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Stem cell engineering of the endoderm: approaches to controlling endoderm induction and differentiation from embryonic stem cells
Descriptive
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Title
Stem cell engineering of the endoderm: approaches to controlling endoderm induction and differentiation from embryonic stem cells
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Title
Approaches to controlling endoderm induction and differentiation from embryonic stem cells
Name
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Parashurama
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Natesh
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Natesh Parashurama
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author
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Yarmush
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Martin
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Advisory Committee
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Martin Leon Yarmush
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chair
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Roth
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Charles
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Advisory Committee
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Charles Michael Roth
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internal member
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Pedersen
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Henrik
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Advisory Committee
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Henrik Pedersen
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internal member
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Berthiaume
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Francois
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Advisory Committee
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Francois Berthiaume
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(authority = RULIB)
outside member
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Tilles
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Arno
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Advisory Committee
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Arno Tilles
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outside member
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Rutgers University
Role
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degree grantor
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Graduate School - New Brunswick
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Text
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theses
OriginInfo
DateCreated
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2008
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2008-01
Language
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English
PhysicalDescription
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electronic
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application/pdf
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text/xml
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xiii, 179 pages
Abstract
Embryonic 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.
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.
Note
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Ph.D.
Note
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Includes bibliographical references.
Subject
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Topic
Chemical and Biochemical Engineering
Subject
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Topic
Stem cells
Subject
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Topic
Embryonic stem cells--Differentiation
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Graduate School - New Brunswick Electronic Theses and Dissertations
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rucore19991600001
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http://hdl.rutgers.edu/1782.2/rucore10001600001.ETD.17190
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ETD_718
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NjNbRU
Identifier
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doi:10.7282/T3RR1ZMJ
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ETD doctoral
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Rights
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The author owns the copyright to this work.
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Copyright protected
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Open
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Name
Natesh Parashurama
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Copyright holder
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Rutgers University. Graduate School - New Brunswick
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Non-exclusive ETD license
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Author Agreement License
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I hereby grant to the Rutgers University Libraries and to my school the non-exclusive right to archive, reproduce and distribute my thesis or dissertation, in whole or in part, and/or my abstract, in whole or in part, in and from an electronic format, subject to the release date subsequently stipulated in this submittal form and approved by my school. I represent and stipulate that the thesis or dissertation and its abstract are my original work, that they do not infringe or violate any rights of others, and that I make these grants as the sole owner of the rights to my thesis or dissertation and its abstract. I represent that I have obtained written permissions, when necessary, from the owner(s) of each third party copyrighted matter to be included in my thesis or dissertation and will supply copies of such upon request by my school. I acknowledge that RU ETD and my school will not distribute my thesis or dissertation or its abstract if, in their reasonable judgment, they believe all such rights have not been secured. I acknowledge that I retain ownership rights to the copyright of my work. I also retain the right to use all or part of this thesis or dissertation in future works, such as articles or books.
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