Defense Date

2010

Document Type

Dissertation

Degree Name

Doctor of Philosophy

Department

Chemical Engineering

First Advisor

Raj Rao

Abstract

In human pluripotent stem cell (hPSC) research and applications, the need for a culture system devoid of non-human components is crucial. Such a system should exhibit characteristics observed in conventional culture systems that have used mouse embryonic fibroblast feeders for hPSC self renewal without the requirement of excessive supplementation with growth factors. To achieve this, we focused on the identification and characterization of extracellular matrix (ECM) substrates for hPSC propagation. ECM substrates derived from mouse and human fibroblasts were assessed for their ability to support self-renewal of hPSCs. Characterization of hPSCs on ECM-based substrates demonstrated maintenance of pluripotent characteristics based on a) high nuclear-cytoplasmic ratio b) immunocytochemical analyses for pluripotent markers (Alkaline phosphatase, AP, Octamer Binding Transcription Factor-4, OCT4 and Specific surface embryonic antigen-4, SSEA4) c) in vitro differentiation potential by embryoid body formation d) Real time RT-PCR analysis for pluripotent and germ-layer specific markers and e) karyotype analysis for chromosome number. Compositional characterization of the ECM substrates using proteomic analysis identified some of the major constituents of the matrix that might contribute to hPSC self-renewal. Based on results from the proteomic analysis, combinatorial ECM substrates were formulated using commercially available proteins and evaluated for applicability in hPSC propagation. Extensive characterization of hPSC propagated on the ECM substrates suggest that a combination of heparan sulfate proteoglycan and fibronectin was sufficient for the promoting hPSC sef-renewal. Finally, an in-direct co-culture system utilizing microporous membranes coated with acellular substrates and a physically separated feeder layer was developed as a microenvironment for hPSC propagation. Real time conditioning of the growth medium and an ECM-based substrate for hPSC adhesion provides a synergy of the biochemical and biophysical cues necessary for hPSC self-renewal. hPSCs cultured in this system demonstrated equivalent pluripotent characteristics as those propagated in conventional culture systems, and provided opportunities for scale up without cell mixing. Overall, these studies could prove to be useful in the development of humanized propagation systems for the production of stable hPSCs and its derivatives for research and therapeutic applications.

Rights

© The Author

Is Part Of

VCU University Archives

Is Part Of

VCU Theses and Dissertations

Date of Submission

May 2010

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