Biology subjects

Smith, A. S. T.

Publications and source records attributed to Smith, A. S. T..

6 recordsLinked to original sources

bioRxiv2026

Designed Minibinders Rewire Receptor Signaling to Enable Functional Human Myogenic Reprogramming

Sarcopenia, loss of muscle mass is a considerable health burden that demands immediate societal attention. Direct myogenic somatic cell reprogramming, a potential muscle regeneration method is constrained by an inability to control the signaling logic that governs cell fate. Here, we show that this barrier can be overcome using AI-designed receptor modulators. Screening de novo minibinders, we identify a synthetic protein cocktail, C6-DPC, that drives efficient human fibroblast-to-muscle transdifferentiation with robust structural and metabolic maturation. C6-DPC reprograms extracellular signaling by activating pro-myogenic FGFR1/2c pathways while suppressing anti-myogenic inputs through ALK1 and TGFBR2; targeted depletion of ALK1 is sufficient to lower the reprogramming barrier. Inflammatory signaling via gp130 emerges as a dominant checkpoint, and its inhibition further enhances conversion. Engineered tissues generate high twitch and tetanic forces in both wild-type and dystrophin-deficient human cells. These findings demonstrate that programmable synthetic ligands can rewrite receptor-level signaling to direct cell fate and enable functional tissue regeneration.

biochemistry↗

bioRxiv2022

Interferon-gamma Elicits Pathological Hallmarks of ALS in Human Motor Neurons

Neuroinflammation is an established factor contributing to amyotrophic lateral sclerosis (ALS) pathology, implicating the possible detrimental effects of inflammatory cytokines on motor neurons. The RNA/DNA-binding protein TDP-43 has emerged as a pivotal actor in ALS, because TDP-43 mutations cause familial ALS and loss of nuclear TDP-43, associated with its redistribution into cytoplasmic aggregates (TDP-43 proteinopathy) in motor neurons occurs in 97% of ALS cases. However, mechanisms linking neuroinflammation to TDP-43 mislocalization have not been described. Programmed death-ligand 1 (PD-L1) is an immune-modulatory protein, highly expressed on cell surfaces following acute inflammatory stress. To determine which inflammatory cytokines might impact motor neuron function, seven cytokines known to be elevated in ALS patients cerebrospinal fluid were tested for their effects on PD-L1 expression in human iPSC-derived motor neurons. Among the tested cytokines, only interferon-{gamma} (IFN-{gamma}) was found to strongly promote PD-L1 expression. Thus, we hypothesized that excessive exposure to IFN-{gamma} may contribute to motor neuron degeneration in ALS. We observed that neuronal populations exposed to IFN-{gamma} exhibited severe TDP-43 cytoplasmic aggregation and excitotoxic behavior correlated with impaired neural firing activity, hallmarks of ALS pathology, in both normal and ALS mutant (TARDB1K+/-) neurons. Single-cell RNA sequencing revealed possible mechanisms for these effects. Motor neurons exposed to IFN-{gamma} exhibited an extensive shift of their gene expression profile toward a neurodegenerative phenotype. Notably, IFN-{gamma} treatment induced aberrant expression levels for 70 genes that are listed in the recent literature as being dysregulated in various ALS subtypes. Additionally, we found that genes related to neuronal electrophysiology, protein aggregation, and TDP-43 misregulation were abnormally expressed in IFN-{gamma} treated cells. Moreover, IFN-{gamma} induced a significant reduction in the expression of genes that encode indispensable proteins for neuromuscular synapse development and maintenance, implying that the continuous cytokine exposure could directly impair signal transmission between motor axons and muscle membranes. Our findings suggest that IFN-{gamma} could be a potent upstream pathogenic driver of ALS and provide potential candidates for future therapeutic targets to treat sporadic forms of ALS, which account for roughly 90% of reported cases.

neuroscience↗

bioRxiv2022

Perpendicular Shear Stresses Drive Transmural Helical Remodeling in Engineered Human Ventricular Models

Tissue engineering with human induced pluripotent stem cell-derived cardiomyocytes enables unique opportunities for creating physiological models of the heart in vitro. However, there are few approaches available that can recapitulate the complex structure-function relationships that govern cardiac function at the macroscopic organ level. Here, we report a down-scaled, conical human 3D ventricular model with controllable cellular organization using multilayered, patterned cardiac sheets. Tissue engineered ventricles whose cardiomyocytes were pre-aligned parallel or perpendicular to the long axis outperformed those whose cardiomyocytes were angled or randomly oriented. Notably, the inner layers of perpendicular cardiac sheets realigned over 4 days into a parallel orientation, creating a helical transmural architecture, whereas minimal remodeling occurred in the parallel or angled sheets. Finite element analysis of engineered ventricles demonstrated that circumferential alignment leads to maximal perpendicular shear stress at the inner layer, whereas longitudinal orientation leads to maximal parallel stress. We hypothesize that cellular remodeling occurs to reduce perpendicular shear stresses in myocardium. This advanced platform provides evidence that physical forces such as shear stress drive self-organization of cardiac architecture.

bioengineering↗

bioRxiv2022

High-throughput, real-time monitoring of engineered skeletal muscle function using magnetic sensing

Engineered muscle tissues represent powerful tools for examining tissue level contractile properties of skeletal muscle. However, limitations in the throughput associated with standard analysis methods limit their utility for longitudinal study, high throughput drug screens, and disease modeling. Here we present a method for integrating 3D engineered skeletal muscles with a magnetic sensing system to facilitate non-invasive, longitudinal analysis of developing contraction kinetics. Using this platform, we show that engineered skeletal muscle tissues derived from both induced pluripotent stem cell and primary sources undergo improvements in contractile output over time in culture. We demonstrate how magnetic sensing of contractility can be employed for simultaneous assessment of multiple tissues subjected to different doses of known skeletal muscle inotropes as well as the stratification of healthy versus diseased functional profiles in normal and dystrophic muscle cells. Based on these data, this combined culture system and magnet-based contractility platform greatly broadens the potential for 3D engineered skeletal muscle tissues to impact the translation of novel therapies from the lab to the clinic.

bioengineering↗

bioRxiv2018

Engineered developmental niche enables predictive phenotypic screening in human dystrophic cardiomyopathy

Directed differentiation of human pluripotent stem cells (hPSCs) into cardiomyocytes typically produces cells with structural, functional, and biochemical properties that most closely resemble those present in the fetal heart. Here we establish an in vitro engineered developmental cardiac niche to produce matured hPSC-derived cardiomyocytes (hPSC-CMs) with enhanced sarcomere development, electrophysiology, contractile function, mitochondrial capacity, and a more mature transcriptome. When this developmental cardiac niche was applied to dystrophin mutant hPSC-CMs, a robust disease phenotype emerged, which was not observed in non-matured diseased hPSC-CMs. Matured dystrophin mutant hPSC-CMs exhibited a greater propensity for arrhythmia as measured via beat rate variability, most likely due to higher resting cytosolic calcium content. Using a custom nanopatterned microelectrode array platform to screen functional output in hPSC-CMs exposed to our engineered developmental cardiac niche, we identified calcium channel blocker, nitrendipine, mitigated hPSC-CM arrhythmogenic behavior and correctly identified sildenafil as a false positive. Taken together, we demonstrate our developmental cardiac niche platform enables robust hPSC-CM maturation allowing for more accurate disease modeling and predictive drug screening.

bioengineering↗

bioRxiv2018

NanoMEA: a versatile platform for high-throughput analysis of structure-function relationships in human stem cell-derived excitable cells and tissues

Somatic cells derived from human pluripotent stem cell (hPSC) sources hold significant potential as a means to improve current in vitro screening assays. However, their inconsistent ability to recapitulate the structural and functional characteristics of native cells has raised questions regarding their ability to accurately predict the functional behavior of human tissues when exposed to chemical or pathological insults. In addition, the lack of cytoskeletal organization within conventional culture platforms prevents analysis of how structural changes in human tissues affect functional performance. Using cation-permeable hydrogels, we describe the production of multiwell nanotopographically-patterned microelectrode arrays (nanoMEAs) for studying the effect of structural organization on hPSC-derived cardiomyocyte and neuronal function in vitro. We demonstrate that nanoscale topographic substrate cues promote the development of more ordered cardiac and neuronal monolayers while simultaneously enhancing cytoskeletal organization, protein expression patterns, and electrophysiological function in these cells. We then show that these phenotypic improvements act to alter the sensitivity of hPSC-derived cardiomyocytes to treatment with arrhythmogenic and conduction-blocking compounds that target structural features of the cardiomyocyte. Similarly, we demonstrate that neuron sensitivity to synaptic blockers is increased when cells are maintained on nanotopographically-patterned Nafion surfaces. The improved structural and functional capacity of hPSC-derived cardiomyocyte and neuronal populations maintained on nanoMEAs may have important implications for improving the predictive capabilities of cell-based electrophysiological assays used in preclinical screening applications.

bioengineering↗