Protocol for combining BMP, MEK, WNT inhibition and iNGN2 to enable rapid neuronal differentiation and regional patterning from hiPSCs

Summary

Neurogenin 2 (iNGN2) overexpression induces human induced pluripotent stem cells (hiPSCs) into neurons but results in a mixed population of peripheral and central nervous system neurons. Pre-differentiation using BMP, MEK, and WNT inhibition (“BMWi”) prior to iNGN2 promotes telencephalic neuron differentiation. We outline neural induction, neural progenitor replating/freezing/thawing, and telencephalic neuron maturation and describe protocols for patterning motor neurons, ventral midbrain, and sensory neurons (sensBMi).

For complete details on the use and execution of this protocol, please refer to Habich et al.¹

Graphical abstract

Highlights

• •A new approach for quickly generating telencephalic neural progenitor cells without dSMADi
• •Enhanced protocol combining BMWi with NGN2 overexpression to produce telencephalic neurons
• •Guiding NPCs during induction to rapidly develop ventral midbrain, motor, and sensory neurons

Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics.

Neurogenin 2 (iNGN2) overexpression induces human induced pluripotent stem cells (hiPSCs) into neurons but results in a mixed population of peripheral and central nervous system neurons. Pre-differentiation using BMP, MEK, and WNT inhibition (“BMWi”) prior to iNGN2 promotes telencephalic neuron differentiation. We outline neural induction, neural progenitor replating/freezing/thawing, and telencephalic neuron maturation and describe protocols for patterning motor neurons, ventral midbrain, and sensory neurons (sensBMi).

Before you begin

Neurodegenerative diseases like Alzheimer’s Disease (AD) and Parkinson’s Disease (PD) are becoming increasingly significant as the population ages.^2,3,4 Although the number of affected individuals continues to grow, there are still no effective therapies that address the root causes of these conditions—most existing treatments only provide symptomatic relief. In the past, research relied mainly on animal models or primary neuron cultures, both of which have ethical and translational limitations. The advent of human induced pluripotent stem cells (hiPSC) offers a promising alternative; their capacity to differentiate into specialized cell types creates new possibilities for disease modeling, drug screening, and developing personalized treatments.^5,6,7

Many neurological disorders affect particular types of neurons, such as dopaminergic neurons in Parkinson’s disease or motorneurons in ALS, so employing the relevant subtype enables more accurate modeling of disease mechanisms, drug responses, and pathophysiology.^8,9,10

Dual inhibition of SMAD2/3 and SMAD1/5/8 signaling pathways (dSMADi) has become a common approach for directing human hiPSCs toward a neuroectodermal fate.¹¹ This method can be further combined with regional patterning signals—for example, WNT inhibition to induce anterior fate,^12,13 SHH signaling for ventralization¹⁴ and retinoic acid (RA) for posteriorization such as in motor neuron differentiation.¹⁵ These protocols result in defined subtypes of neurons but are time-consuming and complicated to implement for inexperienced scientists. Overexpression of the transcription factor neurogenin 2 (NGN2) is used to quickly differentiate hiPSC-derived neurons.^12,16,17,18 Differentiations via NGN2 overexpression results mainly in a telencephalic/cortical neuronal subtype.¹⁷ However, NGN2 neurons express PNS marker genes,^12,17,19 and telencephalic/cortical markers like CUX1/2,¹⁶ while specific forebrain markers such as FOXG1 are absent.^16,20,21

This protocol was optimized using genetically modified hiPSC clones that carry a doxycycline-inducible NGN2 overexpression cassette in the AAVS1 safe harbour locus. One of these clones is available through a cell bank (see key resources table). Alternative strategies for NGN2 overexpression (such as lentiviral delivery) were not evaluated, but can be explored.

Innovation

The BMWi neural induction paradigm presented here offers a robust and broadly applicable approach for differentiating hiPSCs into regionally specified neuroectoderm, without relying on the commonly used SMAD2/3 inhibition.¹¹ Within 4 or 6 days of pre-differentiation followed by 2 days of NGN2 overexpression, it is possible to generate neural precursor cells (NPCs) that can be cryopreserved in large batches and retain high viability after thawing. The pre-differentiation phase can be further modified with additional patterning factors, enabling the differentiation of ventral midbrain- and sensory neuron NPCs within 6 days, and motor neuron NPCs within 8 days. This enables the efficient generation of various neuronal subtypes spanning different regions of the central- and peripheral nervous systems, which will significantly facilitate in vitro modeling of a broad spectrum of neurodevelopmental and neurodegenerative disorders.¹

The coating procedures have been evaluated for long-term and stable neuron cultures and are recommended for establishing the protocol.

The BMWi induction paradigm addresses current research challenges by establishing novel, accelerated differentiation protocols for various neuron types, optimized for large scale experiments and high-throughput screening (HTS) applications.

Preparation of media for cell culture

E8 flex medium

Thaw E8 flex supplement and add to basal medium. Store at 4°C up to 2 weeks.

N2B27 medium

Combine all components and sterile filter medium using a 0.22 μm PES sterile filter. Store at 4°C up to 4 weeks.

Note: Instead of a PES filter you can also use CAA filters.

Neural maturation medium−

Combine all ingredients (except Laminin) and sterile filter medium using a 0.22 μm PES sterile filter. Then add Laminin.

Store at 4°C up to 4 weeks, only warm needed amounts of medium prior to use.

Note: One complete vial of Laminin can be added to a complete bottle of sterile-filtered medium.

Neural maturation medium+

Combine all ingredients (except Laminin) and sterile filter medium using a 0.22 μm PES sterile filter. Then add Laminin.

Store at 4°C up to 4 weeks, only warm needed amounts of medium prior to use.

Note: One complete vial of Laminin can be added to a complete bottle of sterile-filtered medium.

Preparation of Matrigel aliquots

• 1.
  Thaw matrigel (GFR or hESC matrigel) on ice at 4°C overnight.

  • a.
    Keep matrigel bottle in ice.
  • b.
    Prepare 1.5 mL tubes in a −20°C frozen metal block.
  • c.
    Add calculated amount of matrigel in each tube.

Note: Aliquot size of matrigel depends on protein concentration per matrigel batch to dilute to 77 μg/mL for the needed volume of coating. For example, we recommend preparing aliquots for 40 mL of medium (around ∼400 μL aliquots). This would allow to coat, for example, 6 × 96-well plates or 3×6-well plates.

Note: Prechill box of 1000 μL pipette tips at −20°C before aliquoting.

Note: For easier handling, it is possible prepare aliquots directly in 50 mL conical tubes and freeze them. Then dilution (see step 1/4) can be done directly in 50 mL tube, and you avoid the transfer step.

Matrigel hESC-Qualified Matrix coating

Timing: 1 day

The coating described here is used for routine hiPSC culture and neural induction.

• 2.
  hESC-Qualified Matrigel Matrix coating for neural induction or hiPSC culture.

  • a.
    Prepare Matrigel hESC-Qualified Matrix aliquot according to data sheet.
  • b.
    Prepare a Falcon Tube with fridge-cold (4°C) DMEM/F12 to dilute the hESC-Qualified Matrigel Matrix to 77 μg/mL.
  • c.
    Take out the desired number of aliquots.
  • d.
    Immediately thaw the aliquots by pipetting around 300–500 μL of cold medium on it.
  • e.
    Pipetting up- and down around 10 times.
  • f.
    Repeat this procedure 2–3 times until the entire matrigel is thawed with medium.
  • g.
    Immediately transfer the thawed and prediluted hESC-Qualified Matrigel Matrix into the larger vessel.
  • h.
    Mix and directly proceed to coating.
  • i.
    Use volume described in materials and equipment for matrigel coating.
  • j.
    Incubate cell culture ware 1h at 37°C or at least 16h (overnight) at 4°C.

Note: If alternative coatings are used for routine hiPSC culture, these can also be assessed at this stage. It is recommended to observe if possible detachment occurs during the induction.

PLO-Matrigel double coating

Timing: 2 days

Shown here is the preparation of the cell culture ware on which NPC will be replated (step-by-step method details step 8).

• 3.
  Poly-L-ornithine coating.

  • a.
    Add 50 μL 0.01% poly-L-ornithine solution per well of 96 well plate.

    Note: Do not use outer wells. Add 150 μL sterile H₂O to outer wells to prevent evaporation (Figure 1, Example #1). If plating only a subset of wells, surround wells to be plated with cells with a ring of wells containing sterile H₂O (Figure 1, Example #2). 150 μL of water should typically last for the whole period of culture. In case water evaporates with time, refill wells with water.

  • b.
    Gently tap the bottom of the plate to ensure coating across the bottom of each well.
  • c.
    Seal off each plate with parafilm.
  • d.
    Incubate at room temperature for at least 16 h (overnight).

    Note: It is not recommended to leave plates under a closed safety cabinet (without wrapping in parafilm), as the circulation mode can cause heat that will lead to drying out of wells.

    Note: It is recommended to follow up with the next step of the coating around 16 to 24 h later.

• 4.
  Remove PLO and wash with DPBS (−/−).

  • a.
    Completely remove the PLO of the plate by vacuum aspiration.
  • b.
    Quickly change into dispensing mode and dispense 150 μL DPBS (−/−) per 96 well.

Note: It is not necessary to switch pipette tips, all can be done with the same tips.

Note: For other plate or flask types see materials and equipment for used volumes.

• 5.
  Thawing matrigel aliquots.

  • a.
    Prepare a Falcon Tube with fridge-cold (4°C) DMEM/F12 to dilute the matrigel Growth Factor Reduced Matrix to 77 μg/mL.
  • b.
    Take out the desired number of aliquots.
  • c.
    Immediately thaw the aliquots by pipetting around 300–500 μL of cold medium on it, pipetting up- and down around 10 times.
  • d.
    Repeat this procedure 2–3 times until the entire matrigel is thawed with medium, immediately transfer the thawed and prediluted matrigel Growth Factor Reduced Matrix into the larger vessel.
  • e.
    Mix and directly proceed to plating.

Note: Perform this step exactly prior to coating.

CRITICAL: Do not attempt to thaw matrigel aliquots at room temperature or 37°C. This will cause polymerization and result in poor coating.

Note: If handling larger volumes of coating solution, it is recommended to keep it on ice until direct use, still, time on ice should be minimized.

• 6.
  Matrigel Growth Factor Reduced Matrix coating.

  • a.
    Aspirate the DPBS (−/−) washing solution completely.
  • b.
    Immediately add 100 μL diluted matrigel solution to each well.
  • c.
    Wrap dishes in parafilm to avoid evaporation and incubate at 4°C for at least 16 h (overnight).
  • d.
    Store plates up to 1 week at 4°C.

Figure 1.

Example for coating of 96 well plates

To prevent evaporation of medium, all wells used should be surrounded by wells filled with water.

General hiPSC culture

Timing: Variable

For all differentiations shown here we applied hiPSCs with a doxycycline inducible overexpression system for the transcription factor NGN2. Genetically modified clones that allow doxycycline-inducible overexpression through a cassette integrated in the AAVS1 safe harbour locus, were used. Alternative systems for overexpression (such as lentiviral delivery) would need to be individually evaluated.

Note: The medium should be preheated to room temperature or up to 37°C (e.g., using a water bath) for all steps described in this protocol.

• 7.
  Seed hiPSCs for routine culture on matrigel hESC-Qualified Matrix-coated cell culture ware.

  • a.
    Remove matrigel solution from cell culture ware.
  • b.
    Immediately move to the next step to avoid drying out coating.
  • c.
    Seed 50,000 hiPSCs per cm² diluted in 0.2 mL/cm² E8 flex medium supplemented with 10 μM ROCKi on matrigel hESC-Qualified Matrix-coated cell culture ware.

    • i.
      If you want to start from running cell culture split cells in a 1:10 ratio.
• 8.
  1 day after seeding.

  • a.
    Perform a complete medium change to 0.5 mL/cm² E8 flex medium without ROCKi.
• 9.
  When hiPSCs reach 80% confluence (Figure 2) split using Versene/EDTA (0.02%).

  • a.
    Bring matrigel hESC-Qualified Matrix-coated cell culture ware to room temperature (around 1h) for replating.
  • b.
    Remove medium from the cells.
  • c.
    Wash cells once with 2 mL DPBS (−/−) per well of a 6 well plate.
  • d.
    Remove DPBS (−/−).
  • e.
    Add 1 mL Versene/EDTA solution for each well.

    Note: For T75 flask use 5 mL for DPBS (−/−) wash and 3.5 mL Versene/EDTA solution.

    Note: For T175 flask use 10 mL for DPBS (−/−) wash and 8 mL Versene/EDTA solution.

  • f.
    Incubate at RT until the cells start to detach, but without detaching the cells completely.

    Note: Detachment starts typically 3–5 min after adding Versene/EDTA solution.

    Note: If splitting with Versene/EDTA solution causes problems, a split can also be performed using accutase (step 9).

  • g.
    Very carefully, without detaching the cell layer, completely remove the Versene/EDTA solution.
  • h.
    Work well by well to prevent drying out.
  • i.
    Collect hiPSCs at the bottom of the plate by tilting the plate sideways.
  • j.
    Pipet them carefully up- and down 1–2 times to triturate the colony pieces into fragment sizes of ∼50–100 cells.
  • k.
    Add E8 flex medium to cell suspension.
  • l.
    Transfer the suspension carefully into the new wells.
  • m.
    Distribute evenly before placing into incubator.

    Note: If not sure about the splitting ratio, it is recommended to plate 2–3 different densities and adjust these for individual lines. A typical splitting ratio of adapted hiPSC lines in E8 Flex is 1:10.

  • n.
    Change the medium one day later to typically 0.5 mL/cm² if feeding again 2 days later or 0.7 mL/cm² if feeding again 3 days later.

    CRITICAL: Adjust medium amounts if needed. A sign of exhausted medium is a color change to orange/yellow. Metabolic products acidify the medium, causing the phenol red indicator in the medium to turn from red to yellow.

• 10.
  To initiate neural induction, harvest hiPSCs as single cells with accutase.

  • a.
    Prewarm accutase supplemented with 10 μM ROCKi.
  • b.
    Wash hiPSCs with 1.5 mL DPBS (−/−) to each 6 well.
  • c.
    Add 2 mL accutase to each 6 well.
  • d.
    Incubate for 15–20 min at 37°C.
  • e.
    Carefully detach the cell layer by pipetting.
  • f.
    Dilute cell suspension at least 5-fold in N2B27 medium.
  • g.
    Centrifuge at 300 x g for 5 min at room temperature.
  • h.
    Discard supernatant.
  • i.
    Add E8 flex medium supplemented with 10 μM ROCKi.
  • j.
    Resuspend the cells.
  • k.
    Calculate cell count (live dead stain/counting).
  • l.
    Dilute cell suspension.
  • m.
    For neural induction plate 36,000 cells/cm² (Day −1).
• 11.
  Freezing of hiPSCs.

  • a.
    Detach hiPSCs of running cell culture with accutase supplemented with 10 μM ROCKi (6 well 1.5 mL; T175 8 mL).
  • b.
    Dilute cell suspension at least 5-fold in E8 flex medium.
  • c.
    Centrifuge at 300 x g for 5 min at room temperature.
  • d.
    Prepare fridge cold E8 flex medium with 10% DMSO and 10 μM ROCKi (freezing medium).
  • e.
    Discard centrifugation supernatant.
  • f.
    Carefully resuspend the cell pellet in freezing medium (5–7 triturations).
  • g.
    Swiftly aliquot the cell suspension in cryopreservation vials.
  • h.
    Place cryopreservation vials in a controlled cooling rate container.
  • i.
    Freeze them at −80°C.
  • j.
    After one day, transfer cryovials to liquid nitrogen storage.

Note: Instead of N2B27 medium, old E8 Flex medium that is not suitable for culture can be used for centrifugation steps alternatively. Centrifuging in just basal medium (such as DMEM/F12) is not recommended, as it will yield to lower recovery.

Figure 2.

Representative picture of 80% confluent hiPSC culture

Scale bar: 200 μm.

Key resources table

REAGENT or RESOURCE	SOURCE	IDENTIFIER
Chemicals, peptides, and recombinant proteins
Ascorbic Acid	Sigma	A4544
CHIR99021	R&D	4423
Dibutyryl cyclic adenosine monophosphate	Sigma	D0627-1MG
Doxycycline hydrochloride	Sigma	D3072-1ML
IWP2	Tocris	3533
Laminin (mouse)	Merck	114956-81-9
LDN193189	Stemgent	04-0074
Mitomycin C	Sigma	7231816
PD0325901	Tocris	4192
Recombinant Human BDNF Protein	R&D	11166-BD
Recombinant Human GDNF Protein	R&D	212-GD
Retinoic acid	Merck	R2625-50MG
RO4929097	Roche	RO4929097
SAG dihydrochloride	Merck	SML1314
Y-27632	Calbiochem	688000
Experimental models: Cell lines
BIOMEDi001-A (Coriell Fibroblast AG08379)	BioMedX (edited at Bioneer)	N/A
BIOMEDi004-A (Coriell Fibroblast AG08125)	BioMedX (edited at Bioneer)	N/A
BIOMEDi005-A (Coriell Fibroblast AG06869)	BioMedX (edited at Bioneer)	N/A
BIONi010-C-13	Available at EBiSC (www.ebisc.org)	EBiSC Bank acknowledges Bioneer A/S as the source of human induced pluripotent stem cell line BIONi010-C-13 which was generated with support from EFPIA companies and the European Union (IMI-JU)
iPSC0028 MAPT KO	Sigma (edited at Bioneer and internal edit)	N/A
iPSC0028 MAPT TM (iPSC0028 with MAPT P301S/E10+14/E10+16, all biallelic)	EBiSC (edited at Bioneer and internal edit)	N/A
Other
Accutase	Thermo Fisher	A11105-01
Aluminum heating/cooling block	Merck	Z743486-1EA
B27 Plus Neuronal Culture System (consists of Neurobasal Plus Medium and B27Plus supplement 50x)	Thermo Fisher	A3653401
B27 Supplement without VitA (50x)	Thermo Fisher	12587-010
CELL CULTURE MICROPLATE, 96 WELL, PS, F-BOTTOM	Greiner	655090
Corning matrigel Growth Factor Reduced (GFR) Basement Membrane Matrix, LDEV-free, 10 mL	Corning	354230
Corning matrigel hESC-Qualified Matrix, LDEV-free, 5 mL	Corning	354277
Corning® CoolCell™ FTS30	Merck	CLS432006-1EA
DMEM/F12 (+L-glut and Hepes)	Thermo Fisher	11330-057
DMSO for cell culture	ApliChem	A3672
DPBS without Mg2+ and Ca2+ (−/−)	Thermo Fisher	14190-169
Essential 6 medium	Thermo Fisher	A1516401
Essential 8 flex medium Kit	Thermo Fisher	A2858501
Falcon® 96-well Black/Clear Flat Bottom TC-treated Imaging Microplate with Lid	Corning/Falcon	353219
GlutaMAX	Thermo Fisher	21103049
IDT for Illumina RNA UD Indexes Set A, B, C	Illumina	20040553, 20040554, 20040555
Illumina Stranded mRNA Prep, Ligation	Illumina	20040534
Lonza™ BioWhittaker™ Versene™ (EDTA), 0.02%	Thermo Fisher	BW17711E
N2 Supplement (100x)	Thermo Fisher	17502-048
Neurobasal medium	Thermo Fisher	21103049
NextSeq 500/550 High Output Kit v2.5	Illumina	20024906
Nunc 6-well plate	Thermo Fisher	140675
Penicillin/Streptomycin	Thermo Fisher	15140-122
Poly-L-ornithine solution (0.01%)	Sigma	P4957
Synth-a-Freeze Cryopreservation medium	Thermo Fisher	A1254201
T175 flask	Greiner	660175
Taqman assay GAPDH	Thermo Fisher	Hs99999905_m1
Taqman assay MAPT	Thermo Fisher	Hs00902192_m1
Taqman assay PAX6	Thermo Fisher	Hs01088114_m1
Taqman assay PPIA	Thermo Fisher	Hs04194521_s1
Taqman assay RPL13	Thermo Fisher	Hs00744303_s1
Taqman assay TBR1	Thermo Fisher	Hs00232429_m1

Materials and equipment

N2B27 medium

Ingredient	Stock concentration	Dilution	Final concentration	Amount
DMEM/F12	1x	1:2	0.5x	500 mL
Neurobasal medium	1x	1:2	0.5x	500 mL
N2 supplement	100x	1:200	0.5x	5 mL
B27 supplement (w/o vitamin A)	50x	1:100	0.5x	10 mL
Penicillin/Streptomycin	10,000 U/mL/10,000 μg/mL	1:100	10 U/mL/10 μg/mL	10 mL
Glutamax	100 mM	1:200	0.5 mM	5 mL

Neural maturation medium - (NMM-)

Name	Stock concentration	Dilution	Final concentration	Amount
Neurobasal medium	1x	-	1x	500 mL
B27 Supplement without Vitamin A	50x	1:50	1x	10 mL
GlutaMax	100 mM	1:100	1 mM	5 mL
Penicillin/Streptomycin	10,000 U/mL/10,000 μg/mL	1:100	10 U/mL/10 μg/mL	5 mL
BDNF	50 μg/mL	1:5,000	10 ng/mL	100 μL
GDNF	50 μg/mL	1:10,000	5 ng/mL	50 μL
db-cAMP	100 mM	1:500	200 μM	1 mL
Ascorbic Acid	200 mM	1:1,000	200 μM	500 μL
Laminin	∼1 mg/mL	batch-dependent	2 μg/mL	batch-dependent

Neural maturation medium + (NMM+)

Name	Stock concentration	Dilution	Final concentration	Amount
Neurobasal (Plus) medium	1x	-	1x	500 mL
B27 (Plus) Supplement	50x	1:50	1x	10 mL
GlutaMax	100 mM	1:100	1 mM	5 mL
Penicillin/Streptomycin	10,000 U/mL/10,000 μg/mL	1:100	10 U/mL/10 μg/mL	5 mL
BDNF	50 μg/mL	1:5,000	10 ng/mL	100 μL
GDNF	50 μg/mL	1:10,000	5 ng/mL	50 μL
db-cAMP	100 mM	1:500	200 μM	1 mL
Ascorbic Acid	200 mM	1:1,000	200 μM	500 μL
Laminin	∼1 mg/mL	batch-dependent	2 μg/mL	batch-dependent

Volumes for different size of culture dishes

Size	Surface area (cm2)	PLO volume	DPBS (−/−) wash-volume	Matrigel solution volume
384 well	0.056	10 μL	50 μL	20 μL
96 well	0.34	50 μL	150 μL	100 μL
48 well	0.95	125 μL	500 μL	250 μL
24 well	2	250 μL	750 μL	500 μL
12 well	3.8	0.5 mL	1 mL	1 mL
6 well	9.6	1 mL	2 mL	2 mL
T75 flask	75	8 mL	15 mL	12 mL
T175 flask	175	15 mL	25 mL	22 mL

Preparation of small molecules

Small molecule	Referred as	Stock concentration	Final concentration	Solvent
Ascorbic Acid	AA	200 mM	200 μM	sterile, distilled water, sterile filtered
CHIR99021	CHIR	10 mM	500 nM – 10 μM	DMSO
Dibutyryl cyclic adenosine monophosphate	db-cAMP	100 mM	500 μM	sterile, distilled water, sterile-filtered
Doxycycline Hydrochloride	DOX	10 mM	1 μM – 2 μM	DMSO
IWP2	IWP2	5 mM	1 μM	DMSO
LDN193189	LDN	0.5 mM	500 nM	DMSO
Mitomycin C	Mitomycin C	1 mg/mL	1 μM	sterile, distilled water
PD0325901	PD	10 mM	1 μM	DMSO
RO4929097	RO	2 mM	200 nM – 500 nM	DMSO
SAG dihydrochloride	SAG	10 mM	250 nM - 500 nM	DMSO
Y-27632	ROCKi	5 mM	10 μM	sterile, distilled water, sterile-filtered
Retinoic acid	RA	10 mM	1 μM	DMSO

Preparation of proteins

Protein	Referred as	Stock concentration	Final concentration	Preparation
Laminin L2020 (mouse)	Laminin	Depends on batch, ∼ 1 mg/mL	2 μg/mL	Ready to use
Recombinant Human BDNF Protein	BDNF	50 μg/mL	10 ng/mL	first 100 μg/mL DPBS, then diluted in DPBS (−/−) with 0.1% BSA
Recombinant Human GDNF Protein	GDNF	50 μg/mL	5 ng/mL	first 100 μg/mL DPBS, then diluted in DPBS (−/−) with 0.1% BSA

Step-by-step method details

Neural induction for differentiation of telencephalic neurons

Timing: 8 days

Here we describe a method for the neural induction of telencephalic NPC via BMWi pre-patterning followed by NGN2 overexpression. Day 0 to day 6 represent the pre-patterning step before two days (day 7 to day 8) NGN2 overexpression until replating or cryoconservation.

Note: The weekdays suggested below are for the best survival of the cells in the weekend-free protocol.

Day −1—Wednesday

• 1.
  Thawing of hiPSCs.

  • a.
    Bring coated cell culture ware to room temperature.
  • b.
    Bring the needed amount of E8 Flex medium for plating to at least room temperature, up to 37°C.
  • c.
    Thaw hiPSC vial in water bath at 37°C.

    CRITICAL: Make sure that a small ice crystal is left in the vial. Then start diluting cells in fridge-cold E8 flex medium.

  • d.
    Disinfect the vial.
  • e.
    Immediately transfer under a safety cabinet for next steps.
  • f.
    Dilute hiPSC suspension in fridge cold E8 flex medium (at least 5-fold).
  • g.
    Centrifuge at 300 x g for 5 min.
  • h.
    Discard supernatant.
  • i.
    Dilute cell pellet in E8 flex medium supplemented with 10 μM ROCKi.
  • j.
    Count the cells.
  • k.
    Remove matrigel solution from cell culture ware.
  • l.
    Seed 36,000 cells/cm² in 0.5 mL/cm² E8 flex medium supplemented with 10 μM ROCKi.

    Note: For neural induction, use hiPSC dissociated with accutase and stored as single cells (General hiPSC culture steps 9, 10).

    Note: If working from a running culture: seed 30,000 cells/cm².

    Note: For T175 seed 20×10ˆ6 hiPSCs.

Day 0—Thursday

• 2.
  Start of neuronal induction (Figure 3).

  • a.
    Completely remove hiPSC medium.
  • b.
    Add 0.15 mL/cm² E6 medium for washing.
  • c.
    Remove E6 medium.
  • d.
    Add 0.7 mL/cm² E6 medium supplemented with 1 μM IWP2, 1 μM PD0325901 and 500 nM LDN193189.

Note: Washing step with E6 is performed to remove FGFs from E8 flex medium and dead cells from plating.

Note: For T175 wash with 20 mL E6 medium and add 110 mL.

Figure 3.

Differentiation of hiPSCs into telencephalic neurons

3.5·10⁶ hiPSCs were seeded in a 6 well format for neural induction. At day 8 1.5·10⁶ NPCs were replated in one 6 well. For 7 days, neurons were matured in NMM-, then medium was changed to NMM+ for further maturation. Scale bar: 200 μm.

Day 1—Friday

• 3.
  Completely remove the medium.

  • a.
    Slowly add 1.2 mL/cm² N2B27 medium with 1 μM IWP2, 1 μM PD0325901 and 500 nM LDN193189 (troubleshooting 1).

Note: Try to change medium as late as possible, to ensure cells will endure the culture without medium change over the weekend (64 h) (troubleshooting 2).

CRITICAL: If you use 6 well plates for neural induction, 12 mL per well is a high volume. Add medium and handle plate carefully to not spill medium.

Note: If using cell culture flasks, ensure flasks are deep enough to apply sufficient medium (see also key resources table).

Note: For T175 use 210 mL medium.

Day 4—Monday

• 4.
  Completely remove the medium (Figure 3).

  • a.
    Slowly add 1.2 mL/cm² N2B27M with 1 μM IWP2, 1 μM PD0325901 and 500 nM LDN193189 (troubleshooting 2, troubleshooting 3).

CRITICAL: Try to change medium early in the morning, to avoid starvation of NPC after the weekend (around 64 h).

Note: For T175 use 210 mL medium.

Day 5—Tuesday

• 5.
  Completely remove the medium.

  • a.
    Slowly add 1.2 mL/cm² N2B27 medium with 1 μM IWP2, 1 μM PD0325901 and 500 nM LDN193189.

Note: For T175 use 210 mL medium.

Day 6—Wednesday

• 6.
  Completely remove the medium (Figure 3).

  • a.
    Add 0.2 mL/cm² N2B27 medium for washing.
  • b.
    Remove the medium and slowly add N2B27 medium supplemented with 2 μg/mL doxycycline, 1.2 mL/cm² to start overexpression of NGN2.

CRITICAL: If cell detachment is observed, add 1–2 μg/mL Laminin to prevent further detachment.

Note: For T175 use 210 mL medium.

Day 7—Thursday

• 7.
  Completely remove the medium.

  • a.
    Add 1.2 mL/cm² N2B27 medium with 2 μg/mL doxycycline.

Note: In case of replating on Friday prepare PLO/matrigel-coated cell culture ware.

Note: For T175 use 210 mL medium.

Replating of NPCs

Timing: 1 h

The following paragraph describes the procedure of NPC replating after neural induction.

Note: NPCs are replated at a lower density before maturation to ensure they have sufficient space and optimal conditions for their differentiation and neurite outgrowth. A lower cell density prevents the cells from hindering each other's growth, promotes better cell distribution, and enables efficient maturation into mature, functional neurons. Individually spaced neurons are better suited for imaging-based readouts.

Note: Cell densities applied here are recommendations for imaging-based applications or extraction of RNA or protein. Other densities can be applied, too, but need to be evaluated for long-term stability of the culture; potentially medium supply needs to be adjusted for high densities.

Day 8—Friday

• 8.
  Prepare replating medium and prewarm plates.

  • a.
    NMM-supplemented with 10 μM ROCKi, 500 nM RO and 2 μg/mL doxycycline, warm to room temperature, up to 37°C.
  • b.
    Remove the PLO/matrigel-coated plates from 4°C and place at room temperature for around 1 h before plating cells.
• 9.Pre-incubate NPC with 10 μM ROCKi for 60 min prior to detachment.
• 10.
  Completely remove the medium.

  • a.
    Wash briefly with 0.2 mL/cm² DPBS (−/−).
• 11.Add 0.15 mL/cm² pre-warmed accutase supplemented with 10 μM ROCKi.
• 12.
  Incubate at 37°C in the incubator for at least 15 min.

  Note: Cells should be easily pipetted with 1000 μl tip into a complete single cell suspension. Incubate for 5 min longer if needed.

  • a.
    Carefully pipet the cells into single cell suspension with a pipet.
  • b.
    Collect the cells in a conical tube containing 5-fold amount of N2B27 medium compared to accutase.

    Note: Accutase cannot be inhibited by medium and needs to be diluted out.

• 13.For imaging applications, it is recommended to create a single cell suspension by using a cell strainer (40 μm) to remove cell clumps.

Note: The single cell suspension and low plating density enable separation of neuronal cell bodies and easier segmentation in imaging-based assays.

• 14.Centrifuge 5 min at 300 x g to collect the cells.
• 15.
  Replating of NPC.

  • a.
    Resuspend cells in replating medium by carefully pipetting until the pellet is fully resuspended.
  • b.
    Count cells and calculate desired cell numbers.
  • c.
    Replate cells at 27,000 cells/well of a 96 well plate in 150 μL replating medium immediately after removal of coating solution from the recipient PLO/matrigel coated plate.
  • d.
    For a 6 well plate for RNA or protein analysis, recommended plating density is 1×10ˆ6 cells/well in 2 mL replating medium.

Note: Pre-incubation with ROCKi protects the cells and is recommended especially for large cultures.

CRITICAL: Be careful in handling the wells prior to harvesting, as cells could detach easily when removing medium from NPC.

CRITICAL: Do not wash after removing the matrigel coating from the PLO/matrigel coated plate. Do not let the coating dry out because that changes the matrix stiffness which prevents cell attach.

Freezing of NPCs

Timing: 1 h

Here we describe a method to freeze and store large batches of NPC.

Day 8—Friday (alternative)

• 16.
  Freezing of NPC.

  • a.
    Prepare freezing medium and label cryopreservation vials.

    • i.
      Precooled to fridge temperature (4°C) Synth-a-Freeze supplemented with 10 μM ROCKi (referred to as freezing medium).
  • b.
    Follow step 10 to 14.
  • c.
    Carefully take up cells in freezing medium.

    • i.
      Up to 20×10ˆ6 NPCs/mL of freezing medium can be frozen.
  • d.
    Make sure to resuspend the pellet completely in freezing medium.
  • e.
    Count and calculate desired cell numbers.
  • f.
    Add 1 mL cell suspension to each cryovial.

    • i.
      Freeze cryopreservation vials in a cool cell controlled freezing container at −80°C.
  • g.
    The next day, transfer the cells in dry ice to liquid nitrogen storage for long term storage.

Note: Work swiftly to transfer cells in freezing medium to cryopreservation vials and to transfer cryopreservation vials to −80°C freezer to avoid DMSO-induced toxicity. Transfer the cryopreservation vials to liquid nitrogen no more than 3 days after placing them at −80°C.

Note: It is also possible to use N2B27 medium supplemented with 10 μM ROCKi and 10% DMSO as freezing medium. However, lower viability is expected after the thawing process with this procedure.

Note: When cells are counted in freezing medium, they can appear “blurry” in the counting process. Dilution of the sample for counting with N2B27 (5-fold) is recommended in that case.

Thawing from frozen NPC stock

Timing: 1 h

In the following steps, we describe a method how the NPCs can be thawed and seeded with the highest possible viability.

• 17.
  Prepare media and plates.

  • a.
    Prepare fridge cold (4°C) N2B27 medium.
  • b.
    Prepare replating medium: NMM-supplemented with 10 μM ROCKi, 500 nM RO and 2 μg/mL doxycycline, warm to at least room temperature, up to 37°C.

    • i.
      BMWi motorneurons and BMWi DRG will be plated in and cultured throughout the culture time in NMM+ with supplements.
  • c.
    Around 1 h before start thawing, place PLO/matrigel-coated plates at room temperature.
• 18.Transport cells on dry ice from liquid nitrogen storage.
• 19.Thaw cells in water bath at 37°C until only a small piece of ice is left.
• 20.Dilute cell suspension 5-fold in fridge-cold N2B27.
• 21.Centrifuge at 300 x g for 5 min at room temperature.
• 22.
  Resuspend the cells in replating medium.

  • a.
    Plate cells at 27,000 cells/well of a 96 well plate in 150 μL plating medium.
  • b.
    For a 6 well plate for RNA or protein analysis, recommended plating density is 1×10ˆ6 cells/well in 4 mL plating medium.
• 23.Continue as if started at day 8 of the protocol (replating day) for the day of thawing and plating.

Note: Centrifugation at 200 x g can slightly improve survival but will generate a less dense pellet that needs to be handled with more care.

Maturation of telencephalic neurons

Timing: At least 14 days up to 4/6 weeks

This step describes how the maturation of NPC into neurons is carried out.

Day 13—Wednesday

• 24.
  Perform a 50% medium change with NMM-supplemented with 200 nM RO and 10 μM ROCKi and final concentration of 0.15 μg/mL Mitomycin C for mitotic inactivation of single remaining undifferentiated progenitor cells.

  • a.
    Prepare medium with 200 nM RO, 10 μM ROCKi and 0.3 μg/mL Mitomycin C, to obtain a final concentration of 0.15 μg/mL Mitomycin C after the 50% medium change.

Note: 50% medium change means to remove half of the medium and add the same amount of fresh medium as described in step 24 e.g. remove 75 μL per 96 well and add 75 μL fresh medium to each well.

Note: Mitomycin C treatment can be omitted for short term culture (∼7 days), but is recommended for long term culture for most stable results. It is a DNA crosslinking agent which treatment leads to cell cycle arrest in the proliferating cells and induces apoptosis.²² It ensures complete removal of even single proliferative cells (such as remaining NPCs), which could lead to culture overgrowth in long term cultures.

Day 15 —Friday

• 25.Perform a 75% medium change to NMM+ or use NMM+ for further maturation.
• 26.Continue with a feeding schedule of 50% NMM (- or +) medium change every 4–5 days (Mon-Wed-Fri-Mon,…) or, alternatively, twice per week (for example Mo-Fr-Mo,…) (Figure 3) (troubleshooting 4, troubleshooting 5).

Note: It is also possible to cultivate neurons further in NMM-, if required for downstream applications.

Note: In general, it is possible to grow neurons in NMM-. But a switch to NMM+ after 7 days is recommended, as the neurons mature faster (assessed by their electrophysiological activity) and neurons detached less easily during PFA (paraformaldehyde) fixation.

Motor neuron patterning

Timing: 6 days

The following steps show a method to differentiate hiPSC with the BMWi pre-patterning by adding further small molecules to motorneurons.

Day 0—Thursday

• 27.
  Start of neuronal induction with additional motor neuron patterning (start after step 1 and skip step 2 to 6).

  • a.
    Completely remove the medium.
  • b.
    Add 0.15 mL/cm² E6 medium.
  • c.
    Remove the E6 medium.
  • d.
    Add 0.7 mL/cm² E6 medium supplemented with 1 μM IWP2, 1 μM PD0325901, 500 nM LDN193189 and 250 nM SAG.

Note: For T175 wash with 20 mL E6 medium and add 110 mL.

Day 1—Friday

• 28.
  Completely remove the medium.

  • a.
    Slowly add 1.2 mL/cm² N2B27 medium with 1 μM IWP2, 1 μM PD0325901, 500 nM LDN193189, 250 nM SAG and 1 μM RA.

Note: Try to change medium as late as possible, to ensure cells will endure the culture without medium change over the weekend (64 h). The amount of medium mentioned here covers about 64 h (troubleshooting 2).

CRITICAL: 12 mL is a high volume for 6 well. Add medium and handle plate carefully to not spill medium.

Note: For T175 use 210 mL medium.

Day 4—Monday

• 29.
  Completely remove the medium.

  • a.
    Slowly add 1.2 mL/cm² N2B27M with 1 μM IWP2, 1 μM PD0325901, 500 nM LDN193189, 250 nM SAG and 1 μM RA.

Note: When changing the medium completely, work quickly to avoid the cells drying out or suffering.

Note: For T175 use 210 mL medium.

CRITICAL: Try to change medium early in the morning (If you change the medium Friday at 5 PM, the medium change on Monday should be around 9 AM), to avoid starvation of NPCs (troubleshooting 2).

Day 5—Tuesday

• 30.
  Completely remove the medium.

  • a.
    Slowly add 1.2 mL/cm² N2B27M with 1 μM IWP2, 1 μM PD0325901, 500 nM LDN193189, 250 nM SAG and 1 μM RA.
• 31.Next day continue with step 7 (addition of doxycycline).

Note: For T175 use 180 mL medium.

Note: BMWi motorneurons should be replated in NMM+ as replating medium supplemented with ROCKi, RO, DOX (step 17). Continue culture in NMM+ as indicated in steps 24–26 above.

Ventral midbrain patterning

Timing: 4 days

Here we explain how BMWi mDAN NPC can be differentiated by adding further small molecules additional to BMWi pre-patterning.

Day 0—Thursday

• 32.
  Start of neuronal induction with additional ventral midbrain patterning (start after step 1 and skip step 2 to 6).

  • a.
    Completely remove the medium.
  • b.
    Add 0.15 mL/cm² E6 medium to each well.
  • c.
    Remove the E6 medium.
  • d.
    Add 0.7 mL/cm² E6 medium supplemented with 1 μM IWP2, 1 μM PD0325901, 500 nM LDN193189, 500 nM SAG and 0.75 μM CHIR.

Note: Washing step with E6 is performed to remove FGFs from E8 flex medium.

Note: For T175 wash with 20 mL E6 medium and add 110 mL.

Day 1—Friday

• 33.
  Completely remove the medium.

  • a.
    Slowly add 1.2 mL/cm² N2B27 medium with 1 μM IWP2, 1 μM PD0325901, 500 nM LDN193189, 500 nM SAG and 1.5 μM CHIR.
• 34.Three days later (Monday) continue with step 7 (addition of doxycycline).

Note: mDAN NPCs should be replated in NMM- as replating medium supplemented with ROCKi, RO, DOX (step 17). Continue culture in NMM- with possible switch to NMM+ as indicated in steps 24–26 above.

Note: Try to change medium as late as possible (around 5 PM), to ensure cells will endure the culture without medium change over the weekend (64 h) (troubleshooting 2).

CRITICAL: Add medium and handle plate carefully to not spill medium.

Note: For T175 use 210 mL medium.

Sensory neuron patterning (sensBMi)

Timing: 4 days

The following steps show a method for differentiating sensory NPCs using a modified BMWi protocol.

Day 0—Thursday

• 35.
  Start of neuronal induction with additional ventral midbrain patterning (start after step 1 and skip steps 2 to 6).

  • a.
    Completely remove the medium.
  • b.
    Add 1.5 mL/cm² E6 medium to each well.
  • c.
    Remove the E6 medium.
  • d.
    Add 7 mL/cm² E6 medium supplemented with 1 μM IWP2, 1 μM PD0325901, 500 nM LDN193189, 1 μM RA and 3 μM CHIR.

Note: Washing step with E6 is performed to remove FGFs from E8 flex medium.

Note: For T175 wash with 20 mL E6 medium and add 110 mL.

Day 1—Friday

• 36.
  Completely remove the medium.

  • a.
    Slowly add 1.2 mL/cm² N2B27 medium with 1 μM PD0325901, 500 nM LDN193189, 1 μM RA and 3 μM CHIR.
• 37.Three days later (Monday) continue with step 7 (addition of doxycycline).

Note: SensBMi should be replated in NMM+ as replating medium supplemented with ROCKi, RO, DOX (step 17). Continue culture in NMM+ as indicated in steps 24–26 above.

Expected outcomes

With the BMWi protocol, it is possible to differentiate a defined population of telencephalic neurons that express e.g., FOXG1, MAPT, and TBR1 (Figure 4A). Neuroectoderm precursor marker PAX6 is expressed at similar levels in all samples during early differentiation. The pan-neuronal marker MAPT and the cortical excitatory marker TBR1 are present after neural maturation on day 20. By pre-differentiating hiPSCs for 4 or 6 days with BMP, MEK, and WNT inhibition (BMWi) followed by NGN2 overexpression, consistent differentiation results were achieved across 6 different hiPSC donor lines. Repeated differentiations of the same hiPSC line produced consistent gene expression results (Figure 4B). An RNA sequencing experiment was conducted across all stages of the BMWi protocol and compared with iNGN2 neurons, which were derived solely from 2 days of NGN2 expression. Comparison based on markers for various cell types and regions showed that BMWi pre-patterning resulted in a stronger telencephalic population than iNGN2 differentiation (Figure 4C). NPCs of the BMWi protocol expressed regional cortical genes early, with enhanced expression after NGN2 overexpression. BMWi NPCs exhibited significantly higher expression of cortical genes than iNGN2 NPCs, and BMWi neurons showed stronger expression of cortical/telencephalic genes compared to iNGN2 neurons. The transition from NMM-to NMM+ 7 days after final plating did not alter gene expression in BMWi neurons (see step 36). Generally, BMWi neurons displayed stronger expression of glutamatergic than GABAergic genes. The resulting neuron cultures are practically devoid of any non-neural cells after NGN2 induction, the recommended mitotic inactivation step will eliminate all non-neuronal cells, allow long-term cultures of 6 weeks or more after plating and enable (co-)culture experiments in which a pure neuron population is required.

Figure 4.

Transcriptome analysis of BMWi neurons

(A) hiPSC were differentiated according to BMWi protocol (6 days BMWi pre-differentiation, 2 days NGN2 overexpression). Gene expression of relevant marker genes were evaluated via qRT-PCR. (results are shown as mean±SEM, n = 5 hiPSC lines (● =BIONi010-C-13, ▲ = BIOMEDi004-A, ■ = iPSC0028 MAPT TM, ♦ = BIOMEDi001-A, ★ = BIOMEDi005-A, + = iPSC0028 MAPT KO), housekeeping genes: PPIA, RPL13, GAPDH) (B) Results of qRT-PCR experiment. Comparison of 3 independent differentiations of one hiPSC line (results are shown as mean±SEM, n = 1 hiPSC line (● = BIONi010-C-13)).

(C) Bulk RNA sequencing of hiPSCs, BMWi NPC day 4, BMWi NPC day 6, BMWi NPC day 8, 14 days matured BMWi neurons replated in NMM- (BMWi NMM – final) and in NMM-replated BMWi neurons switched after 7 days to NMM+ medium (BMWi NMM - + final). The BMWi differentiation was compared with iNGN2 NPC (2 days doxycycline induced overexpression of NGN2) and 14 days matured iNGN2 neurons replated in NMM- (iNGN2 NMM - final) or NMM+ (iNGN2 NMM + final). Bulk RNA sequencing was performed with Illumina NextSeq 550 System with NextSeq 500/550 High Output Kit v2.5. Library preparation was performed with Illumina Stranded mRNA Prep, Ligation and IDT for Illumina RNA UD Indexes Set A, B, C. The denaturation was carried out according to protocol A (Denature and Dilute Libraries Guide, Illumina) (n = 3 hiPSC lines (BIONi010-C-13, BIOMEDi004-A, iPSC0028 MAPT TM).

In addition to the BMWi telencephalic neuron differentiation protocol, patterning protocols for ventral midbrain neurons, motorneurons, and sensory neurons are provided (Figure 5A). All protocols are weekend-free and easy to perform even for less experienced scientists, as only one replating step is required. On the day of the final replating of the respective protocol, it is also possible to freeze the NPCs in large batches for use in high-throughput screening assays or similar. Viability of the NPC at this stage of all protocols is around 90% (BMWi: 91.85±1.34; BMWi ventral midbrain: 90.87±1.10; BMWi motorneurons: 89.23±2.55; sensBMi: 92.27±1.58) (Figure 5B). The BMWi protocol and all patterning variants thus represent an easily accessible source of defined neuronal populations.

Figure 5.

Protocols to pattern different neuronal subtypes and expected outcomes after NPC thawing

(A) Schematic representation of BMWi (telencephalic neuron – 6 days pre-differentiation), BMWi ventral midbrain, BMWi motorneuron and sensBMi protocol. All NPC are suitable for freezing on day of final replating (day 6 or 8 of respective protocol).

(B) Viability of thawed NPC of BMWi protocol and patterned BMWi neurons (n = 3–6, results are shown as mean±SEM).

Limitations

The BMWi protocol represents a simple protocol for differentiating hiPSC-derived neurons. However, one limitation is the availability of genetically modified hiPSCs required for differentiation. The methods presented require an hiPSC line capable of controlling NGN2 overexpression through the addition of doxycycline.^1,23,24 Other strategies for the overexpression of NGN2, such as lentiviral delivery, need to be assessed. Furthermore, the neurons are more prone to clustering due to only one (re-)plating step. This can be mitigated by utilizing rich coating (as shown here: 77 μg/mL matrigel) and by using a cell strainer during the replating step to ensure seeding of single cells. Clustering is also exacerbated if the mitomycin C inactivation step is omitted.

Troubleshooting

Problem 1

Cell death on day 1 of the protocol.

Potential solution

Some hiPSC lines do not tolerate the transition to the nutrient-poor E6 medium well. In this case, a medium consisting of 1:1 N2B27/E6 medium dilution can improve hiPSC viability. Addition of 5 μM ROCKi can further assist the differentiation of sensitive cell lines.

Problem 2

Cell death after weekend at NPC stage on day 4 to 6 of protocol.

Potential solution

The cell densities obtained by the protocol are very high. Since some cell lines proliferate faster than others, it is advisable to adjust the cell density down for large batches so that the medium in T175 is sufficient to last over the weekend. E.g. one can use 20×10ˆ6 hiPSC to start the differentiation on day −1. It is recommended to perform the medium change as late as possible on Fridays (day 1) e.g., at 5 PM and early on Mondays (day 4) e.g., 9 AM. Try to culture the cells for a maximum of 64 h without changing the medium. Alternatively, an additional medium change on the weekend can be integrated but should not be required.

Problem 3

Detachment of NPC layer of day 4 to day 6 of the BMWi protocol.

Potential solution

At day 4 to 6 it is beneficial to handle cells carefully and just pipet medium as slowly as possible, the medium can be added over the walls of the flask by tilting it, to avoid flushing over the cell layer. Also, a pipet can be used to remove the medium instead than vacuum. Additional laminin (1 μg/mL) in N2B27 can help to avoid detachment.

Problem 4

Clustering of neurons after 2–4 weeks of maturation.

Potential solution

It is recommended to use a cell strainer (40 μm) after harvesting the NPC with accutase. After digestion with accutase, cell clusters remain in the suspension, which are removed. By isolating the NPCs, they grow individually and can thus be more easily segmented in imaging-based assays. Additionally, ROCKi can be added to the NMM for up to 5 days after final replating. The plates mentioned in this protocol have been evaluated to provide best possible adherence, alternative plates should be carefully evaluated. If a very specific plate type is required, the coating procedure might need to be optimized and further (pre-)coatings, such as PEI or PDL, as well as laminin should be assessed. Mitotic inactivation increases stability of the cell layer, NMM+ medium is more supportive than NMM-medium for long term cultures. Telencephalic neurons and mDANs (plated in NMM-) can be switched to NMM+ medium one week after plating. During medium changes, work as gentle as possible, avoid complete medium changes, as it can detach the cell layer.

Problem 5

Detachment of neurons during PFA fixation.

Potential solution

To fix, remove only 50% of the medium from the neurons and add 8% formaldehyde solution in double concentration in DPBS with Ca²⁺ and Mg²⁺. Incubate for 15 min at room temperature. It is important to use DPBS with Ca²⁺ and Mg²⁺ for fixation, storage of fixed plates and for the staining procedure to avoid detachment of cells.

Resource availability

Lead contact

Further information and requests for resources and reagents should be directed to and will be fulfilled by the lead contact, Peter Reinhardt (peter.reinhardt@abbvie.com).

Technical contact

Technical questions on executing this protocol should be directed to and will be answered by the technical contact, Carina Habich (carina.habich@abbvie.com).

Materials availability

This study did not generate new unique reagents.

Data and code availability

The data that support the findings of this study are openly available via GEO (https://www.ncbi.nlm.nih.gov/geo/), under accession numbers GSE253508, GSE253509, and GSE279590.

Acknowledgments

We would like to thank AbbVie employees Elke Käfer and Anja Fink for their technical assistance to provide frozen stocks of hiPSCs. We would also like to thank Prof. Dr. Martin Grininger and Prof. Dr. Jasmin Hefendehl for their supervision. The following figures were created with BioRender.com: graphical abstract and Figures 1 and 5.

Author contributions

Conceptualization, P.R. and C.H.; methodology, P.R. and C.H.; validation, C.H.; formal analysis, C.H. and A.D.; investigation, C.H. and A.D.; data curation, C.H. and A.D.; writing – original draft, P.R. and C.H.; writing – review and editing, C.H., P.R., A.D., J.D.M., and M.C.; visualization, C.H.; supervision, P.R.; project administration, P.R.

Declaration of interests

C.H., A.D., J.D.M., M.C., and P.R. are employees of AbbVie. The design, study conduct, and financial support for this research were provided by AbbVie. AbbVie participated in the interpretation of data, review, and approval of the publication.