• Lasse Hyldgaard Klausen
4. term, Nanotechnology, Master (Master Programme)
Human embryonic stem cell research is a rapidly evolving field with a great potential. Embryonic stem cells can continuously reproduce themselves, and they are capable of forming the entire diversity of cells in an adult. The growth of these cells in vitro requires a high level of control and the culture systems are constantly improving. Embryonic stem cells are distinct from other cells in many aspects, and the presence of the DNA base 5-hydroxymethylcytosine was recently found - a DNA base that in mammalian cells is almost exclusive to embryonic stem cells.
Indolicidin is an antimicrobial peptide with a unique DNA binding capacity that can inhibit the DNA synthesis in bacteria. The effect of binding to mammalian DNA has not yet been properly investigated, and the possibility of an effect from binding to 5-hydroxymethylcytosine in embryonic stem cells was the initial idea for this project.

The effect of indolicidin, the analogue indolicidin-4 and a new peptide IL4-RWT was tested on the embryonic stem cell lines RH1 and T8. The cells were maintained in a culture system of Matrigel and mTeSR. Cytotoxic concentrations were determined, and IL4-RWT, that contains the fluorophore rhodamine WT, was used to test the cellular uptake of the peptide. Flow cytometry showed a slow linear uptake indicating an endocytosis related pathway and this was confirmed by fluorescence confocal laser scanning microscopy showing inclusion bodies.

The effect of IL4 on the pluripotency of RH1 cells was first tested using a short assay. The morphology of the cells was monitored and the expression of pluripotency transcription factors Oct-4 and Nanog was determined using real-time reverse-transcription PCR. No significant effect was observed and a longer assay using the T8 cells was conducted with IL and IL4. A clear positive effect was observed for IL4, but a karyotype test revealed a mosaic making the results void.

DNA was isolated from the adult HDF cells and from the RH1 cells. The DNA was fragmented using ultrasonication or digestion and imaged using AFM. Ultrasonication provided DNA of the desired size without the need for extra cleanup steps as with enzymatic digestion, and this method was used to prepare DNA for testing with IL4. The binding of IL4 to both HDF and RH1 DNA was confirmed, and a fast and easy method for visualisation and analysis of the DNA molecules was described.
Publication date15 Jun 2012
Number of pages100


Phase contrast image of healthy hES cells. (A): RH1 cells (B): WA16 cells.
The expression of GFP by T8 cells during the differentiation assay.
AFM topography of RH1 DNA fragmented by sonication, added IL4.
ID: 65555340