AI Networking Cookbook: Practical recipes for AI-assisted network automation and development
TL;DR
A new strategy to generate human cerebral organoids with vasculature and microglia-like cells using genetic code expansion technology (GCE-T) via site-specific protein engineering is presented, opening a new avenue to advance organoid research and applications.
Modeling neural-immune interactions in neurodegenerative and immune-mediated central nervous system (CNS) diseases requires human 3D models that capture cellular diversity and long-term tissue maturation. Here, we present an enhanced human induced pluripotent stem cell (hiPSC)-derived cerebral organoid (CO) platform optimized to mitigate core hypoxia for over 200 days. Timed pro-myelinating cues established organized neuronal layering and progressive axonal myelination through day 140, while vascular fusion yielded assembloids incorporating endothelial structures and microglia. Extended culture (>500-750 days) spontaneously reproduced hallmark features of human CNS aging, including cellular senescence signatures, neuroaxonal loss, hypomyelination, and the autonomous emergence of a neurotoxic astrocyte transcriptional profile in the complete absence of microglia or immune cells. Co-culture with autologous activated peripheral blood mononuclear cells (PBMC) resulted in transient immune infiltration and a pronounced type II interferon response across CNS lineages. High-plex spatial transcriptomics revealed that immune cell infiltration was associated with oligodendrocyte loss and in aged organoids also with downregulated oligodendrocyte myelin gene transcription. While not fully reproducing adult tissue stoichiometry, this platform enables longitudinal modeling of neural-immune crosstalk in age-related and neuroinflammatory CNS disorders.
This human iPSC-derived tumor–brain organoid platform provides a reliable and scalable system for studying complex tumor-neural interactions and exploring therapeutic approaches that aim to eliminate the tumor while preserving neural function.
Understanding human brain development, function, disease, and drug responses remains a central challenge in neuroscience and medicine. However, detailed analyses of living human brain tissue are technically and ethically difficult, and conventional studies have therefore relied largely on animal models and two-dimensional culture systems. Nevertheless, substantial differences exist between humans and animals in developmental processes, cellular composition, neural circuit, and pharmacological responses. Brain organoids are three-dimensional neural tissues generated from human embryonic stem cells or induced pluripotent stem cells, and they can recapitulate aspects of neural progenitor differentiation, cellular diversity, tissue architecture, and neuronal activity. Owing to these properties, brain organoids provide a valuable platform for human biology research. In this article, we review the development of brain organoid research and discuss recent advances including neuronal activity measurements, neural circuit models using assembloids and connectoids, reconstruction of human-enriched cell types and gene programs, analyses of inter-individual variability, toxicological studies, disease modeling, and drug evaluation. Furthermore, we outline the current status and future perspectives of human biology research using brain organoids, with particular attention to challenges such as heterogeneity, limited maturation, the absence of vascular and immune systems, standardization, quality control, and ethical considerations.
The development of functional human vasculature is essential for tissue engineering, disease modeling, and regenerative medicine. Conventional differentiation protocols of vascular lineages often exhibit lineage heterogeneity and limited control over cellular ratios. Here, we describe a protocol for generating vascular organoids (VOs) via orthogonal forward programming of hPSCs. By utilizing doxycycline-inducible activation of the transcription factors ETV2 and NKX3.1, hPSCs are rapidly directed toward endothelial and mural cell lineages, respectively. This strategy enables the assembly of VOs with precisely tunable cellular compositions within six days. When combined with fluorescent reporter lines (PECAM1-mRuby3 and ACTA2-EGFP), vascular networks can be visualized in real time without the need for tissue clearing or immunostaining. We detail procedures for stable cell line engineering, 3D organoid assembly, in vitro angiogenesis assays for drug screening, and in vivo transplantation under the mouse kidney capsule to form perfusable human vasculature. This platform provides a flexible, standardized, and scalable tool for investigating vascular biology, modeling inherited vasculopathies, and enhancing the vascularization of co-transplant tissues.
The goal is not to replicate the brain in miniature, but to reconstruct its organizing principles in an experimentally accessible system, offering new insights into human neural complexity while advancing neuroscience and medicine.
Microglia-neuron interactions play a key role in a variety of central nervous system disorders. Technologies using human induced pluripotent stem cells (hiPSCs) have been developed to model human brain cells with the goal to understand their function. To effectively study neuro-immune crosstalk and investigate microglial contributions to neuronal network development and function, both microglia and neurons should co-mature allowing for long-term interactions throughout their differentiation. Here, we present a co-maturation protocol that robustly generates glutamatergic neuronal networks containing hiPSC-derived microglia. We validated the long-term co-cultures using single-cell transcriptomics, imaging, and neuronal activity readouts. In this protocol, astrocytes were required for long-term survival of microglia and for their integration into neuronal networks. Our co-maturation approach induced the typical ramified microglia morphology and characteristic microglia-neuron interactions. Homeostatic markers such as P2RY12 and TMEM119 and neuronal remodeling-associated genes were upregulated compared to microglia monocultures, highlighting the necessity of the environment to generate and maintain the context-dependent microglia signature in vitro. In this manuscript, we include the full optimization process of our co-maturation approach, a comprehensive description of the protocol, practical guidelines, and troubleshooting tips. Our co-maturation model provides a powerful tool to assess the role of human microglia in modulating neuronal function and development in health and disease.
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