ORCID

http://orcid.org/https://orcid.org/0000-0003-4664-3936

Date of Award

Summer 8-15-2021

Author's School

Graduate School of Arts and Sciences

Author's Department

Biology & Biomedical Sciences (Developmental, Regenerative, & Stem Cell Biology)

Degree Name

Doctor of Philosophy (PhD)

Degree Type

Dissertation

Abstract

Diabetes mellitus is a chronic and global disease rapidly growing in prevalence. Diabetes can be characterized by the dysfunction or death of the glucose sensing insulin secreting  cell.  cells are located within the islet of Langerhans (islet), a tissue within the pancreas. Human islets are critical for the study and treatment of diabetes. However, they can only be obtained from cadaveric organ donors. These cadaveric islets do not proliferate and can only be maintained in vitro for short periods of time, making their availability rare and fleeting. Stem cell-derived -like cells can be generated in indefinite amounts and are a potential alternative to cadaveric islet cells. Throughout this document stem cell-derived -like cells will be interchangeably referred as SC- cells or SC-islet. A major challenge towards applying SC- cells to disease modeling or cell replacement therapies is their lack of functional maturity and supporting technology. In this thesis, I am to investigate and improve the functional maturity of SC- cells and their supporting technologies. Chapter 1 serves as an introduction to the field of SC- cells. In Chapter 2, by temporally manipulating TGF signaling, I develop a novel differentiation protocol for generation of SC- cells with enhance function. These enhanced SC- cells are generated more efficiently and achieve dynamic insulin secretion with first and second phase insulin secretion kinetics, a critical hallmark of cadaveric islet function. When transplanted into immune compromised diabetic mice, their function is detected within two weeks and cure their diabetes. In Chapter 3, I elucidate the role of transcription factor SIX2 in SC- cells differentiations. Using gene knockdown and knockout techniques, I show SIX2’s necessity for the functional maturation of SC- cells. Importantly, I identify SIX2 positive and negative  cell populations and postulate its use as a marker to guide future  cell maturation efforts. In Chapter 4, I present a method for cryopreserving SC-islets and characterize them relative to their un-cryopreserved counterparts. Cryopreserved SC-islets functionally and transcriptionally resemble un-cryopreserved SC-islets. This advancement will facilitate biobanking of stem cells, a necessary step to increase their accessibility to the research and therapeutic research population. In Appendix A, I describe the application of a luciferase insulin secretion reporter in SC- cells. Luciferase co-secretes in correlation with insulin and a proof-of-concept compound screen is performed identifying several  cell secretagogues. These secretagogues were then assessed with cadaveric islets verifying the potential of SC- cells as biologically relevant screening models. In Appendix B, I describe a method for co-culturing SC- cells with endothelial cells using a hydrogel system. The work in this thesis advances SC- cell technologies and facilitates their use as disease models and cell-therapeutics.

Language

English (en)

Chair and Committee

Jeffrey Millman

Committee Members

David Piston, Maria Remedi, Luis Batista, Thorold Theunissen,

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