A Platform to Study the Effects of Electrical Stimulation on Immune Cell Activation During Wound Healing.

Wound healing is a complex process involving diverse changes in multiple cell types where the application of electric fields has been shown to accelerate wound closure. To define the efficacy of therapies based on electric fields, it would be valuable to have a platform to systematically study the effects of electrical stimulation (ES) upon the inflammation phase and the activation of signaling mediators. Here, an in vivo ES model in which flexible electrodes are applied to an animal model for monitoring inflammation in a wound is reported on. Subcutaneous implants of polyvinyl alcohol sponges elicit inflammation response as defined by the infiltration of leukocytes. The wound site is subjected to electric fields using two types of additively fabricated flexible electrode arrays. The sponges are then harvested for flow cytometry analysis to identify changes in the phosphorylation state of intracellular targets. This platform enables studies of molecular mechanisms, as it shows that an application of low-frequency ES ≤0.5 Hz increases phosphorylation of Erk proteins in recruited leukocytes, identifying a signaling pathway that is activated during the healing process

Programmable systems to engineer human tissues and electrophysiological sensors

To study human development and disease, and develop therapies for regenerative medicine, the ability to create scalable, physiologically relevant human tissues is critical. In this regard, engineering cells and tissues from pluripotent stem cells has led to significant advances in our understanding of human-specific biology and holds promise for therapies. However, several challenges remain, including the ability to differentiate diverse lineages and drive disease states, vascularization to build tissues at scale, and monitoring capabilities to assess function. In this dissertation, I present a multi-faceted approach to address these limitations in creating and assaying complex tissue. To rapidly discover methods to differentiate pluripotent stem cells, I developed a screening method leveraging single cell RNA-sequencing to study the effects of gene overexpression. Using this approach, I assayed both fitness and transcriptomic responses of transcription factors, mutant gene libraries and whole gene families on pluripotent stem cells in multiple culture conditions. From these responses I built gene regulatory networks and found ETV2 as a reprogramming factor toward endothelial cells. I further engineered the system in combination with teratoma formation to develop a multiplexed system to assay the potential of genes and variants to drive oncogenic transformation in a tissue-specific manner. I found that c-MYC alone or together with myristoylated AKT1 drives transformation of neural progenitor lineages, while MEK1 (S218D/S222D) drives proliferative advantage in mesenchymal lineages like fibroblasts. I then harnessed these reprogramming approaches to engineer densely vascularized human tissue. I combined reprogramming and chemically directed differentiation by overexpressing lineage-specifying transcription factors in differentiating vascular organoids to introduce neurons and skeletal muscle into the organoids, demonstrating maintenance of molecular and functional characteristics of the parenchymal and vascular lineages. Finally, I developed flexible, printed electrodes to enable the monitoring of electrophysiological signals and electrical perturbation of tissues. I enabled low noise, high spatial resolution measurement of clinically relevant signals using screen-printed, stretchable concentric ring electrodes. I then applied these screen-printed electrodes to study the effects of electrical stimulation on the wound healing response in vivo. Lastly, I demonstrated preliminary data on a novel fabrication method to print microelectrodes to map cellular electrical activity

Programmable systems to engineer human tissues and electrophysiological sensors

To study human development and disease, and develop therapies for regenerative medicine, the ability to create scalable, physiologically relevant human tissues is critical. In this regard, engineering cells and tissues from pluripotent stem cells has led to significant advances in our understanding of human-specific biology and holds promise for therapies. However, several challenges remain, including the ability to differentiate diverse lineages and drive disease states, vascularization to build tissues at scale, and monitoring capabilities to assess function. In this dissertation, I present a multi-faceted approach to address these limitations in creating and assaying complex tissue. To rapidly discover methods to differentiate pluripotent stem cells, I developed a screening method leveraging single cell RNA-sequencing to study the effects of gene overexpression. Using this approach, I assayed both fitness and transcriptomic responses of transcription factors, mutant gene libraries and whole gene families on pluripotent stem cells in multiple culture conditions. From these responses I built gene regulatory networks and found ETV2 as a reprogramming factor toward endothelial cells. I further engineered the system in combination with teratoma formation to develop a multiplexed system to assay the potential of genes and variants to drive oncogenic transformation in a tissue-specific manner. I found that c-MYC alone or together with myristoylated AKT1 drives transformation of neural progenitor lineages, while MEK1 (S218D/S222D) drives proliferative advantage in mesenchymal lineages like fibroblasts. I then harnessed these reprogramming approaches to engineer densely vascularized human tissue. I combined reprogramming and chemically directed differentiation by overexpressing lineage-specifying transcription factors in differentiating vascular organoids to introduce neurons and skeletal muscle into the organoids, demonstrating maintenance of molecular and functional characteristics of the parenchymal and vascular lineages. Finally, I developed flexible, printed electrodes to enable the monitoring of electrophysiological signals and electrical perturbation of tissues. I enabled low noise, high spatial resolution measurement of clinically relevant signals using screen-printed, stretchable concentric ring electrodes. I then applied these screen-printed electrodes to study the effects of electrical stimulation on the wound healing response in vivo. Lastly, I demonstrated preliminary data on a novel fabrication method to print microelectrodes to map cellular electrical activity

Eustachian and tricuspid valve endocarditis: A rare consequence of the automatic implantable cardioverter-defibrillator placement

Eustachian valve (EV) is usually a rudimentary structure in adults. It is an embryological remnant of sinus venosus that directs oxygenated blood from the inferior vena cava across the foramen ovale and into the left atrium. Intravenous drug use is most commonly associated with infective endocarditis of the right-sided heart structures. Other documented causes of such an occurrence are intracardiac devices like pacemakers and central venous catheters. Patients presenting with concerns of infection and embolic phenomenon should promptly undergo evaluation for infective endocarditis. Although an embryological remnant, the eustachian valve normally regresses after birth, except in a minority of the patients, it persists as a vestigial structure. Here we present an unusual case involving infective endocarditis of the eustachian valve and tricuspid valve both in a patient with recent automatic implantable cardioverter-defibrillator (AICD) placement and history of IV drug abuse and its systemic consequences in a patient with patent foramen ovale

Stretchable Dry Electrodes with Concentric Ring Geometry for Enhancing Spatial Resolution in Electrophysiology.

The multichannel concentric-ring electrodes are stencil printed on stretchable elastomers modified to improve adhesion to skin and minimize motion artifacts for electrophysiological recordings of electroencephalography, electromyography, and electrocardiography. These dry electrodes with a poly(3,4-ethylenedioxythiophene) polystyrene sulfonate interface layer are optimized to show lower noise level than that of commercial gel disc electrodes. The concentric ring geometry enables Laplacian filtering to pinpoint the bioelectric potential source with spatial resolution determined by the ring distance. This work shows a new fabrication approach to integrate and create designs that enhance spatial resolution for high-quality electrophysiology monitoring devices

Stretchable Dry Electrodes with Concentric Ring Geometry for Enhancing Spatial Resolution in Electrophysiology.

The multichannel concentric-ring electrodes are stencil printed on stretchable elastomers modified to improve adhesion to skin and minimize motion artifacts for electrophysiological recordings of electroencephalography, electromyography, and electrocardiography. These dry electrodes with a poly(3,4-ethylenedioxythiophene) polystyrene sulfonate interface layer are optimized to show lower noise level than that of commercial gel disc electrodes. The concentric ring geometry enables Laplacian filtering to pinpoint the bioelectric potential source with spatial resolution determined by the ring distance. This work shows a new fabrication approach to integrate and create designs that enhance spatial resolution for high-quality electrophysiology monitoring devices

Discovery of novel CaMK-II inhibitor for the possible mitigation of arrhythmia through pharmacophore modelling, virtual screening, molecular docking, and toxicity prediction

In the present research, a few well-known artificial intelligence tools were explored for efficient hit selection which could be further utilized for the discovery of CaMK-II inhibitors for the Treatment of arrhythmia. To achieve the desired goals pharmacophore modelling, database retrieval, molecular docking studies, and toxicity prediction were performed. Pharmacophore modelling was performed with the Pharmit open-source database which gave the features viz. Hydrogen Bond Donor, Hydrogen Bond Acceptor, and Hydrophobic. This pharmacophore is generated with the aid of the protein of CaMK-II (PDB ID: 2WEL) and co-crystallized ligand K88. Further, this generated pharmacophore was screened through the various Pharmit databases which include CHEMBL30, ChemDiv, ChemSpace, MCULE, MolPort, NCI Open Chemical Repository, Lab Network, and ZINC. Further, the top two hits from each database that has maximum similarity with the pharmacophore have been selected for the molecular docking and ADMET studies. Among, all the hits CHEMBL 1952032 showed good binding interactions with CaMK-II. Also, it was found to be non-toxic upon evaluation through the OSIRIS property explorer. In the future, it can be explored against the CaMK-II for the development of novel CaMK-II inhibitors which can be used for the mitigation of arrhythmia

Mapping Cellular Reprogramming via Pooled Overexpression Screens with Paired Fitness and Single-Cell RNA-Sequencing Readout.

Understanding the effects of genetic perturbations on the cellular state has been challenging using traditional pooled screens, which typically rely on the delivery of a single perturbation per cell and unidimensional phenotypic readouts. Here, we use barcoded open reading frame overexpression libraries coupled with single-cell RNA sequencing to assay cell state and fitness, a technique we call SEUSS (scalable functional screening by sequencing). Using SEUSS, we perturbed hPSCs with a library of developmentally critical transcription factors (TFs) and assayed the impact of TF overexpression on fitness and transcriptomic states. We further leveraged the versatility of the ORF library approach to assay mutant genes and whole gene families. From the transcriptomic responses, we built genetic co-regulatory networks to identify altered gene modules and found that KLF4 and SNAI2 drive opposing effects along the epithelial-mesenchymal transition axis. From the fitness responses, we identified ETV2 as a driver of reprogramming toward an endothelial-like state