474170 Alginate Capsule Composition Influences the Pancreatic Differentiation of Human Embryonic Stem Cells
Materials and Methods: A single cell suspension of ROCK inhibitor treated hESCs were suspended in 1.1% alginate and 0.2% gelatin followed by encapsulation by drop wise addition into a divalent cation bath. hESCs were encapsulated in barium alginate, crosslinked with 10, 15, 20, 50 or 100 mM BaCl2. The hESCs were differentiated entirely under encapsulation and characterized after completion of pancreatic differentiation. Encapsulated hESCs were induced to the pancreatic islet like state following a stagewise differentiation protocol. Definitive endoderm (DE) was induced with ActivinA and Wnt3Afor 4 days followed by pancreatic progenitor induction with KAAD-cyclopamine and KAAD-cyclopamine with retinoic acid for 2 days each, respectively. DE stage differentiation was characterized by gene and protein expression of SOX17, FOXA2, EOMES, and CER. Pancreatic commitment was analyzed by PDX1 gene and protein expression.
Results: Increasing the concentration of BaCl2 from 10 to 100 mM BaCl2 resulted in an increase in substrate stiffness from 4 to 75 kPA, as determined by AFM analysis. Viability and proliferation of encapsulated hESCs were very sensitive to capsules stiffness, with only 4-7 kPa capsules supporting cell growth and colony formation. Diffusional differences were confirmed to be minimal under the tested conditions. DE stage differentiation was found to be enhanced as capsule stiffness was increased, as shown by both gene and protein analysis of SOX17, FOXA2, EOMES, and CER. Pancreatic commitment was analyzed by PDX1 gene and protein expression. PDX1 gene expression was highest in the 4 kPa stiffness gel (10 mM BaCl2), and was significantly reduced as stiffness increased. The same trend was observed with PDX1 protein expression, which showed the highest number of cells positive for PDX1 in the 10 mM BaCl2 condition.
Discussion: Our study demonstrates that capsule materials properties can significantly modulate the efficiency of chemically induced pancreatic differentiation. Specific encapsulation conditions can significantly enhance the efficiency of chemical induction. In general, higher crosslinking was found to be detrimental to the viability and differentiation of hESCs. Research is currently underway for encapsulating hESCs in a high throughput alginate array platform. This will allow for a more thorough investigation of the effect of substrates stiffness on differentiation, as well as delineate the signaling pathways which govern pancreatic differentiation. Results of these studies will be presented and discussed.
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