Date of Graduation

5-2013

Document Type

Thesis

Degree Name

Bachelor of Science in Biological Engineering

Degree Level

Undergraduate

Department

Biological and Agricultural Engineering

Advisor/Mentor

Jin, Sha

Committee Member/Reader

Carrier, Danielle Julie, 1959-

Committee Member/Second Reader

Griffis, Carl L.

Abstract

Current research regarding stem cells is the premise behind this project. These cells have the potential to develop into any kind of cell in the human body. Due to the scarcity of transplantable pancreas and/or pancreatic beta cells, the production of pancreatic insulin-producing cells from human embryonic stem cells (hESCs) has emerged as one of the most attractive cell therapy strategies for insulin deficient diabetes treatment. The aspect of hESC research that the project focused on was the environment or foundation the hESCs grew in. A key step to pancreatic beta cell production from hESC differentiation or growth is establishing a scaffold or three-dimensional (3D) cell culture system to provide cell suitable microenvironment. Extracellular matrix (ECM) proteins in this 3D microenvironment play a central role in controlling cell growth into the targeted cell needed. Collagen I (Col I), fibronectin (FN), laminin (LM), and vitronectin (VN) are some of many ECM proteins involved in pancreatic beta cell production. In this project, we investigated the effect of different combination of ECM proteins on pancreatic beta cell production from stem cell differentiation. Previous lab studies have already established ECM protein concentrations as: Col I (mg/mL), FN (25 µg/mL), LM(25 µg/mL), and VN(5 µg/mL). The purpose of this research was to see if there was any deviation from this previous set of concentrations. Collagen I is the major structural protein for many scaffolds so this concentration was kept constant throughout all tests. The project split into three phases depending on the ECM protein being tested. Phase I tested FN at varying concentrations. Phase II tested the best FN concentration in the previous phase with varying LM concentrations. Phase III test the best FN and LM concentrations from the previous phases with varying VN concentrations. Three tasks were performed for each phase. 1) Make varied 3D scaffolds by adjusting the composition of ECM proteins and their concentrations; 2) Induce differentiation in 3D gels made from said composition of ECM proteins; 3) Characterize the lineage specification efficacy with varied biological analyses, including live/dead cell stain to detect cell growth/viability, immunofluorescence staining to visualize marker protein expression, and quantitative real-time PCR to detect marker gene mRNA expression. After experimentation and analysis, there will be one combination that can provide maximum production of beta cells from hESCs.

Keywords

stem cell research, genetics

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