Biology subjects

Amin, N. D.

Publications and source records attributed to Amin, N. D..

5 recordsLinked to original sources

bioRxiv2026

RNA isoform-resolved multiplexed sequencing with bioorthogonal barcoding

RNA isoform dysregulation drives disease pathogenesis and is the target of FDA-approved splice-switching therapeutics. However, multiplexed sequencing methods discard splice junction information because only 3' termini are barcoded and counted. Here, we repurpose acylation and click chemistries to conjugate bioorthogonal barcodes (bobcodes) directly onto multiple internal positions along cellular RNAs. Bobcoded RNAs from multiple samples are pooled for multiplexed cDNA synthesis, during which reverse transcriptase switches from each RNA template onto its tethered bobcode with greater than 99% accuracy in species mixing experiments. Bobcode attachment intervals set cDNA insert sizes without a library fragmentation step, and priming with poly(dT) or random hexamers selects between 3'-end counting and full-length isoform capture. A bioorthogonal barcode-sequencing (BOB-seq v0.1) drug screen identifies transcriptome-wide on- and off-target RNA splicing effects and outperforms existing multiplexing RNA sequencing methods in workflow simplicity, sample-to-sample variability, and barcoding accuracy. Bobcodes add isoform resolution to scalable multiplexed RNA sequencing.

genomics↗

bioRxiv2024

Midline Assembloids Reveal Regulators of Human Axon Guidance

Organizers are specialized cell populations that orchestrate cell patterning and axon guidance in the developing nervous system. Although non-human models have led to fundamental discoveries about the organization of the nervous system midline by the floor plate, an experimental model of human floor plate would enable broader insights into regulation of human neurodevelopment and midline connectivity. Here, we have developed stem cell-derived organoids resembling human floor plate (hFpO) and assembled them with spinal cord organoids (hSpO) to generate midline assembloids (hMA). We demonstrate that hFpO promote Sonic hedgehog-dependent ventral patterning of human spinal progenitors and Netrin-dependent guidance of human commissural axons, paralleling non-human models. To investigate evolutionary-divergent midline regulators, we profiled the hFpO secretome and identified 27 evolutionarily divergent genes between human and mouse. Utilizing the hMA platform, we targeted these candidates in an arrayed CRISPR knockout screen and reveal that GALNT2, a gene involved in O-linked glycosylation, impairs floor plate-mediated guidance of commissural axons in humans. This novel platform extends prior axon guidance discoveries into human-specific neurobiology with implications for mechanisms of nervous system evolution and neurodevelopmental disorders.

neuroscience↗

bioRxiv2024

Human assembloid model of the ascending neural sensory pathway

The ascending somatosensory pathways convey crucial information about pain, touch, itch, and body part movement from peripheral organs to the central nervous system. Despite a significant need for effective therapeutics modulating pain and other somatosensory modalities, clinical translation remains challenging, which is likely related to species-specific features and the lack of in vitro models to directly probe and manipulate this polysynaptic pathway. Here, we established human ascending somatosensory assembloids (hASA)- a four-part assembloid completely generated from human pluripotent stem cells that integrates somatosensory, spinal, diencephalic, and cortical organoids to model the human ascending spinothalamic pathway. Transcriptomic profiling confirmed the presence of key cell types in this circuit. Rabies tracing and calcium imaging showed that sensory neurons connected with dorsal spinal cord projection neurons, which ascending axons further connected to thalamic neurons. Following noxious chemical stimulation, single neuron calcium imaging of intact hASA demonstrated coordinated response, while four-part concomitant extracellular recordings and calcium imaging revealed synchronized activity across the assembloid. Loss of the sodium channel SCN9A, which causes pain insensitivity in humans, disrupted synchrony across the four-part hASA. Taken together, these experiments demonstrate the ability to functionally assemble the essential components of the human sensory pathway. These findings could both accelerate our understanding of human sensory circuits and facilitate therapeutic development.

neuroscience↗

bioRxiv2023

Generating human neural diversity with a multiplexed morphogen screen in organoids

Morphogens choreograph the generation of remarkable cellular diversity in the developing nervous system. Differentiation of stem cells toward particular neural cell fates in vitro often relies upon combinatorial modulation of these signaling pathways. However, the lack of a systematic approach to understand morphogen-directed differentiation has precluded the generation of many neural cell populations, and knowledge of the general principles of regional specification remain in-complete. Here, we developed an arrayed screen of 14 morphogen modulators in human neural organoids cultured for over 70 days. Leveraging advances in multiplexed RNA sequencing technology and annotated single cell references of the human fetal brain we discovered that this screening approach generated considerable regional and cell type diversity across the neural axis. By deconvoluting morphogen-cell type relationships, we extracted design principles of brain region specification, including critical morphogen timing windows and combinatorics yielding an array of neurons with distinct neuro-transmitter identities. Tuning GABAergic neural subtype diversity unexpectedly led to the derivation of primate-specific interneurons. Taken together, this serves as a platform towards an in vitro morphogen atlas of human neural cell differentiation that will bring insights into human development, evolution, and disease.

neuroscience↗

bioRxiv2021

Landscape of human spinal cord cell type diversity at midgestation

Understanding spinal cord generation and assembly is essential to elucidate how motor behavior is controlled and how disorders arise. The cellular landscape of the human spinal cord remains, however, insufficiently explored. Here, we profiled the midgestation human spinal cord with single cell-resolution and discovered, even at this fetal stage, remarkable heterogeneity across and within cell types. Glia displayed diversity related to positional identity along the dorso-ventral and rostro-caudal axes, while astrocytes with specialized transcriptional programs mapped onto distinct histological domains. We discovered a surprisingly early diversification of alpha () and gamma ({gamma}) motor neurons that control and modulate contraction of muscle fibers, which was suggestive of accelerated developmental timing in human spinal cord compared to rodents. Together with mapping of disease-related genes, this transcriptional profile of the developing human spinal cord opens new avenues for interrogating the cellular basis of motor control and related disorders in humans.

neuroscience↗