Dissertation

Improving Pancreatic Lineage Commitment of Human Pluripotent Stem Cells in Terms of Pancreas Development and Disease Modelling

Merz, Sarah

2024

Overview

Human stem cell-based differentiation systems are increasingly used to model diseases, advancing molecular research on pathomechanisms by allowing for the introduction of disease-relevant gene modifications and testing potential treatments and treatment outcomes. However, these directed differentiation processes often face limitations, as many resulting cell types remain distinct from their adult counterparts or contain a notable proportion of unwanted contaminating cell types. This underscores the need for ongoing improvement and refinement.In this study, a systematic stage-wise compound testing approach was applied to optimize the pancreatic progenitor differentiation process. The newly generated PPs showed increased levels of the progenitor marker GP2 and a reduced presence of non-pancreatic cell types. Single-cell transcriptomics revealed considerable heterogeneity within the uniformly PDX1/NKX6.1-positive progenitor pool, with subpopulations distinguished by GP2 expression patterns. Close clustering with human fetal tip and trunk cells, along with FACS-based enrichment, identified the GP2high-expressing cluster as a multipotent progenitor group capable of differentiating into acinar, ductal, and endocrine lineages. Incorporating an ex vivo culture method further supported maturation and long-term cultivation of pancreatic cell types, providing an alternative to transplantation studies in mice.Altogether, the generation of this multipotent progenitor population, capable of giving rise to all three lineages of the pancreas, holds significant potential, given that many diseases, such as diabetes, pancreatitis or pancreatic cancer, often impact both the exocrine and endocrine compartments. Understanding the crosstalk, interactions, and mutual influences among different pancreatic cell types is increasingly important for studying complex disease mechanisms and recognizing the pancreas as a multifaceted organ with interconnected endocrine and exocrine functions.In the second part of this doctoral study, the optimized pancreatic progenitor differentiation system was used to model neonatal diabetes disease, providing an ideal platform for investigating inherited diseases affecting pancreas development and function. The case involved a boy suffering from severe neonatal diabetes and carrying a heterozygous mutation in ONECUT1. Although ONECUT1 protein truncating variants typically cause neonatal diabetes in a recessive manner, his phenotype could not be explained by the heterozygous ONECUT1 variant alone. In addition, the patient exhibited a deletion in a non-coding region upstream of ONECUT1. We used the optimized stem cell-based pancreatic differentiation system and deciphered the pathomechanisms, identifying a putative cis-acting enhancer of ONECUT1 within the non-coding region. This system further supported the development of a tailored therapy option for affected patients with similar genetic profiles. Understanding such monogenic disease mechanisms holds broad relevance. Variants in these genes or their regulatory regions are often linked to a predisposition for polygenic diabetes. This genetic overlap between different forms of diabetes suggests shared pathophysiological mechanisms, aiding in better subtyping and comprehension of the syndromic and heterogenous nature of diabetes. Numerous monogenic gene variants were identified in the recent years, but the most are located in the non-coding regions of unknown function. To accurately assess the disease-causing potential of these variants, it is crucial to decipher the regulatory mechanisms - including promoters, enhancers, silencers or lncRNAs - active during pancreatic development and in mature islets.

• Publisher: ProQuest Dissertations & Theses
• Subject: Cellular biology / Epidermal growth factor / Hormones / Kidneys / Oncology / Polyethylene glycol / Stem cells
• ISBN: 9798288848933