This item is under embargo and not available online per the author's request. For access information, please visit http://libanswers.wustl.edu/faq/5640.

ORCID

http://orcid.org/0000-0003-0869-1662

Date of Award

Winter 1-15-2021

Author's School

McKelvey School of Engineering

Author's Department

Biomedical Engineering

Degree Name

Doctor of Philosophy (PhD)

Degree Type

Dissertation

Abstract

Autologous stem cell therapy is a promising treatment for patients with diabetes worldwide. Previous stem cell-derived β (SC-β) cell protocols were unable to efficiently differentiate multiple patient induced pluripotent stem cells (iPSCs) into stem cell-derived islets (SC-islets), containing insulin-secreting SC-β cells. Recent updates targeting the actin cytoskeleton have enabled the differentiation of 14 diabetic and nondiabetic stem cell lines into SC-islets. We used genetic engineering, specifically CRISPR/Cas9, to correct the diabetes-causing mutation in stem cells from patients with Wolfram Syndrome. The genetically engineered SC-β cells functioned and had a composition similar to nondiabetic SC-β cells, unlike the unedited SC-β cells generated from the patient which revealed insulin processing defects and low β cell yields from the differentiation. In addition, the corrected SC-β cells reversed diabetes in mice within 8 days of transplantation, whereas, the diseased SC-β cells maintained hyperglycemic over 8 months. Single-cell RNA sequencing identified differences in the diseased and corrected SC-β cells from patients and was also used to analyze transplanted SC-islets after 6 months in vivo. The transplanted SC-islets had a global transcriptome profile similar to adult human islets, compared to in vitro SC-islets which are comparable to juvenile human islets.

These revelations in transplanted SC-islets led us to study the effect of host mouse sex, diabetic environment, transplantation site, and dose on maturation of SC-islets in mice. SC-islets were only able to reverse pre-existing diabetes in mice at the kidney capsule transplantation location with a dose of 5 million and 2 million SC-islet cells. Alternative intramuscular and subcutaneous, and lower doses of 0.75 million cells were unable to reverse diabetes. SC-islet maturation was dependent on mouse host sex and independent of the diabetic environment.

Next, we defined the transcriptome profile and maturation of SC-islets, stem cell-derived pancreatic progenitors (SC-PPs), and primary cadaveric human islets transplanted under the kidney capsule in mice. SC-islets achieved similar human C-peptide levels compared to human islets in mouse serum after 18 weeks in vivo. SC-PPs were able to mature into SC-β cells in vivo, yet the serum C-Peptide levels only rose to similar levels of SC-islets upon transplantation. Both SC-islets and SC-PPs prevented diabetes in mice following STZ induction. SC-PPs yielded a small portion of SC-β and endocrine cells, and a large number of exocrine cells following in vivo maturation. The transplanted β cells derived from SC-PPs and SC-islets had similar gene expression of β and islet markers. However, there was differential expression of known β cell maturation genes, revealing gene signatures specific to original transplanted cells.

Overall, these studies confirm the ability of SC-islets to reverse diabetes in animal models and define the similarity of transplanted SC-islets to primary human islets. We report differences in transcriptome and maturation of transplanted cells based on cell type and other parameters. The work in this dissertation provides evidence that autologous cell therapy could provide a long term therapy for patients with diabetes.

Language

English (en)

Chair

Jeffrey R. Millman

Committee Members

Fumihiko Urano, Farshid Guilak, Nathaniel Huebsch, Jing Hughes,

Available for download on Wednesday, December 22, 2021

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