Triboelectric Nanogenerator Accelerates Highly Efficient Nonviral Direct Conversion and In Vivo Reprogramming of Fibroblasts to Functional Neuronal Cells

Background

Direct conversion of somatic cells into neurons by lineage-specific transcription factors avoids the use of embryos and pluripotent intermediates, but viral vectors commonly used to deliver these factors raise immunogenicity and tumorigenicity concerns. Nonviral polymer nanoparticles had been used to convert mouse fibroblasts into iN cells, but with low efficiency. The authors therefore asked whether external electrical stimulation could compensate for the low efficiency of nonviral delivery.

Design and approach

The TES generates biphasic pulse-like currents by periodic contact and separation of an aluminium top plate and a micro-pillar polydimethylsiloxane (PDMS) bottom plate (approximately 300 µm thick) backed by Kapton and copper. Contact transfers electrons from aluminium to PDMS, and each release and press drives current in opposite directions through the external load. The TES is connected in series with a titanium-coated silicon culture substrate, so that the cells grow on the stimulating electrode. Genes for Brn2, Ascl1 and Myt1l (BAM) were delivered to primary mouse embryonic fibroblasts (PMEFs) by electroporation and, two days after seeding, again with biodegradable poly(β-amino ester) (PBAE) nanoparticles 100–150 nm in diameter.

How it was evaluated

The authors characterized TES output with flat and micro-pillar PDMS, at contact frequencies of 0.5–3 Hz, by capacitor charging and by cycling. Transfected PMEFs were exposed to TES at 1 Hz for 60 min per day and compared with untreated (NT), TES-only and BAM-only groups using Tuj1 and MAP2 staining, quantitative PCR, neurite length, RNA sequencing, whole-cell patch clamp, calcium imaging and western blotting for phosphorylated ERK1/2. In vivo, PBAE nanoparticles carrying BAM plasmids were injected intradermally in mice on days 0 and 2, and the skin was stimulated through electrocardiogram patches placed 2 cm apart for 30 min per day for 14 d.

Key results

The micro-pillar PDMS layer produced an open-circuit voltage of approximately 30 V and a short-circuit current of approximately 280 nA, compared with approximately 10 V and 110 nA for flat PDMS. Rectified output charged a 22 µF capacitor that lit a blue LED, and cycling caused no noticeable degradation of the biphasic output.

TES shortened and increased conversion. On day 9, 8.85% of seeded cells in the BAM/TES group were Tuj1-positive, compared with 0.13% in the BAM group, and on days 12–14 the values were 14.17% and 6.41%. TES alone produced 0.22% Tuj1-positive cells. On day 12, Tuj1 and MAP2 expression was 1.5–2-fold higher with TES than in the BAM group. In cells maintained for 7 d, a 280 nA stimulus raised Tuj1 and MAP2 expression more than a 110 nA stimulus. The authors reported that TES shortened the time to obtain Tuj1-positive iN cells by 3–5 d and, to their knowledge, gave the highest Tuj1-positive iN efficiency reported with nonviral delivery.

TES also accelerated maturation. No BAM-group cells were MAP2-positive on day 14, whereas some BAM/TES cells were, and on days 16–18 the MAP2-positive fraction of Tuj1-positive cells was 61.2% with TES versus 31.3% without. Average neurite length in the BAM/TES group was 0.17 mm, and individual neurites reached approximately 2.9 mm on day 18. By day 30, the cells showed punctate NeuN and synapsin I staining and were positive for vGlut1 and GAD67. RNA sequencing identified 859 genes with greater than 2-fold changes (P < 0.05) relative to PMEFs, with fibroblast genes repressed and neuronal and synaptic genes upregulated. The cells fired action potentials and showed sodium currents that disappeared with 0.5 × 10−6 M tetrodotoxin, and glutamate raised their intracellular calcium.

During stimulation, intracellular calcium rose persistently on days 9 and 12, and ERK1/2 phosphorylation increased on day 7 in BAM/TES cells, from which the authors inferred a pathway of calcium channel activation, calcium influx and ERK1/2 signaling. In mouse skin, the current measured at 1 Hz was approximately 80 nA, and BAM plus TES generated Tuj1-positive cells with an efficiency of 5.86%, mostly in the dermis, compared with 0.04% for BAM alone.

Open questions

The authors stated that the molecular mechanisms by which electrical stimulation enhances neuronal differentiation are not well understood, and the proposed role of TES frequency in regulating voltage-gated calcium channels was presented as a postulate based on earlier reports. They identified a TES patch that harvests energy from breathing and cardiac motion as the next step.

How it connects to other research

The defined-factor conversion of mouse fibroblasts into functional neurons by Vierbuchen and colleagues used the three factors Ascl1, Brn2 and Myt1l, whereas this study delivers the same factors nonvirally and uses triboelectric stimulation to raise efficiency.