Substrate elasticity does not impact on DNA methylation changes during differentiation of pluripotent stem cells.

Substrate elasticity may direct cell-fate decisions of stem cells. However, it is largely unclear how matrix stiffness impacts on differentiation of induced pluripotent stem cells (iPSCs) and if this is also reflected by epigenetic modifications. We have therefore cultured iPSCs on tissue culture plastic (TCP) and polydimethylsiloxane (PDMS) with different Young´s modulus (0.2 kPa, 16 kPa, or 64 kPa) to investigate the sequel on growth and differentiation towards endoderm, mesoderm, and ectoderm. Immunofluorescence and gene expression of canonical differentiation markers was hardly affected by the substrates. Notably, when we analyzed DNA methylation profiles of iPSCs or after three-lineage differentiation, we did not see any significant differences on the three different PDMS elasticities. Only when we compared DNA methylation profiles on PDMS-substrates versus TCP, we observed clear epigenetic differences, particularly upon mesodermal differentiation. Taken together, stiffness of PDMS-substrates did not impact on directed differentiation of iPSCs, whereas the moderate epigenetic differences on TCP might also be attributed to other chemical parameters. Genomic DNA was isolated using the NucleoSpin Tissue Kit (Macherey-Nagel) and quantified on a NanoDrop ND-1000 spectrophotometer (Thermo Scientific). 1.2 µg of DNA were bisulfite converted and analyzed using the EPIC BeadChip microarray (Illumina, San Diego, USA) by Life & Brain company (Bonn, Germany).