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Cellular and Molecular Bioengineering

Cellular and Molecular Bioengineering - Poster Session B

Poster I10 - Global Methylation Analysis of Thermal Inkjet Bioprinted Human Dermal Fibroblasts and Effects of Bioprinting on Hippo Pathway Effectors YAP/TAZ

Thursday, October 24, 2024
2:45 PM - 3:45 PM EST
Location: Exhibit Hall E, F & G

    Presenting Author(s)

  • Jonah Ayala

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

    • Co-Author(s)

  • PA

    Patricia Ablanedo

    PhD Student
    University of Texas at El Paso, United States

    • Primary Investigator(s)

  • Thomas Boland, PhD

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


Introduction:  Bioprinting has emerged as useful in various biological and medical applications due to its ability to manipulate cells and their substrates. Bioprinting can also be used in cell reprogramming and differentiation. Reprogramming somatic cells to pluripotency is a highly popular area of research due to the potential of induced pluripotent stem cells to be used in several applications such as regenerative medicine, differentiation into other cell types, or development of individualized models, among others. Cells that undergo the bioprinting process experience stress and shear forces that may change their physiological, functional, and genetic characteristics. Bioprinting may affect DNA Methylation, which regulates gene expression by inhibiting the binding of certain transcription factors to DNA and by recruiting specific proteins involved in gene expression. It may also activate or deactivate certain cellular pathways, such as the Hippo pathway, whose members YAP/TAZ have been shown to promote pluripotency reprogramming when localized to the nucleus. The kinase NUAK2 can promote nuclear localization of YAP/TAZ by preventing their phosphorylation. However, when exposed to kinase inhibitor WZ4003, YAP/TAZ has been shown to locate to the cytoplasm. The objective of this work was to assess the global DNA methylation status of thermal inkjet bioprinted (TIB) human dermal fibroblasts in a stem cell environment, as well as to analyze the interplay between YAP/TAZ localization, NUAK2, and stem cell marker expression in TIB fibroblasts.

Materials and

Methods:  To assess global DNA methylation, human dermal fibroblasts were inkjet bioprinted into ECM-coated wells with Pluristem media. Controls were manually seeded into wells with Dulbecco’s Modified Eagle Medium + fetal bovine serum. The DNA was extracted from the samples at various time points - 3, 6, 12, 24, 48, and 72 hours - and a colorimetric assay for the detection of 5-methylcytosine (5-mC) percentages was performed. To assess stem cell marker expression of SOX2, OCT4, and LIN28B and localization of YAP and TAZ, TIB cells were stained with appropriate antibodies at different time points. To assess the effects of NUAK2 suppression on YAP/TAZ expression and consequent stem cell marker expression, printed fibroblasts were incubated with WZ4003 for 48 hours, followed by fixation and staining with previously used antibodies.

Results, Conclusions, and Discussions: Our results showed decreased methylation in TIB fibroblasts at the 3- and 12-hour time points with varying slight changes in methylation at the other time points. The lowest percentage of 5-mC in IJB cells was observed at the 12-hour time point. In contrast, in control cells, the highest methylation was observed at the 3-hour time point. The fluctuation between high methylation status and lower methylation status in TIB cells could be due to the silencing of somatic genes and demethylation at stem cell gene-related promoters, respectively. In addition, the decrease in 5-mC seen post 6 hours could be due to the upregulation of proliferation genes, which play an important role in reprogramming to pluripotency. Expression of stem cell markers OCT4, SOX2, and LIN28B were increased in TIB fibroblasts post-printing. Furthermore, analysis of YAP/TAZ expression in TIB fibroblasts revealed increased YAP and TAZ in the nucleus. TAZ had significantly more nuclear expression from 24 hours to 72 hours post-printing; YAP had significantly increased nuclear expression at 48 hours, although immediately after printing, it exhibited decreased YAP nuclear expression. Furthermore, treatment with WZ4003 post-printing slightly decreased YAP nuclear expression in TIB fibroblasts. It is possible that an increased concentration of WZ4003 could further decrease YAP and TAZ nuclear expression and that decreased YAP nuclear localization as a result of WZ4003 treatment will decrease the expression of stem cell markers. Changes in YAP/TAZ point to the Hippo pathway as a crucial pathway involved in reprogramming to pluripotency. Our results point towards a shift in cell identity and characterization post-printing that shifts towards pluripotency characteristics. The results of this research could pave the way towards developing novel methods of reprogramming.

Acknowledgements (Optional):