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Cwycyshyn, J.

Publications and source records attributed to Cwycyshyn, J..

3 recordsLinked to original sources

An agent-based model of wound healing in fibroblast scratch assays

Wound healing assays are widely used to study fibroblast migration and proliferation in the lab. Although traditional scratch assays provide valuable insight, they can be time-intensive, inflexible, and difficult to reproduce. Computational models provide an efficient and reproducible way to study fibroblast behavior in silico. We developed an agent-based simulation of fibroblast scratch assays that models migration, proliferation, contact inhibition, cell-cell mechanical interactions, and the extracellular matrix (ECM) through a particle representation of fibroblasts and the ECM. To validate our model, we performed two tests: a leave-one-out test, where model parameters were calibrated to 6 imaging time series and tested on the remaining one, and a prediction test, where model parameters were calibrated to the first 24 hours of data and the remaining 66 hours of data were predicted. In our leave-one-out test, our model achieved a mean normalized root mean squared error (mean NRMSE) of 3.34%, and a Pearsons R of > 0.98 across all tested wound geometries time series. In the prediction test, the model predicted the remaining 66 hours of wound closure with an NRMSE of 2.08%. These results demonstrate that experimentally observed wound healing behavior of fibroblasts across different geometries can be modeled by local, agent-based rules. Our model provides an efficient platform for the experimentation and in silico study of fibroblast behavior.

systems biology↗

Data-guided direct reprogramming of human fibroblasts into the hematopoietic lineage

Direct reprogramming of human fibroblasts into hematopoietic stem cells (HSCs) offers a promising strategy for generating autologous cells to treat blood and immune disorders. Current protocols are limited by low efficiency and insufficient tools for evaluating reprogramming outcomes. Although functional assays are the standard for confirming cell identity, they require fully reprogrammed cells, limiting their utility during protocol development. To address this, we assembled a single-cell transcriptomic reference atlas of hematopoietic reprogramming and tested an algorithmically-predicted transcription factor recipe for HSC induction. Long-read single-cell RNA sequencing of CD34+ reprogrammed cells revealed progressive loss of fibroblast identity alongside induction of early hematopoietic and endothelial programs, with reference-atlas benchmarking placing reprogrammed cells in an intermediate transcriptomic state between fibroblasts, endothelial cells, and HSCs. Isoform-level analysis further revealed transcriptional remodeling not captured by gene-level analyses. This experimental-computational framework offers a generalizable strategy for characterizing partially reprogrammed states and guiding optimization of reprogramming protocols.

bioinformatics↗

Automated In Vitro Wound Healing Assay

Restoring the epidermal barrier after injury requires spatial and temporal orchestration of migration, proliferation, and signaling across many cell types. The mechanisms that coordinate this complex process are incompletely understood. In vitro wound assays are common model systems for examining these mechanisms in wound healing. In the scratch assay, a cell-free gap is created by mechanical removal of cells from a monolayer, followed by monitoring cell migration into the gap over time. While simple and low-cost, manual scratch assays are limited by low reproducibility and low throughput. Here, we have designed a robotics-assisted automated wound healing (AWH) assay that increases reproducibility and throughput while integrating automated live-cell imaging and analysis. Wounds are designed as computer-aided design (CAD) models and recreated in confluent cell layers by the BioAssemblyBot (BAB) 3D-bioprinting platform. The dynamics of migration and proliferation in individual cells are evaluated over the course of wound closure using live-cell fluorescence microscopy and our high-performance image processing pipeline. The AWH assay outperforms the standard scratch assay with enhanced consistency in wound geometry. Our ability to create diverse wound shapes in any multi-well plate with the BAB not only allows for multiple experimental conditions to be analyzed in parallel but also offers versatility in the design of wound healing experiments. Our method emerges as a valuable tool for the automated completion and analysis of high-throughput, reproducible, and adaptable in vitro wound healing assays.

bioengineering↗