Sequential Application of Discrete Topographical Patterns Enhances Derivation of Functional Mesencephalic Dopaminergic Neurons from Human Induced Pluripotent Stem Cells

Abstract

Parkinson’s Disease is a progressive neurodegenerative disorder attributed to death of mesencephalic dopaminergic (DA) neurons. Pluripotent stem cells have great potential in the study for this late-onset disease, but acquirement of cells that are robust in quantity and quality is still technically demanding. Biophysical cues have been shown to direct stem cell fate, but the effect of different topographies in the lineage commitment and subsequent maturation stages of cells have been less examined. Using human induced pluripotent stem cells (iPSCs), we applied topographical patterns sequentially during differentiation stages and examined their ability to influence derivation yield and functionality of regionalized subtype-specific DA neurons. Gratings showed higher yield of DA neurons and may be beneficial for initial lineage commitment. Cells derived on pillars in the terminal differentiation stage have increased neuronal complexity, and were more capable of firing repetitive action potentials, showing that pillars yielded better network formation and functionality. Our topography platform can be applied to patient-derived iPSCs as well, and that cells harbouring LRRK2 mutation were more functionally mature when optimal topographies were applied sequentially. This will hopefully accelerate development of robust cell models that will provide novel insights into discovering new therapeutic approaches for Parkinson’s Disease.

Combinatorial approach of binary colloidal crystals (BCCs) and CRISPR activation to improve induced pluripotent stem cell differentiation into neurons

“…In the present study, we showed that BCC surfaces with both 2 μm or 5 μm silica can promote enriched neuronal cultures. Our results expand the findings of previous studies that have used surface topography such as grantings, pillars and nanofiber scaffolds to support the generation of stem cell-derived neurons 13,17–19 . In particular, some of the BCC surfaces are able to improve the generation of iNs with longer neurites and more complex branching, with the most prominent effect observed in BCC9 consisting of 5 μm silica and 100 nm carboxylated polystyrene, the smoothest and most hydrophobic surface among tested in this study.…”

“…For instance, Tan et al . found that grantings can aid early stages of neural differentiation, as they promote commitment into neural progenitors, whereas pillars increase branching and neuronal complexity that are more suitable for maturation 19 . Given that some of the BCCs (BCC9 and BCC13) promoted the generation of longer neurites and/or more complex neurite branching, these surfaces could be adapted to the late stages of existing neuronal differentiation protocols to promote neuronal maturation.…”

“…Indeed, many groups have shown that the substrate topography can influence proliferation, morphology, and gene expression 13–16 . In particular, topographical patterns like gratings and pillars have been employed to support the generation of neurons derived from stem cells, 13,17–19 or neurons derived from fibroblasts using direct reprogramming 20,21 . Our group has previously developed topographically and chemically defined surfaces that are suitable for stem cell culture 15,22 , which are based on binary colloidal crystals (BCCs) 23 .…”

Recent Developments in Surface Topography-Modulated Neurogenesis

“…However, when analyzing the expression of MAP2 and β-III tubulin, the neural differentiation of the iPSCs was higher on the patterned substrate compared to the control, a Matrigel-coated culture dish [43]. More recently, Tan et al used the sequential application of discrete topographical patterns on iPSCs to produce functional neurons [44]. A chemical induction was also necessary, but the effect of topographic cues was not negligible.…”

Current strategies and opportunities to manufacture cells for modeling human lungs

“…In general, grooved topography can provide great insights into PSC fate choice and potentially expedite differentiation protocols. The MARC platform illustrates the importance of combining biophysical and biochemical cues, especially as exposure to topography induced a higher yield of functional and mature progeny within a short time frame as compared to flat substrates or standard directed differentiation protocols [ 230 , 235 ]. This is of extreme importance as current directed differentiation protocols for airway and lung epithelia require longer than 60 days of culture to achieve functional cell types [ 12 , 27 , 28 , 30 , 36 , 119 ].…”