Uniform TitleStem cell engineering of the endoderm: approaches to controlling endoderm induction and differentiation from embryonic stem cells
NameParashurama, Natesh (author), Yarmush, Martin (chair), Roth, Charles (internal member), Pedersen, Henrik (internal member), Berthiaume, Francois (outside member), Tilles, Arno (outside member), Rutgers University, Graduate School - New Brunswick,
SubjectChemical and Biochemical Engineering,
Embryonic stem cells--Differentiation
DescriptionEmbryonic stem (ES) cell technology holds promise for curing innumerable human ailments. However, studies of the endoderm germ layer and its derivatives (liver, pancreas, and lung) are lacking. The overall objective of this thesis is to elucidate the factors that influence endoderm induction and differentiation from ES cells. To improve results in aggregate culture, a microfabricated PDMS (polydimethylsiloxane) stencil was engineered using standard soft lithography techniques used to control ES cells. Precise control over initial aggregate size was obtained, varying the initial aggregate size from 100-500 µm. Analysis of the cells by RT-PCR (Reverse Transcriptase Polymerase Chain Reaction) demonstrated endoderm on Day 10 and hepatocyte-like cells on Day 20, but a mixed population was present. To further enhance endoderm induction, a coculture system was developed. The culture of ES cells on top of collagen-sandwiched mature rat hepatocytes, resulted in a rapid proliferation into a 95% positive endoderm progenitor population by Day 10. Late stage differentiation of these cells and placement in a extracorporeal device to support liver failure in rats resulted in enhanced (50%) survival.
To further understand endoderm induction, a simpler culture system was developed. The culture of ES cells on fibronectin-coated collagen gels resulted in an endoderm fraction of 53% by Day 10 that remained committed upon in vivo implantation. Treatment with activin, an important TGFβ superfamily soluble factor, caused an 80% decrease in the endoderm fraction, while follistatin, an activin inhibitor with unknown function, increased the endoderm fraction to 78%. The activin treated population delayed the induction of endoderm by preventing differentiation of its transient precursors, the epiblast and mesendoderm. Differentiation of activin treated cells to Day 24 resulted in a two-fold reduction in hepatic gene expression and three-fold reduction in hepatic protein expression of when compared to follistatin-treated cells. Subcutaneous transplantation of activin-treated cells resulted in generated a heterogeneous teratoma-like mass, suggesting these cells were primitive. In summary, factors that influence endoderm induction include initial size of aggregate, coculture environment, extracellular matrix, and soluble factors. Two new tools for evaluating clinical applications include in vivo implantation and placement in an extracorporeal device.
NoteIncludes bibliographical references.
CollectionGraduate School - New Brunswick Electronic Theses and Dissertations
Organization NameRutgers, The State University of New Jersey
RightsThe author owns the copyright to this work.