(I-350) Global Methylation Analysis of Inkjet Bioprinted Human Dermal Fibroblasts Reveals Increased Methylation at the 6-hour Time Point

Bioprinting has emerged as a widely utilized method of depositing biological material for various applications, and it also has the potential to be used as a tool for use in cell reprogramming and differentiation. Reprogramming somatic cells to pluripotency is a highly active area of research, due to their potential in regenerative medicine, cell therapy, or the development of individualized disease models, among others. Cells that undergo the bioprinting process experience stress and strain that may change their physiological, functional, and genetic characteristics. DNA Methylation regulates gene expression by inhibiting the binding of certain transcription factors to DNA and by recruiting specific proteins involved in gene expression, and epigenetic changes play an important role in cell reprogramming to pluripotency. The main goal of this work is to assess the global DNA methylation status of thermal inkjet bioprinted (TIB) human dermal fibroblasts (HDFs) in a stem cell environment.

To assess global DNA methylation, primary 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.

Our results – shown in Figure 1 – revealed the highest quantity of methylated sites in TIB fibroblasts at the 6-hour time point with a notable decrease of methylated sites in TIB cells in every subsequent time period. The lowest percentage of 5-mC in TIB cells was observed at the 72-hour time point. In contrast, in control cells, the highest methylation was observed at the 3-hour time point. Variability in 5-mC percentages is more remarkable between the 3 and 12-hour time periods for both sample types. While there is a marked increase in 5-mC percentage in TIB cells from 3 hours to 6 hours, the opposite is true for control cells, where a marked decrease is seen from 3 hours to 6 hours. In addition, in TIB cells, after the 6-hour time point, 5-mC percentages decrease markedly and begin to trend downwards. The presence of abnormally higher methylated sites observed in the TIB cells at 6 hours could signify the silencing of somatic genes and an increase in developmental potential [1]. These results are likely conducive to reprogramming to a pluripotent state due to the inhibition of certain genes. In addition, the decrease in 5-mC seen post 6 hours could possibly be due to the upregulation of proliferation genes, which play an important role in reprogramming to pluripotency.

Boland MJ et al. Circ Res. 2014 Jul 7;115(2):311-24