Neural Assembloids

From Stem Cells to Circuits

September 19-26, 2026

Director: Sergiu Pasca

Stanford University, USA

Co-Organizers: Jimena Andersen & Fikri Birey

Emory University, Atlanta, USA

Faculty:

Sergiu Pasca, Stanford University, USA

Lorenz Studer, Memorial Sloan Kettering Cancer Center, New York, USA

Laurent Nguyen, University of Liège, Belgium

Valentina Fossati, The Jackson Laboratory-NYSCF, New York, USA

Stanislav Zakharenko, St. Jude Children’s Research Hospital, Memphis, USA

Mina Gouti, Max Delbrück Center for Molecular Medicine, Berlin, Germany

Fikri Birey, Emory University, Atlanta, USA (Co-Organizer)

Jimena Andersen, Emory University, Atlanta, USA (Co-Organizer)

Human neural development and circuit assembly rely on precisely timed interactions between diverse cell types and brain regions –processes that have long been inaccessible to longitudinal and functional study. The advent of human induced pluripotent stem (hiPS) cells and 3D culture systems has transformed this landscape, enabling the generation of regionalized neural organoids and their integration into assembloids that recapitulate key steps of human neurodevelopment. These fused systems allow the controlled study of migration, axon guidance, and long-range circuit formation, while also providing unprecedented opportunities to model neurodevelopmental and neurodegenerative disorders in a human context.

This Advanced Course will bring together leaders pioneering the generation of neural assembloids and their applications. Participants will explore cutting-edge methods for generating regionalized organoids, assembling multi-tissue or multi-lineage systems, and interrogating them using imaging, electrophysiology, single-cell and spatial omics, viral tracing, and molecular tools. Through examples spanning cortico-striatal, cortico-motor, thalamo-cortical, sensory-motor, and neuroglial assembloids, the Course will highlight how these models reconstruct complex interactions between neurons, glia, and non-neural tissues, revealing principles of human development.

By providing a comprehensive and forward-looking overview of neural assembloid science from conceptual foundations to practical execution, this Advanced Course aims to equip participants with the knowledge and tools needed to build, analyze, and creatively apply assembloid systems to the next generation of questions in human neurodevelopment.

Sergiu Pasca

Assembloid approaches to reconstruct human neural assembly from pluripotent stem cells

The construction of the human nervous system involves a sequence of complex developmental events that are largely inaccessible in humans. What distinguishes the nervous system is its immense cellular diversity and the extraordinary precision with which diverse cell types are generated, positioned, and interconnected. While many of these cell types must be specified separately through discrete developmental signals, most functions of the brain arise not from individual cell types alone, but from emergent properties generated through cell-cell interactions, long-range connectivity, and circuit assembly.

During the lectures, I will trace the evolution of assembloid approaches as a strategy to reconstruct key steps of human neural assembly from pluripotent stem cells. To build toward this goal, we first introduced instructive signals to generate self-organizing, three-dimensional regionalized neural organoids that resemble distinct domains of the developing central nervous system. These cultures recapitulate many features of human neural development, can be generated reproducibly across many cell lines and experiments, and can be maintained for years in vitro, revealing intrinsic programs of maturation that progress toward postnatal stages.

Building on this capacity to generate regional and cellular diversity, we developed assembloids by combining distinct multicellular neural components to model interactions that cannot be captured in isolated organoids. I will describe how assembloids have enabled the study of human cell migration, neural microcircuit formation, interregional connectivity, midline crossing, neuromodulation, and disease mechanisms. These approaches provide a way to move from the generation of individual cell types and tissue domains toward the reconstruction of emergent properties of human neural circuits.

I will also discuss transplantation as an in vivo assembloid strategy, in which human neural organoids are integrated with living host circuits to advance maturation, connectivity, and functional readouts. Using this approach, engrafted human neurons can respond to sensory stimulation and influence animal behavior, enabling behavioral measurements from patient-derived human cells.

Finally, I will illustrate how assembloids, and their transplantation can be combined with modern neuroscience tools to investigate the cellular, molecular, and circuit-level consequences of mutations and copy number variants associated with neuropsychiatric disorders. Together, these approaches provide a bottom-up framework for studying the assembly, function, and dysfunction of human neural circuits.

Stanislav Zakharenko

Human assembloids to study thalamocortical synaptic transmission and plasticity

The thalamocortical (TC) system links the thalamus and cerebral cortex and plays a key role in sensory processing, attention, and perceptual learning. Dysfunction of the TC system is linked to epilepsy, schizophrenia, and other neuropsychiatric disorders. Long-term synaptic plasticity at TC synapses, in the form of long-term potentiation (LTP) and long-term depression (LTD), controls activity-dependent cortical plasticity, which in turn underlies perceptual learning. This knowledge comes mainly from rodent studies, so our understanding of synaptic plasticity in humans is limited. In my talk, I describe how we modelled the basic human TC system by fusing human induced pluripotent stem cell (hiPSC)–derived thalamic (hThOs) and cortical organoids (hCOs). To build optimal hThOs, we generated a reporter cell line from hiPSCs by inserting a tdTomato-coding sequence into the endogenous TCF7L2 locus. hThOs with high TCF7L2-tdTomato fluorescence showed significant enrichment of thalamic markers. Similarly, to build hCOs, we inserted the tdTomato-coding sequence into the endogenous VGLUT1 locus. Single-nucleus RNA-sequencing revealed that approximately 80% of cells in mature hThOs or hCOs were glutamatergic neurons. When fused to form TC assembloids, hThOs and hCOs formed reciprocal long-range axonal projections. Presynaptic terminal labelling with V5-tagged peroxidase APEX2 in the mitochondrial matrix revealed that hThOs and hCOs formed long-range synapses on opposite sides of the assembloids. Most previous attempts to measure synaptic plasticity in organoids have relied exclusively on extracellular recordings, which integrate signals from a large, heterogeneous population of cells and are not well suited to direct measurement of synaptic currents. Instead, I will describe how we used whole-cell patch-clamp electrophysiology to directly characterise synaptic transmission and plasticity at TC and corticothalamic (CT) synapses. Both TC and CT synapses showed glutamatergic synaptic transmission, producing AMPA receptor– and NMDA receptor–mediated currents. Using two-photon calcium imaging, we identified the postsynaptic sites of synaptic transmission that resembled the dendritic spines observed in animal models. Synaptically-evoked calcium transients in these spines were stronger than those in parent dendritic shafts, suggesting that the dendritic spines are indeed the sites of synaptic transmission that connect to presynaptic terminals, as observed in animal models. Both TC and CT synapses exhibited short-term plasticity (i.e., paired-pulse depression and paired-pulse facilitation, respectively), consistent with findings in animal models. LTP and LTD were reliably induced at both TC and CT synapses in response to either a train of presynaptic stimulations or spike-timing–dependent plasticity protocols. We also found that the mechanisms of LTP and LTD expression in this system differed from those described in rodents. Thus, TC assembloids provide a model system for exploring the basic mechanisms of synaptic plasticity in human circuits.

Valentina Fossati

Scaling human iPSC models for neuroscience: automation, glial biology, and next generation assays

Human induced pluripotent stem cell (iPSC)–derived models are transforming our ability to study neurological diseases, yet most studies remain limited to a small number of manually processed cell lines. This constraint prevents us from capturing the true biological variability arising from individual genetic backgrounds, which increasingly proves essential for understanding disease onset, penetrance, severity, and drug responses. During the lectures, a major focus will be on how cell culture automation enables large-scale, parallel processing of hundreds of iPSC lines, allowing us to systematically incorporate patient-specific genetic diversity into experimental design. Attention will also be devoted to the development of automated, high-throughput differentiation platforms for human glial cells, including astrocytes and microglia, to investigate how these cell types contribute to neurodegenerative diseases such as Alzheimer’s and Parkinson’s. Assay design considerations, quality control strategies, and the challenges of scaling glial models while preserving physiological relevance will be discussed. Finally, our pioneering work on human neural organoids with integrated microglia cultured on the International Space Station will be analysed, where microgravity imposes a radically different set of biological and logistical constraints. These spaceflight studies require unconventional planning, redundant workflows, and operation under suboptimal conditions. Topics of discussion will be how insights from low Earth orbit experiments feed back into terrestrial stem cell biology, providing novel information for optimising traditional culture systems.

Laurent Nguyen

Neuron migration shapes cerebral cortex morphogenesis across species

The cerebral cortex is an evolutionarily expanded brain structure characterized by laminar organization and areal specialization that underpin higher cognitive functions. Its morphogenesis arises from the progressive remodeling of a primordial neuroepithelium through tightly orchestrated developmental steps. Perturbations at any stage of this process can result in cortical malformations associated with a broad spectrum of neurological and neuropsychiatric disorders. Among the critical events shaping cortical assembly, neuronal migration ensures the precise positioning of diverse neural cell types within emerging circuits. Beyond their canonical role in relocation, migrating neurons engage in transient cellular interactions that extend their functional influence during development. Recent findings from my laboratory and others demonstrate that neuronal migration provides instructive cues to neighboring cells, thereby actively contributing to the patterning and maturation of the developing cortex.
During the lectures, I will present our recent work on the migration of cortical interneurons and their dynamic interactions with surrounding cellular populations. By integrating transgenic mouse models with human cerebral assembloids and fetal brain tissue, we examine how these behaviors differ between mouse and human cortices and how they have evolved across species with distinct cortical architectures. We propose that the transient interactions mediated by migrating interneurons represent a fundamental mechanism of developmental self-organization, shaping both cortical morphogenesis and functional circuit assembly.

Lorenz Studer

From induction to assembly: decoding human brain development and disease with stem cell technologies

The ability to generate specific neuronal subtypes from human pluripotent stem cells (hPSCs) has transformed our understanding of human brain development and opened new avenues for disease modelling and cell therapy. In this course, I will trace the arc from foundational neural induction and regional patterning paradigms to advanced organoid and assembloid technologies. I will highlight how increasing biological complexity in stem cell models can unlock new insights into brain development, neurodegeneration, and neuropsychiatric disease, while also discussing limitations of current technologies and how these have stimulated novel basic research aimed at directing fate, maturation, and age of hPSC-derived cell types with improved precision.
We have developed a dual SMAD inhibition paradigm that enables rapid and efficient conversion of hPSCs into neuroectoderm by simultaneously blocking BMP and TGFβ signalling. This simple but powerful platform has become a universal starting point for directed neural differentiation and has enabled the systematic derivation of more than 50 distinct CNS and PNS lineages by combining neural induction with anterior–posterior and dorso–ventral patterning strategies. A key remaining limitation of hPSC-derived neural lineages is their fetal-like maturation state. I will present some of our ongoing efforts to overcome this barrier through strategies that promote accelerated maturation and enable induced ageing in hPSC-derived neural cultures, features essential for faithful modelling of late-onset neurological disorders.
The same developmental logic applies to 3D organoid culture systems, where we have used graded patterning cues to establish regional forebrain identities within a given polarised organoid. Alternatively, organoids can be patterned separately towards specific CNS identities and fused into assembloids that mimic regional interaction, migration, and axonal targeting across brain regions. I will discuss our recent work using an assembloid approach to study the development of parvalbumin (PV)+ cortical interneurons. These late-maturing interneurons are notoriously difficult to generate in vitro, yet we have established assembloid conditions that reliably enable their specification and functional maturation. Using isogenic structural variants associated with increased risk for neuropsychiatric diseases, we identified variant-specific defects in interneuron migration, mitochondrial function, and molecular identity, providing mechanistic insight into how disrupted cortical interneuron development may contribute to schizophrenia risk. Ongoing efforts extend this assembloid paradigm beyond the forebrain by generating four-way assembloids that incorporate striatal and midbrain-like structures. For midbrain specification, I will discuss a recent breakthrough in the lab for generating not just generic midbrain dopaminergic (mDA) neurons but specific A9 (substantia nigra) versus A10 (ventral tegmental area) subtypes on demand, and how these insights may be useful for disease modelling and cell therapy applications.
Finally, I will highlight the power of lineage-tracking technologies to uncover fundamental principles of neural fate specification. In particular, a recent genetic fate-mapping study in the lab has revealed unexpected competency windows at very early stages of hPSC differentiation that influence competence toward forebrain/midbrain versus hindbrain fates. These findings may explain some of the variability commonly observed for regional differentiation paradigms across hPSC lines. Genetic fate-mapping tools include both viral-based barcoding libraries and advanced prime-editing strategies for continuous tracking, which we are currently implementing to track cortical organoid and oligodendrocyte differentiation paradigms. Oligodendrocyte biology represents another frontier where more efficient and accelerated directed differentiation protocols are urgently needed. I will discuss our development of a rapid oligodendrocyte induction protocol that may offer a broadly useful tool for the field.
Together, these studies illustrate how integrating directed differentiation, assembloid engineering, and lineage recording can drive discovery in human developmental neurobiology, disease modelling, and cell therapy applications.

Jimena Andersen

Organoids and assembloids as platforms to study human neurodevelopment and motor system vulnerability

A fundamental challenge in studying the human nervous system is that many cell-cell interactions shaping its development and driving its degeneration are inaccessible in vivo and poorly replicated in traditional in vitro models. Human iPSC-derived organoids and assembloids provide a powerful alternative, enabling the reconstruction of specific cellular interactions within a controlled, human context. Here, I will present work from my laboratory using these platforms to investigate cell-cell interactions across three related areas.
First, I will discuss assembloid-based approaches to dissect the role of extrinsic, partner-derived signals in neuronal development. These signals are inherently absent from single regionalised neural organoids but can be reintroduced by fusing distinct compartments together using assembloids.
Second, I will discuss our efforts to study oligodendrocyte-lineage cells and their interactions with neurons in spinal cord organoids and cortico-motor assembloids. I will describe how we utilise these models to examine the signals that regulate oligodendrocyte lineage progression and capture disease-associated cellular and transcriptional signatures.
Finally, I will discuss how we are using organoids and assembloids to model degeneration in ALS, focusing on cell-type-specific dysfunction and the vulnerability of different cell populations within the motor system.
Together, this work demonstrates how organoid and assembloid systems are transforming our capacity to study human cell-cell interactions – from the earliest stages of spinal cord development to the cellular mechanisms underlying motor neuron disease.

Mina Gouti

Beyond assembly: self-organization as a principle for building advanced human neuromuscular models

The development of human organoid and assembloid technologies has enabled the creation of increasingly complex tissues and circuits in vitro. While many methods depend on the targeted assembly of region-specific organoids, an alternative and powerful approach is the exploitation of intrinsic developmental programmes that drive multi-lineage self-organisation from common progenitor states. Such systems provide unique opportunities to investigate how coordinated interactions between neural, mesodermal, and non-neural cell types develop into functional tissues and how these interactions are disrupted in disease.
During the lectures, I will present our work on human neuromuscular organoids that self-organize from neuromesodermal progenitors into integrated systems comprising spinal cord neurons, skeletal muscle, and supporting cell types, including vascular and immune components. I will discuss how developmental timing, morphogen signaling, and physical cues contribute to the emergence of functional neuromuscular junctions and circuit maturation, highlighting self-organization as a key principle underlying multi-tissue formation.
In addition, how these systems can be scaled and standardized through automation, enabling reproducible generation and high-throughput interrogation of patient-specific organoids will be analyzed and discussed. Combining automated culture platforms with functional assays and single-cell omics allows us to capture disease-relevant phenotypes across genetic backgrounds and to dissect cell-type-specific responses to therapeutic interventions.
Finally, attention will be dedicated to how neuromuscular organoids can be leveraged to model human diseases, with a focus on spinal muscular atrophy and amyotrophic lateral sclerosis. These models reveal early pathological events at the neuromuscular junction and identify distinct mechanisms of drug response, providing a framework for precision medicine approaches.
Together, this work positions self-organizing systems as a complementary strategy to engineered approaches, enabling the study of complex human tissue interactions, the discovery of disease mechanisms, and the development of scalable platforms for therapeutic screening.

Fikri Birey

From disease mechanisms to precision therapeutics using human neural assembloids

Human neural organoids and assembloids have opened unprecedented access to disease-relevant biology that is otherwise inaccessible in model organisms. During the lectures, how we leverage these platforms to transition from disease gene identification to mechanistic understanding and, ultimately, to therapeutic interventions will be discussed.
Initial focus will be on calcium channelopathies caused by mutations in CACNA1C and CACNA1A as illustrative case studies. Using forebrain assembloids, calcium imaging, and single-cell transcriptomics, we have characterised how gain- and loss-of-function variants in voltage-gated calcium channels disrupt neuronal development, migration, and circuit-level activity in a human context. These studies demonstrate the unique capacity of assembloid systems to capture cell-cell interactions and migratory phenotypes that are central to channelopathy pathophysiology but absent from conventional monolayer cultures. The use of these same platforms as testing grounds for genetic therapeutic rescue, including the development of antisense oligonucleotides (ASOs) for calcium channelopathies, will be analysed, showing the potential of organoid systems to support preclinical evaluation of precision nucleic acid therapies. Finally, attention will be devoted to the development of novel assembloid models designed to capture aspects of interregional connectivity and modulatory input that are absent from isolated organoids. These next-generation systems aim to uncover disease-relevant domains that have largely remained unexplored in human cellular models.
Overall, the lectures will seek to illustrate how human organoid systems can help close the translational loop from patient genotype to disease mechanism to candidate therapy within a single, scalable experimental framework rooted in human neurobiology.