Growing Human Parathyroids in a Microphysiological System: A Novel Approach to Understanding and Developing New Treatments for Hyperparathyroidism.

We developed a novel model for studying hyperparathyroidism by growing ex vivo 3-dimensional human parathyroids as part of a microphysiological system (MPS) that mimics human physiology. The purpose of this study was to validate the parathyroid portion of the MPS. We prospectively collected parathyroid tissue from 46 patients with hyperparathyroidism for growth into pseudoglands. We evaluated pseudogland architecture and calcium responsiveness. Following 2 weeks in culture, dispersed cells successfully coalesced into pseudoglands ∼500-700 µm in diameter that mimicked the appearance of normal parathyroid glands. Functionally, they also appeared similar to intact parathyroids in terms of organization and calcium-sensing receptor expression. Immunohistochemical staining for calcium-sensing receptor revealed 240-450/cell units of mean fluorescence intensity within the pseudoglands. Finally, the pseudoglands showed varying levels of calcium responsiveness, indicated by changes in parathyroid hormone (PTH) levels. In summary, we successfully piloted the development of a novel MPS for studying the effects of hyperparathyroidism on human organ systems. We are currently evaluating the effect of PTH on adverse remodeling of tissue engineered cardiac, skeletal, and bone tissue within the MPS.

Hemodynamic Stimulation Using the Biomimetic Cardiac Tissue Model (BCTM) Enhances Maturation of Human Induced Pluripotent Stem Cell-Derived Cardiomyocytes.

Human induced pluripotent stem cell (hiPSC)-derived cardio-myocytes (hiPSC-CMs) hold great promise for cardiovascular disease modeling and regenerative medicine. However, these cells are both structurally and functionally -immature, primarily due to their differentiation into cardiomyocytes occurring under static culture which only reproduces biomolecular cues and ignores the dynamic hemo-dynamic cues that shape early and late heart development during cardiogenesis. To evaluate the effects of hemodynamic stimuli on hiPSC-CM maturation, we used the biomimetic cardiac tissue model to reproduce the hemodynamics and pressure/volume changes associated with heart development. Following 7 days of gradually increasing stimulation, we show that hemodynamic loading results in (a) enhanced alignment of the cells and extracellular matrix, (b) significant increases in genes associated with physiological hypertrophy, (c) noticeable changes in sarcomeric organization and potential changes to cellular metabolism, and (d) a significant increase in fractional shortening, suggestive of a positive force frequency response. These findings suggest that culture of hiPSC-CMs under conditions that accurately reproduce hemodynamic cues results in structural orga-nization and molecular signaling consistent with organ growth and functional maturation.

Biomimetic Cardiac Tissue Model Enables the Adaption of Human Induced Pluripotent Stem Cell Cardiomyocytes to Physiological Hemodynamic Loads.

Induced pluripotent stem cell derived cardiomyocytes (iPSC-CMs) provide a human source of cardiomyocytes for use in cardiovascular research and regenerative medicine. However, attempts to use these cells in vivo have resulted in drastic cell death caused by mechanical, metabolic, and/or exogenous factors. To explore this issue, we designed a Biomimetic Cardiac Tissue Model (BCTM) where various parameters associated with heart function including heart rate, peak-systolic pressure, end-diastolic pressure and volume, end-systolic pressure and volume, and ratio of systole to diastole can all be precisely manipulated to apply hemodynamic loading to culture cells. Using the BCTM, two causes of low survivability in current cardiac stem cell therapies, mechanical and metabolic, were explored. iPSC-CMs were subject to physiologically relevant mechanical loading (50 mmHg systolic, 10% biaxial stretch) in either a low- or high-serum environment and mechanical loads were applied either immediately or gradually. Results confirm that iPSC-CMs subject to mechanical loading in low-serum conditions experienced widespread cell death. The rate of application of stress also played an important role in adaptability to mechanical loading. Under high-serum conditions, iPSC-CMs subject to gradual imposition of stress were comparable to iPSC-CMs maintained in static culture when evaluated in terms of cell viability, sarcomeric structure, action potentials and conduction velocities. In contrast, iPSC-CMs that were immediately exposed to mechanical loading had significantly lower cell viability, destruction of sarcomeres, smaller action potentials, and lower conduction velocities. We report that iPSC-CMs survival under physiologically relevant hemodynamic stress requires gradual imposition of mechanical loads in a nutrient-rich environment.

Hyperglycemic arterial disturbed flow niche as an in vitro model of atherosclerosis.

Type 2 diabetes significantly elevates the risk of cardiovascular disease. This can be largely attributed to the adverse effects of hyperglycemic conditions on normal endothelial cell (EC) function. ECs in both large and small vessels are influenced by hyperglycemic conditions, which increase susceptibility to EC dysfunction and atherosclerotic lesion formation. Fluid shear stress and flow patterns play an essential role in atherogenesis: lesions form only at locations where fluid flow behavior can be classified as "disturbed flow" (i.e., low shear stress recirculation and/or retrograde flow). Since regions of disturbed flow are the focal points of atherosclerotic cardiovascular disease, we hypothesized that the combinatorial effects of high glucose and disturbed flow conditions elicit significantly different responses from ECs than high glucose alone. To validate our hypothesis, we used our endothelial cell culture model (ECCM) to establish vascular niches associated with "normal" and "disturbed" flow conditions typically seen in vivo along with physiological pressure and stretch. We subjected human aortic endothelial cells (HAECs) to hyperglycemic conditions under both "normal" and "disturbed" flow. Our results confirm significant and quantifiable differences in phenotypic and functional markers between cells cultured under conditions of "normal" and "disturbed flow" under hyperglycemic conditions suggesting that elevated glucose in conjunction with "disturbed" flow conditions results in significantly higher level of EC dysfunction. The ECCM can therefore be used as a physiologically relevant model to study early stage hyperglycemia induced atherosclerosis for basic research, drug discovery, and screening and toxicity studies.

Cardiac Tissue Chips (CTCs) for Modeling Cardiovascular Disease.

OBJECTIVE: Cardiovascular research and regenerative strategies have been significantly limited by the lack of relevant cell culture models that can recreate complex hemodynamic stresses associated with pressure-volume changes in the heart. METHODS: To address this issue, we designed a biomimetic cardiac tissue chip (CTC) model where encapsulated cardiac cells can be cultured in three-dimensional (3-D) fibres and subjected to hemodynamic loading to mimic pressure-volume changes seen in the left ventricle. These 3-D fibres are suspended within a microfluidic chamber between two posts and integrated within a flow loop. Various parameters associated with heart function, like heart rate, peak-systolic pressure, end-diastolic pressure and volume, end-systolic pressure and volume, and duration ratio between systolic and diastolic, can all be precisely manipulated, allowing culture of cardiac cells under developmental, normal, and disease states. RESULTS: We describe two examples of how the CTC can significantly impact cardiovascular research by reproducing the pathophysiological mechanical stresses associated with pressure overload and volume overload. Our results using H9c2 cells, a cardiomyogenic cell line, clearly show that culture within the CTC under pathological hemodynamic loads accurately induces morphological and gene expression changes, similar to those seen in both hypertrophic and dilated cardiomyopathy. Under pressure overload, the cells within the CTC see increased hypertrophic remodeling and fibrosis, whereas cells subject to prolonged volume overload experience significant changes to cellular aspect ratio through thinning and elongation of the engineered tissue. CONCLUSIONS: These results demonstrate that the CTC can be used to create highly relevant models where hemodynamic loading and unloading are accurately reproduced for cardiovascular disease modeling.

Mitoquinone ameliorates pressure overload-induced cardiac fibrosis and left ventricular dysfunction in mice.

Increasing evidence indicates that mitochondrial-associated redox signaling contributes to the pathophysiology of heart failure (HF). The mitochondrial-targeted antioxidant, mitoquinone (MitoQ), is capable of modifying mitochondrial signaling and has shown beneficial effects on HF-dependent mitochondrial dysfunction. However, the potential therapeutic impact of MitoQ-based mitochondrial therapies for HF in response to pressure overload is reliant upon demonstration of improved cardiac contractile function and suppression of deleterious cardiac remodeling. Using a new (patho)physiologically relevant model of pressure overload-induced HF we tested the hypothesis that MitoQ is capable of ameliorating cardiac contractile dysfunction and suppressing fibrosis. To test this C57BL/6J mice were subjected to left ventricular (LV) pressure overload by ascending aortic constriction (AAC) followed by MitoQ treatment (2 µmol) for 7 consecutive days. Doppler echocardiography showed that AAC caused severe LV dysfunction and hypertrophic remodeling. MitoQ attenuated pressure overload-induced apoptosis, hypertrophic remodeling, fibrosis and LV dysfunction. Profibrogenic transforming growth factor-ß1 (TGF-ß1) and NADPH oxidase 4 (NOX4, a major modulator of fibrosis related redox signaling) expression increased markedly after AAC. MitoQ blunted TGF-ß1 and NOX4 upregulation and the downstream ACC-dependent fibrotic gene expressions. In addition, MitoQ prevented Nrf2 downregulation and activation of TGF-ß1-mediated profibrogenic signaling in cardiac fibroblasts (CF). Finally, MitoQ ameliorated the dysregulation of cardiac remodeling-associated long noncoding RNAs (lncRNAs) in AAC myocardium, phenylephrine-treated cardiomyocytes, and TGF-ß1-treated CF. The present study demonstrates for the first time that MitoQ improves cardiac hypertrophic remodeling, fibrosis, LV dysfunction and dysregulation of lncRNAs in pressure overload hearts, by inhibiting the interplay between TGF-ß1 and mitochondrial associated redox signaling.

Review: Microfluidics technologies for blood-based cancer liquid biopsies.

Blood-based liquid biopsies provide a minimally invasive alternative to identify cellular and molecular signatures that can be used as biomarkers to detect early-stage cancer, predict disease progression, longitudinally monitor response to chemotherapeutic drugs, and provide personalized treatment options. Specific targets in blood that can be used for detailed molecular analysis to develop highly specific and sensitive biomarkers include circulating tumor cells (CTCs), exosomes shed from tumor cells, cell-free circulating tumor DNA (cfDNA), and circulating RNA. Given the low abundance of CTCs and other tumor-derived products in blood, clinical evaluation of liquid biopsies is extremely challenging. Microfluidics technologies for cellular and molecular separations have great potential to either outperform conventional methods or enable completely new approaches for efficient separation of targets from complex samples like blood. In this article, we provide a comprehensive overview of blood-based targets that can be used for analysis of cancer, review microfluidic technologies that are currently used for isolation of CTCs, tumor derived exosomes, cfDNA, and circulating RNA, and provide a detailed discussion regarding potential opportunities for microfluidics-based approaches in cancer diagnostics.