Date of Graduation

7-2015

Document Type

Thesis

Degree Name

Master of Science in Biomedical Engineering (MSBME)

Degree Level

Graduate

Department

Biomedical Engineering

Advisor/Mentor

Ye, Kaiming

Committee Member

Jin, Sha

Second Committee Member

Beitle, Robert R. Jr.

Keywords

Biological sciences; Applied sciences

Abstract

Embryoid body (EB) formation is a common first step in many human pluripotent stem cell (hPSC) differentiation protocols. Previous work suggests that EBs are sensitive to growth factor withdrawal if they are derived from hPSCs maintained in feeder independent media such as mTeSR1. To promote cell survival, EBs generated from mTeSR1-adapted hPSCs are sometimes cultured in a medium that contains basic fibroblast growth factor (bFGF), a trophic factor often used in hPSC cultures to maintain self-renewal. This distinguishes feeder independent hPSCs from feeder dependent hPSCs. The purpose of this study was to characterize the effects of bFGF as well as small molecule inhibitors of GSK3β on the formation, growth, cell-cell signaling and early fate decisions of human EBs. bFGF or GSK3β inhibitor treated EBs grew in size and showed evidence of cell proliferation. Control EBs that were fed only a basal serum free EB medium without added growth factors tended to fall apart in culture, adopted irregular morphologies and showed no signs of growth or proliferation. Changes in cell-cell adhesion commonly observed in differentiating hPSCs were apparent in the bFGF and BIO treated EBs, while untreated control EBs predominantly expressed E-cadherin, a cell-cell adhesion molecule highly expressed in undifferentiated hPSCs. Expression of pluripotency markers decreased in all EB groups, regardless of treatment, and increased germ layer marker expression was detected in all groups, with a particular bias towards neuroectoderm. Our results suggest a role for GSK3β in the development and survival of EBs derived from feeder independent hPSCs. These results could inform the development of protocols for the directed differentiation of feeder independent hPSCs using defined media and small molecules.

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