Current drug testing methods are unable to accurately predict and study the effects of drugs on human myocardial tissue. These methods are unable to accurately mimic the physiology and maturity of native cardiac tissue, and as a result, the tissues are unable to react to cardiotoxic drugs the same way that native cardiac tissue. Therefore, there is a need for physiologically accurate and mature tissue constructs in order to test clinical drugs for effectiveness, cardiotoxicity, and arrhythmic effects. To fulfill this need, we will use an electroconductive bioink containing decellularized extracellular matrix (dECM) from porcine cardiac tissue, reduced graphene oxide (rGO), and incorporated human induced-pluripotent stem cell (hiPSC)-derived cardiomyocytes to develop a more physiologically accurate cardiac tissue construct. This will result in more adequate cell maturation, and therefore, more accurate preclinical drug screening. The success of this project would lead to the development of high throughput production of biomimetic cardiac tissue models for use in accurate drug screening, along with improving the understanding of cardiac maturation.

Modern disease research utilizes two-dimensional (2D) stem-cell tissue culture models, simplified 3D engineered tissues, and animal organisms to study microenvironments and human physiology. However, in vitro platforms often oversimplify the physical tissue niche and animal models do not always accurately represent the function of human tissues. For instance, 2D tissue platforms used to study cardiac biology are limited because they cannot accurately recapitulate the pumping motion of the human heart which is responsible for the circulation or blood. Furthermore, animal models have an under-representative cardiovascular physiology making them inadequate systems for studying human cardiac biometrics. As such, we propose to develop a 3D tissue culture system that can accurately mimic the hierarchical organization of different human tissues. Using our novel flexible cell-sheet stacking technique, we can precisely stack layers of organized cell-sheets to create 3D laminar tissues. Then, these organized laminar tissues can be manipulated by the flexible scaffold into complex 3D tissue shapes using custom-made molds. For example, the human heart is helically aligned cardiac tissue throughout its structure that allows for the generation of intraluminal pressure during ventricular contraction. Inspired by the intricate architecture of human myocardium, this project aims to analyze how varying degrees of cell orientation can influence intraluminal pressure generating function. To account for the hollow and conical nature of the human heart architecture, we have designed a model for casting closed 3D hydrogel scaffolds with a hollow lumen that allows for intraluminal pressure measurements. The proposed model will allow for the determination of the optimal angle of cell alignment that produces the greatest intraluminal contraction. We will validate this platform using a contractile mouse skeletal muscle cell type. This approach can be adapted to model the organization of any contractile muscle tissue and be used to study the function and microstructure of human tissues.

Vascular remodeling—the modification of preexisting vasculature—often occurs in common diseases such as cardiovascular disease and cancer. Little is known about the factors controlling perivascular remodeling regarding interactions with the endothelium, and current methods of studying this phenomenon in the microvasculature have shown to be inadequate. A small-diameter, single-channel arteriole model was developed to study vascular remodeling under various flow and cellular microenvironments in vitro. The housing device consists of a plexiglass casing that allows for robust and time-efficient fabrication, attachment of flow and pressure, and in situ imaging of the vascular constructs. Type I collagen gel is embedded with human coronary artery smooth muscle cells (HCASMCs), and the lumen is formed using a needle-based subtractive molding method with a 180um acupuncture needle. The original diameter of the lumen is retained while human umbilical vein endothelial cells (HUVECs) are seeded into the lumen by hand-perfusion. Migration and realignment of both the HCASMCs and the HUVECs is observed after applying physiological flow rates of media for 3 days in co-culture. Additionally, endothelial barrier function and smooth muscle contractile function are maintained. The functionality of the model is confirmed through dextran permeability assays, calcium wave propagation, and epinephrine treatments, where dilation of the arteriole occurs upon perfusion of the hormone. These experiments demonstrate the ability of this system to increase both time and efficiency of drug studies, as well as improve understanding of microvascular diseases.

Peripheral neuropathies involve the destruction of peripheral nerves, which leads to impairment of the neuromuscular junction (NMJ). This is the area where the nerve transmits information to muscle. NMJ breakdown will cause this synapse to degrade, generating symptoms such as reduced muscle contraction, respiration, and movement. The problem is that current efforts to develop treatments against peripheral neuropathies are severely dampened by having a lack of human-based models of synapses. Therefore, there is a need for a non-invasive method to explore and characterize synaptic function for future treatments. This research study offers a novel system to investigate the differences in neuromuscular junctions between healthy and disease states by utilizing induced pluripotent stem cells (iPSCs) to derive an in vitro NMJ. Procedures involving the differentiation of healthy-state iPSCs into motor neurons have been optimized. The iPSC cultures were matured until day 45 after induction towards a neural ectoderm lineage, and were analyzed using immunocytochemistry, imaging, patch-clamp electrophysiology, and flow cytometry. Differentiated neurons stained positive for neuronal marker p75, Islet-1 and choline acetyl-transferase (ChAT). Electrophysiology data showed neurons were capable of high degrees of repetitive firing sequences, while flow cytometry showed neuronal purity in culture was roughly around 97%. Human skeletal muscle myoblasts have also been differentiated into myotubes-the precursors for muscle tissue-and imaged for acetylcholine receptor clusters. The culture medium was supplemented with various additives in order to determine which medium promoted greatest acetylcholine receptor (AChR) cluster density and size. It was found that AChR clusters are more prominent and enlarged when the muscle cells are cultured with Agrin supplement. Ongoing studies are looking at combining these cell types into a novel contractility assay in order to investigate NMJ function in vitro. The establishment of this system will enable more effective applications for novel therapeutics and disease progression studies.

The ability to recapitulate the dynamic presentation of signals in a stem cell's microenvironment remains a major hurdle in tissue engineering. By controlling cell growth and differentiation in 4 dimensions (i.e., time and 3D space) through biochemical and biomechanical cues, heterogeneous synthetic tissues could be produced that match the complexity of their native counterparts. By combining strategies in light-programmable hydrogels and recombinant protein engineering, we have demonstrated the ability to control the cellular microenvironment with a UV light mediated reaction that allows a covalent bond to form between a modified protein and the hydrogel. While covalent interactions form strong bonds, they are generally irreversible using mild, cytocompatible chemistries and pose potential temporal limitations. In addition, this technique uses UV light, which may be harmful to cells. Instead, by incorporating LOVTRAP into the photopatterning system, we introduce a greater amount of control by allowing for full reversibility using blue light. In the dark, LOV2 and Zdk1 dimerize to form a protein-protein bond with similar stability to an antibody-antigen interaction. Upon exposure to blue light, the dimer dissociates, allowing for fully reversible control over conjugation. A protein of interest can be tagged with the Zdk1 domain using recombinant protein engineering, while still maintaining its binding affinity for LOV2. LOV2 can then be conjugated throughout a hydrogel to make a light responsive protein binding material. By utilizing a light controlled dimerization event between proteins, the LOVTRAP system is fully reversible using only blue light. Thus far, I have been able to illustrate the photopatterning ability of LOVTRAP and am working towards demonstrating full reversibility. If successful, this technique could be used for tissue engineering applications that require mild treatment conditions and full dynamic control over the presentation of biochemical cues.

Stem-cell derived engineered cardiac tissues are a promising avenue for the research of heart disease, enabling disease-specific modelling and drug screening. However, their validity as a model hinges on the similarity to native cardiac tissue, both in super- and sub-cellular structure and organization. When cultured on nanopattern substrates, myocytes orient the sarcomeres along a similar axis, allowing for greater force production and higher similarity to native cardiac tissue, though the evidence of the finer details are obscured by the diffraction limit of light. Super-resolution microscopy has opened the door to the analysis of diffraction-limited biological structures, and one such recently developed super-resolution technique, Expansion Microscopy (ExM), has made this analysis more accessible. By embedding the fluorophores from an immunostained sample into an expandable hydrogel, sub-diffraction details are physically enlarged and made measurable on standard fluorescence microscopy equipment. In this work, expansion microscopy was performed on engineered cardiac tissue to evaluate the effect of the nanopattern substrate on sub-sarcomeric structure and organization. As expected, it was found that the sarcomeres were more aligned within the cardiomyocytes, and the length between the z-lines were increased when cells were cultured on the nanopattern substrate as compared to the flat substrate. In addition, the width of the Z-disks were significantly different in nanopattern cultured cardiomyocytes as compared to myocytes cultured on flat substrate. The transverse tubules, responsible for a unified action potential, were larger in diameter and better localized with the Z-line, and the myosin heads were closely localized with the actin filament. When examined with super-resolution microscopy, the engineered cardiac tissues display sub-sarcomeric organization that allows for greater and more efficient force production, further demonstrating the efficacy of nanopattern substrates in cardiac cell differentiation and maturation. Though ExM needs further validation, it has proved a powerful sample-side tool for probing diffraction limited features.

Heart failure (HF) has a myriad of negative effects in humans, including fatigue, weakness and reduced ability to exercise. Understanding the mechanisms that lead to this weakened state is critical in order to know how to treat it. There is recent suggestion that HF leads to decreased skeletal muscle force generation and exercise capacity. However, the mechanism responsible for this process is unknown and there are no current pharmacological treatments that target the skeletal muscle. My project aims to use a mouse model of HF called D230N, which have a mutation in the sarcomeric gene tropomyosin that results in progressive systolic dysfunction similar to what is seen in human HF. I will use the D230N mice to test the hypothesis that progressive cardiac dysfunction in a rodent model of HF leads to decreased exercise capacity, reduced skeletal muscle function and structural changes in the skeletal muscle that mimics the changes seen in human HF. I have taken preliminary measurements of 9 month old D230 and WT mice. I used a treadmill apparatus to measure exercise capacity of the mice, which showed that the D230N exercised for a shorter duration compared to WT mice. To measure the maximum force generated in the gastrocnemius muscle I used in vivo muscle stimulations and normalized to the muscle weight. I observed that the D230N generated lower muscle force than WT mice. Based on these results, I propose a longitudinal study to determine how skeletal muscle dysfunction changes as the disease progresses by taking these measurements in mice ranging from 2-12 months. Once we establish that there is skeletal muscle dysfunction in this rodent model of HF, we will move towards more in depth studies to fully understand the underlying mechanism.

The human liver is a unique organ with the ability to regenerate quickly in response to acute injury. During this regeneration process, hepatocytes, i.e. the main cell type of the liver, receive growth factors and other cues and begin to remodel the extracellular matrix (ECM) of the tissue. Liver regeneration has been well characterized in mouse and rat models; however, human liver regeneration remains largely unstudied. In particular, the ECM remodeling process in human liver regeneration is unknown. Here, we use a humanized mouse liver injury model to study changes in the ECM over time during human liver regeneration. To study this, we implanted engineered human liver tissue "seeds" into the fat pad of FNRG-mice. These mice experience liver damage, and liver regeneration cues flood the bloodstream in response. The seeds become exposed to these cues and expand over time, mimicking human liver regeneration inside a mouse host. To study the ECM over time, we sacrificed the animals every other week and then performed special histology stains to characterize ECM components such as collagen I, IV, and fibronectin. We also immunostained for CK18/CK19, markers for hepatocytes and cholangiocytes respectively. Information on the ECM remodeling process is key to understanding human liver regeneration as a whole. Understanding this process could lead to better informed decisions regarding matrix composition in artificial human liver constructs for regenerative medicine.