Date of Graduation

5-2012

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

Costello, Thomas A.

Committee Member/Second Reader

Wochok, Jeffrey C.

Abstract

Embryonic stem cells have the characteristic of self-renewal and the ability to differentiate into tissues from all three germ layers. Many times, the first step in using human embryonic stem cells for regenerative medicine is often initiated by embryoid body (EB) formation. EBs are three-dimensional multicellular aggregates that resemble early post implantation embryos that still maintain the potential to form the three germ layers. Currently, generating EBs from human embryonic stem cells (hESC) that are adapted to feeder-independent medium is difficult, much more so than feeder cultured hESC. Using a chemically defined medium would not only help the application of hESC in research and therapy but also provide the possibility studying the molecular mechanisms of self-renewal and differentiation, without the use of feeder cells. Three comparisons have been done to compare culture conditions: the type of medium, the dosage of bFGF and pretreatment with conditioned MEF medium. By varying the culture conditions, EB formation can be optimized for a based on average total EB count and morphology. The EBs were formed and cultured for 2 weeks in suspension. The effectiveness of completely defined EB medium was compared to commercially available AggreWell medium and proved to be sufficient in forming and maintaining EBs throughout the culture period. Feeder-free and feeder-independent conditions were also compared by conditioning hESC in mouse embryonic fibroblast (MEF) conditioned medium (CM) for 0, 1, 2 or 3 days. Feeder-free culture conditions after 3 days of conditioning showed optimal EB growth. Different concentrations of basic fibroblast growth factor (bFGF) in the EB medium were compared and determined to encourage optimal growth at 40 ng/mL bFGF. Selected EBs were taken up, and quantitative real time PCR amplification was performed to verify the presence of the three germ layers.

Keywords

Human embryonic stem cells; embryoid body; defined medium; basic fibroblast growth factor; feeder-free; feeder-independent

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