Respiratory dysfunction is the leading cause of death in the acute phase of high-cervical spinal cord injuries (SCIs). While survivors of high-cervical SCIs regain some of their respiratory function, many individuals require assisted ventilation. Despite the fact that there are currently many ongoing treatments, more needs to be understood about the anatomy and physiology of the respiratory pathways in the spinal cord in order to find cures for those suffering from high-cervical SCIs. Recent rodent studies using intra-spinal micro-stimulation (ISMS) for forelimb motor recovery has shown moderate functional recovery. In this study we mapped the motor neuron pools of three muscles used in respiration along the cervical and upper thoracic spinal cord. Additionally we explored the extent to which we can activate those muscle with ISMS in spinally intact rats.

This project aims to develop a better model for testing gene therapy vectors in vitro. Gene therapy uses a modified virus to insert a functional copy of a gene into a patient’s cells, but testing new viral vectors on 2D cell culture does not accurately predict success in vivo. To address this need, I have designed a 3D-printed microfluidic device for optimized viral vector testing. Cells are cultured in 3D in one channel, while media and viral vector load can be easily adjusted in a connected channel through a gravity perfusion system. The device is currently undergoing tests for cell viability and demonstrating its utility for gene therapy applications. The primary advantage of this device is that cells cultured in 3D mature more similarly to true skeletal muscle and thus are better models for predicting the success of a viral vector in a patient. The small size and fine tune control over the cell’s environment also save time and money during testing, allowing the maximum number of options to be explored. A new, physiologically relevant model for testing viral vectors would expedite gene therapy research to help bring treatments faster to patients in need of gene replacement therapy.

Spinal cord injury (SCI) can severely impair quality of life. While there are a variety of accommodations for individuals with SCI, there are no clinically available treatments which restore pre-injury levels of function to these patients. The optimization of emerging treatments for SCI will require a large amount of detailed experimental trial data in animal models. Assessments of trained motor behavior in animal models of SCI recovery typically fail to capture information about fine gradations in the course of recovery, which interferes with the development of new restorative treatments. To better characterize these gradations during the study of an emerging method for cervical SCI rehabilitation, I monitored forelimb movements of a cohort of injured rats during a motor behavior task using a high-precision haptic interface device - a machine which simulates physical interactions with virtual objects or events by applying forces to its user. I used the motion information gathered by this device to trigger electrical stimulation of the spinal cord to enhance attempted movements and encourage Hebbian plasticity within the spinal cord. Functional recovery of strength and range of motion was assessed relative to pre-injury baseline, demonstrating the utility of 3D haptic interfaces for the assessment of promising SCI treatments. Our goal is to help individuals with disabilities due to spinal cord injury regain function and independence by quantifying, optimizing, and the accelerating the translation of potential new treatments for SCI using these precise and clinically relevant recovery measures.

X-Link Myotubular Myopathy (XLMTM) is a progressive congenital pediatric disease that affects one in every 50,000 male births. XLMTM is caused by a mutation of the MTM1 gene. MTM1 codes for myotubularin, an enzyme that is involved in the excitation-contraction coupling of muscle cells. XLMTM is characterized by a severe phenotype marked with muscle weakness and hypotonia that can result in many life-threatening complications, including diaphragmatic weakness leading to respiratory failure. Adeno-associated virus (AAV)-mediated gene therapy has reversed muscle pathology in murine and canine models. Treatment of the canine models with an AAV8-MTM1 vector indicated improved muscle strength as well as prolonged life. The field of muscle disease research would benefit greatly if an in vitro patient-specific induced pluripotent stem cell (iPSC) model were utilized to generate XLMTM-null muscle myotubes. It is necessary to develop an in vitro model of the disease using specific patient-derived cells because the patient-specific disease model has the predictive utility to determine if there are aspects of the disease not mitigated by gene therapy. This will help pinpoint the areas the disease affects in order to come up with a cure. To do this, MTM1-mutant iPSCs were differentiated stepwise to myogenic precursors and then to multinucleated myotubes using a small molecule approach that has been shown to mimic embryonic muscle specification. Extent of myotube differentiation was qualified with immunochemistry, using muscle-specific markers such as Myogenin, MyoD, and MF20. Experiments are currently underway to identify the disease phenotype in vitro and correlate with known pathology observed in the patients with XLMTM.

Over a quarter of a million individuals live with Spinal Cord Injury (SCI) in the United States and experience debilitating chronic conditions, reduction of quality of life, and an accumulation of high healthcare costs. Recent clinical studies have shown that epidural spinal stimulation can lead to improvements in motor function, weight bearing, and autonomic dysreflexia among patients with SCI. It is thought that epidural stimulation creates an excitatory environment in the spinal cord and allows descending signals that were previously too weak to effect a motor output to reach a supra-threshold level. While the results are promising, both the mechanism of action and optimal parameters for stimulation and rehabilitation remain unknown. Therefore, in order to further develop this potential therapy, it is necessary to create a clinically relevant research model for the study of the treatment in question. This project addresses this through the development of an automated rehabilitative device for animal subjects following spinal cord injury, designed to mimic the extensive rehabilitation received by human patients with SCI and measure functional recovery following injury and during epidural stimulation treatment. Initial results show that subjects interact with the automated device, indicating a potential for more widespread implementation. Specifically, this high-throughput and automated system will be initially utilized for the study of the effects of epidural spinal stimulation in the rat cervical cord following contusion injury in order to quantify both transient and lasting motor improvements.

Patient specific individualized pluripotent (iPS) cells can be differentiated into cardiomyocytes that beat spontaneously, express sarcomeric proteins, and contain ion channels. However, these differentiated cardiomyocytes express a “fetal” like phenotype in that they are smaller, irregularly shaped, have a shortened sarcomere length, and lowered excitation-contraction rates that prevents their clinical applicability. In addition to executing their intrinsic genetic program, functional cardiomyocytes also receive inductive cues from their microenvironment. In the intact heart, those cues are supplied by the extra cellular matrix (ECM). The marriage of iPSC-derived cardiomyocyte technology with ECM differentiation cues will result in more mature cardiomyocytes that better represent natural human cardiomyocytes. Therefore, we are using a “native” cardiac matrix derived from a decellularized pig heart to try and promote maturation in cardiomyocytes using no mechanical inductive cues. We are particularly interested in the fibronectin to laminin ratio because the work of Wang, Lei, and Weighton (2015) suggests a proper ratio of these ECM components promotes maturation. Additionally, we are interested in the stiffness of the ECM coating as the cardiac environment has an optimal stiffness level. The iPSC will be differentiated into cardiomyocytes by using chemicals that alter the WNT signaling pathway. Some cells were exposed to the ECM during differentiation to measure the extent of cardiomyocyte maturation in the presence of ECM. Other cells were transferred to the ECM after the differentiation process. The extent of maturation will be measured by gene expression profiling and RNA sequencing. We hypothesize that the presence of cECM will enhance maturation based on the work of Oberwallner et al. (2014). If successful, this work will lead to improved drug screening and allow for tissue engineering for cell replacement therapies.

Autism Spectrum Disorder (ASD) describes a set of neurodevelopmental conditions characterized by behavioral and cognitive phenotypes with multiple gene involvement. There is a strong association between bowel disorders and ASD, making the enteric nervous system (ENS) a potential target for drug treatment and therapy. The ENS innervates the gastrointestinal system and regulates its function. Current studies of the ENS are limited to the invasive procedure of removal of gut tissue, or animal models that do a poor job of recapitulating the human disease. The work presented here uses induced pluripotent stem cells (iPSCs) from ASD patients that have been differentiated into enteric neurons to model the ENS for study. Fully differentiated enteric neurons pass through an intermediate cell type called neural crest (NC). Here, we report the successful differentiation of iPSCs to neural crest progenitor cells (NCPCs). Treatment of iPSCs with two inductive factors, SB431542 (a TGF-B Inhibitor) and BIO (a GSK-3 inhibitor) generated a high yield of NCPCs. The expression of NCPC markers by TaqMan® PCR gene expression assays, was used to quantitate the extent of NCPC differentiation. To demonstrate the specificity of the differentiation protocol, immunocytochemistry results between NCPCs and forebrain neurons were compared. Together, these findings support the conclusion that iPSCs can be efficiently differentiated to neural crest progenitors as an intermediate in the generation of an “ENS in a dish” to study the chronic constipation suffered by patients with ASD.

Duchenne muscular dystrophy (DMD) is a neuromuscular disorder caused by mutations in the gene that encodes the protein dystrophin. This disease is incurable and causes severe muscle weakness, respiratory failure, cardiomyopathy, and early death. Understanding the causes of DMD and developing new treatment strategies relies on in vitro models. However, current animal and cell based disease models of the cardiomyopathy experienced by patients with DMD do not accurately recapitulate the human presentation of the disease. Stem cell derived cardiomyocytes provide a platform for improved and personalized disease modeling, however the cells are functionally immature, exist in two dimensional cultures, and do not experience the mechanical stresses of the human heart. There is a need, therefore, for a way to generate three-dimensional constructs of cardiomyocytes and subject them to physiologically relevant mechanical loads for the purpose of promoting maturation and disease phenotype testing. Induced pluripotent stem cells (iPSC) derived from the urine of healthy and diseased patients were used to generate cardiomyocytes in vitro (iPSC-cardiomyocytes). Differentiations of stem cells were enriched using metabolic methods, allowing us to generate cardiomyocyte cultures with greater than 90% purity. Three-dimensional cardiomyocyte constructs were generated in polydimethylsiloxane (PDMS) molds using a cardiac-stromal co-culture method with a combination of collagen and geltrex. In order to further replicate the human heart, a device was designed and fabricated that was capable of applying controllable, uniaxial mechanical loads to the three-dimensional cardiomyocytes and was used to measure the force generation of DMD or healthy iPSC-cardiomyocytes. Future studies involving the device will increase our understanding of the pathology of DMD and allow for novel, personalized treatments to be developed.