Human Forebrain Assembloids
Project Overview
This research combines genetically engineered human forebrain assembloids with polymeric nanofiber scaffolds to create a model system for studying interneuron migration disorders. These disorders are associated with various neurodevelopmental conditions including epilepsy, autism spectrum disorder, and schizophrenia.
The study knocks out ERBB4, a receptor on migrating interneurons whose variants are linked to schizophrenia, epilepsy and intellectual disability, and asks how its loss changes interneuron migration. I did the study’s computational and statistical analysis: the RNA-seq analysis of knockout against control assembloids (differential expression, GO and pathway enrichment, and the overlap with epilepsy and intellectual-disability genes), a protein-interaction network of the neuron-migration genes, the pathway analysis of the sorted interneurons’ proteomics, the statistical tests on the imaging and migration measurements and, in the submitted manuscript, a pan-tissue correlation of the control assembloids.
Forebrain Assembloids
Dorsal and ventral forebrain organoids fused, so that reporter-labelled interneurons migrate from one into the other
Transcriptomic Profiling
RNA-seq of ERBB4-knockout and control assembloids, with GO and pathway enrichment
Network Biology
A protein-interaction network of the neuron-migration genes that the knockout downregulates
Pan-tissue Correlation
Control assembloid transcriptomes correlated with a panel of human tissues (submitted manuscript)
Scientific Context
Interneuron migration is a critical process during brain development where inhibitory neurons born in the ventral forebrain migrate long distances to reach their final positions in the cortex. Disruptions in this process lead to an imbalance between excitation and inhibition, contributing to various neurological and psychiatric disorders.
Forebrain Assembloids
These are fusion organoids that combine dorsal (cortical) and ventral (subcortical) brain regions, allowing researchers to study interneuron migration in a human-relevant 3D model system. They provide unique insights into human-specific aspects of brain development that cannot be studied in animal models.
Nanofiber Scaffolds
The integration of polymeric nanofiber scaffolds provides physical guidance cues that influence cell migration patterns. This biomaterial interface allows for controlled studies of how physical and molecular cues interact to guide neuron migration.
Clinical Relevance
Understanding interneuron migration disorders is crucial for developing therapies for epilepsy, autism, and schizophrenia. This model system provides a platform for drug screening and testing potential therapeutic interventions.
Computational Analysis Pipeline
RNA-seq Data Processing
Bulk RNA-seq of knockout and control assembloids (in the preprint, day 60, four biological replicates each): read quality control and trimming, and transcript quantification with Kallisto.
Differential Expression Analysis
Genes differentially expressed between knockout and control assembloids, found with DESeq2 under false-discovery-rate control.
Pathway Enrichment
GO and KEGG enrichment of the differentially expressed genes in DAVID, and a Fisher’s exact test of their overlap with epilepsy and intellectual-disability gene sets. In the preprint, genes for axon guidance and the actin and microtubule cytoskeleton were among those the knockout downregulated.
Protein-Interaction Network
A STRING network of the 25 neuron-migration genes downregulated in the knockout; it centres on RHOA, NTRK2 and FYN.
Proteomics Pathways
Pathway enrichment of the proteins that differ between knockout and control interneurons sorted from the assembloids, which pointed to SLIT/ROBO and RHO GTPase signalling.
Pan-Tissue Correlation
In the submitted manuscript (not in the preprint), the transcriptomes of control assembloids were correlated with those of a panel of human tissues, to see which they most resemble.
Findings in the Preprint
Interneurons Migrate Less
ERBB4 loss did not stop interneurons forming, but ventral organoids without it sent out fewer neuronal projections, and fewer of their interneurons migrated 100 µm or more in an outgrowth assay.
RHOA/RAC Signalling
Knockout assembloids downregulated neuron-migration and cytoskeleton genes; a network of the 25 downregulated neuron-migration genes centres on RHOA, NTRK2 and FYN, pointing to dysregulated RHOA/RAC signalling.
Interneuron Proteome
Proteomics of interneurons sorted from the assembloids pointed to SLIT/ROBO and RHO GTPase signalling.
Technical Details
Model & Data
- Cells: IMR90 iPSCs with inducible Cas9 and a GAD1-mClover interneuron reporter; ERBB4 exon 2 deleted
- RNA-seq (preprint): Day-60 assembloids, four knockout and four control replicates; NovaSeq 6000, 150 bp paired-end
- Proteomics: SWATH-MS of FACS-sorted mClover-positive interneurons
Analysis Tools
- FastQC, Trimmomatic, MultiQC: Read quality
- Kallisto, tximport: Quantification
- DESeq2: Differential expression
- DAVID: GO and KEGG enrichment
- STRING: Protein-interaction network
Statistical Approach
- Differential expression: DESeq2, Benjamini-Hochberg false discovery rate
- Gene-set overlap: Two-sided Fisher’s exact test
- Imaging measures: Student’s t-test and Mann-Whitney test
Clinical & Therapeutic Implications
This research has potential implications for understanding and treating neurodevelopmental disorders.
Disease Modeling
The assembloid system provides a human-relevant platform for modeling neurodevelopmental disorders, allowing researchers to study disease mechanisms that cannot be fully recapitulated in animal models or 2D cell cultures.
Drug Screening Platform
This system can be used for high-throughput screening of potential therapeutic compounds. The identified molecular signatures serve as readouts for assessing drug efficacy in rescuing migration defects.
Personalized Medicine
Patient-derived assembloids can be generated to study individual-specific disease mechanisms and test personalized therapeutic strategies, advancing precision medicine approaches for neurodevelopmental disorders.
Biomarker Development
The molecular signatures identified could serve as biomarkers for early diagnosis or treatment monitoring, potentially enabling earlier intervention in at-risk individuals.
Collaborative Research Team
This project brings together stem-cell biology, genome engineering, bioengineering, imaging, proteomics and computational biology to study how human cortical interneurons migrate.
Lead PI: Dr. Debojyoti Chakraborty, IGIB CSIR
Collaborators: Bioengineering and imaging groups
Computational and Statistical Analysis: Vishal Bharti (RNA-seq, GO and pathway enrichment, the protein-interaction network, the proteomics pathway analysis, the statistics and, in the submitted manuscript, the pan-tissue correlation)
Duration: 2023 - Present
Status: Research Square preprint; under revision at Stem Cell Reports
Publication
An interface of genetically engineered human forebrain assembloids and polymeric nanofiber scaffolds for multiscale profiling of interneuron-migration disorders
Research Square preprint; under revision at Stem Cell Reports. DOI: 10.21203/rs.3.rs-3831019/v1
Summary: ERBB4 variants are linked to schizophrenia, epilepsy and intellectual disability, but its role in the migration of human cortical interneurons was unknown. The study knocks out ERBB4 with CRISPR in human iPSCs that carry an interneuron reporter, derives dorsal and ventral forebrain organoids and fuses them into assembloids, and follows interneuron migration in the assembloids and on nanofiber scaffolds that mimic the fibrous extracellular matrix. ERBB4 loss did not stop interneurons forming but reduced their migration, and the RNA-seq and proteomics pointed to RHOA/RAC and SLIT/ROBO signalling.
Future Research Directions
Single-Cell Resolution
Expanding analysis to single-cell RNA sequencing to understand cell-type-specific effects and heterogeneity in migration disorders. This will reveal rare cell populations and transitional states during migration.
Spatial Transcriptomics
Implementing spatial transcriptomics to map gene expression patterns in 3D within assembloids, providing insights into how spatial organization affects interneuron migration and integration.
Drug Discovery Pipeline
Developing automated screening platforms using the molecular signatures as readouts to identify compounds that can rescue migration defects, accelerating therapeutic development.
Clinical Translation
Validating findings in patient samples and developing diagnostic tools based on the identified biomarkers, moving towards clinical implementation of assembloid-based personalized medicine.
Interested in Neurodevelopmental Research?
If you're interested in collaborating on brain organoid research, computational neuroscience, or neurodevelopmental disorders, I'd be happy to discuss potential opportunities.