DOI

10.17077/etd.v54456jv

Document Type

Thesis

Date of Degree

Fall 2014

Degree Name

MS (Master of Science)

Degree In

Biomedical Engineering

First Advisor

Nicholas Zavazava

First Committee Member

Michael Mackey

Second Committee Member

Edward Sander

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.

Public Abstract

Type 1 diabetes (T1D) is caused by the destruction of pancreatic β-cells. This process results in loss of the β-cell mass and reduced production of insulin. Lack of insulin, the key regulator of glucose, leads to elevated blood glucose levels, which can lead to the complications of diabetes such as blindness, cardiovascular disease, neuropathy and kidney failure. Our goal was to establish an alternative source of pancreatic β-cells. First, human skin cells were reprogrammed to generate induced pluripotent stem cells. These cells are very similar to embryonic stem cells in that they can be driven to generate any desired tissues such as pancreatic β-cells. We established a protocol that utilizes Matrigel to create the three dimensional (3D) environment which facilitates cell differentiation.

During the differentiation process, the cells were first differentiated to endodermal cells, which express CXCR4. Past this stage, the cells transitioned into pancreatic precursor cells, characterized by the expression of Pdx-1, a key pancreatic nuclear factor. At the end of the differentiation procedure, the cells formed three characteristic types of clusters—tight clusters, cyst-like clusters that had a thick edge and cyst like clusters that contained compact clusters at the core. On characterization of the cells at the end of the differentiation process, the tight cell clusters were positive for insulin, C-peptide, Nkx6.1, and Nkx2.2. On glucose stimulation, the cells responded by secreting insulin. However, when compared to human pancreatic islets, the response to glucose stimulation by our cells was very low.

Thus, we have established a method for generating insulin producing cells from human skin cells. However, the protocol needs further improvement to enhance insulin secretion and we need to test our cells in vivo to determine whether they can correct glucose levels.

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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