Novel Cross-Species Salivary Gland-Parasympathetic Neuron Coculture System

Materials and reagents

Biological materials

1. Human embryonic stem cell line (WiCell, WA09, female, NIH 0062)

2. Sox10-Cre mice (Jackson Laboratory, stock 025807)

3. RFP Cre reporter mice B6.Cg-Gt(ROSA)26Sor^{tm14(CAG-tdTomato)Hze}/J (Jackson Laboratory, stock 007914)

1. Geltrex (Invitrogen, catalog number: A1413202)

2. Poly-L-ornithine hydrobromide (PO) (Sigma, catalog number: P3655)

3. Mouse laminin I (LM) (R&D Systems, catalog number: 3400-010-01)

4. Human fibronectin (FN) (VWR/Corning, catalog number: 47743-654)

5. Essential 8 medium (E8) (Gibco, catalog number: A15169-01)

6. Essential 8 supplement (E8) (Gibco, catalog number: A15171-01)

7. Essential 6 medium (E6) (Gibco, catalog number: A15165-01)

8. Neurobasal media (Gibco, catalog number: 21103-049)

9. Stem cell banker (Amsbio, catalog number: 11924)

10. DMEM/F12 (Thermo Fisher/Life Technologies, catalog number: 11330-057)

11. DMSO (Thermo Fisher/Life Technologies, catalog number: BP231-100)

12. Fetal bovine serum (FBS) (Atlanta Biologicals, catalog number: S11150)

13. B27 supplement (Thermo Fisher/Life Technologies, catalog number: 12587-010)

14. N2 supplement (Thermo Fisher/Life Technologies, catalog number: 17502-048)

15. L-Glutamine (Thermo Fisher/Gibco, catalog number: 25030-081)

16. GlutaMAX (Gibco, catalog number: 35050061)

17. Antibiotic-antimycotic (Fisher, catalog number: 15 240 096)

18. Accutase (Innovation Cell Technologies, catalog number: AT104500)

19. EDTA (Sigma, catalog number: ED2SS)

20. Trypsin-EDTA (Gibco, catalog number: 25200056)

21. Phosphate-buffered saline (PBS) (Gibco, catalog number: 14190-136)

22. SB431542 (Tocris/R&D Systems, catalog number:1614), make 10 mM stock

23. BMP4 (R&D Systems, catalog number: 314-BP), make 10 µg/mL stock

24. CHIR99021 (R&D Systems, catalog number: 4423), make 6 mM stock

25. Y27632 (R&D Systems, catalog number: 1254), make 10 mM stock

26. FGF2 (R&D Systems, catalog number: 233-FB/CF), make 10 µg/mL stock

27. NRG1 (PeproTech, catalog number: 100-03), make 100 µg/mL stock

28. GDNF (PeproTech, catalog number: 450), make 10 µg/mL stock

29. BDNF (R&D Systems, catalog number: 248-BD), make 10 µg/mL stock

30. CNTF (R&D Systems, catalog number: 257-NT), make 100 µg/mL stock

31. Ascorbic acid (Sigma, catalog number: A8960), make 100 mM stock

32. dbcAMP (Sigma, catalog number: D0627), make 100 mM stock

33. Retinoic acid (Sigma, catalog number: R2625), make 1 mM stock

34. Collagenase A (Tribioscience, catalog number: TBS2116-01), make 2 mg/mL stock

35. Dispase II (Tribioscience, catalog number: TBS2117-01), make 5 mg/mL stock

36. DPBS without Ca⁺² and Mg⁺² (Thermo Fisher/Life Technologies, catalog number: 14190144)

37. 70% ethanol

1. NCC induction medium, D0, 1 (see Recipes)

2. NCC induction medium, D2-10 (see Recipes)

3. SCP differentiation medium, D10-16 (see Recipes)

4. ParasymN differentiation medium, D16 (see Recipes)

5. Salivary gland cell medium (see Recipes)

6. Tongue tissue separation solution (see Recipes)

7. Salivary gland cell dissociation solution (see Recipes)

8. Dissociation termination solution (see Recipes)

1. NCC induction medium, D0, 1 (50 mL)

   Reagent	Final concentration	Quantity or Volume
   E6	n/a	50 mL
   SB431542 (10 mM stock)	10 µM	50 µL
   BMP4 (10 µg/mL stock)	0.2–1 ng/mL	1–5 µL
   CHIR99021 (6 mM stock)	300 nM	2.5 µL
   Y27632 (10 mM stock)	10 µM	50 µL

   Note: Please see Troubleshooting 1 for more information.




2. NCC induction medium, D2-10 (100 mL)

   Reagent	Final concentration	Quantity or Volume
   E6	n/a	100 mL
   SB431542	10 µM	100 µL
   CHIR99021	0.75 µM	12.53 µL




3. SCP differentiation medium, D10-16 (100 mL)

   Reagent	Final concentration	Quantity or Volume
   Neurobasal media	n/a	100 mL
   N2 supplement	1%	1 mL
   B27 supplement	2%	2 mL
   L-glutamine	1%	1 mL
   CHIR99021	3 µM	50 µL
   FGF2 (10 µg/mL stock)	10 ng/mL	100 µL
   NRG1 (100 µg/mL stock)	10 ng/mL	10 µL




4. ParasymN differentiation medium, D16 (100 mL)

   Reagent	Final concentration	Quantity or Volume
   Neurobasal media	n/a	100 mL
   N2 supplement	1%	1 mL
   B27 supplement	2%	2 mL
   GlutaMAX	1%	1 mL
   FBS	1%	1 mL
   GDNF (10 µg/mL stock)	25 ng/mL	250 µL
   BDNF (10 µg/mL stock)	25 ng/mL	250 µL
   Ascorbic acid (100 mM stock)	200 µM	200 µL
   CNTF (100 µg/mL stock)	25 ng/mL	10 µL
   dbcAMP (100 mM stock)	200 µM	200 µL
   Retinoic acid (1 mM stock)	0.125 µM	12.5 µL

   Note: Retinoic acid should be added to the medium freshly every feeding.




5. Salivary gland cell medium (10 mL)

   Reagent	Final concentration	Quantity or Volume
   DMEM/F12	1×	9.6 mL
   FBS	1%	100 µL
   B27	1×	200 µL
   Antibiotic-antimycotic	1×	100 µL




6. Tongue tissue separation solution (2 mL)

   Reagent	Final concentration	Quantity or Volume
   Collagenase A (2 mg/mL stock)	1 mg/mL	1.0 mL
   Dispase II (5 mg/mL stock)	2.5 mg/mL	1.0 mL
   PBS	1×




7. Salivary gland cell dissociation solution (3 mL)

   Reagent	Final concentration	Quantity or Volume
   Trypsin-EDTA	0.25%	3 mL




8. Dissociation termination solution (2 mL)

   Reagent	Final concentration	Quantity or Volume
   DMEM/F12	1×	1800 µL
   FBS	10%	200 µL

Laboratory supplies

1. TC-treated 6-well tissue culture plate (Corning, catalog number: 3516)

2. TC-treated 24-well tissue culture plate (Corning, catalog number: 3526)

3. TC-treated 10 cm tissue culture plate (Corning, catalog number: 430167)

4. Ultra-low attachment plate (Corning, catalog numbers: 07 200 601 and 07 200 602)

5. 15 mL conical tissue culture tubes (VWR/Corning, catalog number: 89039-664)

6. 50 mL conical tissue culture tubes (VWR/Corning, catalog number: 89039-656)

7. Cryovial (Thermo Fisher/Life Technologies, catalog number: 375353)

8. Trypan blue (Corning, catalog number: MT-25-900-CI)

9. P2-10, 20, 200, 1000 pipettes and tips (Eppendorf)

10. Parafilm (ULINE)

11. 5 mL low retention vials (Eppendorf, catalog number: 30122348)

12. 35 mm dish (Genesee Scientific, catalog number: 32-103G)

13. 100 mm culture dishes (Genesee Scientific, catalog number: 32-107G)

14. 3 mL syringes (BD, catalog number: 8194938)

15. 30-G needle (BD, catalog number: 9193532)

16. 70 µm cell strainer (Fisher Scientific, catalog number: 352350)

17. 35 µm cell strainer (Electron Microscopy Science, catalog number: 64750-25)

18. 100% CO₂ gas (Airgas, CD USP50)

19. Plastic CO₂ euthanasia chamber (Length: 10 cm, wide: 7 cm, and height: 5 cm)

Equipment

1. Water bath (VWR)

2. CellDrop automated cell counter (DeNovix)

3. Racks

4. Liquid nitrogen tank (Custom Biogenic Systems)

5. Freezer (-20 °C)

6. Refrigerator (2–8 °C)

7. Water bath (37 °C)

8. Centrifuge (Eppendorf)

9. 37 °C incubator with 5% CO₂

10. Class II biological safety hood

11. Lionheart FX microscope (BioTek)

12. Dissecting microscope (Olympus, model: SZX16 upright fluorescent and brightfield)

13. Biosafety cabinet (NUAIRE, model: GellGard, Class II, Type A2)

14. CO₂ incubator (NUAIRE)

15. Centrifuge (Thermo Scientific, model: LEGEND XTR)

16. Light microscope (EVOS XL Core)

17. Tank with CO₂ (Airgas)

Instruments

1. Surgical and fine scissors (Moria, catalog number: 9601; Fine Science Tool, catalog number: 91604-09)

2. Surgical and fine forceps (Inox electronic, catalog number: 91150-20; Fine Science Tool, catalog number: 112900)

3. Spatula (Fine Science Tool, catalog number: 10360-13)

Software and datasets

1. Gen5 (BioTek)

2. Prism v10.0.3 (GraphPad, 9/25/2023)

3. Fiji (Image J)

4. Adobe Photoshop

5. CellSens Dimension

Procedure

Caution: The procedures for sections A and B will take 1.5–2.0 h without an opportunity to pause. The experiments need to be done as efficiently as possible for harvesting healthy cells. Please make sure you have all the instruments, solutions, and equipment ready before euthanizing the animals.

1. Salivary gland dissection

   1. Euthanize the 8-week-old Sox10-Cre::RFP mice by placing them in a CO₂ chamber. Ensure mouse death by cervical dislocation.

   2. Place the mouse in the surgical area. Wet the mouse head using 70% ethanol to disinfect the surface and prevent fur from getting into the oral cavity.

   3. Decapitate the mouse. Hold the head in one hand and open the mouth cavity by pulling the skin over the skull and below the mandible (Figure 1A).

   4. Cut the corners of the mouth along the cheek using dissecting scissors to further open the oral cavity (Figure 1B).

      
      

      
      Figure 1. Opening the mouse oral cavity. (A) Image of ventral view of a mouse head. (B) Image of a mouse head with the mouth cavity widely open.

      
      

   5. Dissect the tongue with the mandible and remove the skin covering the mandible (Figure 2). Transfer the tongue on the mandible to 30 mL of fresh sterile DPBS (Ca⁺² and Mg⁺² free) in a 50 mL tube. Wash five times using DPBS.

      
      

      
      Figure 2. Dissected tongue with the mandible. A representative image of the dorsal view of an adult mouse tongue sitting on the mandible. Black dotted line encircles the single circumvallate taste papilla in the midline of posterior tongue. Scale bar: 500 μm.

   
   

   6. Using surgical forceps to hold the tongue under a dissecting microscope, inject ~0.5 mL of the tongue tissue separation solution (a mixture of Collagenase A and Dispase II enzymes) into the subepithelial space of posterior tongue as described [17] (Figure 3A and B) and incubate for 30 min at 37 °C (Figure 3C).

      Note: See Troubleshooting #3 and #5 for precautions.

      
      

      
      Figure 3. Intralingual injection of tongue tissue separation solution. (A) An image to illustrate the entry and location of the needle for injecting the solution into the posterior part of the tongue. (B) An image of dorsal view of the posterior tongue after 0.5 mL solution injection. (C) An image of dorsal view of the posterior tongue after 15 min of incubation at 37 °C. Black dotted lines encircle the single circumvallate taste papilla. Scale bars: 500 μm.

   
   

   7. After the incubation, dissect the posterior tongue region with RFP⁺ von Ebner’s glands. Use small scissors to cut the epithelium of the circumvallate papilla region (Figure 4A), then use fine forceps to peel off the epithelium of the circumvallate papilla region (Figure 4B).

      
      

      
      Figure 4. Peeling the circumvallate epithelium. (A) Brightfield image of the dorsal view of the posterior tongue region after removing part of the epithelium. Black dotted line outlines the cutting edge of epithelium in the circumvallate papilla area with the epithelium removed. (B) Brightfield image of the ventral view of the peeled epithelium. White dotted line encircles the circumvallate papilla epithelium. Scale bars: 500 μm in A; 100 μm in B.

   
   

   8. Under a fluorescent stereomicroscope, we were able to see the RFP⁺ von Ebner’s glands in the circumvallate region (Figure 5A). Dissect and collect ~0.1 mL of von Ebner’s salivary gland tissue in the surrounding of the circumvallate papilla under the fluorescent microscope (Figure 5B).

      Caution: If you collect more gland tissues for more cells, you will need to use more solution of 0.25% trypsin-EDTA accordingly.

      
      

      
      Figure 5. Dissection of von Ebner’s gland tissue from Sox10-Cre::RFP mice. (A) RFP fluorescent image of the dorsal view of the posterior tongue region under the fluorescent stereomicroscope. Bright RFP⁺ glands were readily visible in the regions surrounding and under the circumvallate papilla. White dotted line in A encircles the circumvallate papilla. White dashed squares encircle the circumvallate papilla-adjacent regions from which glands were collected. (B) RFP fluorescent image of two pieces of dissected tissue enriched RFP⁺ Ebner’s glands (white arrowheads). Scale bars: 500 μm.

   
   

   9. Wash the salivary glands in 15 mL of fresh sterile DPBS in a 100 mm sterile culture dish.




2. Salivary gland cell dissociation

   1. Transfer salivary glands into a 35 mm dish.

   2. Use fine-tip scissors to cut the salivary gland tissues into small pieces (Figure 6).

      
      

      
      Figure 6. Tissue pieces of Ebner’s glands after mincing. RFP⁺ Ebner’s gland tissue (Figure 5B) was cut into smaller pieces using fine scissors under a fluorescent stereomicroscope. Arrows point to a few pieces of RFP⁺ Ebner’s gland tissue after mincing. Scale bar: 200 μm.

   
   

   3. Incubate the tissue pieces in 3 mL of salivary gland cell dissociation solution (0.25% trypsin-EDTA) for 30 min at 37 °C. Gently agitate the tissues every 5 min using 1 mL pipette tips. Do not cut the tip, as the sharp edge of the cutting end may injure the cells.

      Note: See Troubleshooting 4 for precautions.

   4. Add 1 mL of dissociation termination solution to stop the reaction and transfer all of the solution with cells into a 5 mL low-binding vial.

   5. Centrifuge the cell suspension at 450× g for 10 min at 4 °C and remove the supernatant as much as possible. Be careful not to disturb the cell pellet.

   6. To completely remove the remaining trypsin-EDTA, repeat step B4, adding 1 mL of dissociation termination solution to suspend the cells. Then, repeat step B5 for the centrifugation.

   7. Remove the supernatant and leave one-fourth of the medium (250–300 mL) without disturbing the pellet and filter the cells using a 70 µm cell strainer, followed by a 35 µm cell strainer.

   8. Collect the filtered cell suspension and proceed straight to Section C (Figure 7).

      
      

      
      Figure 7. Cell suspension. Photomicrographs of the dissociated cells and some small cell clusters from RFP⁺ Ebner’s glands. Scale bar: 100 μm.




3. Salivary cell passaging

   1. Centrifuge the cell suspension at 200× g for 5 min.

   2. Aspirate the medium.

   3. Wash cells twice with PBS (1 mL/24-well, 3 mL/6-well, and 4 mL/10 cm plates).

   4. Add prewarmed (5 min in a water bath) trypsin-EDTA (1 mL/24-well, and 3 mL/10 cm plates).

   5. Incubate for 5 min in a 37 °C incubator.

   6. Pipette gently with a P1000 pipette 3–5 times to fully dissociate the cells.

   7. Transfer the trypsin-EDTA and cell solution to a conical tissue culture tube that contains at least 5 times higher volume of salivary gland cell medium.

   8. Centrifuge at 200× g for 5 min.

   9. Resuspend the cells with 1 mL of salivary gland cell medium.

   10. Count the cells.

   11. Replate the cells on desired tissue culture plates at 15 × 10³/cm² density with salivary gland cell medium (0.5 mL/24-well, 2 mL/6-well, and 7 mL/10 cm plates).

   12. Change the medium the next day, and every 2–3 days after (1 mL/24-well, 3 mL/6-well, and 10 mL/10 cm plates).

   13. Cells should reach >80% confluency in one week (Figure 8) and will be ready for replating/freezing.

       The ideal passage number of the cells for the experiment is P3–8. Please see also troubleshooting 2.

       
       

       
       Figure 8. Cell morphology of salivary gland cells after replating. Brightfield images showing the isolated salivary gland cells after (A and A’) 24 h, and (B and B’) 4–6 days. Cell density in B is ideal for making frozen stocks or replating the SCPs to start the coculture. Scale bars = 200 µm.




4. (Optional pause point) Salivary cell cryo-banking

   1. After passage 3, resuspend 80% confluent and dissociated cells from a 10 cm dish into 1 mL of salivary gland cell medium that contains 10% DMSO.

   2. Transfer the medium with the cells into a cryogenic tube.

   3. Freeze the vial at -80 °C fridge in a wrapped Styrofoam box.

   4. Next day, move the vial to a LN tank for long-term storage. The cells can be safely preserved at this stage until the hPSC-derived cells are ready.




5. NCC induction

   1. Prepare Geltrex-coated plates.

      1. Thaw Geltrex at 4 °C overnight.

      2. Dilute Geltrex with ice-cold DMEM/F12 at 1:100 dilution.

      3. Coat the plates with Geltrex (1 mL/24-well, 2 mL/6-well plates) and seal with parafilm.

      4. Incubate the plates at 4 °C at least overnight before use.

   2. Plating hPSCs on day 0

      1. Resuspend dissociated hPSCs (15 min by EDTA in order to gain single cells) in 1 mL d0, 1 media. hPSC is maintained in E8 medium. For details of hPSC replating, please also check our previous protocols [10,18].

      2. Aspirate the Geltrex solution from the plates coated overnight.

      3. Plate hPSCs at 125k cells/cm² density (0.5 mL/24-well, 2 mL/6-well plates).

      4. Incubate cells at 37 °C.

      5. Next day (day 1), fully change the medium with NCC induction, d0, 1 media (1 mL/24-well, 3 mL/6-well plates).

      6. On day 2, fully change the medium with NCC induction, D2–10 medium (1 mL/24-well, 3 mL/6-well plates).

      7. From now on, feed the cells by full medium change every two days (days 2, 4, 6, 8) until day 10 (1 mL/24-well, 3 mL/6-well plates).

      8. On day 10, differentiated NCCs should actively aggregate and form the condensed “dark ridge” structure (Figure 9) [10].

         Caution: If you fail to see such structures, it is not recommended to proceed to the next step.

      
      

      
      Figure 9. Cell morphology during Schwann cell precursor (SCP) differentiation. Brightfield images showing the differentiated D10 neural crest cell (NCC) “dark ridges” (indicated by the yellow arrow). Scale bar = 200 µm.




6. (Optional pause point) Dissociate NCCs for cryobanking

   1. On day 10, aspirate the culture medium.

   2. Wash cells twice with PBS (1 mL/24-well, 3 mL/6-well, and 4 mL/10 cm plates).

   3. Add Accutase (1 mL/24-well, and 2 mL/10 cm plates).

   4. Incubate for 20–30 min in a 37 °C incubator.

   5. Pipette gently with a P1000 pipette 3–5 times and fully dissociate the cells.

      Test if cells are ready for dissociation by gently pipetting 1–2 times. If more times are needed, incubate 5 more minutes each time and test for up to 30 min.

   6. Transfer the Accutase and cell solution to a conical tissue culture tube that contains at least 5 times higher volume of PBS.

   7. Centrifuge at 200× g for 5 min.

   8. Resuspend the cells with ice-cold stem cell banker.

   9. Count the cells.

   10. Transfer the cells (8 × 10⁶ cells/1 mL/vial) in ice-cold stem cell banker to cryogenic tubes.

   11. Freeze the vial at -80 °C freezer in a wrapped Styrofoam box.

   12. Next day, move the vial to a LN tank for long-term storage. The differentiation can be safely paused at this stage.




7. Dissociate NCCs for SCP differentiation

   1. On day 10, aspirate the culture medium.

   2. Wash cells twice with PBS (1 mL/24-well, 3 mL/6-well, and 4 mL/10 cm plates).

   3. Add Accutase (1 mL/24-well, and 2 mL/10 cm plates).

   4. Incubate for 20–30 min in a 37 °C incubator.

   5. Pipette gently with a P1000 pipette 3–5 times and fully dissociate the cells.

      Test if cells are ready for dissociation by gently pipetting 1–2 times. If more times are needed, incubate 5 more minutes each time and test for up to 30 min.

   6. Transfer the Accutase and cell solution to a conical tissue culture tube that contains at least 5 times higher volume of PBS.

   7. Centrifuge at 200× g for 5 min.

   8. Resuspend the cells with day 10–16 medium.

   9. Count the cells.

   10. Replate the cells on 24-well ultra-low attachment tissue culture plates at 1 × 10⁶/well density with SCP differentiation, D10–16 medium (0.5 mL/well).

   11. Add day 10–16 medium on the next day (0.5 mL/well), which makes the total volume 1 mL.

   12. Feed cells on days 13 and 15 by full medium change.

       In order to keep all the cells, tilt the plate and aspirate the medium slowly. Always keep the tip on the surface of the culture medium away from the cells (Figure 10A).

   13. On day 16, SCPs should be differentiated in suspending spheroids (Figure 10B).

       Caution: If you fail to see such structures, it is not recommended to proceed to the next step.

       
       

       
       Figure 10. Cell morphology during Schwann cell precursor (SCP) differentiation. (A) Cartoon illustration of the method to change medium in suspension cultures. (B) Brightfield images showing the differentiated D16 SCP spheroids. Scale bar = 200 µm.




8. SCP replating for parasymN differentiation/coculture

   1. Please make sure you have 80% confluent salivary cells at passages 3–8 before proceeding.

   2. On day 16, tilt the plate and collect all the SCP spheroids in the culture medium into a conical tissue culture tube and fill up the tube with PBS (at least the same volume of cell suspension).

   3. Centrifuge at 200× g for 3 min.

   4. Aspirate the supernatant and add Accutase to the spheroids (1 mL for less than 12 wells, 2 mL for less than 24 wells).

   5. Transfer the Accutase that contains the spheroids back to a low attachment well.

   6. Incubate for 20–30 min in 37 °C incubator.

   7. Gently pipette the spheroids with a P1000 pipette, transfer the cells to a conical tissue culture tube, and fill up the tube with PBS.

   8. Centrifuge at 200× g for 5 min.

   9. Resuspend the cells with 1 mL day 16 parasymN medium.

   10. Count the cells.

   11. Replate SCP cells at a density of 1 × 10⁵/cm² on top of 80% confluent salivary cells (passage 3–8) in a 24-well plate containing 0.5 mL parasymN differentiation medium.

   12. Fully change the medium on the next day and every 2–3 days after (1 mL/24-well plates).

   13. After 7–10 days, aggregated cell bodies of parasymNs should be observed on the salivary cell layer with well-developed neurites (Figure 11).

       
       

       
       Figure 11. Cell morphology of the coculture. Brightfield images showing the cell bodies (indicated by the yellow arrows) and the axons (indicated by the red arrows) of hPSC-parasymNs on the salivary cells (indicated by the white arrow) 10 days after the coculture. Scale bar = 200 µm.

Data analysis

After 7–10 days, well-developed parasymNs should be observed on the salivary cell layer. Cell bodies of parasymNs should be clearly distinguishable from the salivary tissue, given that parasymNs will aggregate with bright and round cell bodies (Figure 11, yellow arrows). The development of neurons and their connectivity with salivary cells can be clearly observed by immunostaining with specific markers as desired (for instance, PRPH in Figure 12A) [19]. Secretory granules in salivary cells are autofluorescent and should be observable at high resolution (Figure 12B). Both total salivary cell number and granule+ salivary cells are increased with parasymNs, suggesting improved development and maturation (Figure 12C). We can also monitor Ca²⁺ dynamics in the salivary cells (Figure 12D). The timeline of the entire process is summarized in Figure 13.

Figure 12. Sample result of functional activity of the coculture. A. Immunofluorescent staining for peripheral neural marker PRPH shows parasymNs growing on the salivary tissue with extensive neurites and aggregating cell bodies. This feature can be mostly seen in peripheral neuron cultures since it might resemble the ganglion formation in the body. B. RFP signals representing SOX10+ salivary cells as well as the autofluorescent granules. C. Representative quantification result showing the improved proliferation and maturation of the salivary tissues when cocultured with parasymNs. D. Representative Ca²⁺ image (using fluo-4) for the salivary cells shows the functional connectivity between parasymNs and salivary cells upon parasymN activation by nicotine in the coculture. Scale bars = 200 µm.

The replating density of D16 SCP is critical for parasymN differentiation especially in neural culture alone. However, given that current understanding suggests that there might be mutual effects on the development and maturation of cocultured cells [8], the cell density in cocultures might be adjusted; however, we have not tested this. Nevertheless, if the density is too high, the chance that the cells detach after coculture will be higher. It is also worth mentioning that, while hPSC-derived parasymNs allow an unlimited neuron source for research, the nature of small amounts and limited passage numbers of primary salivary tissues still restrain the scale-up option of this coculture model system. Establishing a coculture model with both hPSC-derived parasymNs and hPSC-derived salivary tissues would be an important future task.

Figure 13. Timeline of the coculture

Validation of protocol

This protocol or parts of it has been used and validated in the following research article(s):

1. Wu et al. [12]. Parasympathetic neurons derived from human pluripotent stem cells model human diseases and development. Cell Stem Cell (Figure 6, panel H, and supplementary figure 9).

2. Yu et al. [16]. The duct of von Ebner’s glands is a source of Sox10⁺ taste bud progenitors and susceptible to pathogen infections. Frontiers in Cell and Developmental Biology (Figure 1, panel A and B).

General notes and troubleshooting

General notes

1. Efficient and successful early NCC induction for the first 10 days of differentiation is critical. For more detailed methods and tips, please also see our previous protocols [10,18].

2. It is always recommended to replate a neuron-only group and a salivary gland-only group in parallel to the cocultures to validate the neuron differentiation efficiency and functionality as well as the salivary gland cell integrity of each batch. When differentiating parasymNs alone, coat the plate with PO (15 μg/mL in DPBS)/LM (2 μg/mL in DPBS)/FN (2 μg/mL in DPBS).

Troubleshooting

Problem 1: D16 SCP spheroid is poorly formed.

Possible cause: D10 NCC is not properly differentiated (very few or no NC ridges can be observed).

Solution: It is critical to make sure that each stage of the differentiation, including D0 hPSC and D10 NCC, has passed the quality control steps as previously described [10]. If NCC differentiation is not ideal, a BMP4 titration test is highly recommended, as detailed in [18]. In general, concentrations between 0.2 and 1 ng/mL are ideal. In our case, for example, we had to drop the concentration from 1 to 0.4 ng/mL due to the batch-to-batch variability of BMP4.

Problem 2: ParasymNs in the cocultures do not regulate the activity of the salivary tissues.

Possible cause: The parasymN or salivary cell batch is bad.

Solution: Only use salivary cells between passages 3 and 8. Always replate a neuron-only group in parallel to the coculture to validate the neuron differentiation efficiency and functionality of each batch.

Problem 3: Difficulty in injecting tongue tissue separation solution into subepithelial space.

Possible cause: Incorrect injection technique or improper handling of the tongue.

Solution: Practice the injection technique to ensure accurate delivery of solution. Use a dissecting microscope for better visualization and precision. Be careful not to poke and injure the tongue epithelium; otherwise, the solution will leak, leading to incomplete enzyme digestion.

Problem 4: Incomplete or over-enzyme digestion for tongue tissue separation.

Possible cause: Insufficient enzyme incubation time, incorrect enzyme concentration, or improper injection of enzyme. Solution: Verify the concentration and activity of Collagenase A and Dispase II. Ensure proper mixing and incubation at the correct temperature for the specified duration.

Problem 5: Primary cell clumping or low viability.

Possible cause: Over-digestion of tissues for the separation or excessive mechanical stress during pipetting.

Solution: Control the incubation time ≤ 30 min at 37 °C after the injection of tongue tissue separation solution. Agitate gently during the incubation with salivary gland cell dissociation solution. Consider optimizing the trypsin-EDTA concentration and incubation time.

Acknowledgments

This work was supported by 1R01NS114567-01A1 to NZ and R21DC018910 and R21DC018089 to HXL. This protocol was adapted from Wu et al. [12] and Yu et al. [15].

Competing interests

The authors declare no competing interests.

Ethical considerations

This study employed human embryonic stem cell lines (WA09), the use of which by the Zeltner lab was approved by WiCell. All iPSCs employed in this work were reprogrammed from human samples obtained through the public repository Coriell Research Institute. The use of animals was approved by the Institutional Animal Care and Use Committee at The University of Georgia (AUP# A2022 06-030-A6) and complied with the National Institutes of Health Guidelines for the care and use of animals for research.

References

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