Biology subjects

Layne, M. D.

Publications and source records attributed to Layne, M. D..

3 recordsLinked to original sources

bioRxiv2025

A Bio-inspired Latent TGF-β Conjugated Scaffold Improves Neocartilage Development

In cartilage tissue engineering, active TGF-{beta} is conventionally supplemented in culture medium at highly supraphysiologic doses to accelerate neocartilage development. While this approach enhances cartilage extracellular matrix (ECM) biosynthesis, it further promotes tissue features detrimental to hyaline cartilage function, including the induction of tissue swelling, hyperplasia, hypertrophy, and ECM heterogeneities. In contrast, during native cartilage development, chondrocytes are surrounded by TGF-{beta} configured in a latent complex (LTGF-{beta}), which undergoes cell-mediated activation, giving rise to moderated, physiologic dosing regimens that enhance ECM biosynthesis while avoiding detrimental features associated with TGF-{beta} excesses. Here, we explore a bio-inspired strategy, consisting of LTGF-{beta}-conjugated scaffolds, providing TGF-{beta} exposure regimens that are moderated and uniformly administered throughout the construct. Specifically, we evaluate the performance of LTGF-{beta} scaffolds to improve neocartilage development with bovine chondrocyte-seeded agarose constructs compared to outcomes from active TGF-{beta} media supplementation (MS) at a physiologic 0.3 ng/mL dose (MS-0.3), supraphysiologic 10 ng/mL dose (MS-10), or TGF-{beta} free. For small-size constructs ({emptyset}3x2 mm), LTGF-{beta} scaffolds yield neocartilage that achieves native-matched mechanical properties (800-925 kPa) and sGAG content (6.6%-7.1%), while providing a cell morphology and collagen distribution more reminiscent of hyaline cartilage. LTGF-{beta} scaffolds further afford an optimal chondrogenic phenotype, marked by a 12-to 28-fold reduction of COL-I expression relative to TGF-{beta}-free and a 7-to 17-fold reduction of COL-X expression relative to MS-10. Further, for large-size constructs, which approach the dimensions needed for clinical cartilage repair, LTGF-{beta} scaffolds significantly reduce mechanical and biochemical heterogeneities relative to MS-0.3 and MS-10. Overall, the use of LTGF-{beta} scaffolds improves the composition, structure, material properties, and cell phenotype of neocartilage.

bioengineering↗

bioRxiv2024

Direct interrogation of context-dependent GPCR activity with a universal biosensor platform

G protein-coupled receptors (GPCRs) are the largest family of druggable proteins in the human genome, but progress in understanding and targeting them is hindered by the lack of tools to reliably measure their nuanced behavior in physiologically-relevant contexts. Here, we developed a collection of compact ONE vector G-protein Optical (ONE-GO) biosensor constructs as a scalable platform that can be conveniently deployed to measure G-protein activation by virtually any GPCR with high fidelity even when expressed endogenously in primary cells. By characterizing dozens of GPCRs across many cell types like primary cardiovascular cells or neurons, we revealed new insights into the molecular basis for G-protein coupling selectivity of GPCRs, pharmacogenomic profiles of anti-psychotics on naturally-occurring GPCR variants, and G-protein subtype signaling bias by endogenous GPCRs depending on cell type or upon inducing disease-like states. In summary, this open-source platform makes the direct interrogation of context-dependent GPCR activity broadly accessible.

biochemistry↗

bioRxiv2023

Adrenergic Reprogramming of Preexisting Adipogenic Trajectories Steer Naive Mural Cells Toward Beige Differentiation

In adult white adipose tissue, cold or {beta}3-adrenoceptor activation promotes the appearance of thermogenic beige adipocytes. Our comprehensive single-cell analysis revealed that these cells arise through the reprogramming of existing adipogenic trajectories, rather than from a single precursor. These trajectories predominantly arise from SM22-expressing vascular mural progenitor cells. Central in this transition is the activation of Adrb3 in mature adipocytes, leading to subsequent upregulation of Adrb1 in primed progenitors. Under thermoneutral conditions, synergistic activation of both Adrb3 and Adrb1 recapitulates the pattern of cold-induced SM22+ cell recruitment. Lipolysis-derived eicosanoids, specifically docosahexaenoic acid (DHA) and arachidonic acid (AA) prime these processes and in vitro, were sufficient to recapitulate progenitor cells priming. Collectively, our findings provide a robust model for cold-induced beige adipogenesis, emphasizing a profound relationship between mature adipocytes and mural cells during cold acclimation, and revealing the metabolic potential of this unique cellular reservoir.

cell biology↗