Microarray with micro- and nano-topographies enables identification of the optimal topography for directing the differentiation of primary murine neural progenitor cells.

During development and tissue repair, progenitor cells are guided by both biochemical and biophysical cues of their microenvironment, including topographical signals. The topographical cues have been shown to play an important role in controlling the fate of cells. Systematic investigation of topographical structures with different geometries and sizes under the identical experimental conditions on the same chip will enhance the understanding of the role of shape and size in cell-topography interactions. A simple customizable multi-architecture chip (MARC) array is therefore developed to incorporate, on a single chip, distinct topographies of various architectural complexities, including both isotropic and anisotropic features, in nano- to micrometer dimensions, with different aspect ratios and hierarchical structures. Polydimethylsiloxane (PDMS) replicas of MARC are used to investigate the influence of different geometries and sizes in neural differentiation of primary murine neural progenitor cells (mNPCs). Anisotropic gratings (2 µm gratings, 250 nm gratings) and isotropic 1 µm pillars significantly promote differentiation of mNPCs into neurons, as indicated by expression of ß-III-tubulin (59%, 58%, and 58%, respectively, compared to 30% on the control). In contrast, glial differentiation is enhanced on isotropic 2 µm holes and 1 µm pillars. These results illustrate that anisotropic topographies enhance neuronal differentiation while isotropic topographies enhance glial differentiation on the same chip under the same conditions. MARC enables simultaneous cost-effective investigation of multiple topographies, allowing efficient optimization of topographical and biochemical cues to modulate cell differentiation.

Enhanced differentiation of neural progenitor cells into neurons of the mesencephalic dopaminergic subtype on topographical patterns.

Parkinson's disease (PD) is a neurodegenerative disease attributed to the loss of midbrain dopaminergic (DA) neurons. The current lack of predictive models for this disease has been hampered by the acquirement of robust cells, posing a major barrier to drug development. Differentiation of stem cells into subtype specific cells may be guided by appropriate topographical cues but the role of topography has hitherto not been well understood. We used a Multi-Architecture (MARC) chip with various topographical structures and identified three topographies, which generate DA neurons from murine hippocampal neural progenitor cells with the highest percentage of neuronal (ß-III-tubulin positive) and dopaminergic (tyrosine hydroxylase positive) populations. Analysis on single pattern structures showed that 2 µm gratings with 2 µm spacing and 2 µm height (2 µm gratings) and 2 µm gratings with hierarchical structure produced cells with the highest gene expression of TH and PITX3, with the longest neurite and highest percentage of alignment. Quantitative image analysis showed the 2 µm gratings produced cells with the highest expression of pituitary homeobox 3 (PITX3), LIM homeobox transcription factor 1 alpha (LMX1a), aldehyde dehydrogenase 1 family member A1 (ALDH1a1) and microtubule associated protein 2 (MAP2), as compared to nano-gratings and unpatterned controls. These patterns also enhance DA neuron differentiation on different substrate rigidities, as seen on both poly-dimethylsiloxane (PDMS) and tissue culture polystyrene (TCPS) substrates. These results show the use of topographical influence for neuronal subtype specification, which could be translated into a wide range of clinical applications for PD.

Extending neurites sense the depth of the underlying topography during neuronal differentiation and contact guidance.

The topography of the extracellular microenvironment influences cell morphology, provides conduct guidance and directs cell differentiation. Aspect ratio and dimension of topography have been shown to affect cell behaviours, but the ability and mechanism of depth-sensing is not clearly understood. We showed that murine neural progenitor cells (mNPCs) can sense the depth of the micro-gratings. Neurite elongation, alignment and neuronal differentiation were observed to increase with grating depth. We proposed a mechanism for depth-sensing by growing neurites: filopodial adhesion in the growth cones favour elongation but the bending rigidity of the neurite cytoskeleton resists it. Thus, perpendicular extension on deeper grooves is unfavourable as neurites need to bend over a larger angle. A quantitative model was developed and its prediction of neurite growth on gratings fit well with the experimental data. The results indicated that mNPC fate can be directed by appropriately designed patterned surfaces.