Biology subjects

Whye, D.

Publications and source records attributed to Whye, D..

3 recordsLinked to original sources

bioRxiv2025

An open-source pipeline for calcium imaging and all-optical physiology in human stem cell-derived neurons

High-throughput profiling of neuronal activity at single-cell resolution is essential for advancing our understanding of brain function, enabling large-scale functional screens, and modeling neurological disorders. However, existing approaches are limited by scalability, manual data processing, and variability, thus restricting their ability to detect disease-associated phenotypes. Here, we present a scalable, open-source platform that integrates optogenetic stimulation, calcium imaging, automated data acquisition, and a fully integrated analysis pipeline. By combining spontaneous and evoked activity profiling, the system enables robust quantification of dynamic neuronal responses across hundreds of stem cell-derived human neurons and multiple timepoints, facilitating phenotyping at both cellular and network levels. We validated the platform by recapitulating established activity phenotypes in neurodevelopmental disorders including CDKL5 Deficiency Disorder and SSADH deficiency. In addition, we generated CRISPR-Cas9 knock-in human induced pluripotent stem cell (hiPSC) lines stably expressing the genetically encoded calcium indicator GCaMP6s to model network dysfunction in Tuberous Sclerosis Complex (TSC). Using this system, we further demonstrated functional rescue of the altered neuronal activity observed in the TSC following pharmacological intervention. By linking single-cell dynamics to population-level phenotypes, this framework provides a powerful and broadly applicable tool for disease modeling, mechanistic studies, and therapeutic screening across a range of neurological disorders.

neuroscience↗

bioRxiv2024

Excitatory Cortical Neurons from CDKL5 Deficiency Disorder Patient-Derived Organoids Show Early Hyperexcitability Not Identified in Neurogenin2 Induced Neurons

CDKL5 deficiency disorder (CDD) is a rare developmental and epileptic encephalopathy resulting from variants in cyclin-dependent kinase-like 5 (CDKL5) that lead to impaired kinase activity or loss of function. CDD is one of the most common genetic etiologies identified in epilepsy cohorts. To study how CDKL5 variants impact human neuronal activity, gene expression and morphology, CDD patient-derived induced pluripotent stem cells and their isogenic controls were differentiated into excitatory neurons using either an NGN2 induction protocol or a guided cortical organoid differentiation. Patient-derived neurons from both differentiation paradigms had decreased phosphorylated EB2, a known molecular target of CDKL5. Induced neurons showed no detectable differences between cases and isogenic controls in network activity using a multielectrode array, or in MAP2+ neurite length, and only two genes were differentially expressed. However, patient-derived neurons from the organoid differentiation showed increased synchrony and weighted mean firing rate on the multielectrode array within the first month of network maturation. CDD patient-derived cortical neurons had lower expression of CDKL5 and HS3ST1, which may change the extracellular matrix around the synapse and contribute to hyperexcitability. Similar to the induced neurons, there were no differences in neurite length across or within patient-control cell lines. Induced neurons have poor cortical specification while the organoid derived neurons expressed cortical markers, suggesting that the changes in neuronal excitability and gene expression are specific to cortical excitatory neurons. Examining molecular mechanisms of early hyperexcitability in cortical neurons is a promising avenue for identification of CDD therapeutics. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=79 SRC="FIGDIR/small/622878v1_ufig1.gif" ALT="Figure 1"> View larger version (29K): org.highwire.dtl.DTLVardef@9d84b8org.highwire.dtl.DTLVardef@1745073org.highwire.dtl.DTLVardef@14dbd20org.highwire.dtl.DTLVardef@bba058_HPS_FORMAT_FIGEXP M_FIG C_FIG

neuroscience↗

bioRxiv2023

Cortical neurons obtained from patient-derived GNAO1 iPSCs show altered differentiation and functional properties

Pathogenic variants in the GNAO1 gene, encoding the alpha subunit of an inhibitory heterotrimeric guanine nucleotide-binding protein (Go) highly expressed in the mammalian brain, have been linked to encephalopathy characterized by different combinations of neurological symptoms, including developmental delay, hypotonia, epilepsy and hyperkinetic movement disorder with life-threatening paroxysmal exacerbations. Currently, there are only symptomatic treatments, and little is known about the pathophysiology of GNAO1-related disorders. Here, we report the characterization of a new in vitro model system based on patient-derived induced pluripotent stem cells (hiPSCs) carrying the recurrent p.G203R amino acid substitution in Go, and a CRISPR-Cas9-genetically corrected isogenic control line. RNA-Seq analysis highlighted aberrant cell fate commitment in neuronal progenitor cells carrying the p.G203R pathogenic variant. Upon differentiation into cortical neurons, patients cells showed reduced expression of early neural genes and increased expression of astrocyte markers, as well as premature and defective differentiation processes leading to aberrant formation of neuronal rosettes. Of note, comparable defects in gene expression and in the morphology of neural rosettes were observed in hiPSCs from an unrelated individual harboring the same GNAO1 variant. Functional characterization showed lower basal intracellular free calcium concentration ([Ca2+]i), reduced frequency of spontaneous activity, and a smaller response to several neurotransmitters in 40- and 50-days differentiated p.G203R neurons compared to control cells. These findings suggest that the GNAO1 pathogenic variant causes a neurodevelopmental phenotype characterized by aberrant differentiation of both neuronal and glial populations leading to a significant alteration of neuronal communication and signal transduction.

neuroscience↗