Human pluripotent stem cells (hPSCs) are cells capable of self-renewal while differentiating into any cell type in the body. The differentiation into skeletal muscle progenitor cells (SMPCs), results in mature skeletal muscle cells. As the potential of deriving SMPCs from hPSCs has been further researched for medical application, the problem encountered is how to generate fully functional skeletal muscle cells from hPSCs experimentally. Currently, there are many existing protocols (e.g., HX, JC, MS Protocols) for such differentiation, known as myogenesis, but each has problems that need to overcome (e.g., time-consuming, not fully functional cells, resulted in other cell types). Our goal is to figure out what genes and metabolites are involved in the natural process of differentiation (from prenatal skeletal muscle progenitors to satellite cells) and apply the knowledge experimentally. To address this, we present the use of computational methods for the biological network-based integration of transcriptomic and metabolomic data. By analyzing data for both in vivo process and in vitro protocol results, we can find potential genes and metabolites differences, relating from the transcriptomic level to metabolomic level, revealing the inadequacy in the experimental protocols, presenting probable genes to improve the results. This involves RNA sequencing data analysis (e.g., single-cell, microarray) of human and mouse cells from embryonic to postnatal stages, with Seurat (pseudo-time analysis), Monocle, and AFFY, which processes/analyzes the data revealing potential genes. Then further analysis with UKIN, guided network propagation, to rank and identify the most probable genes, and perturb-Met to analyze metabolites involved. This is a time and cost-efficient way to find most probable genes directing the differentiation, which can then be tested and verified experimentally. If successful, it can be further developed into potential treatments much more effective and efficient than available medications and technologies for presently incurable musculoskeletal diseases.

Alzheimer’s Disease (AD) is a neurodegenerative condition that affects more than 50 million individuals worldwide. The progression of AD is hallmarked by the buildup of beta-amyloid plaques and neurofibrillary tangles, leading to neuronal death on a large scale. In the early stages of AD, the hippocampus is disproportionately affected by this heavy neuronal loss. The genetic elements and pathways contributing to this AD pathology are still poorly understood. Excitatory synapse assembly (ESA) processes have been previously shown to be affected by the pathology of AD, leading us to investigate the expression patterns of genes involved in ESA in different brain regions. A gene ontology (GO) analysis was conducted to isolate individual genes involved in ESA and analyze their relation to beta-amyloid plaque and pTau neurofibrillary tangle severity in patients who presented signs of dementia due to AD. ESA gene expression was shown to be strongly correlated with increasing levels of pTau in all brain regions except for the hippocampus, where there was no correlation, in a linear regression analysis. This implies that the hippocampus has a unique response to AD pathology with regards to ESA gene expression among the different brain regions. The inability of hippocampal cells to express neuronal repair genes in the presence of severe neurofibrillary tangles requires further analysis and could eventually confer a novel target for AD therapeutics.

Amyloid β (Aβ) plaques are a hallmark of Alzheimer’s disease (AD), the most common form of dementia that afflicts over 5 million Americans. Soluble proteins, including Aβ, are cleared from the brain by the glymphatic system, a brain wide network of perivascular spaces that facilitates the intermixing of cerebrospinal fluid and interstitial fluid. Prior studies report that aquaporin-4 (AQP4), a water channel polarized to perivascular astrocyte endfeet, supports glymphatic clearance of soluble proteins from the brain. In the aging brain, glymphatic clearance becomes impaired and AQP4 becomes depolarized from astrocytic endfeet. Such loss of perivascular AQP4 localization is correlated with AD status and Aβ plaque burden in the human brain. In the present study, we test whether such AQP4 depolarization promotes Aβ plaque formation. AQP4 is anchored to astrocytic endfeet via the dystrophin protein complex that includes the adaptor protein α-syntrophin (α-Syn). We crossed the α-Syn knockout mouse, which lacks perivascular AQP4 localization, with the 5XFAD mouse line which spontaneously develops Aβ plaques. Our preliminary analysis suggests that loss of perivascular AQP4 localization with α-Syn knockout increases Aβ burden relative to controls. These findings demonstrate that loss of perivascular AQP4 localization, such as occurs in the human brain in the setting of AD, contributes to the development of Aβ pathology. In the future, it may be possible that targeting the localization of AQP4 may be the basis for new therapeutics that can slow or even reverse AD pathology.

Alzheimer’s Disease (AD) is a neurodegenerative disease, characterized by amyloid-ß plaque deposition in the brain, that affects more than 5 million Americans. The glymphatic system is a network of perivascular spaces that facilitates fluid movement and solute clearance from the brain, and its dysfunction in aging has been implicated in the development of AD. The water channel aquaporin-4 (AQP4), located in astrocytic endfeet bordering the perivascular spaces, supports glymphatic function. In the aging rodent and human AD brain, loss of perivascular AQP4 localization is associated with impaired glymphatic function and increased amyloid-ß deposition. Yet the molecular basis for this loss of perivascular AQP4 localization is unknown. Aquaporin-4ex (AQP4ex) is a novel translational readthrough variant of AQP4. Selective deletion of AQP4ex results in the mislocalization of AQP4 all over the astrocytic membrane, indicating that AQP4ex is a crucial element in the perivascular localization of AQP4. In this study, we quantitatively analyze the expression and localization of AQP4ex to determine whether changes in AQP4ex associate with aging, AD status, or AD pathology. Using immunofluorescent double-labeling, confocal microscopy, and custom digital image analysis techniques, we define AQP4ex expression and localization between young and aged mice, and compare these changes between wild-type animals and transgenic animals that spontaneously form amyloid-ß plaques. Using a case series of post mortem human frontal cortical tissue, we compare AQP4ex expression between healthy young adults, cognitively intact aged subjects, and aged subjects with an AD diagnosis. This is the first characterization of AQP4ex expression in the murine brain and in a human case series, and these data will contribute to the small but growing body of research on AQP4ex and its relationship with AQP4 localization, creating opportunities to identify a new novel mechanism and novel target in AD pathology.

Breast milk is essential to the health and development of a child, containing antibodies that protect infants from common illnesses. However, exclusive breastfeeding is not always possible, and no infant formula substitutes for maternal antibodies. Previous studies on mice showed high germinal center (GC) B cell levels in response to the absence of maternal antibodies from breast milk early in life. Germinal centers B cells are involved in the adaptive immune system by secreting high affinity antibodies. However, little is known about the consequence of this, making characterizing the isotype, location, and duration of the antibody response in neonates (newborns) lacking maternal breast milk antibodies essential. For this project, I designed and optimized a tissue preparation and flow cytometry panel to assess memory B cells, GC B cells, and plasma cells (cells that secrete antibodies to fight infections and disease). The flow panel uses the cell markers CD138 and B220 to identify plasma cells by isolating the cells that are positive for CD138 and negative for B220. However, the marker CD138 can be sensitive to collagenase, resulting in the potential failure to identify plasma cells successfully. For this reason, I tested a range of collagenases, including Collagenase A, D, and IV. Concluding that CD138 was being cleaved off by all tested collagenases, I then used TACI as a new type of plasma cell differentiation marker. I evaluated these protocols by the viability of cells and the plasma cells' frequency. This protocol allows for the determination of localization, persistence, and isotype of early life B cells activated in the absence of breast milk.

Adoptive immunotherapies with engineered T cells offer great promise as safe and effective treatments to prevent and treat relapse of leukemia. T cells can be genetically modified to express T cell receptors (TCR-T) that target diverse types of tumor-associated antigens, including neoantigens created from abnormal, malignancy-restricted proteins. TCR-T immunotherapies targeting neoantigens have the advantage of being highly specific for malignant cells and thus should cause little damage to normal tissues. A critical step in developing neoantigen-targeting TCR-T immunotherapies is confirming that the engineered T cells recognize cells bearing the mutation(s) of interest. However, for uncommon mutations, few or no leukemia cell lines may exist that naturally contain mutations of interest. CRISPR/Cas9 offers to be an efficient and reliable method to model relevant mutations expressed under near-physiological conditions in cell lines relative to traditional gene editing tools. We will use CRISPR/Cas9-mediated gene editing to introduce missense mutations into genes encoding proteins involved in RNA splicing. Knock-in mutations will be introduced in acute myeloid leukemia (AML) cell lines by homology directed repair (HDR). These cell lines will then be used in in vitro assays to validate TCR-T cells recognition of neoantigen-bearing leukemia cells and in vivo assays to generate a xenograft model of human leukemia in mice. This project, in conjunction, with the continued work on antigen discovery will contribute to the development of a library of leukemia-antigen specific TCRs to provide T cell immunotherapy options for diverse patients in the future.