Valle, V. | Search bioRxiv

bioRxiv2025

Electroporation-Based Gene Delivery and Whole-Organoid Imaging in Human Retinal Organoids

Human retinal organoids (hRetOrg) derived from human induced pluripotent stem cells (hiPSCs) have emerged as powerful in vitro systems for studying retinal development, modeling retinal diseases, and evaluating therapeutic strategies. However, current genetic manipulation approaches, such as stable hiPSC line generation and viral transduction, are laborious, costly, and inefficient, with limited spatial specificity and high variability. Here, we report a rapid, scalable, and spatially precise electroporation-based platform for efficient plasmid-based gene delivery in early-stage hRetOrg. This method enables tunable and region-specific transfection of retinal progenitor cells without viral vectors or clonal selection. Coupled with resonant-scanning two-photon microscopy, this approach allows fast live cell imaging of whole organoids with subcellular resolution. This versatile system supports high-throughput genetic manipulation and imaging in intact hRetOrg, advancing studies of human retinal development, gene function, and disease. MotivationhRetOrgs offer an unprecedented platform for functional genetic studies of human retinal development and disease. However, existing methods for gene manipulation in hRetOrg are limited by low throughput, inefficiency, and lack of scalability, hindering systematic analysis of gene function and regulatory elements. To address these limitations, we developed a streamlined, high-efficiency pipeline that enables spatially targeted electroporation of hRetOrg during early retinogenesis, combined with fast, high-resolution imaging of whole organoids using two-photon microscopy, allowing studies at both tissue and subcellular scales.

developmental biology↗

bioRxiv2024

Striatal output regulates the postnatal maturation of cortical circuits

The dorsomedial prefrontal cortex (dmPFC) is interconnected with the basal ganglia (BG) through large-scale circuit loops that regulate critical motor and cognitive functions. In mice, these circuits undergo extensive postnatal maturation with marked changes in neural activity and expansion of synaptic connectivity. While cortical activity is known to regulate the development of downstream striatal circuits, the role of the basal ganglia in cortical maturation remains unknown. Here, we used mesoscale two-photon microscopy and whole-cell electrophysiology to examine whether striatal output during early postnatal development impacts the activity and maturation of upstream dmPFC circuits. We found that ablating spiny projection neurons of the direct or indirect pathways of the striatum during the first two postnatal weeks causes bidirectional changes in dmPFC neural activity, similar to observations in mature circuits. In addition, these manipulations alter the maturation of synaptic connectivity of dmPFC layer 2/3 pyramidal neurons, shifting the balance of excitation and inhibition of cortical circuits. These findings demonstrate that striatal output modulates the activity of cortical circuits during early postnatal development and suggest a regulatory role of the basal ganglia in the establishment of cortical circuits.

neuroscience↗