Response of Adipose Tissue Derived Stem Cells to 5-azacytidine and Zebularine Treatment, Culturing in Methylcellulose-based Medium and Matrix Elasticity
Author
Petersen, Morten
Term
4. term
Publication year
2009
Pages
76
Abstract
This thesis examines whether human adipose-derived stem cells (ASCs) can be directed toward the cardiomyogenic lineage using three literature-based strategies: (1) treatment with the cytidine analogues 5-azacytidine and zebularine added to standard growth medium, (2) culture in a methylcellulose-based medium to probe spontaneous differentiation, and (3) culture on substrates with controlled elasticity, produced as polyacrylamide gels crosslinked with collagen type I or fibronectin at stiffness levels designed to mimic neural, muscle, and bone tissue. Cell morphology was monitored by phase-contrast microscopy, early cardiac marker expression was quantified by real-time PCR, and gel substrates were produced and characterized to ensure relevant elastic properties. The results show that neither 5-azacytidine nor zebularine treatment, nor the methylcellulose-based medium, induced differentiation of ASCs into cardiomyocyte-like cells. Collagen- and fibronectin-coated polyacrylamide gels could be fabricated, but ASCs did not proliferate on these substrates, indicating that further optimization of the fabrication process is needed and limiting assessment of matrix elasticity effects. Overall, the findings suggest that the tested protocols are insufficient to trigger cardiomyogenic differentiation of ASCs under the conditions used, and that enhanced materials and culture conditions are required.
Denne opgave undersøger, om humane fedtvævsafledte stamceller (ASCs) kan styres mod kardiomyogen differentiering ved tre tilgange udledt af litteraturen: (1) behandling med de to cytidinanaloger 5-azacytidin og zebularin tilsat standard vækstmedium, (2) dyrkning i et methylcellulose-baseret medium for at afprøve spontan differentiering, og (3) dyrkning på substrater med kontrolleret elasticitet, fremstillet som polyacrylamidgeler tværbundet med kollagen type I eller fibronectin med stivheder designet til at efterligne nerve-, muskel- og knoglevæv. Cellemorfologi blev fulgt med fasekontrastmikroskopi, og udtryk af tidlige hjertemarkører blev kvantificeret med real-time PCR; de mekaniske egenskaber af gelerne blev fremstillet og karakteriseret for at sikre relevante elasticiteter. Resultaterne viste, at hverken 5-azacytidin- eller zebularinbehandling, og heller ikke dyrkning i methylcellulose-baseret medium, inducerede differentiering af ASCs til hjertemuskel-lignende celler. Det var muligt at fremstille kollagen- og fibronectinbelagte polyacrylamidgeler, men ASCs kunne ikke proliferere på disse underlag, hvilket kræver yderligere procesoptimering og begrænsede vurderingen af matrixelasticitetens effekt. Samlet peger arbejdet på, at de afprøvede protokoller ikke er tilstrækkelige til at udløse kardiomyogen differentiering af ASCs under de testede betingelser, og at der er behov for forbedringer af både materialer og kulturforhold.
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