Modulation of murine embryonic stem cell differentiation and gene expression by scaffold surface modification

Objectives: Cardiac progenitor cells derived from pluripotent cells are a promising cell source for cardiac regenerative therapy. The differentiation of embryonic stem (ES) cells is modulated by a network of molecular parameters but can also be influenced by properties of the surrounding substrate. Therefore, we have tested the hypothesis whether surface modification of the substrate the cells grow on can possibly affect the differentiation of ES cells into cardiac progenitors.

Methods: Murine Nkx2.5-CE-GFP ES cells, in which GFP is driven by a cardiac specific Nkx2.5 enhancer were differentiated into GFP-positive cardiac progenitor cells according to established “hanging drop” protocols. After two days embryoid bodies were collected and distributed onto normal culture dishes and on dentin slices (1.5 cm x 1.5 cm x 700 µm) half of which were subjected to a surface treatment using hydrochloric acid. This leads to demineralization of the surface and concomitant uncovering of the embedded collagen. After seven days the occurrence of GFP-positive cardiac progenitors was quantitated by FACS analysis. Gene expression of candidate genes was evaluated by qRT-PCR on a LightCycler 1.5.

Results: Nkx2.5-CE-GFP ES cells plated on normal culture dishes yielded approximately 1.1 ± 0.1% GFP-positive cardiac progenitor cells after seven days. A similar frequency (1.3 ± 0.1%, p= 0.22) was detected when the cells were plated on untreated dentin chips. On the demineralized surface, the fraction of GFP-positive cardiac progenitors was dramatically reduced to 0.3 ± 0.1% (p< 0.0001). Likewise, the expression of the essential cardiac transcription factor Nkx2.5 was strongly downregulated in these cells (p< 0.001) compared to cells differentiated on untreated dentin chips or on normal culture dishes. Comparable results were obtained with Islet1. In contrast, the expression of Hand2 remained virtually similar under all differentiation conditions. Finally, we analyzed the expression of Tnnt2 as a marker for terminal cardiac differentiation. Again in the population differentiated on the demineralized surface Tnnt2 expression was significantly reduced (p< 0.001).

Conclusions: Our results show that a change in substrate surface can heavily influence the differentiation of ES cells into cardiac precursors. The data suggest an important role of either collagen binding or mechanosensing of ES cells which may possibly be reflected by a selective modulation of certain molecular intracellular components.