Micropatterning Facilitates the Long-Term Growth and Analysis of iPSC-Derived Individual Human Neurons and Neuronal Networks.

Micropatterned substrate fabrication(a) Scheme showing the microcontact printing of gold-coated substrates. A polydimethylsiloxane (PDMS) stamp is used to pattern octadecanethiol onto a gold-coated glass substrate, rendering the desired areas protein-adhesive. Backfilling the substrate with an ethylene glycol-terminated molecule renders the surrounding areas resistant to protein deposition (and therefore resistant to cell adhesion). Coating the backfilled substrate with laminin makes the stamped “islands” cell-adhesive. (b) Immunofluorescent images of different micropatterned substrates showing surfaces presenting flower (1250μm2), line (10μm diameter) and star (2500μm2) patterns. Scale bar, 50μm.

Advantage of micropatterning technology over conventional protocol for iPSC-derived neuronal culturing(a) Schematic of the differentiation of midbrain dopaminergic neurons from iPSCs, which have been reprogrammed from adult somatic cells. Culturing iPSC-derived neurons on conventional glass coverslips usually results in a less defined growth of neurons and neuronal processes, while culturing neurons on micropatterns enables the analysis of individual neurons and neuronal networks under controlled conditions. (b) Immunofluorescence image of iPSC-derived neurons grown on unpatterned PDL/laminin-coated coverslips following the conventional protocol. Cells were labeled with the neuronal marker β-III-tubulin (red) and DAPI (blue). Scale bar, 100μm. (c) Left: Bright field image taken 20h after plating iPSC-derived neurons onto a substrate with a flower-shaped pattern. Scale bar, 100μm. Right: Immunofluorescence image of iPSC-derived neurons on a substrate representing a flower-shaped pattern. Neurons were labeled with the neuronal marker β-III-tubulin (green) and DAPI (blue). Scale bar, 100μm. (d) Quantification of TH-positive neurons as percent of total number of cells cultured on micropatterned substrates or on conventional unpatterned PDL/laminin-coated coverslips at day 70 of differentiation; n.s.: not significant. (e) Immunofluorescence images of iPSC-derived neurons grown on a substrate with a line-shaped micropattern at day 70 of differentiation. Cells were labeled with the dopaminergic marker TH (green), the neuronal marker β -III-tubulin (red) and DAPI (blue). Examples of TH-positive (arrows with open arrowhead) and TH-negative (arrows with closed arrowhead) neurons are indicated. Scale bar, 100μm. Enlarged view of the boxed region shows a TH-positive neuron. Scale bar, 10μm.

Analysis of mitochondrial dynamics in long-term neuronal cultures(a) Image series showing mitochondrial trafficking as fusion (solid arrow; upper panel) and fission (dashed arrow, lower panel) events in one neuronal process over 160 seconds. iPSC-derived neurons were plated onto a live cell imaging substrate representing a flower-shaped micropattern. Mitochondria were visualized by transduction of mito-GFP BacMam 2.0. (b) Immunofluorescence images of iPSC-derived neurons grown on a substrate with line-shaped pattern shown in low magnification (10×; Scale bar, 100μm) or higher magnification (40×; Scale bar, 20μm). Cells were labeled with neuronal marker β-III-tubulin (green), mitochondrial marker TOM20 (red) and DAPI (blue). TOM20 signal was converted into a binary image for analysis of mitochondrial structures. (c) Mitochondrial length and interconnectivity in individual neuronal processes of iPSC-derived neurons grown on substrates with line-shaped pattern was analyzed after 40 (d40) and 100 days (d100) of differentiation using ImageJ software. *p<0.05; ***p<0.001. Scale bar, 5μm. (d) Image series showing retrograde moving (arrowhead), anterograde moving (arrow with open arrowhead) or stationary (arrow with closed arrowhead) mitochondria over 180 seconds. Corresponding kymograph is shown at the bottom of the image series. Scale bar, 10μm. Mitochondrial trafficking was followed after 40, 70 and 100 days of differentiation and retrograde moving, anterograde moving (≥ 10μm/3 minutes in either direction) and stationary mitochondria were analyzed using ImageJ software. The percentage motility of mitochondria along the neuronal process was calculated as a percentage of the total number of mitochondria imaged per process. *p<0.05.

Dynamic network formation of iPSC-derived neurons(a) Bright field images showing neurite outgrowth towards a nearby cell body over 25 minutes. iPSC-derived neurons were seeded onto a live cell imaging substrate with a flower-shaped micropattern on day 30 of differentiation and bright field images were taken 50 hours after seeding. Scale bar, 10μm. (b) Immunofluorescence images of iPSC-derived neurons grown on a substrate with a star-shaped micropattern. Cells were fixed and labeled with the axonal marker tau (red), the dendritic marker MAP2 (red) and DAPI (blue) at day 40 (top panel) or day 100 (bottom panel) of differentiation. Enlarged views of the boxed regions show tau-positive axons (arrows with open arrowheads) and MAP2-positive dendrites (arrows with closed arrowheads). Scale bar, 100μm. (c) Schematic of the potential use of micropatterned substrates for studies of cell dynamics, such as mitochondrial dynamics (a), neuronal outgrowth (b), synaptic transmission (c) and cellular degradation pathways, e.g. lysosomal degradation of aggregated proteins or cellular contents (d).