Protocol for generating postganglionic sympathetic neurons using human pluripotent stem cells for electrophysiological and functional assessments

Summary

Assessing the development and function of the sympathetic nervous system in diseases on a large scale is challenging. Here, we present a protocol to generate human pluripotent stem cell (hPSC)-derived postganglionic sympathetic neurons (symNs) differentiated via neural crest cells (NCCs), which can be cryopreserved. We describe steps for hPSC replating, NCC replating and cryobanking, and symN differentiation. We then demonstrate the functionality of the hPSC-derived symNs, focusing on electrophysiological activity, calcium flux, and norepinephrine dynamics.

For complete details on the use and execution of this protocol, please refer to Wu et al.^1,2

Graphical abstract

Highlights

• •Steps for generating postganglionic sympathetic neurons (symNs) from hPSCs
• •Instructions on cryopreservation of progenitor neural crest cells
• •Guidance on functional assessments of hPSC-symNs

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

Assessing the development and function of the sympathetic nervous system in diseases on a large scale is challenging. Here, we present a protocol to generate human pluripotent stem cell (hPSC)-derived postganglionic sympathetic neurons (symNs) differentiated via neural crest cells (NCCs), which can be cryopreserved. We describe steps for hPSC replating, NCC replating and cryobanking, and symN differentiation. We then demonstrate the functionality of the hPSC-derived symNs, focusing on electrophysiological activity, calcium flux, and norepinephrine dynamics.

Before you begin

hPSC maintenance

Timing: 7–10 days

The purpose of hPSC maintenance is to culture stem cells such that they remain in a self-renewing, undifferentiated state. For this protocol, stem cells are maintained in chemically defined, feeder-free conditions. At this stage, media conditions and appropriate passaging prevent spontaneous differentiation in the stem cell cultures, allowing for future specific directed differentiation into the cell type of interest, here symNs. Before starting the differentiation, stem cells should exhibit classical, healthy stem cell morphology, including round, dense, flat colonies with bright edges as shown in Figure 1A. To start the differentiation, colonies are fully dissociated and plated with even distribution across the whole plate. The differentiation begins directly at day 0.

• 1.
  Thawing hPSCs.

  • a.
    Prepare one Vitronectin (VTN) coated 100 mm tissue culture dish.

    • i.
      For each 100 mm dish, prepare 7 mL of 1x Dulbecco’s phosphate buffer saline without cations (DPBS) with 0.5 mg/mL VTN.
    • ii.
      Add 7 mL VTN solution to the dish and incubate at room temperature for 1 h.

      Note: If plates will not be used immediately after incubation, remove VTN solution, replace with DPBS and leave at 37°C for up to 5 days.

  • b.
    Thaw cells.

    • i.
      Remove cell vial from liquid nitrogen and gently thaw in 37°C water bath, carefully swirling until thawed.
    • ii.
      Gently transfer thawed hPSCs to a 15 mL conical tube with 10 mL DPBS.
    • iii.
      Centrifuge at 200xg for 5 min.
    • iv.
      Gently aspirate the supernatant, careful not to disturb the cell pellet.
    • v.
      Use a p1000 pipet to resuspend pellet in 1 mL Essential 8 (E8) medium. Once resuspended, add 9 mL of E8 medium.
  • c.
    Plate cells.

    • i.
      Aspirate solution from VTN coated plate.
    • ii.
      Transfer hPSC mixture to the plate. Make sure cells are distributed evenly by gently shaking the plate side-to-side and up-and-down (not in circles).
    • iii.
      Incubate at 37°C in 5% CO₂.
    • iv.
      One day after thawing, aspirate media from plate and replace with 10 mL fresh E8 media. Feed this way every day until the undifferentiated colonies look as in the example in Figure 1A, day 0 (3–4 days).
• 2.
  Splitting hPSCs.

  • a.
    hPSCs will be ready to split when the plate shows observable big, round, bright edged colonies (Figure 1A, day 0). Splitting should occur before colonies begin to grow into one another.
  • b.
    Detach colonies from plate.

    • i.
      Carefully aspirate E8 media from the plate.
    • ii.
      Wash the plate 2x with 10 mL DPBS.
    • iii.
      Aspirate DPBS from plate and add 4 mL 0.25 M Ethylenediaminetetraacetic acid (EDTA).
    • iv.
      Incubate for 2 min at 37°C in 5% CO₂.
  • c.
    Dissociate colonies.

    • i.
      Aspirate EDTA, the colonies should remain sticking to the dish.
    • ii.
      Strongly pipette 10 mL fresh E8 media into the plate to lift the colonies of the surface of the dish.
    • iii.
      Briefly pipette up and down to break up large colonies.
  • d.
    Split hPSCs.

    • i.
      Collect medium and cells in a 15 mL conical tube.
    • ii.
      Depending on confluency, hPSCs should be split by 1:10-1:20. For example, to split by 1:10 in 100 mm dish, combine 1 mL cell mixture with 9 mL fresh E8 medium.
    • iii.
      Plate in 100 mm VTN-coated dish.

Note: Optimal splitting density should be determined for each researcher and cell line independently.

CRITICAL: hPSCs should be passaged a minimum of 2–3 times after thawing before differentiation.

Figure 1.

Differentiation of postganglionic symNs through NCCs using hPSCs

(A) Schematic illustration of the symN differentiation timeline.

(B) Demonstration of BMP4 titration during day 0–2 of differentiation.

(C) Day 14 symN progenitor spheroids derived from fresh or thawed day 10 NCCs.

(D) Early (day 20) and differentiated (day 30+) symNs. Scale bars represent 200 μm.

Coating geltrex plates

Timing: 2 days

Plates coated in Geltrex, a basement membrane matrix, provide the necessary extracellular support for arising NCCs. Plates must be prepared ahead of time.

• 3.Thaw 1 mL Geltrex vial overnight on ice at 4°C.
• 4.
  Dilute Geltrex 1:100 in cold DMEM/F12.

  • a.
    Prepare 99 mL of cold DMEM/F12 in a 150 mL bottle.
  • b.
    Transfer 1 mL Geltrex to DMEM/F12 and mix.
• 5.Coat 6-well plates with 2 mL Geltrex solution per well (or 24-well plates with 1 mL per well).
• 6.Keep overnight at 4°C. If storing for more than 2 days, wrap plates in parafilm. Coated plates can be kept at 4°C for several weeks, as long as they do not dry out.
• 7.Plates will be used to start the differentiation at day 0.

CRITICAL: Work quickly and keep the Geltrex on ice as much as possible.

Coating PO/LM/FN plates

Timing: 2 days

During symN differentiation and maturation, plates coated with Poly-L Ornithine hydrobromide (PO), mouse Laminin-1 (LM), and human Fibronectin (FN) provide matrix support to growing neurons and ensure strong attachment to culture dishes. PO/LM/FN coated plates must be prepared ahead of time.

• 8.
  Coat plates with PO.

  • a.
    Prepare 7.5 μg/mL PO solution in DPBS.
  • b.
    Distribute solution to desired wells: 6-well plate: 2 mL/well, 24-well plate: 1 mL/well, 96-well plate: 200 μL/well.
  • c.
    Incubate the dish over night at 37°C.
• 9.
  Coat plates with LM and FN.

  • a.
    Prepare LM/FN solution with 1 μg/mL LM and 1 μg/mL FN in DPBS.
  • b.
    Aspirate the PO solution from the plates.
  • c.
    Using DPBS, rinse the plates three times.
  • d.
    Distribute LM/FN solution to wells: 6-well plate: 3 mL/well, 24-well plate: 1.5 mL/well, 96-well plate: 300 μL/well.

CRITICAL: For best results, be sure to thaw laminin slowly, first overnight at ‒20°C, then moving to 4°C until fully thawed.

CRITICAL: PO is toxic to cells. Be sure to rinse your plates well with DPBS at least 3 times after removing PO solution.

CRITICAL: You must begin preparing the PO/LM/FN coated plates two days ahead of time.

Optional: In emergency cases, LM/FN can be incubated for 2–4 h only, however this may lead to suboptimal differentiation or survival results.

Key resources table

REAGENT or RESOURCE	SOURCE	IDENTIFIER
Antibodies
Sox10 (mouse)	Santa Cruz	Cat# SC365692
PRPH (mouse)	Santa Cruz	Cat# SC377093
Tuj1 (rabbit)	BioLegend	Cat# 802001
CHRNA3 (rabbit)	Proteintech	Cat# 10333-1
CHRNB4 (mouse)	Santa Cruz	Cat# 514315
NET (mouse)	Mab Technology	Cat# NET17-1
alphaADR (rabbit)	Abcam	Cat# ab85570
bADR (mouse)	Santa Cruz	Cat# 271322
TrkA (mouse)	R&D	Cat# MAB1751R
NPY (mouse)	Santa Cruz	Cat# 133080
cFos	Santa Cruz	Cat# 166940
VMAT1 (rabbit)	Novus	Cat# NBP2-58895
VMAT2 (mouse)	R&D	Cat# MAB8327
TH (rabbit)	ABclonal	Cat# a12756
AF647 (donkey anti mouse)	Invitrogen	Cat# A31571
AF488 (donkey anti rabbit)	Invitrogen	Cat# A21206
AF647 (donkey anti rabbit)	Invitrogen	Cat# A31573
AF488 (donkey anti mouse)	Invitrogen	Cat# A21202
Chemicals, peptides, and recombinant proteins
Recombinant human protein vitronectin (VTN)	Fisher Scientific	Cat# A31804
Dulbecco’s phosphate-buffered saline (DPBS)	VWR	Cat# 45000-436
Gibco’s Essential 8 medium (E8)	Fisher Scientific	Cat# A1517001
0.5 M EDTA	Fisher Scientific	Cat# AM9262
Geltrex	Fisher Scientific	Cat# A1413202
DMEM/F12	Fisher Scientific	Cat# 11330057
Poly-L ornithine hydrobromide	Sigma	Cat# P3655
Mouse laminin-I	R&D Systems	Cat# 3400-010-01
Human fibronectin	VWR	Cat# 47743-654
Gibco’s Essential 6 medium	Fisher Scientific	Cat# A1516401
SB 431542	Bio-Techne	Cat# 1614/50
BMP4	Bio-Techne	Cat# 314-BP-050/CF
Chir99021	Bio-Techne	Cat# 4423/50
Neurobasal medium	Fisher Scientific	Cat# 21103049
N2 supplement	Fisher Scientific	Cat# 17502048
B27 supplement	Fisher Scientific	Cat# 12587001
L-glutamine 200 mM	Fisher Scientific	Cat# 25030081
FGF2	Bio-Techne	Cat# 233-FB/CF
Human GDNF	PeproTech	Cat# 450-10-500UG
Recombinant human BDNF	Bio-Techne	Cat# 248-BDB-250
L-ascorbic acid 2 phosphate magnesium	Sigma	Cat# A8960-5G
Human beta NGF	PeproTech	Cat# 450-01-250UG
Dibutyryl cAMP	Sigma	Cat# D0627
Accutase	Fisher Scientific	Cat# NC9464543
Retinoic acid (RA)	Sigma	Cat# R2625
(+)- Aphidicolin	Cayman	Cat# 1400700
Fluo-4	Tocris	Cat# 6255
Fluorescent norepinephrine probe NS510	Zhang et al. (Zhang, 2019 #3)	N/A
Stem-CellBanker	AMSBIO	Cat# 11924
Experimental models: Cell lines
H9	WiCell	WAe009-A
Software and algorithms
GraphPad Prism	GraphPad Software	https://www.graphpad.com/features
Fiji	ImageJ	https://imagej.net/software/fiji/
Time Series Analyzer	ImageJ	https://imagej.nih.gov/ij/plugins/time-series.html
Axis Navigator 1.4	Axion BioSystems	N/A
Gen5	Agilent BioTek	N/A
Other
24-well Corning Costar ultra-low attachment microplates	Fisher Scientific	Cat# 7200602
BioCircuit MEA 96 well plates	Axion BioSystems	Cat# M768-BIO-96-50
MaestroPro	Axion BioSystems	https://www.axionbiosystems.com/products/mea/maestro-pro
Lionheart FX automated live cell imager	Agilent BioTek	https://www.agilent.com/en/product/cell-analysis/cell-imaging-microscopy/automated-cell-imagers/biotek-lionheart-fx-automated-microscope-1623188

Materials and equipment

day 0–1 media

Reagent	Final concentration	Amount
E6	N/A	50 mL
SB	10 μM	50 μL
BMP4	0.2–1 ng/mL	1–5 μL
Chir	300 nM	2.5 μL
Total	N/A	50 mL

[Note on storage conditions: store at 4°C for up to one month]

day 2–10 media

Reagent	Final concentration	Amount
E6	N/A	100 mL
SB	10 μM	100 μL
Chir	0.75 μM	12.53 μL
Total	N/A	100 mL

[Note on storage conditions: store at 4°C for up to one month]

Spheroid Media

Reagent	Final concentration	Amount
Neurobasal media	N/A	100 mL
N2 supplement	1%	1 mL
B27 supplement	2%	2 mL
L-glutamine	1%	1 mL
Chir	3 μM	50 μL
FGF2	10 ng/mL	100 μL
Total	N/A	100 mL

[Note on storage conditions: store at 4°C, use within 2 weeks]

symN media

Reagent	Final concentration	Amount
Neurobasal	N/A	100 mL
N2 supplement	1%	1 mL
B27 supplement	2%	2 mL
L-glutamine	1%	1 mL
GDNF	25 ng/mL	250 μL
BDNF	25 ng/mL	250 μL
Ascorbic Acid	200 μM	200 μL
NGF	25 ng/mL	100 μL
dbcAMP	200 μM	200 μL
Total	N/A	100 mL

[Note on storage conditions: store at 4°C, use within 2 weeks]

Fluo-4 Staining media

Reagent	Final concentration	Amount
symN media	N/A	50 mL
Fluo-4 stain	2 μM	100 μL
RA	0.125 μM	6.25 μL
Total	N/A	50 mL

[Note on storage conditions: store at 4°C, use within 2 weeks]

NE Staining media

Reagent	Final concentration	Amount
symN media	N/A	50 mL
NE probe NS510	0.5 μM	50 μL
RA	0.125 μM	6.25 μL
Total	N/A	50 mL

[Note on storage conditions: store at 4°C, use within 2 weeks]

Note: The staining media has the same composition as symN media, you need to add fresh dye every time in the recommended amount.

CRITICAL: Always keep your dye/stain on ice and protected from light

Step-by-step method details

Neural crest induction

Timing: 10 days

The first ten days of this protocol seek to induce differentiation of NCCs from hPSCs. Successful differentiation will be marked by the appearance of dense cell ridges (Figure 1A, day 10). Visible ridges should appear from day 3 and continue to darken to day 10.

• 1.Prepare day 0–1 media.
• 2.
  Plate hPSCs for differentiation.

  • a.
    Allow geltrex plates to adjust to room temperature.
  • b.
    Dissociate hPSCs.

    • i.
      Aspirate media from 60‒70% confluent plate of hPSCs.
    • ii.
      Rinse the plate of hPSCs with DPBS twice.
    • iii.
      Add 7 mL EDTA to the dish and incubate at 37°C for 15 min.
    • iv.
      Carefully pipet off cells and transfer to a 50 mL conical tube. You should be able to dissociate the cultures into single cells easily.
    • v.
      Rinse plates with an equal volume of DPBS and add to conical tube.
    • vi.
      Centrifuge for 4 min at 120–200x g.
    • vii.
      Discard supernatant.
  • c.
    Plate hPSCs.

    • i.
      Resuspend in 1 mL day 0–1 media to disrupt pellet. Dilute with 9 mL day 0–1 media.
    • ii.
      Take 10 μL of cell mixture and count the cells.
    • iii.
      Aspirate the geltrex solution from the previously coated plates.
    • iv.
      Plate your cells at a density of 1.25∗10⁶ cells/cm² in a low volume, such as 2 mL for a 6-well or 500 μL for a 24-well.
    • v.
      Incubate cells at 37°C.
• 3.
  Maintain Neural Crest Cells Problem 1.

  • a.
    Day 1.

    • i.
      Feed cells with day 0–1 media: 1 mL/24-well or 3 mL/6-well.
  • b.
    Day 2.

    • i.
      Prepare day 2–10 media.
    • ii.
      Feed cells with day 2–10 media: 1 mL/24-well or 3 mL/6-well.
  • c.
    Day 4-Day 10.

    • i.
      Feed neural crest cells day 2–10 media every-other day until day 10: 1 mL/24-well or 3 mL/6-well.

Note: Formation of ridges may lead to cells lifting off the plates and floating as aggregates. Problem 2.

Neural crest cell cryopreservation and replating

Timing: 1 h

On day 10, differentiated cells express NCC markers such as SOX10 (Figure 1B) and are ready for further differentiation into neural crest derivatives (Figure 1A). At this time, cells may be cryopreserved allowing for NCC banking, transportation, and storage (Figure 1C).

• 4.Prepare spheroid media.
• 5.
  Dissociate.

  • a.
    On day 10 of differentiation, remove media from cells and wash once with DPBS.
  • b.
    Add accutase just enough to cover the well: 1 mL/6-well or 300 μL/24-well.
  • c.
    Incubate the cells in accutase for 20 min at 37°C.
  • d.
    Prepare 50 mL conical tubes with a large volume of DPBS to dilute accutase.
  • e.
    Pipette accutase/cell mixture from wells, making sure to collect all the cells. Transfer the cell/accutase mixture into the prepared conical tube. Additional rinses of the wells with DPBS may be used to get any remaining cells from the wells.
  • f.
    Centrifuge cells for 4 min at 200x g.
• 6.
  Cryopreservation.

  • a.
    Discard supernatant and resuspend cells in 1 mL spheroid media.
  • b.
    Dilute cells further with 9 mL day 10 media.
  • c.
    Take 10 μL cell mixture to count cells.
  • d.
    Neural Crest Cells should be frozen at a density of 8∗10⁶ cells/mL.
  • e.
    Centrifuge cells for 4 min at 200x g.
  • f.
    Discard supernatant and resuspend in appropriate amount of Stem Cell Banker.
  • g.
    Distribute cell mixture into labeled cryotubes, 1 mL/vial, and freeze at ‒80°C overnight, followed by transfer to LN for long-term storage.
• 7.
  Replating as spheroids.

  • a.
    Discard supernatant and resuspend cells in 1 mL spheroid media.
  • b.
    Dilute cells further with 9 mL of day 10 media.
  • c.
    Take 10 μL cell mixture to count cells.
  • d.
    Dilute the cells such that 0.5∗10⁶ cells are present in every 500 μL of media.
  • e.
    Plate the cells in ultra-low attachment 24-well plates as 500 μL/24 well.
  • f.
    Incubate the cells at 37°C.

Note: After accutase incubation, NCCs might be aggregating and become hard to fully dissociate. Troubleshooting problem 3.

Pause point: NCC frozen vials can be stored in LN for long-term preservation.

Neural crest spheroid expansion

Timing: 4 days

The spheroid stage allows NCCs to expand and be purified (because the media is selectively supporting NCCs) to ensure successful symN differentiation. During this stage, NCCs are in 3D culture and will begin to form smooth, round spheroids (Figures 1A and 1C, day 14). Any non NCCs will remain outside of the spheroids.

• 8.On day 11, one day after replating, add 500 μL fresh spheroid media to the wells.
• 9.
  Spheroid maintenance.

  • a.
    On day 13, tilt plates such that the aggregating spheroids sink to one side of the well.
  • b.
    Carefully remove as much spent media from the wells as you can without removing spheroids, leaving behind <200 μL of media.
  • c.
    Add 1 mL fresh media to each well.
  • d.
    Very gently pipette up and down to disrupt any large or irregular spheroid clumps.

Troubleshooting problem 4.

Optional: Spheroid cultures can be kept for up to two weeks. For extended spheroid culture, feed spheroids every other day with spheroid media containing 0.5 μM Retinoic Acid (RA), added fresh at each feed.¹

Differentiation of mature sympathetic neurons

Timing: 7–16 days (day 20–30 of differentiation)

During this stage symN differentiation takes place. Neurites should be visible from day 18 and will continue to grow into visible axons (Figure 1D). Differentiated neurons should express markers associated with symN function (Figure 2). SymNs are ready to be assessed via functional assays from day 30 on, with spontaneous electrophysical activity noticeable as early as day 20.

• 10.Prepare symN media.
• 11.
  Dissociate spheroids.

  • a.
    On day 14, collect the media containing the spheroids from all wells into conical tubes.
  • b.
    Centrifuge cells for 4 min at 200x g.
  • c.
    Discard supernatant.
  • d.
    Resuspend spheroids in 1 mL accutase, and place in an empty well of 24-well ultra-low attachment plate.
  • e.
    Incubate cells for 20 min at 37°C.
  • f.
    After incubation, pipette to break the spheroids into single cells and transfer cell/accutase mixture to conical tubes filled with DPBS to dilute the accutase.
  • g.
    Centrifuge cells for 4 min at 120–200x g.
  • h.
    Remove supernatant and resuspend the cells in 1 mL symN media.
  • i.
    Take 10 μL cell mixture to count the cells.
• 12.
  Plate on PO/LM/FN coated plates.

  • a.
    Remove coating from prepared PO/LM/FN coated plates.
  • b.
    Plate cells at a density 1.0∗10⁵ cells/cm² in a low volume of symN media: 500 μL for 24-well, 200 μL for 96-well.
• 13.
  symN Maintenance.

  • a.
    On day 15, one day after replating, feed symNs with fresh symN media with RA (0.125 μM) added fresh: 1 mL for 24-well, 300 μL for 96 well.
  • b.
    Feed every 3 days from day 15 to day 24 with symN media containing RA added fresh at each feed. Neurites should be visible from day 18, with axon density increasing over time (Figure 1D).
  • c.
    From day 24, feed weekly with symN media adding RA fresh at each feeding. Weekly feedings allow for gentle handling of cells that may easily lift off, without any observed effects of insufficient nutrients.

Figure 2.

Characterization of hPSC symNs via immunofluorescent staining of day 40 symNs

In all panels red is either peripherin (PRPH) or neuron specific beta tubulin (Tuj1).

(A) Positive expression of markers necessary for reception of acetylcholine signaling: cholinergic nicotinic receptor alpha-3 (CHRNA3) and beta-4 (CHRNB4).

(B) Positive expression of neurotrophin receptor tropomyosin receptor kinase A (TRKA).

(C) Positive expression of markers associated with norepinephrine feedback: adrenergic receptors alpha-2 (αADR) and beta-2 (βADR).

(D) Positive expression of markers necessary for norepinephrine reuptake and signal termination: norepinephrine transporter protein 1 (NET).

(E) Positive expression of markers associated with norepinephrine vesicular trafficking: vesicular monoamine transporter 1 (VMAT1) and 2 (VMAT2).

(F) Positive expression of enzymes involved in norepinephrine synthesis: tyrosine hydroxylase (TH).

(G) Positive expression of immediate early response gene c-Fos, indicating neuronal activation.

(H) Positive expression of cotransmitter neuropeptide Y (NPY). Scale bars represent 100 μm. White arrows indicate representative points of positive expression, although not all instances of positive expression are denoted in each figure.

Troubleshooting problem 5, problem 6.

Optional: On day 14, spheroids may be landed, plated as spheroids, instead of dissociated as the cells will migrate out of spheroids and differentiate. In this case, remove spheroid media, resuspend the spheroids in symN media, and expand 1:4 into PO/LM/FN coated plates (1 well of spheroids divided into 4 PO/LM/FN coated wells of same area). It may be suitable to land spheroids for experiments that do not require an exact cell density, i.e. axon outgrowth, RNA or gDNA collection, etc.

Optional: Aphidicolin (0.5 μM) can be added day 20–30 to ensure the purity of differentiation.² This can be added fresh at each feeding or added to a stock of symN media for use from day 20–30.

Optional: SymNs may be maintained in long term cultures. We have kept them up to 120 days and they may be able to be maintained even longer. From day 35, symNs can easily detach from the plates. Be sure to remove and replace media when feeding very gently or remove and replace half of spent media only.

Note: Expect to expand spheroids 1:4 when replating.

Note: From day 14, land or replate spheroids on plates which will be necessary for your functional assays of choice, i.e., MEA plates.

Functional assays-MEA analysis

Timing: 15 min

A characteristic trait of mature sympathetic neurons is spontaneous electrical activity (Figure 3). Multi-Electrode Array (MEA) can be used to analyze neuronal firing and activity. This protocol produces symNs that will spontaneously fire as early as day 21.

• 14.Start-up MEA instrument.
• 15.Take MEA plate with plated symNs to the recording chamber of the MEA instrument.
• 16.Be sure the instrument is set for neuronal recording.
• 17.Allow symNs to acclimate to the recording chamber for 5 min prior to recording.
• 18.Once acclimated, record the spontaneous neuronal activity of cultures for 10 min.
• 19.Frequency of MEA recordings will depend on your experimental design. SymN activity should increase from day 21.

Note: To prepare cultures for MEA recording, cells should be plated from day 14 dissociated neural spheroids. They should be plated at a cell density of 1 × 10⁵ cells/cm².

Note: Unless using MEA plates with transparent bottoms, additional wells on a regular cell culture plate with a transparent bottom should be prepared and cultured under the same conditions in parallel. This will allow for monitoring of sympathetic neuron growth.

Note: MEA system may also be used to electrically invoke activity in cultures.

Figure 3.

Electrophysiological analysis of hPSC-derived symNs using MEA

(A) Day 30 symNs on MEA plates. Black lines are the electrodes.

(B) Representative spike map on day 20 and day 30+. Each horizontal line represents an electrode, each vertical line represents an action potential, black bars represent individual spikes, blue bars represent bursts.

(C‒H) Firing characteristics, including increased mean firing rate (MFR.€), number of spikes (D), number of active electro.€ (E), weighted mean firing rate (wMFR, MFR by the total number of active electrodes in the well) (F), and number of bursts (G) indicated increased symN differentiation and maturation from day 20 to day 30, while burst duration (H) is not significantly changed. However, the variability was dramatically narrowed, suggesting the symNs maturating. Unpaired t-test. Scale bars represent 200 μm.

Functional assays-calcium imaging

Timing: 2 h

This section outlines the use of fluorescence imaging to analyze the spontaneous calcium (Ca²⁺) flux in differentiated symNs after day 30, when neuron activity has increased and should be clearly detectable (Figure 4A). Spontaneous Ca²⁺ flux is an intrinsic characteristic of mature and functional neurons.³ Before you start, make sure you have symN media with fresh RA (0.125 μM) and calcium imaging dye Fluo-4 (2 μM).

• 20.
  Dye symNs for calcium imaging.

  • a.
    On day 30 or later, replace existing media in the plate with symN media containing Fluo-4 dye at 2 μM concentration.
  • b.
    Incubate the cells for 20 min at 37°C. We use a black 96 well plate with a clear bottom with 200 μL media/well.
  • c.
    After 20 min wash the wells with DPBS once.
  • d.
    Replace DPBS with symN media containing freshly added RA for at least 30 min.
  • e.
    The cells are ready for calcium imaging. We have used a fluorescent microscope at 10x magnification to observe the spontaneous flux of Ca²⁺ (Methods video S1).
  • f.
    Here we have used ∼5 frames per second and BioTek Lionheart to for live imaging. The frame rate may vary depending on the type of experiment, kinetics of the dye or indicator, type of microscope etc.

Figure 4.

Functional assays of mature symNs on day 30 or day 30+ using Ca²⁺ and NE probe imaging

(A) Fluorescent image of live, mature symN stained with Fluo-4 (green). Regions of interest (ROI) are indicated with respective colored circles from representative traces from b.

(B) Representative traces showing the relative change of Ca²⁺ over time in ROI’s indicated in a.

(C) Phase contrast (left), fluorescent (middle), merged (right) image of live mature symNs stained with the NS510 NE probe⁴ in C.

(D) Bar graph showing fluorescence intensity of NE stained cell bodies (unstained (NA) and stained conditions).

• 21.
  Calcium imaging analysis.

  • a.
    Add the Time series analyzer plugin to Fiji. Available here: https://imagej.nih.gov/ij/plugins/time-series.html.
  • b.
    Open the image sequence in Image J, then select the region of interest (ROI), meaning cells in the frame you want to acquire the fluorescent intensities from.
  • c.
    You will see the data point of change of fluorescence in individual ROI’s. Randomly selected representative ROI’s/cells are indicated in Figure 4A with same color as traces in Figure 4B.
  • d.
    Export it to Excel and process the data as ΔF/F_min, where ΔF is the fluorescence intensity of a chosen cell minus the minimum intensity of the same cell throughout the recording.
  • e.
    The ΔF/F values from Excel were plotted on graphs using Prism (Figure 4B).

Troubleshooting problem 7, problem 8, problem 9.

Functional assays-norepinephrine imaging

Timing: 1 h

In this section we are determined to show norepinephrine (NE) in mature symN after day 30 (Figures 4C and 4D). Here we employed the NS510 probe, used to track NE inside the live cells.⁴ This staining should be ideally performed on day 30 or afterwards. Before starting, prepare symN media with freshly added RA (0.125 μM) as well as a working solution of NS510 (0.5 μM).

• 22.On day 30 or afterwards, replace the existing media with the fresh symN media containing the NS510 dye (0.5 μM).
• 23.Incubate the cells for 45 min at 37°C. Here we have used black 96 well plate with clear bottoms, with 200 μL media/well.
• 24.After 45 min wash the well once with DPBS and replace it with symN media with freshly added RA (0.125 μM).
• 25.The cells are now ready for imaging.
• 26.We used a fluorescent microscope BioTek Lionheart at 10X to take the images (Figure 4C).
• 27.After images were taken, they were processed to make publication-quality graphs using software ImageJ.
• 28.The mean fluorescence intensity of the individual cell in unstained and stained cells was calculated in ImageJ and the values were plotted in Prism (Figure 4D).

Troubleshooting problem 7, problem 8, problem 9.

Expected outcomes

In this protocol, we present a method to efficiently differentiate symNs from NCCs using hPSCs. Therefore, our protocol is not only suitable for functional analysis, but also for developmental studies of symNs. To ensure the reproducibility, it is important carefully check the differentiation in the following critical stages/checkpoints. (1) To start with, hPSCs need to form nice colonies with bright and smooth edges. The density if hPSCs should not be too high, meaning most colonies should not be fully merging together. (2) A good symN differentiation relies on the efficient NC induction on day 0–10. Since NCCs are highly migratory and proliferative, the cells on day 10 should form clear and dark “ridge” structures (Figure 1B). (3) NCCs maintained as 3D spheroids will be further induced toward the sympathoblast progenitor cell fate from day 10–14. We have previously shown that the spheroid culture stage in this protocol is highly selective, meaning mainly SOX10+/CD49d+ NCCs will survive and form spheroids, and thus the cell sorting step, which is time consuming, expensive and harmful to the cells can be omitted.¹ Furthermore, day 10 NCCs can be frozen, and yield about 80% survival after thawing (Figure 1C).² This “cryopause” step allows scalable production of neurons and ensures the reproducibility using our protocol.⁵ On day 14, healthy NCCs should generate firm, round, and smooth spheroids (Figure 1A). (4) After replating, differentiated symNs will visibly emerge after day 20 and become well developed after day 30. As a result, clear neurite outgrowth and development should be observed (Figure 1D). Please refer to Figure 1 for representative pictures at each critical stage/checkpoints.

On day 30, we further demonstrated the evaluation of symN cell fate by detecting essential functional protein expression, CHRNA3/B4 (nicotinic receptors, Figure 2A), TrkA (NGF receptor, Figure 2B) , α/β-ADRs (adrenergic receptors, Figure 2C), NET (norepinephrine transporter, Figure 2D), VMAT1/2 (vesicular monoamine transporter, Figure 2E), TH (tyrosine hydroxylase, Figure 2F), c-Fos (neural activation, Figure 2G), and NPY (neuropeptide Y, Figure 2H), using immunofluorescent staining. The presence of certain markers is the very first step to ensure that the differentiated neurons may display necessary sympathetic functions, such as preganglionic cholinergic stimulation, neural activation, NE synthesis, release and recycling. We then examined the electrophysiological activity of hPSC-derived symNs using MEA (Figure 3A). Signals should be detectable as early as day 20 and get stronger and stabilized after day 30 (Figures 3B–3H), along with neural differentiation and maturation. Lastly, we show that the Ca²⁺ flux as well as NE synthesis can be monitored in a time-lapse manner using live cell imaging in. Both Ca²⁺ (Figures 4A and 4B) and NE (Figures 4C and 4D) should be detectable spontaneously. Their levels may be further induced or suppressed after specific pharmacological treatments, or in various diseased conditions, as we showed previously.^2,6,7

Limitations

There are several factors that may affect the success of symN differentiation. It is important to start the differentiation with hPSCs that are healthy and in excellent condition. hPSCs on day 0 should show smooth edges and round, dense colonies (Figure 1A, day 0). The ideal seeding density on day 0 could slightly vary when using different ESC or iPSC lines and may affect the efficiency of NC induction. Therefore, adjustments to the day 0 seeding density might be needed. A good NC induction should result in thick and abundant “ridge” structures (Figure 1B, right) on day 10 of differentiation, which is a critical checkpoint to decide whether the differentiation should be proceeded or aborted. This is determined by the BMP4 concentration on day 0–2 of differentiation. Since BMP4 is highly brand and even lot dependent, it is important to perform titration tests when using a new batch of BMP4. On day 14, the spheroid stage, symN progenitor spheroids should be round and firm with smooth edges, and a size of around 100–500 μm (Figure 1C). The efficiency of spheroid formation should be considered as another checkpoint of the differentiation. More details about the checkpoints during differentiation can be found in our previous publication.¹

The efficiency of symN differentiation after day 30 is high (about 80%). However, it still contains low numbers of contaminating smooth muscle cells, fibroblasts, and placode-derived non-neural cells,² which may affect the result of certain functional studies. However, TH+/PRPH+ symNs derived from this protocol yield over 90% of all neurons in the cultures, and we barely detect potential contaminating neural types that could interrupt the interpretation of symN signaling² (such as CNS neurons, cholinergic sensory or parasympathetic neurons, for example). Finally, a limitation of this protocol is that hPSC-derived symNs are not as mature as cultured primary neurons or neurons in living organisms. In this protocol, we provide two replating methods on day 14, spheroid plating or dissociated plating. Spheroid plating induces neural differentiation faster than dissociated plating. However, when performing functional comparisons that require precise cell density, such as the MEA assay, dissociated plating is recommended. Further efforts to improve the maturity of hPSC-derived symNs are an important task in the future.

Overall, this protocol provides an efficient method to differentiate postganglionic symNs. Compared with other similar protocols,^{8,9,10,11,12,13} our NC differentiation is free of FACS sorting process (which is needed in the protocol by Oh et al.¹¹), and can be cryopreserved (which is only described by Kirino et al.¹⁰), which ensure the scalability of the differentiation. Although this protocol is established and demonstrated here using H9 ESCs, similar to others,^8,9,10,11,12 we have shown previously that it can be reproduced in a wide variety of embryonic stem cell (ESCs) and iPSCs.^2,6 This confirms the reproducibility of the protocol. Functionality wise, others’^8,9,10,11,12 and our symNs express most essential functional markers, display spontaneous neural activity, which is manipulatable by treatments of symN activators or inhibitors.^1,2 Like those protocols,^8,9,10,11,12 we also have confirmed that the differentiated symNs are able to regulate their target tissue, such as cardiomyocyte, in co-culture models.² This may suggest certain level of neural maturation. However, among those protocols, our symNs are one of the first to be practically used for disease modeling, including genetic disorder Familial Dysautonomia,² diabetes,⁷ and COVID-19 infection.⁶

Troubleshooting

Problem 1

NC ridges can barely be observed on day 10 (Figure 1B). Step 3.

Potential solution

• •Perform a BMP4 titration test during day 0–2.
• •We recommend starting with 1 ng/mL, and test concentrations below 1 ng/mL to find the ideal dosage. Example: 0 ng/mL, 0.4 ng/mL, 0.8 ng/mL, 1 ng/mL.

Problem 2

NC ridges form, but lift up before day 10. Step 3.

Note: Lifting of ridges is a sign that the NC differentiation has been very efficient.

Potential solution

• •When feeding the cells, tilt the plates and aspirate old media from one corner carefully, and try to not aspirate the floating NC ridges.
• •Alternatively, collect the old media, spin down, and put the lifted cells back to the plates.

Problem 3

Dead cells clump after NC dissociation and resuspension. Step 5.

Potential solution

Add DNase (0.1 mg/mL) with accutase and proceed as described above.

Problem 4

Spheroids aggregate together during day 10–14. Step 9.

Potential solution

Pipette the spheroids for up to four times after feeding to break up the clumps.

Problem 5

During dissociation, spheroids become sticky and/or get stuck in pipette tips. Step 11.

Potential solution

When adding accutase or DPBS, have an aliquot of the accutase or DPBS available to use to rinse your pipette tip when transferring spheroids.

Alternatively, add DNase (0.1 mg/mL) when dissociating with accutase and proceed as described above.

Problem 6

Spheroids cannot be dissociated after accutase incubation. Step 11.

Potential solution

• •Typical dissociation time is 20–30 min. However, it might need optimization when using different ESC or iPSC lines.
• •Use multiple wells to test how much time it takes to fully dissociate the spheroids. Increase the incubation time gradually. Check every 10 more min.

Problem 7

Mature neurons in old culture are sensitive and sometimes get detached from the plate during washing steps while staining them for Ca²⁺/NE imaging. Step 20. Step 24.

Potential solution

• •Ideally, there should be a DPBS washing step before imaging but if significant detachment is observed in mature cultures this washing step could be omitted.
• •Remove stain/dye media completely from the cells if you are skipping the washing.

Problem 8

If you do not see Ca²⁺/NE-stained neurons under the microscope, this could be the breakdown of your staining dye Step 20. Step 25.

Potential solution

• •To avoid this situation, you ideally want to decrease your freeze and thaw cycle of the dye/stain.
• •You can aliquot your staining dyes.
• •Always keep it protected from light by covering the dye-containing vials or tubes with aluminum foil.
• •Do not keep your stains long at room temperature, try to keep them on ice if possible.
• •Use black colored plates for imaging instead of transparent ones.

Problem 9

You start seeing signals fading, while imaging. This is potentially due to photo-bleaching Step 20. Step 25.

Potential solution

• •Scan the plate at a low laser intensity of microscope until you find the area you want to the image.

Resource availability

Lead contact

Further information and requests for resources and reagents should be directed to and will be fulfilled by the lead contact, Dr. Nadja Zeltner (nadja.zeltner@uga.edu).

Technical contact

Hsueh-Fu Wu (hsuehfu.wu@uga.edu).

Materials availability

We do not have newly generated materials associated with this protocol; all reagents are commercially available. hPSC lines can be obtained from us upon proper Material Transfer Agreements and hESCs used here can be obtained via WiCell Repository.

Data and code availability

The protocol does not include unique datasets generated or analyzed during this study.