Date of Graduation


Document Type


Degree Name

Doctor of Philosophy in Cell & Molecular Biology (PhD)

Degree Level



Biomedical Engineering


Kaiming Ye

Committee Member

Sha Jin

Second Committee Member

Douglas Rhoads

Third Committee Member

Uchechukwu Wejinya

Fourth Committee Member

Christa Hestekin


3D, Differentiation, Embryonic Stem Cells, Scaffold


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.