Pyrolytic Carbon Micro and Nano Topographies for Stem Cell Differentiation

Abstract

Stem cells are unspecialized cells that play a fundamental role in the maintenance and regeneration of cells and tissues located in a microenvironment, defined as a niche. A discrete array of cellular niche factors contributes to the overall stem cell functions such as self-renewal, quiescence, morphology, migration, proliferation, differentiation, phenotype preservation, or apoptosis. To augment the efficiency of cell-based regenerative therapies particularly for transplanting cells into patients, it is essential to understand the significance of cellular microenvironment that potentially regulates stem cell behavior and fate. Advancements in new technologies have offered insights into an interaction of human neural stem cells with extracellular matrix and nanotopographies. However, much research is required to sufficiently characterize and fine-tune the functions of micro and nano topographies (MNTs) in different materials for stem cell research. This research aims to explore the potential impact of nanoengineered pyrolytic carbon-based MNTs for human neural stem cell differentiation (hNSCs), mainly into dopaminergic neurons (DAn). Firstly, the fabrication of random carbon nanograss (CNG) MNTs has been reported to demonstrate their ability to be applied as cellsubstrate enhancing hNSCs adhesion, survival, and differentiation into DAn. These MNTs are expected to play an essential role in stem cell-based cell replacement therapy (CRT) and neural implants for Parkinson’s disease (PD). The unique combination of random CNG MNTs and biocompatible pyrolytic carbon material enhanced hNSCs neurogenesis up to 2.3 folds and dopaminergic differentiation up to 3.5 folds both in presence and absence of poly-lysine (PLL) biocoating. Moreover, the influence of different ECMs and pyrolytic CNGs has been studied for hNSCs differentiation into DAn compared to flat surfaces in both conditions of ECMs coated and uncoated substrates. The results show that PLL is a most favorable biocoating among other ECMs for enhancing DAn generation when used with CNG substrates. The uncoated CNGs were found to equally outperform for boosting dopaminergic differentiation suggesting the favorable elimination of biocoatings for future in vitro and in vivo neuronal stem cell studies. Secondly, the multifunctional pyrolytic CNG MNTs have been optimized and fabricated as CNG electrode chips to exploit their use in electrochemical detection of dopamine (DA) compared to planar 2D electrodes. Thirdly, the periodic and spatially organized pyrolytic carbon nanopillar (CNP) MNTs have been fabricated using maskless lithography, followed by the development of CNP electrode chips for electrochemical detection of DA. Lastly, a Au-based ultramicroelectrode array (UMEA) containing 54 individually addressable microelectrodes were developed and tested for real-time detection of dopamine exocytosis from single cells in a population of PC-12 cells. In the future, the periodic pyrolytic CNP MNTs can be used as cell-platform to characterize hNSCs adhesion, morphology, proliferation, and dopaminergic differentiation, and can be compared with random pyrolytic CNG MNTs for both cellular and molecular research aspects.