Engineering pluripotent stem cell heterogeneity and its effects on differentiation

Pluripotent stem cells (PSCs) have the potential to revolutionize both fundamental and applied bioscience studies, yet their applications have been limited due to their heterogeneity. Stem cell heterogeneity can lead to several challenges, such as differentiation into undesired cell types, compromised functionality, and the growth of cancerous tumors. Therefore, despite nearly a century of stem cell research, the FDA has only approved hematopoietic stem cells for therapeutic applications. It is essential to understand and regulate the mechanism of heterogeneity for enhanced stem cell engineering. To this end, we developed and optimized a reliable and cost-effective method using flow cytometry, allowing us to analyze multiple samples with various treatments at the single-cell level for population heterogeneity. With the established quantification methodology, we implemented a heuristic approach to identify potential mechanisms that may influence heterogeneity. The screened mechanisms spanned a broad range of disciplines, from modulating the growth substrate's stiffness to inhibiting DNA gene expression regulatory mechanisms to controlling cell properties such as cell cycle. We are the first to demonstrate that modulation of the cell cycle could induce a consistent and significant effect on the pluripotent gene expression. PSCs with modulated cell cycles were then differentiated into cardiac and motor neuron lineages. We present a novel finding: partial cell cycle arrest, compared to untreated and near-complete arrest conditions, significantly enhanced the functionality of differentiated tissues. In addition to demonstrating a novel methodology for enhancing stem cell differentiation, these findings suggest that much remains to be understood about how heterogeneity influences differentiation. The work presented lays a foundational methodology for further investigating and engineering pluripotent heterogeneity and investigating its effects on differentiation.

Name thesis:degree_name

Ph.D.

Level thesis:degree_level

Dissertation

Discipline thesis:degree_discipline

Bioengineering

Grantor

University of Illinois at Urbana-Champaign

Year dc:date

2024

Author dc:creator

• Wang, Jason Xiao

Contributors dc:contributor

• Kong, Hyunjoon
• Dar, Roy
• Underhill, Gregory
• Golding, Ido

• Stem Cell
• Heterogeneity
• Stochastic Gene Expression
• Differentiation
• Cardiac Tissue
• Motor Neuron
• Small Molecule Inhibitors

Statement dc:rights

• Copyright 2024 Jason Wang

Language dc:language

en, eng

Handle dc:identifier

https://hdl.handle.net/2142/124561

OAI record oai:identifier

https://www.ideals.illinois.edu/oai-pmh?verb=GetRecord&metadataPrefix=etdms&identifier=oai%3Awww.ideals.illinois.edu%3A2142%2F124561