Date of Award

2025-08-01

Degree Name

Doctor of Philosophy

Department

Biomedical Engineering

Advisor(s)

Thomas Boland

Abstract

Thermal inkjet bioprinting (TIB) has emerged as a powerful tool with many potential applications, such as organ regeneration, drug testing, and cell differentiation, among others. Despite the forces and stress that cells are subjected to during the printing process, there is little research that investigates in detail the combined effects of the TIB process and the surrounding environment on cells. Furthermore, a cell's biological environment greatly influences its behavior. Therefore, understanding the effects of bioprinting on cells in a particular environment at a genetic level can provide clues regarding changes in cell characteristics. Bioprinting could potentially be used as a tool for reprogramming or differentiation into different cell types. We hypothesize that using a thermal inkjet bioprinter to bioprint human fibroblasts into induced pluripotent stem cell (iPSC) media will stress the cells, induce disruption of their membranes, and encourage them to upregulate stem-cell-related genes and proteins in response to their environment. Apart from assessing TIB-related changes in gene and protein expression, this work also assesses changes seen in the Hippo pathway effectors YAP/TAZ, as nuclear localization of these has been shown to have positive effects in reprogramming to pluripotency.In this study, we investigated changes in thermal inkjet bioprinted human dermal fibroblasts printed into a stem cell environment using different techniques. We performed immunocytochemistry to assess changes in stem related proteins, lamin A/C, and Hippo pathway effectors. RNA sequencing analyses were conducted at different time points to assess changes in gene expression dynamics at different time points. Finally, DNA methylation studies and analyses on DNA integrity were conducted to examine the impacts of TIB on DNA. Results from RNA sequencing experiments showed differential expression of genes involved in pluripotency-relevant pathways and upregulation of some genes associated with stemness. Immunocytochemistry experiments revealed increases in stem cell markers, particularly for the earlier stem cell marker Lin28b in bioprinted cells, which was particularly prominent at the 24-hour time point post-printing. Analysis of YAP and TAZ localization behavior revealed increased nuclear expression of both YAP and TAZ in TIB cells compared to control cells. Further analyses on YAP dynamics as a result of TIB revealed that stresses on the cell during the TIB process could be influencing YAP phosphorylation at serine 128 and allowing it to enter the nucleus. Finally, DNA methylation analysis revealed decreases in DNA methylation right after printing. The results of this study reveal fundamental changes in gene and protein expression that should be taken into account when using TIB cells in other experiments and that could be used to our benefit to develop new methods of cell engineering and cell reprogramming.

Language

en

Provenance

Received from ProQuest

File Size

85 p.

File Format

application/pdf

Rights Holder

Patricia Ablanedo Morales

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Biomedical Commons

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