Carbon Nano Tubes as a Scaffold for hESC Differentiation into Photoreceptor Precursor Cells

Purpose: Carbon Nanotubes (CNTs) have been introduced as an efficient scaffold and interface in various biological applications due to their physical (mechanical and thermal) and electrical properties. Their physical properties are known to influence stem cell proliferation and differentiation, and are therefore used as a scaffold for in vitro differentiation studies and as scaffold for implantation in vivo. Here we introduce our approach of utilizing CNTs as a scaffold for the differentiation of human embryonic stem cells (hESCs) into photoreceptor precursor cells (PRPs) and report preliminary results of the investigation of cell morphology and cell-surface interface and the effect of the CNTs on the differentiation process.

Methods: Three different CNTs types were produced :multiwalled- vertical, aligned, and Carbon Nano Fibers (CNFs) `spaghetti.` CNTs were characterized using SEM imaging. PRPs were obtained from GFP-expressing hESC, following a 24 day differentiation protocol, recently optimized by our group. hESC spheres were seeded on the various CNTs type at day 7 and differentiated until day 24. Biocompatibility of CNTs was assed using propidiom iodie (PI) staining for viability examination. Morphological analysis was achieved through confocal microscopy and focal adhesion of cells was characterized using actin staining (Phalloidin).

Results: When comparing hESC differentiation on standard coverslips to differentiation on the various CNTs types, we observed that cells differentiated on CNTs demonstrated comparable viability to those differentiated on coverslips. Interestingly, morphological analysis revealed these cells preferentially grew from the spheres in the directionality of the CNT surface.

Conclusions: These results highlight the feasibility of using CNTs as an efficient scaffold for cell differentiation and could form the basis of an innovative approach for vision restoration in patients suffering from outer retinal degeneration in which a directionality in transplanted cells is desired.