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In This Article

  • Summary
  • Abstract
  • Introduction
  • Protocol
  • Results
  • Discussion
  • Disclosures
  • Acknowledgements
  • Materials
  • References
  • Reprints and Permissions

Summary

The electroporation of primate cerebral organoids provides a precise and efficient approach to introduce transient genetic modification(s) into different progenitor types and neurons in a model system close to primate (patho)physiological neocortex development. This allows the study of neurodevelopmental and evolutionary processes and can also be applied for disease modeling.

Abstract

The cerebral cortex is the outermost brain structure and is responsible for the processing of sensory input and motor output; it is seen as the seat of higher-order cognitive abilities in mammals, in particular, primates. Studying gene functions in primate brains is challenging due to technical and ethical reasons, but the establishment of the brain organoid technology has enabled the study of brain development in traditional primate models (e.g., rhesus macaque and common marmoset), as well as in previously experimentally inaccessible primate species (e.g., great apes), in an ethically justifiable and less technically demanding system. Moreover, human brain organoids allow the advanced investigation of neurodevelopmental and neurological disorders.

As brain organoids recapitulate many processes of brain development, they also represent a powerful tool to identify differences in, and to functionally compare, the genetic determinants underlying the brain development of various species in an evolutionary context. A great advantage of using organoids is the possibility to introduce genetic modifications, which permits the testing of gene functions. However, the introduction of such modifications is laborious and expensive. This paper describes a fast and cost-efficient approach to genetically modify cell populations within the ventricle-like structures of primate cerebral organoids, a subtype of brain organoids. This method combines a modified protocol for the reliable generation of cerebral organoids from human-, chimpanzee-, rhesus macaque-, and common marmoset-derived induced pluripotent stem cells (iPSCs) with a microinjection and electroporation approach. This provides an effective tool for the study of neurodevelopmental and evolutionary processes that can also be applied for disease modeling.

Introduction

Investigating the (patho)physiological development and evolution of the cerebral cortex is a formidable task that is hampered by the lack of suitable model systems. Previously, such studies were confined to two-dimensional cell culture models (such as primary neural progenitor or neuronal cell cultures) and evolutionarily distant animal models (such as rodents)1,2. While these models are useful for addressing certain questions, they are limited in modeling the complexity, cell type composition, cellular architecture, and gene expression patterns of the developing human neocortex in healthy and diseased states. These limitations lead, for example, to the poor translatability of mouse models of human diseases to the human situation, as described for certain cases of microcephaly (e.g., Zhang et al.3). Recently, transgenic non-human primates, which are an evolutionarily, functionally, and morphologically closer model of human neocortex development, have come into focus4,5,6,7,8 as they overcome many limitations of cell culture- and rodent-based models. However, the use of non-human primates in research is not only highly expensive and time-consuming but also raises ethical concerns. More recently, the development of brain organoid technology9,10 has emerged as a promising alternative that solves many of the limitations of previous models11,12,13,14,15,16.

Brain organoids are three-dimensional (3D), multicellular structures that emulate the main features of the cytoarchitecture and cell-type composition of one or multiple brain regions for a defined developmental time window11,12,13,14,17. These 3D structures are generated either from induced pluripotent stem cells (iPSCs) or, if available for the species of interest, from embryonic stem cells (ESCs). In general, two types of brain organoids can be distinguished based on the methodology used: unguided and regionalized (guided) brain organoids18. In generating the latter type of organoids, small molecules or factors are provided that guide the differentiation of the pluripotent stem cells to organoids of a particular brain region (e.g., forebrain organoids)18. By contrast, in unguided organoids, the differentiation is not guided by the addition of small molecules but rather relies exclusively on the spontaneous differentiation of the iPSCs/ESCs. The resulting brain organoids consist of cell types representing different brain regions (e.g., cerebral organoids)18. Brain organoids combine many key features of brain development with relatively cost- and time-efficient generation from any species of interest for which iPSCs or ESCs are available11,12,13,14. This makes brain organoids an excellent model for many kinds of neurobiological studies, ranging from evolutionary and developmental questions to disease modeling and drug testing15,16. However, addressing such questions using brain organoids strongly depends on the availability of different methods for genetic modification.

One key aspect of studying neocortex (patho)physiological development and its evolution is the functional analysis of genes and gene variants. This is usually achieved by (ectopic) expression and/or by knock-down (KD) or knock-out (KO) of those genes. Such genetic modifications can be classified into stable and transient genetic modification, as well as into the modifications being temporally and spatially restricted or not restricted. Stable genetic modification is defined by the introduction of a genetic alteration into the host genome that is passed on to all subsequent cell generations. Depending on the time point of genetic modification, it can affect all the cells of an organoid or can be restricted to certain cell populations. Most frequently, stable genetic modification is achieved in brain organoids at the iPSC/ESC level by applying lentiviruses, transposon-like systems, and the CRISPR/Cas9 technology (reviewed by, e.g., Fischer et al.17, Kyrousi et al.19, and Teriyapirom et al.20). This has the advantage that all cells of the brain organoid carry the genetic modification and that it is not temporally or spatially restricted. However, the generation and characterization of these stable iPSC/ESC lines are very time-consuming, often taking several months until the first modified brain organoids can be analyzed (reviewed by e.g., Fischer et al.17, Kyrousi et al.19, or Teriyapirom et al.20).

In contrast, transient genetic modification is defined by the delivery of genetic cargo (e.g., a gene expression plasmid) that does not integrate into the host genome. While this modification can, in principle, be passed on to subsequent cell generations, the delivered genetic cargo will be progressively diluted with each cell division. Therefore, this type of genetic modification is usually temporally and spatially restricted. Transient genetic modification can be carried out in brain organoids by adeno-associated viruses or by electroporation (reviewed by, e.g., Fischer et al.17, Kyrousi et al.19, and Teriyapirom et al.20), with the latter being described in detail in this article. In contrast to stable genetic modification, this approach is very fast and cost-efficient. Indeed, electroporation can be performed within minutes, and, depending on the target cell population(s), electroporated organoids are ready for analysis within days (reviewed by, e.g., Fischer et al.17 and Kyrousi et al.19). However, gross morphological changes of the brain organoid, such as differences in size, cannot be detected using this method, as this type of genetic modification is temporally and spatially restricted. This restriction can also be an advantage, for example, in the case of studying individual cell populations within the organoid or the effects on brain organoids at specific developmental time points (reviewed by, e.g., Fischer et al.17 and Kyrousi et al.19).

A classical approach to study gene function during brain development and evolution is in utero electroporation. In utero electroporation is a well-known and useful technique for the delivery of gene expression constructs into rodent21,22,23 and ferret24,25 brains. First, a solution containing the expression construct(s) of interest is microinjected through the uterine wall into a certain ventricle of the embryonic brain, depending on the region to be targeted. In the second step, electric pulses are applied to transfect the cells directly lining the targeted ventricle. This approach is not only limited to ectopic expression or the overexpression of genes, as it can also be applied in KD or KO studies by microinjecting short hairpin (shRNA) or CRISPR/Cas9 (in the form of expression plasmids or ribonucleoproteins [RNPs]), respectively26,27. However, the in utero electroporation of mouse, rat, and ferret embryos has the same limitations as described above for these animal models.

Ideally, one would like to perform in utero electroporation directly in primates. While this is, in principle, technically possible, in utero electroporation is not conducted in primates due to ethical concerns, high animal maintenance costs, and small litter sizes. For certain primates, such as great apes (including humans), this is not possible at all. However, these primates have the greatest potential for the study of human (patho)physiological neocortex development and its evolution. One solution to this dilemma is to apply the electroporation technique to primate brain organoids28.

This paper presents a protocol for the electroporation of a subtype of primate brain organoids, primate cerebral organoids. This approach allows the fast and cost-efficient genetic modification of cell populations within the ventricle-like structures of the organoids. Specifically, we describe a unified protocol for the generation of primate cerebral organoids from human (Homo sapiens), chimpanzee (Pan troglodytes), rhesus macaque (Macaca mulatta), and common marmoset (Callithrix jacchus) iPSCs. Moreover, we describe the microinjection and electroporation technique in detail and provide "go" and "no-go" criteria for performing primate cerebral organoid electroporation. This approach is an effective tool for studying (patho)physiological neocortex development and its evolution in a model especially close to the human situation.

Protocol

1. Culture of primate iPSCs

NOTE: Due to its robustness, the method presented here can be applied to any primate iPSC line. In this article, we describe cerebral organoid production from human (iLonza2.2)29, chimpanzee (Sandra A)30, rhesus macaque (iRh33.1)29, and common marmoset (cj_160419_5)31 iPSC lines. The culture conditions are summarized in Table 1. See the Table of Materials for details related to all the materials, reagents, and equipment used in this protocol.

  1. For culturing the respective iPSCs, follow the originally described culture conditions. In general, for the successful generation and electroporation of cerebral organoids, use iPSC lines that have not been cultured for more than 90 passages. Additionally, at the start of cerebral organoid generation, ensure that iPSCs exhibit features of pluripotency with no signs of differentiation.

2. Generation of cerebral organoids from primate iPSCs

NOTE: The protocol for cerebral organoid generation is based on a modified version28,30,32,33 of the original cerebral organoid protocol10,34 with some species-specific modifications (detailed below).

  1. Seeding of iPSCs to generate embryoid bodies (EBs)
    1. Once the iPSCs have reached 80%-90% confluency, wash them with Dulbecco's phosphate-buffered saline (DPBS), and add 500 µL of recombinant trypsin substitute or 1 mL of proteolytic and collagenolytic mixture.
      NOTE: Typically, iPSCs are cultured in a 60 mm cell culture dish to obtain approximately 900,000 cells, which is enough to generate 96 cerebral organoids. The cell numbers can be adjusted depending on the number of organoids needed. Be aware that not all the generated cerebral organoids might be suitable for electroporation.
    2. Incubate the dish at 37 °C for 2 min to detach the cells.
      NOTE: Up to an additional 2 min of incubation at 37 °C might be needed depending on the iPSC line or enzyme used. It is advised to examine the dish under a microscope to ensure that the cells have started detaching.
    3. Add 1.5 mL of prewarmed (37 °C) iPSC culture medium to stop the reaction, and pipette up and down 7x-10x (not more than 10x) to dissociate the cells from the cell culture dish and to obtain a single-cell suspension.
    4. Transfer the cell suspension to a 15 mL conical centrifuge tube, and centrifuge the cells at 200 × g for 5 min at room temperature.
    5. Aspirate the supernatant, and resuspend the pellet in 2 mL of iPSC culture medium supplemented with either 50 µM Y27632 or 50 µM pro-survival compound.
    6. Use 10 µL of the cell suspension to count the cells using a Neubauer chamber.
    7. Adjust the cell suspension to a concentration of 9,000 cells per 150 µL (60,000 cells/mL) using iPSC culture medium supplemented with 50 µM Y27632 or 50 µM pro-survival compound.
    8. To generate embryoid bodies (EBs), seed 150 µL of the cell suspension into each well of an ultra-low attachment 96-well plate. While pipetting, gently shake the tube containing the cell suspension to prevent the cells from sedimenting.
    9. Culture the EBs in a humified atmosphere of 5% CO2 and 95% air at 37 °C (0 days post seeding [dps]). Do not disturb the EBs within the first 24 h after seeding.
      NOTE: EBs generated from marmoset iPSCs need to be cultured in a humified atmosphere under hypoxic conditions (5% CO2, 5% O2, and 90% N2) at 37 °C.
    10. After ~48 h (2 dps), change the medium to iPSC culture medium without Y27632/pro-survival compound. Remove 100 µL of medium per well, and add 150 µL of prewarmed (37 °C) fresh medium without Y27632. Go row by row.
    11. Perform further medium changes every other day; remove 150 µL of medium from each well, and add 150 µL of prewarmed (37 °C) fresh medium without Y27632/pro-survival compound per well.
      NOTE: After 4-5 dps, the periphery of the EBs should become translucent.
  2. Induction of neuroectoderm
    NOTE: In general, good-quality EBs should have smooth contours and translucent borders at this stage. The time points of neural induction slightly differ between primate species and iPSC lines. In the case of the cell lines used here (see section 1 and Table of Materials), neural induction for marmoset EBs usually needs to be started at 4 dps, for rhesus macaque at 5 dps, and for human and chimpanzee EBs at 4-5 dps (Figure 1A), depending on the state of the EBs (see above).
    1. Remove 150 µL of medium from each well of the first row of the 96-well plate, and add 150 µL of prewarmed (37 °C) neural induction medium (see Table 2) per well in the same row.
    2. Continue changing the medium as described above row by row for the whole 96-well plate. Perform further neural induction medium (NIM) changes every other day by removing 150 µL of NIM from each well and adding 150 µL of prewarmed (37 °C) fresh NIM.
      NOTE: From this point on, the marmoset EBs should be cultured under the same conditions as the other primate EBs (humified atmosphere of 5% CO2 and 95% air at 37 °C).
  3. Embedding in basement membrane matrix
    NOTE: Once the EBs have developed a pronounced, translucent, radially organized neuroepithelium on the surface, structural support needs to be provided for the development of ventricle-like structures. This is achieved by embedding the EBs into a basement membrane matrix. Due to differences in development rates, marmoset and rhesus macaque EBs are ready for embedding already at 7 dps, while human and chimpanzee EBs are usually embedded at 8-9 dps. For simplicity, basement membrane matrix refers only to Matrigel in this protocol. However, Geltrex can be used as a replacement.
    1. In preparation for embedding, UV-sterilize scissors, forceps, a small rack for 0.2 mL tubes, and three to six squares of parafilm treated with 70% (vol/vol) ethanol under the laminar flow hood for 15 min. Let the basement membrane matrix thaw on ice for several hours (~1.5 mL of basement membrane matrix is usually enough for 96 EBs).
      NOTE: Always keep the basement membrane matrix on ice.
    2. Create a 4 x 4 dimple grid on the parafilm. Place the parafilm grid on the 0.2 mL tube rack so that the paper-enveloped side is facing up, and gently press a gloved finger against each hole of the rack.
    3. Remove the paper, and cut the dimple grid out of the parafilm square using scissors to adjust its size to fit into a 60 mm cell culture dish. Place the dimpled parafilm back on the 0.2 mL tube rack to provide a basis for the basement membrane matrix droplet generation.
    4. Using a pipette with a cut 200 µL pipette tip, carefully transfer the EBs one after another from the well of the culture dish to the parafilm dimples.
    5. After moving 16 EBs to the grid, take a new 200 µL pipette tip, and remove the remaining medium from the dimples.
    6. Pipette one drop (~15 µL) of basement membrane matrix onto each dimple containing one EB.
    7. Take a 10 µL pipette tip and quickly move the EBs into the center of each droplet without disturbing the droplet borders.
    8. Place the dimpled parafilm with the basement membrane matrix drops in a 60 mm cell culture dish, and incubate for 15-30 min at 37 °C to allow the matrix to polymerize.
    9. To detach the matrix-embedded EBs from the parafilm, add 5 mL of differentiation medium (DM) without vitamin A (see Table 2) to the dish, and turn the parafilm square upside-down using forceps so that the side with the EBs is facing the bottom of the dish.
    10. Carefully shake the dish to make the basement membrane matrix drops containing the EBs detach from the parafilm. If some of them are still attached, take an edge of the parafilm square using forceps, and rapidly roll it up toward the center of the dish multiple times.
    11. Culture the cerebral organoids on an orbital shaker at 55 rpm in a humified atmosphere of 5% CO2 and 95% air at 37 °C. Keep them in DM without vitamin A with medium changes every other day. To induce the production of neurons, switch to DM with vitamin A (retinoic acid, RA) after 13 dps for marmoset and rhesus macaque cerebral organoids or 14-15 dps for human and chimpanzee cerebral organoids (Figure 1A). From this point on, change the medium every 3-4 days.
      NOTE: To support neuronal survival, DM with vitamin A can be supplemented with 20 µg/mL human neurotrophin 3 (NT3), 20 µg/mL brain-derived neurotrophic factor (BDNF), and 1 µL/mL basement membrane matrix from 40 dps on.

3. Electroporation of primate cerebral organoids

NOTE: From a technical point of view, the electroporation of cerebral organoids can be conducted as soon as the ventricle-like structures are pronounced enough to be targeted by microinjection. The optimal electroporation time window depends on the biological question and on the cell population(s) of interest. For example, if apical progenitors (APs) are the main target, then cerebral organoids at around 30 dps are already suitable. If basal progenitors (BPs) or neurons are the main targets, older cerebral organoids of more than 50 dps should be used (see, for example, Fischer et al.28).

  1. Preparation of the electroporation setup
    NOTE: The electroporation efficiency is strongly affected by the size and the concentration of the electroporated plasmid(s) (see the discussion section for details).
    1. Prepare a sufficient amount of electroporation mix for the control and gene of interest (GOI), for example, 10 µL of electroporation mix for each control and GOI to electroporate approximately 30 cerebral organoids per condition.
      NOTE: For the composition of the electroporation mixes, see Table 3.
    2. Prewarm (non-sterile) Dulbecco's modified Eagle medium/nutrient mixture F-12 (DMEM/F12) and DM with vitamin A to 37 °C. Prepare a small spatula and fine and normal scissors, and spray the instruments with 70% (vol/vol) ethanol.
      NOTE: The following steps can be performed either under sterile or non-sterile conditions as the DM contains antibiotics (see Table 2). In our experience, the absence of sterility has never caused any contamination.
    3. Prepare 35 mm cell culture dishes, and connect the Petri dish electrode chamber to the electroporator.
      NOTE: Petri dish electrode chambers are commercially available. However, they can be easily produced in a cost-efficient way (see Supplemental File 1).
    4. Using microloader tips, fill microinjection needles with 8 µL of each electroporation mix. Cut the tips of the needles using fine scissors before the first use to achieve a stable flow. However, remove only a small part of the tip, as a blunt and wide tip can severely damage the organoids.
      NOTE: Microinjection needles are either commercially available as pre-pulled microinjection needles or can be pulled in the lab if a needle puller is available. Follow the needle puller manufacturer's instructions to generate microinjection needles with a long taper and a 10 µm tip diameter.
  2. Microinjection and electroporation
    1. Under a microscope, choose five cerebral organoids with smooth borders and clearly visible ventricle-like structures. Move them to a 35 mm cell culture dish containing prewarmed (37 °C) DMEM/F12 using a cut 1,000 µL pipette tip.
      NOTE: Choose cerebral organoids with pronounced and accessible ventricle-like structures (see Figure 1B).
    2. To inject a ventricle-like structure, carefully insert the needle through its wall, and infuse it with the electroporation mix until visibly filled. Do not apply excessive pressure on ventricle-like structures to avoid them bursting. Proceed with six to eight ventricle-like structures of each cerebral organoid in this manner.
      NOTE: If the needle becomes clogged during the microinjection process, the tip needs to be slightly trimmed.
    3. Transfer one microinjected cerebral organoid to the Petri dish electrode chamber with a small amount of DMEM/F12. Arrange the organoid in a way that the surfaces of the microinjected ventricle-like structures face toward the electrode connected to the positive pole of the electroporator.
      NOTE: Orienting the structures in this way ensures that the cells are transfected on the side of the ventricle-like structure that is not affected by an adjacent structure.
    4. Electroporate the cerebral organoids one by one using the following settings: 5 pulses of 80 V, a pulse duration of 50 ms, and an interval of 1 s. Move the electroporated organoids to a new 35 mm cell culture dish filled with prewarmed (37 °C) DMEM/F12.
      NOTE: The electroporation settings might depend on the available square wave electroporator. These settings are optimized for the referenced electroporation system. Increasing the voltage can lead to the displacement of the cells.
    5. Proceed in the same manner with the next five cerebral organoids using the second electroporation mix (for example, GOI).
      NOTE: Repeat steps 3.2.1-3.2.5 until the desired number of electroporated cerebral organoids is reached.
    6. If the microinjection and electroporation were conducted under non-sterile conditions, transfer the electroporated organoids to a sterile 35 mm cell culture dish under a laminar flow hood while moving as little DMEM/F12 as possible to the new cell culture dish.
  3. Further culture and fixation of cerebral organoids
    1. Culture the electroporated organoids in DM with vitamin A on an orbital shaker at 55 rpm in a humified atmosphere of 5% CO2 and 95% air at 37 °C.
    2. On the next day after electroporation, check the cerebral organoids for successful electroporation under a conventional inverted fluorescence microscope.
      NOTE: Depending on the length of the further culture after electroporation, different cell populations within the cerebral organoid are affected (see also the representative results section).
    3. Proceed with downstream applications after culturing the electroporated cerebral organoids for an amount of time suitable for the biological question of interest.
      NOTE: Electroporated cerebral organoids can be processed for different downstream applications (e.g., fixation for immunofluorescence staining or snap-frozen for RNA isolation and qRT-PCR). Here, we describe the fixation of electroporated cerebral organoids.
    4. Transfer the electroporated organoids to a 15 mL conical centrifuge tube using a cut 1,000 µl pipette tip, and remove excess medium.
    5. Add a sufficient amount of 4% paraformaldehyde (PFA) in DPBS (pH 7.5), and incubate for 30 min at room temperature.
      CAUTION: PFA is classified as a human carcinogen and may cause irreparable health damage. Additional precaution measures including nitrile gloves and goggles are strongly recommended.
    6. Aspirate the PFA, add 5 mL of DPBS, shake a little, and aspirate the DPBS. Repeat this 2x. Store the organoids in DPBS at 4 °C until further use.
      NOTE: The protocol can be paused here, as PFA-fixed cerebral organoids can be stored at 4 °C for several months. PFA-fixed electroporated organoids can be analyzed by cryosectioning and immunofluorescence staining10,28 or by whole-mount staining and clearing35,36. For example images, see the representative results section (see Table 4 for antibody details).

Results

The protocol described here allows the efficient generation of cerebral organoids from human, chimpanzee, rhesus macaque, and common marmoset iPSC lines with minimal timing alterations required between species (Figure 1A). These organoids can be electroporated in the range of 20 dps to 50 dps, depending on the accessibility of the ventricle-like structures and the abundance of the cell population(s) of interest. However, prior to electroporation, it is important to determine whether the cere...

Discussion

The procedures described here represent a unified protocol for the generation of cerebral organoids from different primate species with a targeted electroporation approach. This allows the ectopic expression of a GOI in a model system that emulates primate (including human) (patho)physiological neocortex development. This unified protocol for the generation of primate cerebral organoids uses the same materials (e.g., media) and protocol steps for all four primate species presented. Developmental differences between these...

Disclosures

The authors declare that they have no conflicts of interest.

Acknowledgements

We apologize to all the researchers whose work could not be cited due to space limitations. We thank Ulrich Bleyer of the technical services at DPZ and Hartmut Wolf of the workshop at MPI-CBG for the construction of the Petri dish electrode chambers; Stoyan Petkov and Rüdiger Behr for providing human (iLonza2.2), rhesus macaque (iRh33.1) and marmoset (cj_160419_5) iPSCs; Sabrina Heide for the cryosectioning and immunofluorescence staining; and Neringa Liutikaite and César Mateo Bastidas Betancourt for critically reading the manuscript. Work in the laboratory of W.B.H. was supported by an ERA-NET NEURON (MicroKin) grant. Work in the laboratory of M.H. was supported by an ERC starting grant (101039421).

Materials

NameCompanyCatalog NumberComments
20 µL MicroloaderEppendorf5242956003
2-MercaptoethanolMerck8.05740.0005
35 mm cell culture dishesSarstedt83.3900
60 mm cell culture dishesCytoOneCC7682-3359
Activin ASigma-AldrichSRP3003
AOC1SelleckchemS7217
Axio Observer.Z1 Inverted Fluorescence MicroscopeZeissreplacable by comparable fluorescent microscopes
AZD0530Selleckchem S1006
B-27 Supplement with Vitamin A (retinoic acid, RA) (50x)Gibco17504-044
B-27 Supplement without Vitamin A (50x)Gibco12587-010
BTX ECM 830 Square Wave Electroporation SystemBTX45-2052
CGP77675Sigma-AldrichSML0314
Chimpanzee induced pluripotent stem cell line Sandra Adoi: 10.7554/elife.18683 
Common marmoset induced pluripotent stem cell line cj_160419_5doi: 10.3390/cells9112422
Dulbecco's Modified Eagle Medium/Nutrient Mixture F-12 (DMEM/F12)Gibco11320-033
Dulbecco's phosphate-buffered saline (DPBS)Gibco14190-094pH 7.0−7.3; warm to room temperature before use
Fast GreenSigma-AldrichF7252-5G
ForskolinSelleckchem2449
GlutaMAX Supplement (100x)Gibco35050-061glutamine substitute supplement
Heparin (1 mg/mL stock)Sigma-AldrichH3149
Human induced pluripotent stem cell line iLonza2.2doi: 10.3390/cells9061349
Human Neurotrophin-3 (NT-3)PeproTech450-03
InsulinSigma-Aldrich19278
IWR1Sigma-AldrichI0161
Leica MS5 stereomicroscope (MDG 17 transmitted-light base)Leica10473849replacable by comparable stereomicroscopes
MatrigelCorning354277/354234basement membrane matrix; alternatively, Geltrex (ThermoFisher Scientific, A1413302) can be used
MEM Non-Essential Amino Acids Solution (100x)Sigma-AldrichM7145
N-2 Supplement (100x)Gibco17502-048
Neurobasal mediumGibco21103-049
ParafilmSigma-AldrichP7793
Paraformaldehyde Merck818715handle with causion due to cancerogenecity
Penicillin/Streptomycin (10,000 U/mL)PanBiotechP06-07100
Petri dish electrode chamberself-produced (see Supplemental File 1)also commertially available
Pre-Pulled Glass PipettesWPITIP10LTborosilicate glass pipettes with long taper, 10 µm tip diameter
Pro-Survival CompoundMerckMillipore529659
Recombinant Human/Murine/RatBrain-Derived Neurotrophic Factor (BDNF)PeproTechAF-450-02
Rhesus macaque induced pluripotent stem cell line iRh33.1doi: 10.3390/cells9061349
StemMACS iPS-Brew XFMiltenyi Biotech130-104-368
StemPro Accutase Cell Dissociation ReagentGibcoA1110501proteolytic and collagenolytic enzyme mixture
TrypLEGibco12604-013recombinant trypsin substitute; warm to room temperature before use
Ultra-Low Attachment 96-well platesCostar7007
Y27632Stemcell Technologies72305

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