Junday, K. | bioRxiv Science

bioRxiv2025

Robotic Handling Preserves Induced Pluripotent Stem Cell Derived Vascular Smooth Muscle Cells Differentiation using a Weekend-free, Automatable, Low-variability, Low-cost (WALL) Protocol

Vascular disease modeling and tissue engineering require scalable, efficient, and reproducible methods for differentiating cells, however no studies have validated whether automation, essential for scale-up, preserves differentiation quality in cells. Here, we developed a differentiation protocol for induced pluripotent stem cell (iPSC)-derived vascular smooth muscle cells (iVSMCs) that is applicable to both manual, and automated robotic culture. iPSC lines generated from male and female donors across various ages were used to optimize the generation of iVSMCs. iVSMCs had consistent morphology and protein expression was largely similar to primary VSMCs, including expression of myosin heavy chain-11 (MYH11), -smooth muscle actin (-SMA), transgelin (TAGLN) and calponin (CNN1). Functionally, these iVSMCs could respond to the vasoconstrictor carbachol. Coupling this protocol with an automated Hamilton liquid-handling robotics system allowed the generation of iVSMCs in large quantities, morphologically similar to manually differentiated iVSMCs. Comparative proteomic analysis confirmed protein expression did not differ between automated and manual differentiation methods. Automation markedly reduced manual labor and facilitated increased production without sacrificing cell quality. This study demonstrates the feasibility of automating iVSMC differentiation, marking a significant step towards scalable VSMC manufacturing for three-dimensional applications, and for the ever-growing demands of organoid and tissue engineering applications. Statement of significanceThe rapidly expanding fields of tissue engineering and high-throughput disease modeling require large quantities of viable cells, making automation essential. Robotic liquid handling systems are increasingly employed for generating large quantities of induced pluripotent stem cell (iPSC)-derived cells, requiring the development of scalable protocols. No studies have yet evaluated whether the use of robotic liquid handling alters the differentiation of cells compared to traditional manual handling. Here, we report a scalable differentiation protocol that robustly generates iPSC-derived VSMCs (iVSMCs) from a cohort of female and male donors of varying age (n=5 lines). We demonstrate for the first time that iVSMCs, differentiated through an automated robotic pipeline, had no significant differences in morphological or proteomic phenotype compared to manual differentiation. The ability to automate this protocol facilitates robust and reproducible differentiation of large quantities of iVSMC, setting a precedent for the application of automation in generating iPSC-derived cells. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=57 SRC="FIGDIR/small/690512v1_ufig1.gif" ALT="Figure 1000"> View larger version (18K): org.highwire.dtl.DTLVardef@673d61org.highwire.dtl.DTLVardef@1eead89org.highwire.dtl.DTLVardef@c3089eorg.highwire.dtl.DTLVardef@6af2cd_HPS_FORMAT_FIGEXP M_FIG C_FIG

developmental biology↗

bioRxiv2019

Bayesian population receptive field modeling in human somatosensory cortex

Somatosensation is fundamental to our ability to sense our body and interact with the world. Our body is continuously sampling the environment using a variety of receptors tuned to different features, and this information is routed up to primary somatosensory cortex. Strikingly, the spatial organization of the peripheral receptors in the body are well maintained, with the resulting representation of the body in the brain being referred to as the somatosensory homunculus. Recent years have seen considerable advancements in the field of high-resolution fMRI, which have enabled an increasingly detailed examination of the organization and properties of this homunculus. Here we combined advanced imaging techniques at ultra-high field (7T) with a recently developed Bayesian population receptive field (pRF) modeling framework to examine pRF properties in primary somatosensory cortex. In each subject, vibrotactile stimulation of the fingertips (i.e., the peripheral mechanoreceptors) modulated the fMRI response along the post-central gyrus and these signals were used to estimate pRFs. We found the pRF center location estimates to be in accord with previous work as well as evidence of other properties in line with the underlying neurobiology. Specifically, as expected from the known properties of cortical magnification, we find a larger representation of the index finger compared to the other stimulated digits (middle, index, little). We also show evidence that the little finger is marked by the largest pRF sizes. The ability to estimate somatosensory pRFs in humans provides an unprecedented opportunity to examine the neural mechanisms underlying somatosensation and is critical for studying how the brain, body, and environment interact to inform perception and action.

neuroscience↗