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Cellular and Molecular Bioengineering

Cellular and Molecular Bioengineering - Poster Session E

Poster V12 - Thermal inkjet Bioprinting of Human Fibroblasts into Stem Cell Environment Reveals Changes in Protein Expression and Changes in the Hippo Pathway

Saturday, October 26, 2024

10:00 AM - 11:00 AM EST

Location: Exhibit Hall E, F & G

Presenting Author(s)

PA

Patricia Ablanedo Morales

research associate
University of Texas at El Paso
El Paso, Texas, United States

Co-Author(s)

Jonah Ayala

Undergraduate Student
The University of Texas at El Paso
El Paso, Texas, United States
KM

Kayla Molina

Doctoral Student
University of Minnesota, United States

Primary Investigator(s)

Thomas Boland, PhD

Professor
The University of Texas at El Paso, Texas, United States

Introduction: 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 effects of the TIB process 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.

Materials and

Methods: In this study, we thermal inkjet bioprinted human dermal fibroblasts into iPSC qualified media onto iPSC qualified ECM gel coating and seeded control unprinted fibroblasts into Dulbecco’s Modified Eagle Medium + fetal bovine serum. We performed immunocytochemistry for well-established stem cell markers, SOX2, OCT4, and LIN28B on the bioprinted fibroblasts and manually seeded controls. Furthermore, we performed immunocytochemistry for Hippo pathway effectors YAP and TAZ at different time points in both TIB and control fibroblasts. In order to further expand on the role of YAP/TAZ in the effects of TIB on cells, we treated printed cells with WZ4003, a kinase inhibitor known to inhibit NUAK2 – a kinase that prevents YAP/TAZ phosphorylation and allows them to enter the nucleus.

Results, Conclusions, and Discussions: Previous results from RNA sequencing experiments conducted on a fibroblast cell line showed differential expression of genes involved in pluripotency relevant pathways and upregulation of some genes associated with stemness. Immunocytochemistry experiments revealed expression of NANOG, Sox-2, Oct-4, and Lin28b in bioprinted cells. Overall, printed cells seem to adopt some behavior attributed to iPSC, signifying that they may be beginning possible differentiation into stem cells. These results point to wide ranging changes in gene and protein expression that should be analyzed further. Analysis of YAP and TAZ expression revealed increased nuclear expression of both YAP and TAZ in TIB cells compared to control cells. YAP had a significant difference at 48 hours post-TIB and TAZ expression was significantly different at 24 hours to 72 hours. These results suggest that the Hippo pathway may be de-activated only at certain time points post printing. Increased nuclear expression of YAP and TAZ in TIB cells is promising since this has been shown to have positive effects on reprogramming to pluripotency. The activity of YAP/TAZ in the nucleus as a result of TIB could lead to expression of stem cell related genes and lead to an overall shift in cell characteristics. Furthermore, treatment with WZ4003 led to slightly decreased YAP expression in the nucleus, which is likely the result of NUAK2 inhibition. Understanding how genes are up- or downregulated and how cellular and metabolic pathways are affected when cells are subjected to the bioprinting process can provide valuable information that may allow manipulation of cells using bioprinting in order to achieve transformation into different cell types.

Acknowledgements (Optional):