Transcriptomic Mapping of Neural Diversity, Differentiation and Functional Trajectory in iPSC-Derived 3D Brain Organoid Models

Experimental models of the central nervous system (CNS) are imperative for developmental and pathophysiological studies of neurological diseases. Among these models, three-dimensional (3D) induced pluripotent stem cell (iPSC)-derived brain organoid models have been successful in mitigating some of the drawbacks of 2D models; however, they are plagued by high organoid-to-organoid variability, making it difficult to compare specific gene regulatory pathways across 3D organoids with those of the native brain. Single-cell RNA sequencing (scRNA-seq) transcriptome datasets have recently emerged as powerful tools to perform integrative analyses and compare variability across organoids. However, transcriptome studies focusing on late-stage neural functionality development have been underexplored. Here, we combine and analyze 8 brain organoid transcriptome databases to study the correlation between differentiation protocols and their resulting cellular functionality across various 3D organoid and exogenous brain models. We utilize dimensionality reduction methods including principal component analysis (PCA) and uniform manifold approximation projection (UMAP) to identify and visualize cellular diversity among 3D models and subsequently use gene set enrichment analysis (GSEA) and developmental trajectory inference to quantify neuronal behaviors such as axon guidance, synapse transmission and action potential. We showed high similarity in cellular composition, cellular differentiation pathways and expression of functional genes in human brain organoids during induction and differentiation phases, i.e., up to 3 months in culture. However, during the maturation phase, i.e., 6-month timepoint, we observed significant developmental deficits and depletion of neuronal and astrocytes functional genes as indicated by our GSEA results. Our results caution against use of organoids to model pathophysiology and drug response at this advanced time point and provide insights to tune in vitro iPSC differentiation protocols to achieve desired neuronal functionality and improve current protocols.

Imaging Three-Dimensional Brain Organoid Architecture from Meso- to Nanoscale across Development

Organoids are human stem cell-derived three-dimensional cultures offering a new avenue to model human development and disease. Brain organoids allow studying various aspects of human brain development in the finest details in vitro in a tissue-like context. However, spatial relationships of subcellular structures such as synaptic contacts between distant neurons are hardly accessible by conventional light microscopy. This limitation can be overcome by systems that quickly image the entire organoid in three dimensions and in super-resolution. To that end we have developed a setup combining tissue expansion and light sheet fluorescence microscopy for imaging and quantifying diverse spatial parameters during organoid development. This technique enables zooming from a mesoscopic perspective into super-resolution within a single imaging session, thus revealing cellular and subcellular structural details in three spatial dimensions, including unequivocal delineation of mitotic cleavage planes as well as the alignment of pre- and postsynaptic proteins. We expect light sheet fluorescence expansion microscopy (LSFEM) to facilitate qualitative and quantitative assessment of organoids in developmental and disease-related studies.Summary statementThe combination of light sheet fluorescence and expansion microscopy enables imaging of mature human brain organoids in toto and down to synaptic resolution

Methodologies for Generating Brain Organoids to Model Viral Pathogenesis in the CNS

(1) Background: The human brain is of interest in viral research because it is often the target of viruses. Neurological infections can result in consequences in the CNS, which can result in death or lifelong sequelae. Organoids modeling the CNS are notable because they are derived from stem cells that differentiate into specific brain cells such as neural progenitors, neurons, astrocytes, and glial cells. Numerous protocols have been developed for the generation of CNS organoids, and our goal was to describe the various CNS organoid models available for viral pathogenesis research to serve as a guide to determine which protocol might be appropriate based on research goal, timeframe, and budget. (2) Methods: Articles for this review were found in Pubmed, Scopus and EMBASE. The search terms used were “brain + organoid” and “CNS + organoid” (3) Results: There are two main methods for organoid generation, and the length of time for organoid generation varied from 28 days to over 2 months. The costs for generating a population of organoids ranged from USD 1000 to 5000. (4) Conclusions: There are numerous methods for generating organoids representing multiple regions of the brain, with several types of modifications for fine-tuning the model to a researcher’s specifications. Organoid models of the CNS can serve as a platform for characterization and mechanistic studies that can reduce or eliminate the use of animals, especially for viruses that only cause disease in the human CNS.

Induction of inverted morphology in brain organoids by vertical-mixing bioreactors

Organoid technology provides an opportunity to generate brain-like structures by recapitulating developmental steps in the manner of self-organization. Here we examined the vertical-mixing effect on brain organoid structures using bioreactors and established inverted brain organoids. The organoids generated by vertical mixing showed neurons that migrated from the outer periphery to the inner core of organoids, in contrast to orbital mixing. Computational analysis of flow dynamics clarified that, by comparison with orbital mixing, vertical mixing maintained the high turbulent energy around organoids, and continuously kept inter-organoid distances by dispersing and adding uniform rheological force on organoids. To uncover the mechanisms of the inverted structure, we investigated the direction of primary cilia, a cellular mechanosensor. Primary cilia of neural progenitors by vertical mixing were aligned in a multidirectional manner, and those by orbital mixing in a bidirectional manner. Single-cell RNA sequencing revealed that neurons of inverted brain organoids presented a GABAergic character of the ventral forebrain. These results suggest that controlling fluid dynamics by biomechanical engineering can direct stem cell differentiation of brain organoids, and that inverted brain organoids will be applicable for studying human brain development and disorders in the future.

Human ALS/FTD brain organoid slice cultures display distinct early astrocyte and targetable neuronal pathology

Amyotrophic lateral sclerosis overlapping with frontotemporal dementia (ALS/FTD) is a fatal and currently untreatable disease characterized by rapid cognitive decline and paralysis. Elucidating initial cellular pathologies is central to therapeutic target development, but obtaining samples from presymptomatic patients is not feasible. Here, we report the development of a cerebral organoid slice model derived from human induced pluripotent stem cells (iPSCs) that recapitulates mature cortical architecture and displays early molecular pathology of C9ORF72 ALS/FTD. Using a combination of single-cell RNA sequencing and biological assays, we reveal distinct transcriptional, proteostasis and DNA repair disturbances in astroglia and neurons. We show that astroglia display increased levels of the autophagy signaling protein P62 and that deep layer neurons accumulate dipeptide repeat protein poly(GA), DNA damage and undergo nuclear pyknosis that could be pharmacologically rescued by GSK2606414. Thus, patient-specific iPSC-derived cortical organoid slice cultures are a reproducible translational platform to investigate preclinical ALS/FTD mechanisms as well as novel therapeutic approaches.

Unraveling Human Brain Development and Evolution Using Organoid Models

Brain organoids are proving to be physiologically relevant models for studying human brain development in terms of temporal transcriptional signature recapitulation, dynamic cytoarchitectural development, and functional electrophysiological maturation. Several studies have employed brain organoid technologies to elucidate human-specific processes of brain development, gene expression, and cellular maturation by comparing human-derived brain organoids to those of non-human primates (NHPs). Brain organoids have been established from a variety of NHP pluripotent stem cell (PSC) lines and many protocols are now available for generating brain organoids capable of reproducibly representing specific brain region identities. Innumerous combinations of brain region specific organoids derived from different human and NHP PSCs, with CRISPR-Cas9 gene editing techniques and strategies to promote advanced stages of maturation, will successfully establish complex brain model systems for the accurate representation and elucidation of human brain development. Identified human-specific processes of brain development are likely vulnerable to dysregulation and could result in the identification of therapeutic targets or disease prevention strategies. Here, we discuss the potential of brain organoids to successfully model human-specific processes of brain development and explore current strategies for pinpointing these differences.

A new brain organoid model to study Parkinson’s Disease

Human midbrain organoid models represent a new tool to study the underlaying etiology of Parkinson’s disease in physiological conditions.