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Browsing by Author "Reutzel, Bryan"

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    Using CRISPR to Model MCT1 Deficiency in Pluripotent Stem Cells.
    (2024) Reutzel, Bryan
    Monocarboxylate transporter 1 (MCT1) plays a key role in transporting monocarboxylates such as lactate, pyruvate, and ketone bodies across the neurovascular unit/blood brain barrier (NVU/BBB). Human MCT1 is a highly conserved 500 amino acid protein embedded in the plasma membrane which contains twelve transmembrane segments with its amino and carboxyl termini in the cytoplasm (Figure). It acts as a facilitative carrier that transports a monocarboxylate and H + in equimolar amounts down a concentration gradient. Some carboxylate drugs may enter the brain via MCT1 and because of its role in metabolism, it has become the target for transport inhibitors (α-cyano-4-hydroxycinnamate, MD-1, AZD3965). Despite the important function of MCT1 in moving metabolically relevant substrates, there have been reports of mutations in the SLC16A1 gene, which codes for MCT1, and render the protein product dysfunctional. Patients with these mutations present with metabolic ketoacidosis that sometimes include seizures, vomiting, and mild to moderate developmental delay. There are currently no in vitro models of MCT1 deficiency in human cells. We hypothesized that creating a cell line of human stem cells in which the SLC16A1 gene is altered would be useful for characterizing the role of MCT1 in the metabolism of cells of the NVU. We therefore used CRISPR/Cas9 editing of human induced pluripotent stem cells (iPSCs). We were able to isolate a heterozygous KO cell line, and were further able to use the isolated line to attempt to create a full/homozygous knockout. The characterization of the resulting cell lines of this study will provide insight into how this pathology may affect the NVU, as well as provide a model system to further investigate this genetic disease.
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    Vascularization of Glioblastoma: Role of Cancer Stem Cells (2022-04-08)
    (2022) Reutzel, Bryan; University of Minnesota Duluth. Department of Chemistry and Biochemistry
    Glioblastoma (GBM) is the most common primary brain tumor in humans, but even with the best treatment currently available, the prognosis is dismal. This difficulty in treating GBM is multifactorial, owing to both properties of brain tissue, as well as properties specific to GBM. One property of GBM that is of particular interest is the presence of cancer stem cells, which are thought to play a role in tumor recurrence and chemotherapy resistance. These stem cells are also thought to play a role in alternative tumor vascularization processes by adopting an endothelial cell-like phenotype to enhance tumor growth. This cancer stem cell to endothelial transition is the subject of the present research. My hypothesis is that an established protocol to differentiate pluripotent stem cells into endothelial-like cells will induce a similar transition in the cancer stem cells. To test this, mouse glioma line GL261 cells were treated with growth factors as described in established protocols, and messenger RNA (mRNA) was extracted. This mRNA was reverse transcribed to complementary DNA (cDNA) for detection of endothelial- specific transcripts by quantitative PCR (qPCR) analysis. qPCR probes for endothelial markers Claudin 5 and P-glycoprotein were used to measure changes in expression relative to cells grown without growth factors. If the increased expression is observed in mouse cells, a future experiment using human GBM samples is planned using probes for additional endothelial marker genes.