(453c) Effect of Early Endoderm Induction On Late Stage Pancreatic Maturation of Differentiating Human Embryonic Stem Cells

Pancreatic differentiation of mouse and human embryonic stem cells has received considerable attention over the last decade. While there has been some success in deriving insulin positive cells from embryonic stem cells, typically the differentiated cells are limited in yield and functionality. Most of the successful differentiation protocols involve a step-wise directed differentiation strategy starting with definitive endoderm (DE) induction; followed by pancreatic progenitor induction and finally islet maturation. While extensive studies have established multiple pathways of endoderm induction, the effect of these pathways on late stage pancreatic maturation remains elusive. The aim of this study is to develop a thorough understanding of the effect of pathway of early endoderm induction on late stage pancreatic maturation.

The human embryonic stem cells were differentiated to mature insulin producing cells following a directed differentiation approach. The first stage of definitive endoderm was induced following four alternative pathways, based on most promising reports by different groups. Namely, we used Activin A in combination with one of the following molecules and growth factors: Fgf2, Bmp4, Wnt3a or PI3K Inhibitor. DE derivatives obtained from these four pathways were subjected to same maturation protocol: pancreatic progenitor induction using sonic hedgehog inhibition and retinoid signaling; islet maturation through notch inhibition. The differentiation potential of each of the endoderm derivatives were compared at different stages of differentiation.

Our results clearly indicate that late stage pancreatic maturation is distinctly governed by the initial pathway of endoderm commitment. From the results obtained from 6 independent studies, we found that DE induction by treatment with PI3KI resulted in at least two times higher upregulation of DE markers than any other conditions; however, at later stages of differentiation these cells obtained a moderate 800-fold upregulation of insulin. Conversely, cells induced towards DE by treatment with Wnt3a and Fgf2 showed between 10,000 and 20,000 fold upregulation of insulin. Consistent with PCR analysis, Wnt3a led to highest yield of C-peptide expressing cells with around 25% positive cells. Interestingly, while the Bmp4 derived DE cells resulted in only about 2-fold increase in insulin expression, they showed the highest upregulation of glucagon: around 4,000 times higher than undifferentiated cells, leading to the conclusion that inducing DE with Bmp4 leads to more alpha cells than beta cells. Detailed analysis using computational methods showed Wnt3a and Fgf2 cultures to show high similarity in gene patterning, and Wnt3a to follow closer resemblance to pancreatic organogenesis in respect to pancreatic marker progression.

We conclude that using Activin A in combination with Wnt3a at the definitive endoderm stage leads to the highest insulin upregulation as well as higher yield of C-peptide expressing cells. Moreover, our analysis shows that mature cell type specification is affected by early differentiation events. Namely our results suggest that while Wnt3a derivatives differentiate into beta-like cells, the use of Bmp4 preferentially directs cells toward alpha cell commitment. To the best of our knowledge, no other study compares methods of definitive endoderm induction and their effect at later stages of development. This study is important since it provides a better understanding of pathways involved in early events of pancreatic differentiation and allows for standardization of these stages in order to achieve better yield and functionality of differentiated human embryonic stem cells