Date of Graduation

8-2014

Document Type

Dissertation

Degree Name

Doctor of Philosophy in Cell & Molecular Biology (PhD)

Degree Level

Graduate

Department

Cell & Molecular Biology

Advisor/Mentor

Ye, Kaiming

Committee Member

Jin, Sha

Second Committee Member

Rhoads, Douglas D.

Third Committee Member

Wejinya, Uche C.

Fourth Committee Member

Hestekin, Christa N.

Keywords

3D; Differentiation; Embryonic stem cells; Scaffold

Abstract

The potential of human embryonic stem cells (hESCs) to differentiate into insulin producing beta cells offers great hope for cell-based therapy for diabetes treatment. However, in vitro pancreatic differentiation of hESCs remains challenging. In the past decade, most protocols for differentiating pancreatic cells have been focused on the use of signaling molecule cocktails on 2D substrates. Studies on embryonic development biology strongly suggest that extracellular matrix (ECM) plays a critical role on hESCs behavior. In this work, we first established a 3D collagen scaffold culture system for hESCs differentiating into definitive endoderm (DE), which is the first and most important step for coaxing hESCs into transplantable beta cells. Collagen scaffold have shown to promote the attachment, proliferation and DE differentiation of hESCs in 3D microenvironment. Furthermore, we optimized the 3D scaffold compositions by integrating various ECM proteins into collagen scaffold. Our data showed that compared to collagen with single ECM protein, collagen combined with fibronectin, laminin, and vitronectin can greatly enhance DE differentiation generating up to 93% SOX17 positive DE population. Finally, we demonstrated that mature insulin-producing cells can be achieved by differentiating hESCs in 3D biomimic scaffold made of collagen and Matrigel, combined with a modified step-wise protocol. More mature insulin-producing cells were generated with our 3D scaffold compared to traditional 2D culture. The 3D differentiated pancreatic endocrine cells were assembled into tissue-like structure and displayed greater similarities in phenotype and gene expression profile to adult human islets. These hESC-induced pancreatic cells comprised 20% insulin positive cells in 3D culture and can response to high glucose and release four-fold insulin as compared to 2D culture. Insulin-secreting granules were also observed, which confirmed the generation of insulin-producing cells in 3D scaffold.

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