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Ramalingam, J.

Publications and source records attributed to Ramalingam, J..

2 recordsLinked to original sources
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↗
bioRxiv2018

Reaction of Hydrogen sulfide homeostasis genes under biotic and abiotic stress condition in rice -- computational approach

Gaseous molecules are widespread signaling compounds, regulating the cell development process in all major plant parts. For many decades, hydrogen sulfide molecule is considered mainly for its deleterious effects on plant system. The increasing recent experimental evidence and phenomenal concepts on H2S molecule further advance our understanding of H2S interaction with plant tissues. In addition, the H2S messenger molecule is found to have positive effects on plant growth, in limited condition, to maintain the balanced homeostasis. To meet the increasing demand, and to sustain the crop yield, various crop improvement programs have been followed. However, there is a concern that traditional plant improvement method and increasing climate change has a negative impact on crop production. A major approach to combating plant stress is to evaluate and explore the alternate source mechanism(s). Towards this aim, it will be valuable to characterize the genes involved in H2S homeostasis in the staple food crop rice pan-genome. In this research, we identified 15 H2S homeostasis genes in rice and used it for the ~3k rice pan-genome analysis to find out the genetic relatedness based on single nucleotide polymorphism data. Multidimensional scale plot of 15 H2S homeostasis genes among the rice cultivars, and RNA-seq experimental data analysis under various biotic and abiotic stress shows the functional genes involved in biotic and abiotic stress. This study provides new insights into plant stress management in crop breeding and suggests how H2S gene(s) can be utilized to improve the agronomic traits in rice and other food crops.

plant biology↗