Project description:Background: Three-dimensional (3D) in vitro culture systems using human induced pluripotent stem cells (hiPSCs) represent impactful platforms to model neurodegenerative disease biology in physiologically relevant microenvironments. Though many successful biomaterials-based 3D model systems have been established for other neurogenerative diseases, such as Alzheimer’s Disease, relatively few exist for Parkinson’s Disease (PD) research. Methods: We employed tissue engineering approaches to construct a 3D silk scaffold-based platform for the culture of hiPSC-dopaminergic (DA) neurons derived from healthy individuals and PD patients harboring LRRK2 G2019S or GBA N370S mutations. We then compared results from protein, gene expression and metabolic analyses obtained from two-dimensional (2D) and 3D culture systems. Results: The 3D platform enabled the formation of dense dopamine neuronal network architectures and developed biological profiles both similar and distinct from 2D culture systems in healthy and PD disease lines. 3D PD cultures showed elevated levels of α-synuclein and alterations in purine metabolite profiles. Furthermore, computational network analysis of transcriptome networks nominated several novel molecular interactions occurring in neurons from patients with mutations in LRRK2 and GBA. Conclusion: The brain-like 3D system presented here is a realistic platform to interrogate molecular mechanisms underlying PD biology. The key advantages of silk-based bioengineering technology include long-term culture and the ability to incorporate multiple brain-relevant cell types to parse cell-cell interactions in development, disease, and aging.
Project description:Transcriptional profiling of the Fischer Rat Thyroid (FRT) cells comparing polarizing cells grown as a confluent two-dimensional monolayer (2D culture system) with cells grown in matrigel where they acquire a three-dimensional follicular structure (3D culture system).The goal was to identify regulators of 3D epithelial thyroid polarization and follicle formation.
Project description:To better understand and develop treatments for lung cancer, it is important to have reliable and physiologically relevant culture models. Traditional methods of growing lung cancer cells in two-dimensional monolayers have limitations in mimicking the complex architecture and microenvironment of lung tumors in vivo, limiting their value as reliably informative disease models. In this study, we introduce a new cell culture platform called "tumoroids," which involves growing HCC827 lung cancer cells in three-dimensional configurations for more accurately predicting how cell-cell orientation and signaling affects the cancer-driving properties of lung cancer. By comparing transcriptional profiles of HCC827 cells grown as tumoroids versus the same cells grown in two-dimensional monolayers, we investigate how cell-cell orientation and signaling impact the cancer-driving properties of lung cancer. We examine key features associated with cancer progression, such as epithelial mesenchymal transition, replicative ability, and induction of angiogenesis. Additionally, we assess the functional characteristics of the 3D tumoroid culture system by subjecting the cells to irradiation. Through comparing the gene expression profiles of the 3D tumoroid cultures with those of primary human lung adenocarcinoma, we find that the 3D tumoroid cultures closely resemble the characteristics of primary human lung cancer. This suggests that the 3D tumoroid culture platform can serve as a valuable in vitro model for studying lung cancer, offering greater clinical relevance compared to traditional 2D cultures. Overall, this study highlights the importance of using advanced culture models like 3D tumoroids to improve our understanding of lung cancer and facilitate the development of effective treatments.
Project description:Background: Alzheimer’s disease (AD) is an incurable neurodegenerative disorder with a rapidly increasing prevalence worldwide. Current approaches targeting hallmark pathological features of AD have had no consistent clinical benefit. Neuroinflammation is a major contributor to neurodegeneration and hence, microglia, the brain’s resident immune cells, are an attractive target for potentially more effective therapeutic strategies. However, there is no current in vitro model system that captures AD patient-specific microglial characteristics using physiologically relevant and experimentally flexible culture conditions. Methods: To address this shortcoming, we developed novel 3D Matrigel-based monocyte-derived microglia-like cell (MDMi) mono-cultures and co-cultures with neuro-glial cells (ReNcell VM). We used single-cell RNA sequencing (scRNAseq) analysis to compare the transcriptomic signatures of MDMi between model systems (2D, 3D and 3D co-culture) and against published human microglia datasets. To demonstrate the potential of MDMi for use in personalized preclinical strategies, we generated and characterized MDMi models from sixteen AD patients and matched healthy controls, and profiled cytokine responses upon treatment with anti-inflammatory drugs (dasatinib and spiperone). Results: MDMi in 3D exhibited a more branched morphology and longer survival in culture compared to 2D. scRNAseq uncovered distinct MDMi subpopulations that exhibit higher functional heterogeneity and best resemble human microglia in 3D co-culture. AD MDMi in 3D co-culture showed altered cell-to-cell interactions, growth factor and cytokine secretion profiles and responses to amyloid-β. Drug testing assays revealed patient- and model-specific cytokine responses. Conclusion: Our study presents a novel, physiologically relevant and AD patient-specific 3D microglia cell model that opens avenues towards improving personalized drug development strategies in AD.
Project description:Neural Stem Cells (NSCs) of biological behaviors are frequently regulated by the three-dimensional (3D) niches that they located in. The impact of cell density cue inside niche on cellular outcomes are usually underestimated and understudied. In this study, a facile NSCs of 3D culture system was developed using collagen self-assembled fibril hydrogel. We used RNA sequencing and molecular biology technologies to investigate the NSCs of cell contacts and differentiations in developed 3D hydrogel culture system with low (0.75 million/mL), medium (1.5 million/mL), and high (3 million/mL) cell packing density. Compared with low cell density system, cytoskeletal spreading, gap junction, and tight junction mediated cell contacts were significantly improved in high cell density culture. Moreover, high cell density increased NSCs differentiation into immature (Tuj1) neuron, while there is no discrepancy of NSCs differentiation into astrocytes in different cell densities of 3D culturing system. This study provides new understanding in the regulation of 3D cultured NSCs behaviors through niche of cell density cue.
Project description:Microarray analysis was used to identify genes that were differentially expressed when epithelial cells were grown in 3D Matrigel culture with stromal co-culture compared to without stroma.
Project description:Microarray analysis was used to identify genes that were differentially expressed when epithelial cells were grown in 3D Matrigel culture with stromal co-culture compared to without stroma.
Project description:Lymphangioleiomyomatosis (LAM) is a rare disease involving cystic lung destruction by invasive LAM cells. These cells harbor loss-of-function mutations in TSC2, conferring hyperactive mTORC1 signaling. Here, tissue engineering tools are employed to model LAM and identify new therapeutic candidates. Biomimetic hydrogel culture of LAM cells is found to recapitulate the molecular and phenotypic characteristics of human disease more faithfully than culture on plastic. A 3D drug screen is conducted, identifying histone deacetylase (HDAC) inhibitors as anti-invasive agents that are also selectively cytotoxic toward TSC2−/− cells. The anti-invasive effects of HDAC inhibitors are independent of genotype, while selective cell death is mTORC1-dependent and mediated by apoptosis. Genotype-selective cytotoxicity is seen exclusively in hydrogel culture due to potentiated differential mTORC1 signaling, a feature that is abrogated in cell culture on plastic. Importantly, HDAC inhibitors block invasion and selectively eradicate LAM cells in vivo in zebrafish xenografts. These findings demonstrate that tissue-engineered disease modeling exposes a physiologically relevant therapeutic vulnerability that would be otherwise missed by conventional culture on plastic. This work substantiates HDAC inhibitors as possible therapeutic candidates for the treatment of patients with LAM and requires further study.