(489e) Endothelial Cells Mediate Maturation of Human Embryonic Stem Cell Dervied Pancreatic Progenitors Into Insulin Expressing Cells

Diabetes and its complications affect 25.8 million people in the US and is a growing healthcare problem worldwide.  While transplantation of pancreatic islets offer potential therapy, it is restricted by scarcity of donor islets. Effective use of hESC could overcome this limitation, however current approaches to differentiating hESCs to mature islets have been restricted in yield and functionality of the differentiated phenotype. In our work we propose to mimic in-vivo development during maturation of hESC derived pancreatic progenitor cells. Over past years developmental studies have established the critical involvement of endothelial cells in maturation and functionality of pancreatic islets, which is not surprising given the islet’s dense vasculature.

We have developed a multi-stage protocol for directed differentiation of hESCs involving definitive endoderm induction, followed by pancreatic progenitor specification and final maturation into insulin producing cells. For the last step, we propose to co-culture the hESC-derived pancreatic progenitor cells with endothelial cells in accordance with developmental studies. We used two different cell types in our studies: Rat Heart Microvascular Endothelial Cells (RHMVEC) and Human Umbilical Vein Endothelial Cells (HUVEC). As a control experiment we co-cultured the pancreatic progenitor cells with NIH3T3 fibroblast to verify sensitivity to cell types. All direct contact co-cultures were FACS sorted and analyzed for gene and protein expression levels. Two different non-contact co-culture configurations were also investigated: transwell co-culture and endothelial cell conditioned media. Results were evaluated with respect to conventional maturation using notch inhibition by DAPT.

We evaluated each stage of differentiation using qRT-PCR, immunostaining and flow cytometry. Our protocol resulted in 89% Foxa2 positive endoderm and high, uniform nuclear staining for Pdx-1 at pancreatic progenitor stage with roughly 70% of the cells being Pdx1 positive as observed by flow cytometry. Maturation with DAPT resulted in 17,500 fold upregulation of insulin mRNA with 8% commitment, while parallel RHMVEC co-culture resulted in 20,000 fold upregulation of insulin mRNA with 34% commitment. HUVEC co-culture resulted in an even higher insulin expression, 7 times higher than RHMVEC. Control cultures with fibroblast did not show appreciable insulin upregulation, indicating the effect to be specific to endothelial cells. Alternative co-culture configurations also show promising results, with transwell dominating over conditioned media, but still lower than direct contact configuration.

Our results establish an alternate strategy for hESC maturation into islet cell types. Current investigation is underway to elucidate the mechanism of endothelial cell mediated maturation.