Mammalian stem cells reprogramming in response to terahertz radiation

Abstract

We report that extended exposure to broad-spectrum terahertz radiation results in specific changes in cellular functions that are closely related to DNA-directed gene transcription. Our gene chip survey of gene expression shows that whereas 89% of the protein coding genes in mouse stem cells do not respond to the applied terahertz radiation, certain genes are activated, while other are repressed. RT-PCR experiments with selected gene probes corresponding to transcripts in the three groups of genes detail the gene specific effect. The response was not only gene specific but also irradiation conditions dependent. Our findings suggest that the applied terahertz irradiation accelerates cell differentiation toward adipose phenotype by activating the transcription factor peroxisome proliferator-activated receptor gamma (PPARG). Finally, our molecular dynamics computer simulations indicate that the local breathing dynamics of the PPARG promoter DNA coincides with the gene specific response to the THz radiation. We propose that THz radiation is a potential tool for cellular reprogramming.

Figure 1. THz irradiation of mouse stem cells.

a) THz radiation was generated by a frequency-doubling BBO crystal, in Argon at 600 torr pressure, with 1 KHz repetition rate . The irradiated and control cells were in thermal contact and the temperature of both samples was monitored by themosensors. b) Mouse stem cells were monitored by light microscopy for morphological changes in response to THz exposure. A significant accumulation of lipid-like droplets in the cellular cytoplasm was visible in response to 6 hours of THz irradiation. Representative photographs are shown with 100x magnification for: Cells without THz exposure; cells after 2 hours of THz exposure; and cells after 6 hours THz exposure. Cells with an increased number of lipid-like droplets inclusions in the cytosol are indicated with black arrow; orange arrows – undifferentiated cells; white arrows – initial stage of adipogenesis.

Figure 2. Gene specific effect of THz irradiation in mouse stem cell cultures.

a) Gene expression profiling of MSC cells in response to 9 hours of THz exposure. Of ∼21,000 annotated genes represented in the Affymetrix mouse genome microarray, 1050 were underexpressed (red) and 1154 were overexpressed (green) with statistical significance p<0.05 as compared to the non-irradiated parallel control; b) RT-PCR measurement for selected genes. The relative level of gene expression in response to 9 hours exposure of MSC to THz radiation normalized to the TBP gene. The identity of the genes is shown below the bars. Cells without THz treatment served as control. The experimental results are consistent between three independent RT-PCR measurements in duplicates and in two different sets of irradiation. Brown bars – THz irradiated cells; blue bars – control cells. The table lists the S-Scores (representing the change in expression level in response to THz irradiation compared to the nonirradiated control) for these genes derived from the Affymetrix mouse genome microarray. c) The temperature was monitored using an IR detector, and separately using thermo-sensors glued to the outside of the petri dish lids. The temperature at the end of irradiation for the control plate with cells (c-79.4⁰F) and the THz irradiated cells (THz-79.9⁰F) is shown above the snap shots panels (snap shots are from the IR detectors in Fahrenheit). d) RT-PCR measurements for selected genes in response to 2, 4, and 9 hours irradiation. RNA levels are normalized to the TBP gene. The identity of the gene is shown at the top. The duration of irradiation is shown below the bars in hours (h). The number of specific transcripts is shown on the vertical axis in relative units [R.U.]. The RT-PCR results are consistent between two independent sets of measurements in duplicates.

Figure 3. Computer simulation data.

EPBD based Langevin molecular dynamics showing the intrinsic breathing dynamics in the promoter regions of PPARG and PPARA genes: vertical axis - size of bubbles [base pairs, bp] with defined average lifetimes; horizontal axis - nucleotide position [base pair] where the TSS is labeled ‘+1’. The color bar on the right indicates the bubble lifetimes in pico seconds [ps]. The promoter sequences obtained from the DBTSS (http://dbtss.hgc.jp), are shown above the simulation data panel. The red letter indicates the TSS position. The identity of the gene sequence is shown above the plots.