A framework for neural organoids, assembloids and transplantation studies.

As the field of neural organoids and assembloids expands, there is an emergent need for guidance and advice on designing, conducting and reporting experiments to increase the reproducibility and utility of these models. In this Perspective, we present a framework for the experimental process that encompasses ensuring the quality and integrity of human pluripotent stem cells, characterizing and manipulating neural cells in vitro, transplantation techniques and considerations for modelling human development, evolution and disease. As with all scientific endeavours, we advocate for rigorous experimental designs tailored to explicit scientific questions as well as transparent methodologies and data sharing to provide useful knowledge for current research practices and for developing regulatory standards.

Glioblastoma Cortical Organoids Recapitulate Cell-State Heterogeneity and Intercellular Transfer.

Glioblastoma (GBM) is characterized by heterogeneous malignant cells that are functionally integrated within the neuroglial microenvironment. In this study, we model this ecosystem by growing GBM into long-term cultured human cortical organoids that contain the major neuroglial cell types found in the cerebral cortex. Single-cell RNA sequencing analysis suggests that, compared with matched gliomasphere models, GBM cortical organoids more faithfully recapitulate the diversity and expression programs of malignant cell states found in patient tumors.

Transcriptional dynamics orchestrating the development and integration of neurons born in the adult hippocampus.

The adult hippocampus generates new granule cells (aGCs) with functional capabilities that convey unique forms of plasticity to the preexisting circuits. While early differentiation of adult radial glia-like cells (RGLs) has been studied extensively, the molecular mechanisms guiding the maturation of postmitotic neurons remain unknown. Here, we used a precise birthdating strategy to study aGC differentiation using single-nuclei RNA sequencing.

scX: a user-friendly tool for scRNAseq exploration.

Motivation: Single-cell RNA sequencing (scRNAseq) has transformed our ability to explore biological systems. Nevertheless, proficient expertise is essential for handling and interpreting the data.

Results: In this article, we present scX, an R package built on the Shiny framework that streamlines the analysis, exploration, and visualization of single-cell experiments.

Fetal brain response to maternal inflammation requires microglia.

In utero infection and maternal inflammation can adversely impact fetal brain development. Maternal systemic illness, even in the absence of direct fetal brain infection, is associated with an increased risk of neuropsychiatric disorders in affected offspring. The cell types mediating the fetal brain response to maternal inflammation are largely unknown, hindering the development of novel treatment strategies.

Making Ramón y Cajal proud: Development of cell identity and diversity in the cerebral cortex.

Since the beautiful images of Santiago Ramón y Cajal provided a first glimpse into the immense diversity and complexity of cell types found in the cerebral cortex, neuroscience has been challenged and inspired to understand how these diverse cells are generated and how they interact with each other to orchestrate the development of this remarkable tissue. Some fundamental questions drive the field's quest to understand cortical development: what are the mechanistic principles that govern the emergence of neuronal diversity? How do extrinsic and intrinsic signals integrate with physical forces and activity to shape cell identity? How do the diverse populations of neurons and glia influence each other during development to guarantee proper integration and function? The advent of powerful new technologies to profile and perturb cortical development at unprecedented resolution and across a variety of modalities has offered a new opportunity to integrate past knowledge with brand new data. Here, we review some of this progress using cortical excitatory projection neurons as a system to draw out general principles of cell diversification and the role of cell-cell interactions during cortical development.

Transcriptional dynamics orchestrating the development and integration of neurons born in the adult hippocampus.

The adult hippocampus generates new granule cells (aGCs) that exhibit distinct functional capabilities along development, conveying a unique form of plasticity to the preexisting circuits. While early differentiation of adult radial glia-like neural stem cells (RGL) has been studied extensively, the molecular mechanisms guiding the maturation of postmitotic neurons remain unknown. Here, we used a precise birthdating strategy to follow newborn aGCs along differentiation using single-nuclei RNA sequencing (snRNA-seq).

scX: A user-friendly tool for scRNA-seq exploration.

Single-cell RNA sequencing (scRNA-seq) has transformed our ability to explore biological systems. Nevertheless, proficient expertise is essential for handling and interpreting the data. In this paper, we present scX, an R package built on the Shiny framework that streamlines the analysis, exploration, and visualization of single-cell experiments.

A family portrait of human brain cells.

A cell census provides information on the source of human brain specialization.

Cell-type specific defects in PTEN-mutant cortical organoids converge on abnormal circuit activity.

De novo heterozygous loss-of-function mutations in phosphatase and tensin homolog (PTEN) are strongly associated with autism spectrum disorders; however, it is unclear how heterozygous mutations in this gene affect different cell types during human brain development and how these effects vary across individuals. Here, we used human cortical organoids from different donors to identify cell-type specific developmental events that are affected by heterozygous mutations in PTEN. We profiled individual organoids by single-cell RNA-seq, proteomics and spatial transcriptomics and revealed abnormalities in developmental timing in human outer radial glia progenitors and deep-layer cortical projection neurons, which varied with the donor genetic background.

Proper acquisition of cell class identity in organoids allows definition of fate specification programs of the human cerebral cortex.

Realizing the full utility of brain organoids to study human development requires understanding whether organoids precisely replicate endogenous cellular and molecular events, particularly since acquisition of cell identity in organoids can be impaired by abnormal metabolic states. We present a comprehensive single-cell transcriptomic, epigenetic, and spatial atlas of human cortical organoid development, comprising over 610,000 cells, from generation of neural progenitors through production of differentiated neuronal and glial subtypes. We show that processes of cellular diversification correlate closely to endogenous ones, irrespective of metabolic state, empowering the use of this atlas to study human fate specification.

Engineering multicellular living systems-a Keystone Symposia report.

The ability to engineer complex multicellular systems has enormous potential to inform our understanding of biological processes and disease and alter the drug development process. Engineering living systems to emulate natural processes or to incorporate new functions relies on a detailed understanding of the biochemical, mechanical, and other cues between cells and between cells and their environment that result in the coordinated action of multicellular systems. On April 3-6, 2022, experts in the field met at the Keystone symposium "Engineering Multicellular Living Systems" to discuss recent advances in understanding how cells cooperate within a multicellular system, as well as recent efforts to engineer systems like organ-on-a-chip models, biological robots, and organoids.

A nomenclature consensus for nervous system organoids and assembloids.

Self-organizing three-dimensional cellular models derived from human pluripotent stem cells or primary tissue have great potential to provide insights into how the human nervous system develops, what makes it unique and how disorders of the nervous system arise, progress and could be treated. Here, to facilitate progress and improve communication with the scientific community and the public, we clarify and provide a basic framework for the nomenclature of human multicellular models of nervous system development and disease, including organoids, assembloids and transplants.

Pyramidal neuron subtype diversity governs microglia states in the neocortex.

Microglia are specialized macrophages in the brain parenchyma that exist in multiple transcriptional states and reside within a wide range of neuronal environments. However, how and where these states are generated remains poorly understood. Here, using the mouse somatosensory cortex, we demonstrate that microglia density and molecular state acquisition are determined by the local composition of pyramidal neuron classes.

Brain organoids: the quest to decipher human-specific features of brain development.

The development of the human brain occurs largely in utero over long periods of time and is thus experimentally inaccessible; therefore, tractable experimental models are needed. Human brain organoid have emerged as powerful model systems to investigate human-specific features of brain development. Focusing on the cerebral cortex, here, we discuss how brain, and more specifically cortical, organoid models have newly enabled discovery of aspects of progenitor biology and cortical-cell diversification that are unique to humans.

Dissecting cellular diversity of cortical GABAergic cells across multiple modalities: A turning point in neuronal taxonomy.

Decoding the complexity of the brain requires an understanding of the architecture, function, and development of its neuronal circuits. Neuronal classifications that group neurons based on specific features/behaviors have become essential to further analyze the different subtypes in a systematic and reproducible way. A comprehensive taxonomic framework, accounting for multiple defining and quantitative features, will provide the reference to infer generalized rules for cells ascribed to the same neuronal type, and eventually predict cellular behaviors, even in the absence of experimental measures.

Autism genes converge on asynchronous development of shared neuron classes.

Genetic risk for autism spectrum disorder (ASD) is associated with hundreds of genes spanning a wide range of biological functions. The alterations in the human brain resulting from mutations in these genes remain unclear. Furthermore, their phenotypic manifestation varies across individuals.

Optogenetic axon guidance in embryonic zebrafish.

Axons form the long-range connections of biological neuronal networks, which are built through the developmental process of axon guidance. Here, we describe a protocol to precisely and non-invasively control axonal growth trajectories in live zebrafish embryos using focal light activation of a photoactivatable Rac1. We outline techniques for photostimulation, time-lapse imaging, and immunohistochemistry.

Genetic dissection of the glutamatergic neuron system in cerebral cortex.

Diverse types of glutamatergic pyramidal neurons mediate the myriad processing streams and output channels of the cerebral cortex, yet all derive from neural progenitors of the embryonic dorsal telencephalon. Here we establish genetic strategies and tools for dissecting and fate-mapping subpopulations of pyramidal neurons on the basis of their developmental and molecular programs. We leverage key transcription factors and effector genes to systematically target temporal patterning programs in progenitors and differentiation programs in postmitotic neurons.