Jul 13, 2023

Public workspaceProtocol for neurophysiological imaging in human iPSC-derived neurons

DOI
dx.doi.org/10.17504/protocols.io.81wgbxp8ylpk/v1
  • Natalia Garcia Perez1,2,
  • Yi-Ning Kang1,2,
  • Emma Moles-Garcia1,2,
  • Tom Venneman1,2,
  • Pieter Vanden Berghe1,2
  • 1KU Leuven;
  • 2LENS
Natalia Garcia Perez
Icon indicating open access to content
QR code linking to this content
DOI: dx.doi.org/10.17504/protocols.io.81wgbxp8ylpk/v1
Protocol Citation: Natalia Garcia Perez, Yi-Ning Kang, Emma Moles-Garcia, Tom Venneman, Pieter Vanden Berghe 2023. Protocol for neurophysiological imaging in human iPSC-derived neurons. protocols.io https://dx.doi.org/10.17504/protocols.io.81wgbxp8ylpk/v1
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License,  which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: In development
The protocol works but we are still developing and optimizing this protocol.
Created: July 05, 2023
Last Modified: July 13, 2023
Protocol Integer ID: 84533
Funders Acknowledgement:
Aligning Science Across Parkinson’s
Abstract
The protocol describes how to perform neurophysiological imaging experiments on different types of human iPSC-derived neurons.
Materials
Solutions:

HEPES buffer consists of: (in mM) 140 NaCl, 5 KCl, 10 HEPES, 2 CaCl2, 2 MgCl2, and 10 d-glucose, adjusted to pH = 7.4 using NaOH (Li et al. 2022).

Electrodes:

  • Focal electrodes were constructed by soldering a 50 µm tungsten wire to a gold connector inserted into a glass pipette that was pulled using a Sutter instruments pipette puller. The tip of the glass pipetted was sealed by melting the glass in the open flame of a lighter. This electrode was connected to a Grass stimulator and (30V) pulses were given at 1-20 Hz. A silver electrode was used as a reference electrode. (Li et al. 2019)


  • For field stimulation:  two Pt wire electrodes (0.5 mm diameter) were flatted and cut to from two planar side which were spaced 1 mm apart in a plexiglass rod. This rod was positioned using a Narishige 3D manipulator such that the 1 mm space covered the field of view that was under investigation. The electrode was gently put on the coverslip and current (5-10 mA) stimuli were given to depolarize the neuronal networks.

iPSC culture and maintenance
iPSC culture and maintenance
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.
iPSC-derived neurons generation
iPSC-derived neurons generation
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.
Live-cell imaging
Live-cell imaging
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 Concentration50-100 nanomolar (nM) for Duration00:10:00 at Temperature37 °C .
  • The cells are washed 3x with HEPES and left to incubate atTemperature37 °C for Duration00:05:00 .
  • 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 Duration01:00:00 .

1h 15m
For ENS-like neurons:
  • The cells are loaded with the calcium dye Fluo-4-AM at a concentration of Concentration1 micromolar (µM) for Duration00:10:00 at TemperatureRoom 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 Duration01:00:00 .

1h 10m
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 inConcentration5-7.5 nanomolar (nM) (for dopaminergic-like neurons) or Concentration20 nanomolar (nM) (for ENS-like neurons) MitoTracker for Duration00:10:00 at Temperature37 °C .
  • The cells are washed 3x with HEPES and left to incubate at Temperature37 °C for Duration00:05:00 .
  • 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 toDuration01:00:00 .
1h 15m
Stimulation
Stimulation
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+ (Concentration75 millimolar (mM) )
  • Substance P (Concentration1 micromolar (µM) )
  • DMPP (Concentration10 micromolar (µM) )


Microscopy
Microscopy
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.
Protocol references