Abstract:  Hematopoietic stem cells (HSCs) play a critical role in repopulating the blood and have immense therapeutic potential. Over 20,000 Americans require bone marrow transplants yearly, and the major limitation to improving HSC transplants has been the inability to maintain and expand functional HSCs in vitro. Since HSCs initially expand in the fetal liver during development, and since they can migrate to the adult liver and regenerate during injury, we tested the hypothesis that engineered in vitro liver models could expand functional HSCs ex vivo and maintain their multipotency. We found that the human liver microenvironment can expand functional HSCs. We are now developing a fetal liver model in order to study the expansion and maturation of HSCs in the fetal liver and the microenvironmental cues they receive from fetal hepatoblasts. We are comparing 2D and 3D engineered liver platforms for their stability and ability to expand HSCs in serum-free medium, and we have assessed the hepatoblasts’ differentiation over time in these models. Our next steps include incorporation of fetal liver sinusoidal endothelial cells (LSECs) into the fetal liver model to study their effects on HSCs’ migration to and expansion.

Abstract:  Vascular Endothelial Growth Factor (VEGF) is a pro-angiogenic factor and a strong modulator of cell-cell interactions. Under physiological conditions, VEGF mediates the formation of healthy and intact blood vessels. However, in disease states, dysregulation of VEGF signaling is responsible for developing disorganized and leaky vasculature, as observed in tumors. How VEGF leads to both the disruption and development of vasculature is unknown. VE-cadherin is the key component of adhering junctions, multiprotein structures controlling endothelial permeability. Although endothelial adherens junctions have been extensively studied, the dynamic changes in adherens junction protein complexes that follow VEGF stimulation have not been systemically characterized. Current technology makes examining dynamic interactions challenging. Immunoprecipitation is the most common method for dissecting protein interactions, yet it lacks the ability to discern weak interactions and heavily depends on the quality of the specific antibody. Biotin proximity labeling is a powerful technique that overcomes many limitations of immunoprecipitation and provides high sensitivity even for weak interactions. However, the existing proximity labeling tools lack temporal resolution and do not allow for the detection of dynamic changes in protein interactions. Thus, to examine the dynamic effects of VEGF stimulation on VE-cadherin interactions, traditional methods are not applicable. To address this issue, we have developed a light-regulated biotin ligase, LightR-TurboID, that provides high sensitivity and temporal resolution. We aim to use LightR-TurboID to examine VEGF-induced changes in the VE-cadherin interactome. Specifically, how does VEGF stimulation disrupt and amend barrier function, and how is this balance between leakiness and angiogenesis achieved? We hypothesize that there are previously unrecorded dynamic changes in the VE-cadherin interaction following VEGF stimulation occurring on a short time scale. Using our VE-cadherin-LightR-TurboID tool, we can determine what dynamic interactions are occurring and how they are affecting barrier integrity.

Abstract:  

Traditional antifibrotic therapies often struggle with patient adherence and limited efficacy, highlighting the need for advanced treatment strategies. Enhancing cell-based therapies for pulmonary fibrosis requires strategies that improve cellular retention and functions within target tissues after delivery. Single-cell encapsulation in gel coatings, designed with specific cues and immune checkpoint molecules, offers a promising strategy to formulate cell-based therapeutics for improved clinical outcomes. This approach aims to extend the residence time of encapsulated cells and modulate immune responses critical for effective tissue remodeling. Our study investigates the modification of soft alginate gel coatings around single mesenchymal stromal cells (MSCs) to minimize innate immune recognition, enabling them to modulate macrophage subpopulations in the lungs via paracrine mediators over an extended period. Our approach shows significant enhancements in resolving both transient and persistent lung fibrosis in experimental models.

Additionally, nano-based therapies, such as lipid-based nanoparticles like liposomes and biological nanoparticles naturally secreted by cells (extracellular vesicles, EVs), have emerged as promising, complex, biocompatible drug delivery systems. These advancements have attracted global attention among researchers, prompting rapid development of more effective drug delivery methods. The ideal nanoparticle safely transports its drug payload to a predetermined target, facilitating intracellular or extracellular release for direct internalization and desired therapeutic effects. Along the way, nanoparticles protect the drug from non-target tissues, prolonging circulation time and enabling sustained release. While encapsulated MSC therapy remains broad, nanovesicles functionalized with specific ligands offer a more precise approach.

Abstract:  The omentum is a visceral adipose tissue that contains numerous fat-associated lymphoid clusters (FALC) surrounded by blood capillaries. These clusters consist of large aggregates of B cells, T cells, dendritic cells, macrophages, and innate lymphoid cells that participate in the clearance of pathogens. In the omentum, at least two distinct macrophages are located in distinct compartment: LYVE1hi Tim4(+) embryonically-derived macrophages and CX3CR1hi Tim4(-) monocyte-derived macrophages. Here, we  examine the role of CX3CR1hi Tim4(-)  macrophages that are imbedded within the FALC. Using confocal imaging with reporter/conditional knock-out mice, we have characterized that CX3CR1+ Tim4- highly interact with blood capillaries and fibroblastic reticular cells (FRCs) within FALC. In addition, we examine how CCL19+ FRCs and blood endothelial cells interact with CX3CR1+ macrophages in steady state and inflammatory conditions.

Abstract: Mammals typically maintain their body temperature within 0.5°C of their mean core temperature, around 37°C. However, harsh environments or lack of food can trigger a torpor or hibernation-like state, during which the body temperature drops by 5-10°C to conserve energy and allow survival. A recent study (Takahashi et al., Nature 2020) developed a genetically engineered mouse model that can temporarily induce this hypometabolic state with a small molecule called clozapine N-oxide. This state can also be induced via regulation of mice body temperature and food intake. This process is now known to be beneficial for medical applications where slowing down metabolic processes limits tissue damage, for example after heart attack or stroke, as well as being of relevance to the future possibility of long-distance space exploration. However, it is unclear how this state affects the hematopoietic and immune system. In this T32 application, we aim to investigate the effects of inducing and recovering from a hibernation-like hypometabolic state on the immune response to inflammation and the hematopoietic stem cell niche.

Abstract:  Circadian rhythm is an important regulator of numerous physiological processes, and its disruption is associated with increased risk of cardiovascular disease. Heart failure with preserved ejection fraction (HFpEF) is associated with endothelial dysfunction, but the relationship between rhythm disturbances, endothelial dysfunction, and HFpEF remains unclear. Using a systems pharmacology approach, we recently identified and obtained preliminary results that time-dependent but not chronic daily dosing of a class of drugs prevent the development of HFpEF which is associated with an improvement in microvascular function. Here, we will study the underlying mechanisms of disrupted endothelial circadian rhythm in the context of HFpEF and how this disruption can potentially be pharmacologically targeted as a therapeutic option.