(453b) Alginate Encapsulation of Human Embryonic Stem Cells Enhances Its Directed Differentiation to Pancreatic Islet-Like Cells

Introduction: Type 1 diabetes (T1D) is an autoimmune disease affecting myriadpeople worldwide whereinthe beta cells ofthe pancreasare destroyed resulting in insulin dependence. The Edmonton Protocol of islet transplantation affords the feasible option for treatment of diabetes. However shortage of donor islets combined with immune rejection limits islet transplantation from becoming a viable therapy. We propose alginate encapsulation of insulin producing cells derived from human embryonic stem cells (hESC) is a potentially viable therapy for type 1 diabetes, due to the unlimited supply of hESCs and the immunoisolation capability of alginate capsules.

Materials and Methods: We developed a stage wise directed differentiation protocol to derive islet-like cells from hESCs. Definitive endoderm was induced with ActivinA and Wnt3A for 4 days followed by pancreatic progenitor induction with Cyclopamine and Cyclopamine with Retinoic Acid for 2 days each; respectively. Maturation was induced by Nicotinamide for 2 days and Nicotinamide with DAPT for 7 days.  A single cell suspension of ROCK inhibitor treated hESCs weresuspended in 1.1% alginate and 0.2% gelatin followed by subsequent encapsulation by drop wise addition into a 100mM CaCl₂ bath with 10mM HEPES. hESCs were encapsulated at 3 different stages: after maturation to islet-like cells, the definitive endoderm (DE) stage or as undifferentiated (UD) hESCs. The hESCs were then continued with remaining differentiation protocol based on the encapsulation stage. Islet maturation was analyzed by gene expression of insulin, MAFA, glucagon, and PDX1. Further characterization was done by immunostaining, flow cytometry, and protein content of islet-specific markers.  Additionally, the gene expression of extracellular matrix (ECM) and cell adhesion molecules (CAM) was analyzed by qRT-PCR array.  Finally, key molecules of the TGFβ and WNT pathway were analyzed at the DE stage to further investigate the effect of alginate encapsulation on hESC differentiation.

Results: Our results to date show that encapsulation of fully matured cells result in low viability, and significantly lower gene expression of mature markers compared to tissue culture plastic controls (TCP), prior to encapsulation. However, when hESC derived DE cells were encapsulated and further induced to islet like cells; the cells exhibited strong maturation markers significantly higher than TCP controls. The viability of the encapsulated cells, however, still remained low. Finally, we evaluated the encapsulation of UD hESCs and allowed them to progress through all stages of differentiation under encapsulation.  The encapsulated UD hESCs remained viable and grew into large colonies towards the end of the differentiation protocol. Very encouragingly, many of the differentiation markers analyzed were even stronger when cells were encapsulated in contrast to TCP controls. To further analyze the maturation of hESCs fully differentiated under encapsulation, we measured the intracellular protein content of the islet-specific hormone c-peptide.  The encapsulated cells showed significantly higher c-peptide (0.28pg c-pep /ug total-protein) than TCP controls (0.085pgc-pep/ug total-protein). Upon glucose challenge, the hESCs fully matured under encapsulation secreting approximately 19 pg/ml of c-peptide into the media.  The ECM and CAM array was analyzed at the DE and PP stage for encapsulated cells, normalized to cells differentiated on TCP.  While most genes were down regulated, NCAM1 and VCAM1 were upregulated at the DE stage and MMP7, 15 were upregulated at the PP stage.  Investigation of the TGFβ pathway showed an increased ratio of pSMAD3/pAKT and pSMAD2/pAKT under encapsulation, compared to the TCP control. 

Discussion: Our results indicate that islet-specific maturation of hESCs is not only a feasible option in the 3D encapsulated alginate system but is alsoa configuration that appears to significantly enhance maturation of hESCs. This is enhanced maturation is likely due to higher levels of pSMAD3/pAKT under encapsulation. C-peptide secretion upon glucose stimulation shows that the islet-like cells are able to fully cleave proinsulin into mature c-peptide and insulin for secretion. Further studies are currently underway to adapt this protocol to microencapsulated cells to further mature and test the in-vivo functionality of these encapsulatedcells in diabetic mice.