Bioengineered 3D In Vitro Strategies to Investigate Phenotypic and Genotypic Differences in Lymphatic Network Sprouting
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Gonzalez-Vargas, Yarelis
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Abstract
This dissertation explored bioengineered 3D in vitro models to study lymphatic network sprouting and lymphatic malformations (LMs). Traditional in vivo studies have limitations; however, reproducible tissue-engineered models using primary lymphatic cells offer promising alternatives. Tissue engineering models were created by embedding lymphatic cells from rat, sheep, and human sources into poly (ethylene glycol) (PEG)-based hydrogels to replicate the lymphatic microenvironment. These models elucidate the mechanisms of lymphatic endothelial cell (LEC) sprouting and the effects of factors, such as extracellular matrix composition and mechanical compression. These findings demonstrate the potential of these models to mimic in vivo conditions and serve as platforms for testing therapeutic interventions. Methods were refined for the precision management of LMs using patient-derived organoids (PDOs). Lymphatic malformations, which are rare vascular anomalies caused by somatic PIK3CA mutations, lead to lymphatic dysfunction and cyst formation, affecting 1 in 4000 live births. Symptoms are typically evident at birth because of their developmental origin. Current treatment options are limited and lack standardization. The second and third aims of this dissertation address these limitations using tissue engineering with patient-derived tissues. Tissue samples from patients with LM were cultured in PEG hydrogels for up to 30 days to yield high numbers of patient-derived cells (PDCs) to assemble lymphatic malformation organoids (LMOs). The genetic fidelity of PDCs to the LM was confirmed using single-cell RNA sequencing and whole-exome sequencing. The efficacy of PI3K/AKT/MTOR pathway inhibitors, especially the selective PI3K inhibitor alpelisib, was tested in LMOs, which showed significant variability in drug responses among patients, with gene expression analyses indicating upregulation of pro-apoptotic genes after treatment. These results highlight the potential of targeted therapies and the value of patient-derived models in advancing the understanding of LM biology and developing precision medicine approaches. Overall, this dissertation underscores the innovative use of bioengineered 3D in vitro models for investigating lymphatic network sprouting and managing lymphatic malformations. These findings emphasize the importance of accounting for tissue heterogeneity and genetic variability when developing effective in vitro models and designing targeted therapeutic interventions.
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2024-07-24
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Dissertation