthermore, transcriptomic profiles suggest a significant enrichment of extracellular matrix (ECM) in dynamic-derived cerebellar organoids, which can better mimic the neural microenvironment and support a consistent neuronal network. Thus, an efficient generation of organoids with cerebellar identity was achieved for the first time in a continuous process using a dynamic system without the need of organoids encapsulation in ECM-based hydrogels, allowing the possibility of large-scale production and application in high-throughput processes. The presence of factors that favors angiogenesis onset was also detected in dynamic conditions, which can enhance functional maturation of cerebellar organoids. We anticipate that large-scale production of cerebellar organoids may help developing models for drug screening, toxicological tests, and studying pathological pathways involved in cerebellar degeneration.
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Developmental xenocortication using human-derived organoids in mice.
The inaccessibility of human brain tissue limits the study of human development and function, a challenge that human stem-cell-derived neural models are beginning to address1,2. Transplantation of neural organoids into rodent hosts enables the in vivo study of aspects of human neurodevelopment and circuit function, alongside behavioural phenotyping of the host animals. However, spatial limitations and competition with host circuits constrain the integration of neural organoids, which is critical for studying disease. Here we establish a transplantation platform using a genetic strategy to effectively deplete glutamatergic neurons from mouse neocortex and hippocampus (apallial) and neonatally engraft the cortical cavity with human stem-cell-derived cortical organoids (hCO) to generate xenocortical mice. This leads to robust graft growth with hCOs occupying most of the cortical volume and generating a diversity of human cortical cell types, including layer 5 extratelencephalic projection neurons. Human cortical neurons integrate with the mouse nervous system, and in vivo cortical graft-wide calcium imaging and electrophysiological analyses revealed patterns of organized activity resembling developing circuits. Behavioural analyses of apallial and xenocortical mice revealed broadly preserved locomotion alongside selective differences in limb coordination and altered organization of spontaneous behaviour. Lastly, this platform enabled behavioural readouts in a model of injury to developing human cortical cells. We envision that xenocortication will be useful for obtaining circuit- and behaviour-level readouts using human neurons to study neurodevelopment, model disease and develop therapeutics.
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Assessing in-vitro models for microglial development and fetal programming: a critical review.
This review evaluates in-vitro models for studying how maternal influences during pregnancy impact the development of offspring microglia, the immune cells of the central nervous system. The models examined include primary microglia cultures, microglia cell lines, iPSC-derived microglia, PBMC-induced microglia-like cells, 3D brain organoids derived from iPSCs, and Hofbauer cells. Each model is assessed for its ability to replicate the in-vivo environment of the developing brain, with a focus on their strengths, limitations, and practical challenges. Key factors such as scalability, genetic and epigenetic fidelity, and physiological relevance are highlighted. Microglia cell lines are highly scalable but lack genetic and epigenetic fidelity. iPSC-derived microglia provide moderate physiological relevance and patient-specific genetic insights but face operational and epigenetic challenges inherent to reprogramming. 3D brain organoids, derived from iPSCs, offer an advanced platform for studying complex neurodevelopmental processes but require extensive resources and technical expertise. Hofbauer cells, which are fetal macrophages located in the placenta and share a common developmental origin with microglia, are uniquely exposed to prenatal maternal factors and, depending on fetal barrier maturation, exhibit variable epigenetic fidelity. This makes them particularly useful for exploring the impact of maternal influences on fetal programming of microglial development. The review concludes that no single model comprehensively captures all aspects of maternal influences on microglial development, but it offers guidance on selecting the most appropriate model based on specific research objectives and experimental constraints.
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Isolation of coated vesicles, plain synaptic vesicles, and flocculent material from a crude synaptosome fraction of guinea pig whole brain.
Two vesicular fractions and one nonvesicular fraction were prepared from crude synaptosomes by differential centrifugation and salting out with ammonium sulfate. Fraction 1 contained a mixture of coated vesicles, material thought to be derived from breakdown of the coats (shell fragments), and plain synaptic vesicles. Fraction 2 contained a mixture of plain synaptic vesicles and flocculent material. Fraction 3 contained flocculent material only. Fractions 1 and 3 were partially purified by passage through a Sephadex column. Fraction 3 contained no shell fragments but contained finer flocculent material which, it is suggested, is composed of unit particles either occurring singly or linked together into chainlike or amorphous aggregates. Each unit particle appears to have four subunits and is here referred to as a tetrasome. Tetrasomes sometimes appear to be attached to the surfaces of the plain synaptic vesicles. Also, it is possible that aggregates of tetrasomes form part of the structure of the presynaptic dense projections.
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Annulate lamellae, lamellar bodies and subsurface cisternae in neurons of the avian hyperstriatum accessorium.
Structures identified as annulate lamellae, lamellar bodies and subsurface cisternae were found in neurons of the hyperstriatum accessorium of the avian forebrain. Annulate lamellar arrays with up to six lamellae were present in the larger somata. The lamellae were made up of fused smooth-surfaced cisternae forming pores or annuli and were surrounded by a dense filamentous to granular material. Stacks of nonfenestrated, parallel, regularly spaced cisternae, designated as lamellar bodies, also appeared in the cytoplasm. When flattened they were reminiscent of the electron dense subsurface cisternae. Continuity could be demonstrated between peripherally located subsurface cisternae and lamellar bodies. The dense filamentous to finely granular substance was also located between these structures. Annulate lamellae, lamellar bodies and subsurface cisternae were always observed in conjunction with the rough endoplasmic reticulum. The functional significance of these structural associations is considered.
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Novel human iPSC models of neuroinflammation in neurodegenerative disease and regenerative medicine.
The importance of neuroinflammation in neurodegenerative diseases is becoming increasingly evident, and, in parallel, human induced pluripotent stem cell (hiPSC) models of physiology and pathology are emerging. Here, we review new advancements in the differentiation of hiPSCs into glial, neural, and blood-brain barrier (BBB) cell types, and the integration of these cells into complex organoids and chimeras. These advancements are relevant for modeling neuroinflammation in the context of prevalent neurodegenerative disorders, such as Alzheimer's disease (AD), Parkinson's disease (PD), and multiple sclerosis (MS). With awareness of current limitations, recent progress in the development and application of various hiPSC-derived models shows potential for aiding the identification of candidate therapeutic targets and immunotherapy approaches.
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Silicate Microfiber Scaffolds Support the Formation and Expansion of the Cortical Neuronal Layer of Cerebral Organoids With a Sheet-Like Configuration.
Cerebral organoids (COs) are derived from human-induced pluripotent stem cells in vitro and mimic the features of the human fetal brain. The development of COs is largely dependent on "self-organization" mechanisms, in which differentiating cells committed to cortical cells autonomously organize into the cerebral cortex-like tissue. However, extrinsic manipulation of their morphology, including size and thickness, remains challenging. In this study, we discovered that silicate microfiber scaffolds could support the formation of cortical neuronal layers and successfully generated cortical neuronal layers, which are 9 times thicker than conventional COs, in 70 days. These cortical neurons in the silicate microfiber layer were differentiated in a fetal brain-like lamination pattern. While these cellular characteristics such as cortical neurons and neural stem/progenitor cells were like those of conventional COs, the cortical neuronal layers were greatly thickened in sheet-like configuration. Moreover, the cortical neurons in the scaffolds showed spontaneous electrical activity. We concluded that silicate microfiber scaffolds support the formation of the cortical neuronal layers of COs without disturbing self-organization-driven corticogenesis. The extrinsic manipulation of the formation of the cortical neuronal layers of COs may be useful for the research of developmental mechanisms or pathogenesis of the human cerebral cortex, particularly for the development of regenerative therapy and bioengineering.
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Thiaminepyrophosphatase activity in the plasma membrane of microglia.
An intense thiaminepyrophosphatase (TPPase) activity was demonstrated in glial cells and blood vessels in the central nervous system (CNS), when incubation was carried out with thiaminepyrophosphte (TPP, cocarboxylase), using the method of Novikoff and Goldfischer (1961). Glial cells with TPPSase activity were identified as microglia because they were morphologically similar to microglial cells in the sections stained with silver impregnation. TPPase activity was localized in the microglial perikaryon and in the processes, as viewed under a light microscope. Electron microscopically, enzyme activity was localized in the plasma membrane of microglia. We consider this activity to be a true TPPase activity hydrolyzing TPP, and we then went on to examine the substrate specificity, optimum pH, effect of chemical inhibitors and activators, and the effect of glutaraldehyde fixation. Our data are reported herein.
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Ependyma and ependymal protrusions of the lateral ventricles of the rabbit brain.
The ependymal lining of the lateral ventricles of the rabbit brain was studied by means of scanning (SEM) and transmission electron microscopy (TEM). There exist cells devoid of cilia in the anterior horn over the region of the caudate nucleus, in the inferior horn over the hippocampus and on the opposite side over cortical regions. On the surface of some of these ependymal cells, accumulations of cytoplasmic folds and globules can be found. They bulge at different height over the ependymal cells. Clots of these cell particles are tied off from the cell, coming to lie as globules either on or between the cilia of the ependyma. TEM reveals that these protrusions are cell debris consisting of different sized vesicles, cell organelles, tubuli and cell filaments. They originate from the ependymal layer but may reach down to subependymal cells. Multivesicular protrusions into the ventricular lumen are also observed. Possible causes of these protrusions are discussed; they are likely to be related to the age of the animals. On the ependyma of the caudate nucleus cilia, microvilli, microblebs and supraependymal neuronal cell processes are distributed unevenly over the surface. Within regions where cilia predominate there are cells which are tightly covered with microvilli. A certain direction of the course of the supraependymal neuronal fibers could not be found.
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DSCAM/PAK1 pathway suppression reverses neurogenesis deficits in iPSC-derived cerebral organoids from patients with Down syndrome.
Down syndrome (DS), caused by trisomy of chromosome 21, occurs in 1 of every 800 live births. Early defects in cortical development likely account for the cognitive impairments in DS, although the underlying molecular mechanism remains elusive. Here, we performed histological assays and unbiased single-cell RNA-Seq (scRNA-Seq) analysis on cerebral organoids derived from 4 euploid cell lines and from induced pluripotent stem cells (iPSCs) from 3 individuals with trisomy 21 to explore cell-type-specific abnormalities associated with DS during early brain development. We found that neurogenesis was significantly affected, given the diminished proliferation and decreased expression of layer II and IV markers in cortical neurons in the subcortical regions; this may have been responsible for the reduced size of the organoids. Furthermore, suppression of the DSCAM/PAK1 pathway, which showed enhanced activity in DS, using CRISPR/Cas9, CRISPR interference (CRISPRi), or small-molecule inhibitor treatment reversed abnormal neurogenesis, thereby increasing the size of organoids derived from DS iPSCs. Our study demonstrates that 3D cortical organoids developed in vitro are a valuable model of DS and provide a direct link between dysregulation of the DSCAM/PAK1 pathway and developmental brain defects in DS.