Document Type

Thesis

Date of Degree

Fall 2014

Degree Name

MS (Master of Science)

Degree In

Biomedical Engineering

First Advisor

Nicholas Zavazava

Abstract

Type 1 Diabetes (T1D) is an autoimmune disorder in which the pancreatic β-cells are destroyed by the body's immune system. The reduced number of β-cells leads to inadequate insulin secretion and high glucose levels in the body. The requirement of insulin injection throughout life and lack of donors for islet transplantations has prompted a search for more accessible and available sources of insulin producing cells that can be transplanted in T1D patients. To that end, the discovery of induced pluripotent stem (iPS) cells has provided a potential source of precursors for cell therapy for T1D. iPS cells are reprogrammed somatic cells which can be transplanted back into the patient from whom the somatic cells were initially derived, thus potentially avoiding immune rejection when transplanted. As a potential therapy for T1D, we aim to derive insulin producing cells (IPCs) from human iPS cells. In contrast to the conventional two dimensional (2D) cell culture systems used in many iPS derived IPC studies, the inner cell mass (ICM) from which various organs differentiate during embryogenesis is a cluster of cells that enables signaling crosstalk between cells of different types. Three dimensional (3D) cell culture systems allows cells to form cell clusters that promote cell - cell signaling. Hence, we hypothesized that 3D cell culture systems will yield better efficiency of differentiation to functional IPCs in vitro than 2D cultures.

Initially, the synthetic polymers sodium alginate and matrigel were analyzed for their ability to enable cell clustering to establish 3D cell culture systems. The 3D cell environment established using matrigel was used for the differentiation of human iPS cells to Insulin Producing Cells (IPC). The cells were first converted to endodermal cells. A mixture of growth factors then induced the differentiation of endodermal cells to pancreatic cells. The pancreatic cells were converted to IPCs that resemble pancreatic β-cells. Our 3D differentiated IPCs strongly expressed pancreatic endocrine transcription factors and pancreatic hormones. The IPCs also produced insulin when exposed to a high glucose environment. But the number of IPCs obtained at the end of the differentiation was low.

Hence, our results demonstrate that 3D differentiation generates functional IPCs in vitro unlike 2D differentiation. In the future we aim to improve the percentage of IPCs that we generate from the 3D differentiation. Our expectation is that these cells will be able to cure hyperglycemia in diabetic mice more rapidly compared to the 2D differentiated cells owing to their proven insulin production in the presence of a high glucose environment in vitro.

Keywords

3D cultures, Differentiation, Induced Pluripotent Stem Cells, Insulin Producing Cells, Scaffolds, Stem cells

Pages

x, 38 pages

Bibliography

Includes bibliographical references (pages 34-38).

Comments

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Copyright

Copyright © 2014 Pavana Gururaj Rotti

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