279081 Alginate Encapsulation of Human Embryonic Stem Cells: Promising Platform for Pancreatic Maturation
Type 1 diabetes is an autoimmune disease affecting people worldwide wherein insulin producing beta cells of the pancreas are destroyed resulting in insulin dependence. The Edmonton Protocol of pancreatic islet transplantation affords the feasibility for treatment of diabetes. However shortage of donor islets combined with immune rejection problems limits islet transplantation from becoming a viable therapy. We propose alginate encapsulated insulin producing cells derived from human embryonic stem cells (hESC)/ induced pluripotent cells (iPSC) to be a viable therapy for type 1 diabetes, due to the virtually unlimited supply of hESCs/ iPSCs and the immunoisolation capability of alginate capsules.
Confluent H1 hESC colonies were treated with 10uM of the ROCK inhibitor Y-27632 for 2 h followed by Accutase treatment to dissociate the cells. Single cell hESC suspension in 1.1% Ca alginate and 0.2% gelatin were encapsulated by drop wise addition via a 21 guage syringe into a bath of 100mM CaCl2 containing 10mM HEPES. Either undifferentiated hESCs or cells pre-differentiated to definitive endoderm were used prior to encapsulation. Encapsulated cells were further matured towards the pancreatic islet cell types following a directed differentiation protocol. Viability and proliferation analysis was conducted using the LIVE/DEAD assay and AlamarBlue at each stage of differentiation. Definitive endoderm was induced with 100 ng/ml Activin A and 25 ng/ml Wnt3A for 4 days followed by pancreatic progenitor induction with 0.2 uM Cyclopamine and 0.2uM Cyclopamine with 2uM Retanoic Acid for 2 days each respectively. Maturation was induced by 10uM Nicotinamide for 2 days and 10uM Nicotinamide with 30uM DAPT for 7 days. Islet maturation was analyzed by gene expression levels of Insulin, MAFA, Glucagon, and PDX1. The differentiated population was further characterized by immunostaining, flowcytometry, protein content and release for relevant islet-specific proteins.
Our preliminary results indicate the potential of pancreatic maturation of encapsulated hESCs. Encapsulated single cells remain viable and grow into colonies towards the end of the differentiation protocol. Comparison of encapsulated single cell versus colony encapsulation clearly demonstrated the higher differentiation potential of the single cell configuration. While significant dead cells were observed after initial encapsulation, dead cells were less prominent towards the end of the protocol when small colonies of viable differentiated cells become dominant. Each stage of differentiation was confirmed by gene and protein analysis of the relevant markers. Very encouragingly, many of the differentiation markers analyzed were even stronger than parallel control cultures in conventional tissue culture plates. For example, very strong insulin expression thousands of folds higher than the control culture was observed at the end of the protocol. It is also worth mentioning that the control cultures themselves exhibit high levels of insulin protein content.
Our results to date indicate that islet-specific maturation of hECSs is not only feasible in the encapsulated 3D alginate capsule system but also such a configuration is significantly enhancing the maturation of hESCs. Further studies are currently underway to understand the mechanism contributing to such a noticeable enhancement. Furthermore, plans are in place to test the in-vivo functionality of these encapsulated hESC derived islet cells upon implantation into diabetic mice.
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