Biology subjects

Leible, D.

Publications and source records attributed to Leible, D..

3 recordsLinked to original sources

bioRxiv2025

Human brain organoids record the passage of time over multiple years in culture

The human brain develops and matures over an exceptionally prolonged period of time that spans nearly two decades of life. Processes that govern species-specific aspects of human postnatal brain development are difficult to study in animal models. While human brain organoids offer a promising in vitro model, they have thus far been shown to largely mimic early stages of brain development. Here, we developed human brain organoids for an unprecedented 5 years in culture, optimizing growth conditions able to extend excitatory neuron viability beyond previously-known limits. Using module scores of maturation-associated genes derived from a time course of endogenous human brain maturation, we show that brain organoids transcriptionally age with cell type-specificity through these many years in culture. Whole-genome methylation profiling reveals that the predicted epigenomic age of organoids sampled between 3 months and 5 years correlates precisely with time spent in vitro, and parallels epigenomic aging in vivo. Notably, we show that in chimeric organoids generated by mixing neural progenitors derived from "old" organoids with progenitors from "young" organoids, old progenitors rapidly produce late neuronal fates, skipping the production of earlier neuronal progeny that are instead produced by their young counterparts in the same co-cultures. The data indicate that human brain organoids can mature and record the passage of time over many years in culture. Progenitors that age in organoids retain a memory of the time spent in culture reflected in their ability to execute age-appropriate, late developmental programs.

neuroscience↗

bioRxiv2025

Combinatorial protein barcodes enable self-correcting neuron tracing with nanoscale molecular context

Mapping nanoscale neuronal morphology with molecular annotations is critical for understanding healthy and dysfunctional brain circuits. Current methods are constrained by image segmentation errors and by sample defects (e.g., signal gaps, section loss). Genetic strategies promise to overcome these challenges by using easily distinguishable cell identity labels. However, multicolor approaches are spectrally limited in diversity, whereas nucleic acid barcoding lacks a cellfilling morphology signal for segmentation. Here, we introduce PRISM (Protein-barcode Reconstruction via Iterative Staining with Molecular annotations), a platform that integrates combinatorial delivery of antigenically distinct, cell-filling proteins with tissue expansion, multi-cycle imaging, barcode-augmented reconstruction, and molecular annotation. Protein barcodes increase label diversity by >750-fold over multicolor labeling and enable morphology reconstruction with intrinsic error correction. We acquired a [~]10 million {micro}m3 volume of mouse hippocampal area CA2/3, multiplexed across 23 barcode antigen and synaptic marker channels. By combining barcodes with shape information, we achieve an 8x increase in automatic tracing accuracy of genetically labelled neurons. We demonstrate PRISM supports automatic proofreading across micron-scale spatial gaps and reconnects neurites across discontinuities spanning hundreds of microns. Using PRISMs molecular annotation capability, we map the distribution of synapses onto traced neural morphology, characterizing challenging synaptic structures such as thorny excrescences (TEs), and discovering a size correlation among spatially proximal TEs on the same dendrite. PRISM thus supports selfcorrecting neuron reconstruction with molecular context.

neuroscience↗

bioRxiv2022

Expansion Sequencing of RNA Barcoded Neurons in the Mammalian Brain: Progress and Implications for Molecularly Annotated Connectomics

Mapping and molecularly annotating mammalian neural circuits is challenging due to the inability to uniquely label cells while also resolving subcellular features such as synaptic proteins or fine cellular processes. We argue that an ideal technology for connectomics would have the following characteristics: the capacity for robust distance-independent labeling, synaptic resolution, molecular interrogation, and scalable computational methods. The recent development of high-diversity cellular barcoding with RNA has provided a way to overcome the labeling limitations associated with spectral dyes, however performing all-optical circuit mapping has not been demonstrated because no method exists to image barcodes throughout cells at synaptic-resolution. Here we show ExBarSeq, an integrated method combining in situ sequencing of RNA barcodes, immunostaining, and Expansion Microscopy coupled with an end-to-end software pipeline that automatically extracts barcode identities from large imaging datasets without data processing bottlenecks. As a proof of concept, we applied ExBarSeq to thick tissue sections from mice virally infected with MAPseq viral vectors and demonstrated the extraction of 50 barcoded cells in the visual cortex as well as cell morphologies uncovered via immunostaining. The current work demonstrates high resolution multiplexing of exogenous barcodes and endogenous synaptic proteins and outlines a roadmap for molecularly annotated connectomics at a brain-wide scale.

neuroscience↗