Browsing by Author "Friguglietti, Jefferson"
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Item Mico Patterned Substrates for Differentiating Mesenchymal Stem Cells into Insulin Producing Cells(2017) Friguglietti, Jefferson; Ngo, Michelle; Deneke, Ruth; Le, Phi; Fraga, Daniel; Merchant, Fatima AzizType 1 Diabetes is a global epidemic on the rise, effecting nearly 1.25 million Americans and rising. Currently the only therapeutic option for patients besides traditional insulin shots is transplantation of donor islet which carries insulin producing beta cells. However, this procedure requires lifelong immunosuppressant drugs and large quantity of donor cells. The lack of viable donor islets for patient transplantation makes alternative cell based therapy vital for future patients. Mesenchymal stem cells (MSCs) derived from patient?s bone marrow has been shown to be a potential therapeutic cell line for various diseases including Diabetes. Aggregating stem cells before differentiation plays a crucial role in increasing the secretion of a wide array of therapeutic factors and protecting its multipotent potential.Item Micro-Patterned Substrates for Differentiating Mesenchymal Stem Cells into Insulin Producing Cells(2017-12) Friguglietti, Jefferson; Merchant, Fatima Aziz; Zagozdzon-Wosik, Wanda; Balan, Venkatesh; Ganapathy, Sivakumar; Flavier, Albert B.; Zouridakis, GeorgeConventional insulin therapy for Type 1 diabetes mellitus is often accompanied by long-term complications such as heart disease and kidney damage, if patients do not follow a very strict and controlled regime of taking insulin shots. Transplantation of pancreatic islets is a therapeutic option available for Type 1 Diabetes, where in donor islets are transplanted into patients for controlling glucose levels without the need of insulin shots. Although the current islet transplantation Edmonton protocol has made progress in successfully treating diabetic patients, a lack of viable donor cells and side effects associated with immunosuppressant drugs make alternative therapeutic options critical. Cell replacement therapy via differentiation of adult stem cells into glucose-responsive insulin producing cells (IPCs) has recently provided hope for Type I diabetes. However, inadequate functional performance of the differentiated cells with poor long-term insulin production has slowed further progress. Thus, there is a critical need for improving the total yield of differentiated cells and their functional performance. In this study we investigated the potential of a novel substrate of micro-patterned Titanium diboride (TiB2) on Silicon (Si) wafers for culturing adult human bone marrow mesenchymal stem cells (hBM-MSCs) and differentiating them into insulin producing cells (IPCs). The hypothesis is that these substrates enable formation of aggregates, thereby enabling a 3D micro-environment for differentiation. Stereomicroscopy showed MSCs preference for TiB2 patterns over Si and the formation of uniform aggregates only on the TiB2 after the differentiation protocol. Moreover, MSCs not only remained at 80% or more viable when aggregated, but phenotyping analysis for the presence of biomarker CD105 demonstrated conserved multi-lineage potential throughout the 9 day pre-differentiation incubation period. More importantly, our results suggest a 2-3 fold increase of insulin secretion from MSCs differentiated on the micro-patterned substrates when compared against differentiation in conventional tissue culture flasks.Item NOVEL 3D CELL CULTURE TECHNIQUE UTILIZING A SILICON TITANIUM DIBORIDE MICROPATTERNED SUBSTRATE FOR DIFFERENTIATING MESENCHYMAL STEM CELLS INTO INSULIN PRODUCING CELLS(2023-12) Friguglietti, Jefferson; Merchant, Fatima Aziz; Akay, Yasemin M.; Wu, Tianfu; Al-Ubaidi, Muayyad R.; Sabek, Omaima3D cell culture techniques are increasingly used in stem cell tissue engineering since they better mimic the in vivo environments compared to conventional 2D culture. Current 3D culture methods include less complex suspension methods such as hanging well and ultra-low attachment plate, to more advance methods incorporating scaffold designs to support cell-cell and cell-extracellular matrix interaction (ECM). In this study we investigate a novel microfabricated silicon-titanium diboride (Si-TiB2) substrate’s efficacy in increasing function of differentiated mesenchymal stem cells (MSCs) into insulin producing cells (IPCs). This substrate provides a 3D microenvironment (aggregates) and geometric (pattern shape), mechanical (stiffness gradients) and biochemical (selectively adsorbed proteins) cues that are critical for cell differentiation. Differentiated IPCs are essential, providing an alternative tissue bank for future transplantation into diabetic patients. Utilizing photolithography technique, e-beam deposited TiB2 layers were fabricated on Si, wherein differences in their surface properties (hardness, stiffness, wetness, and electrical charge), enable selective adsorption of specific proteins on the micropatterns. MSCs from adult bone marrow were cultured on the substrate, and following a seven-day culture period subjected to a differentiation protocol. Functional analysis included quantification of c-peptide following a glucose stimulated insulin response (GSIR) assay. Morphological analysis of the cytoskeleton through immunofluorescence staining (f-actin, green) revealed increased rounded morphology of cells within the multicellular aggregates confirming a 3D culture environment on the SiTiB2 substrate. Additionally, immunofluorescence staining for biomarkers (n - cadherin, red) showed increased expression within the rounded core indicating cell-cell interaction with the aggregates. Further BioAFM analysis indicated cells with higher elastic modulus in the center of aggregates compared to cells on the edges of the micropatterns (p < 0.05). GSIR assay showed MSCs differentiated on the Si-TiB2 substrate had a better response to high glucose stimulation when compared to those differentiated in 2D monolayers in tissue culture plates (p < 0.05). Finally, co – culture with HUVEC and hBM-MSCs revealed unique formation of aggregation with HUVEC located primarily in the center of the aggregate. The TiB2 substrate provides a unique culture platform to better understand differentiation of MSCs into insulin producing cells when compared to ULP and traditional 2D flask.