Protocol for neurophysiological imaging in human iPSC-derived neurons

The iPSC cell lines (WTC11 and KOLF2.1J) are cultured in Essential 8 medium on Geltrex-coated plates. The cultures are passaged every 3 days with 0.5mM EDTA.

Cells were differentiated from human iPSCs (WTC11, KOLF2.1 cell lines) according to published protocols, with some modifications to allow for easier live-cell imaging.

To generate midbrain dopaminergic-like neurons, we followed the Kriks et al. (2011) protocol, with the following modifications:

• Simplified basal medium: Days 1-20: Neurobasal-A + 1% GlutaMAX + 2% B27 Supplement + 1% N2 Supplement + 1% PenStrep

Days 20+: Neurobasal-A + 1% GlutaMAX + 2% B27 Supplement + 1% PenStrep

• Additional 1:2 replating step at Day 8 of differentiation to avoid cell detachment due to overgrowth.

• For the replating step at Day 20, the cells are replated under low-density conditions (30 x10³ - 100 x10³ cells per cm²) as opposed to the recommended high-density conditions (300 x10³ - 400 x10³ cells per cm²)

• If there are non-neuronal cells present in the culture after Day 20, a treatment with 2 µM Ara-C for 24-48h is carried out.

To generate enteric nervous system-like neurons, we followed the Barber et al. (2019) protocol, with the following modifications:

• Additional replating step at Day 20-22. We plate 60 x10³ - 100 x10³ cells per cm².

• If there are non-neuronal cells present in the culture after Day 20, a treatment with 2 µM Ara-C for 24-48h is carried out.

Using calcium as a proxy for neuronal activity, we assess calcium dynamics during basal conditions, as well as during stimulations.

For midbrain dopaminergic-like neurons:

• The cells are loaded with the calcium dye Fluo-4-AM at a concentration of 50-100nanomolar (nM) for 0h 10m 0s at 37°C.
• The cells are washed 3x with HEPES and left to incubate at37°C for 0h 5m 0s.
• The coverslips are mounted into an imaging holder and the cells covered with HEPES. As an easy alternative, glass bottom culture dishes can be used.
• The cells can be imaged for up to 1h 0m 0s.

For ENS-like neurons:

• The cells are loaded with the calcium dye Fluo-4-AM at a concentration of 1micromolar (µM) for 0h 10m 0s at Room temperature, on a shaker (<50 rpm).
• The cells are washed 1x with HEPES and mounted into an imaging holder. The cells are covered with HEPES. As an easy alternative, glass bottom dishes can be used.
• The cells can be imaged for up to 1h 0m 0s.

Using mitochondrial dyes such as MitoTracker (MitoTracker Green/Red/Red CMXRos), we investigate neuritic mitochondrial transport in these cells (Van Steenbergen et al. 2022).

• The cells are loaded in5-7.5nanomolar (nM) (for dopaminergic-like neurons) or 20nanomolar (nM) (for ENS-like neurons) MitoTracker for 0h 10m 0s at 37°C.
• The cells are washed 3x with HEPES and left to incubate at 37°C for 0h 5m 0s.
• The coverslips are mounted onto an imaging holder and covered with HEPES. As an easy alternative, glass bottom culture dishes can be used.
• The cells can be imaged for up to1h 0m 0s.

Depending on the experiment, we use a combination of different stimuli to assess neuronal physiology.

Electrical stimulation

• Electrodes (custom field electrodes and focal electrodes; see Materials for details)

• Stimulator: Master8 (A.M.P.I) driving the A385 Stimulus Isolator (World Precision Instruments)

• Stimulation protocols: ‣ Field electrode

         **•**  3 - 100 pulses at 20 Hz - 100 Hz
  
  
  
         **•** Current: 5 - 10 mA
  
  
  
    ‣ Focal electrode
  
         **•** Train duration: 2x1000 ms
  
  
  
         **•** Rate: 2x10 pps
  
  
  
         **•** Delay: 1x1ms
  
  
  
         **•** Duration: 3x0.1 ms
  
  
  
         **•** Volts: 3x10 volts
  

Local perfusion (for ENS-like cells, as in Boesmans et al. 2019)

• High-K⁺(75millimolar (mM))
• Substance P (1micromolar (µM))
• DMPP (10micromolar (µM))

A variety of microscopes can be used. We use wide-field systems (Zeiss Axiovert, Nikon coupled to Till Vision image acquisition hard/software) and confocal systems (Zeiss LSM 780-NLO, Zeiss LSM 880 AiryScan). The image acquisition speed should be at least 1-2 Hz in order to capture the relevant physiological events.

Laser powers and exposure times (generally ~ 2 mWatt for widefield, 20-80ms exposure per 500 ms) should be adjusted to avoid bleaching and to preserve neuronal health.