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Podsumowanie

In this protocol, we describe a stable, highly efficient differentiation strategy for the generation of postganglionic sympathetic neurons from human pluripotent stem cells. This model will make neurons available for the use of studies of multiple autonomic disorders.

Abstract

Human pluripotent stem cells (hPSCs) have become a powerful tool for disease modeling and the study of human embryonic development in vitro. We previously presented a differentiation protocol for the derivation of autonomic neurons with sympathetic character that has been applied to patients with autonomic neuropathy. However, the protocol was built on Knock Out Serum Replacement (KSR) and feeder-based culture conditions, and to ensure high differentiation efficiency, cell sorting was necessary. These factors cause high variability, high cost, and low reproducibility. Moreover, mature sympathetic properties, including electrical activity, have not been verified. Here, we present an optimized protocol where PSC culture and differentiation are performed in feeder-free and chemically defined culture conditions. Genetic markers identifying trunk neural crest are identified. Further differentiation into postganglionic sympathetic neurons is achieved after 20 days without the need for cell sorting. Electrophysiological recording further shows the functional neuron identity. Firing detected from our differentiated neurons can be enhanced by nicotine and suppressed by the adrenergic receptor antagonist propranolol. Intermediate sympathetic neural progenitors in this protocol can be maintained as neural spheroids for up to 2 weeks, which allows expansion of the cultures. In sum, our updated sympathetic neuron differentiation protocol shows high differentiation efficiency, better reproducibility, more flexibility, and better neural maturation compared to the previous version. This protocol will provide researchers with the cells necessary to study human disorders that affect the autonomic nervous system.

Introduction

Postganglionic sympathetic neurons (symNs) belong to the autonomic nervous system (ANS) and have multiple important roles in responding and regulating homeostasis of the body independent of consciousness. For example, stress stimulates symNs and evokes the fight-or-flight response that leads to an increase in heart rate, blood pressure, and sweating. SymNs are affected in multiple human disorders due to genetics, toxicity/injury, or as companions to other diseases. An example of a genetic neuropathy is the childhood disorder Familial Dysautonomia (FD), where a severe dysregulation of symNs causes dysautonomic crisis, evident by sweating, blotching of the skin, vomiting attacks, hypertension, and anxiety¹. An example of toxicity is chemotherapy treatment, which has been reported to have toxic side effects on autonomic neurons². It is known that autonomic denervation and hyper-innervation can both lead to, or accompany, diseases such as Parkinson’s disease or hypertensive renal disease^3,4. Thus, being able to conduct research and understand the mechanisms of symN biology and defects in the context of disease is beneficial for the search of novel and effective treatments.

Anatomy
The peripheral nervous system branches into sensory and autonomic divisions. The afferent nerves of the sensory nervous system are responsible for sensation of pain and touch, whereas the ANS is responsible for relaying information from all organs to the brain. The ANS is divided into the enteric nervous system, innervating the gastrointestinal tract, the parasympathetic nervous system, which is important for relaxation, and the sympathetic nervous system (SNS), which is important for activation/regulation of organs. The SNS adapts a two-neuron system⁵. Preganglionic sympathetic neural axons in the spinal cord first project to the sympathetic ganglia, where postganglionic symN cell bodies are located. These neurons then send long projections to innervate the target tissues of every organ in the body. Signals transmitted by preganglionic neurons are cholinergic, whereas postganglionic symNs are adrenergic and thus express norepinephrine (NE) as their main neurotransmitter. There are few notable exceptions of postganglionic, sympathetic neurons that are cholinergic, including the ones innervating blood vessels. Adrenergic postganglionic neurons express the enzymes tyrosine hydroxylase (TH), aromatic L-amino acid decarboxylase (AAAD), dopamine β-hydroxylase (DBH), and monoamine oxidase (MAO-A), all responsible for generating and metabolizing NE. Furthermore, they express the NE recycling transporters and/or receptors α-adrenergic receptor (ADRA2), β-adrenergic receptor (ADR2B), norepinephrine transporter (NET1), and vesicular monoamine transporter (VMAT1/2).

Development
During embryonic development symNs are derived from the neural crest (NC), which emerges between the neural tube and overlaying ectoderm⁶, and can differentiate into multiple cell lineages, including melanocytes, osteoblasts, adipocytes, glia, enteric neurons, sensory neurons, and autonomic neurons⁷. Neural crest cells (NCCs) are highly migratory cells that take several routes through the embryo. At this early stage of NC development, the cells express the markers SNAIL1/2, FOXD3, and SOX10^8,9,10,11. The migration route together with the axial location they adopt determines the NC subtype into which they will develop. These NC subtypes can be distinguished by their specific HOX gene expression: Cranial NCCs do not express HOX genes, vagal NCCs express HOX 1–5, trunk NCCs express HOX 6–9, and sacral NCCs express HOX 10–11¹². Among them, trunk NCCs are recognized as the main source of symNs. SymN precursors express the transcription factor MASH1/ASCL1¹³, which promotes expression of PHOX2B¹⁴ and INSM1¹⁵. The GATA family of transcription factors is expressed during late sympathetic development. GATA2 and GATA3 are expressed in the symNs, which in turn activates DBH¹⁶. The transcription factor HAND2 is also important for the expression and maintenance of DBH and TH¹⁷.

HPSCs (e.g., embryonic and induced pluripotent stem cells) are a powerful tool¹⁸ to recapitulate developmental paradigms and generate symNs that can then be employed for disease modeling of various human disorders. Thus, while generating symNs from hPSCs, it is crucial to follow developmental guidelines and assess expression of appropriate markers along the differentiation process.

Previous symN protocol
Few research groups have previously reported the generation of symNs from hPSCs^19,20,21. The direct comparison of these protocols to each other and ours was reviewed recently²². In 2016²³, we published a differentiation protocol for the generation of autonomic neurons with symN character (Figure 1A). This protocol used KSR-based medium, which was used in both the maintenance of undifferentiated hPSCs and cell differentiation. Furthermore, hPSCs were maintained on mouse embryonic fibroblasts (MEF feeder cells). We employed this protocol and PSCs from patients with FD to model the disorder²³. In 2019, we described a more detailed version of this older protocol²⁴. In summary, the neural fate was induced by dual SMAD inhibition²⁵ to block TGF-β and BMP signaling in the first 2 days. WNT activation using CHIR99021 promoted neural progenitors to become NC cells. On day 11, cells were sorted by FACS for CD49D⁺ or SOX10⁺ populations^26,23, which yielded about 40% NC generation efficiency. Thus, sorting was needed to ensure the efficiency and purity for the next steps of differentiation. The NCCs were maintained and amplified as spheroids with the combined treatment of FGF2 and CHIR. After 4 days, the NC spheroids of maintenance were plated and given BDNF, GDNF, and NGF to finish the symN maturation. Although these symNs expressed strong symN markers such as ASCL1, TH, DBH, and PHOX2A, markers for more mature symNs, including expression of the nicotinic acetylcholine receptor (CHRNA3/CHRNB4) and vesicle transporter (VMAT1/2), were low even after 70 days of differentiation. HOX genes in this protocol were not formally tested, and mature neural properties, including electrophysiological activity of the cells, were not verified.

Here, we present an optimized protocol to generate symNs (Figure 1B). HPSCs are maintained in feeder-free conditions, on vitronectin (VTN)-coated dishes, using Essential 8 (E8) media²⁷. The formula of the differentiation media has been modified at each stage, thereby increasing the percentage of the NC population²⁸. The symN maturation can be done on CD49D⁺/SOX10⁺ sorted or unsorted bulk NCC populations. Both show high levels of symN marker expression by day 30. Moreover, the symNs generated with this protocol are responsive to electrophysiological recording and to treatments with symN activator and inhibitor compounds.

Protokół

NOTE: The H9 PHOX2B:GFP reporter line was provided by Oh et al.19. Some qPCR primers used in this paper were obtained from OriGene Technologies, while a few sequences are obtained from Frith et al.20,30. 1. Set-up for dish coating, media preparation, and hPSC maintenance Dish coating Vitronectin (VTN) coating Place vials of VTN in a 37 °C water bath until fully thawed, then mix thoroughly. For a 100 mm Petri dish, mix 7 mL of 1x phosphate buffered saline (PBS) with 0.5 mg/mL VTN, add VTN solution to the dish, and incubate at room temperature (RT) for 1 h. Basement membrane matrix coating Thaw vials of basement membrane matrix (see Table of Materials) on ice at 4 °C overnight. For one well of a 6 well plate, mix 2 mL of DMEM/F12 with 20 µL of 100x basement membrane matrix, add basement membrane matrix solution to the dish, wrap the dish with paraffin film, and store in a clean container at 4 °C overnight. Work as quickly as possible. Coated dishes can be stored in 4 °C for up to 2 weeks. Polyornithine (PO)/laminin (LM)/fibronectin (FN) coating On the first day, for one well of a 24 well plate, mix 15 µg/mL of PO with 1 mL of 1x PBS, incubate at 37 °C, 5% CO2 overnight. Thaw both LM and FN at -20 °C overnight and store at 4 °C until fully thawed. On the second day, aspirate PO solution, wash the wells 2x with 1x PBS, add 1 mL of 1x PBS containing 2 µg/mL of LM and 2 µg/mL of FN and incubate at 37 °C in 5% CO2 overnight. At this point the dish with the LM/FN solution can be kept in the incubator for months as long as it does not dry out. Add more 1x PBS to prevent the dish from drying out. Media preparation Prepare the Essential 8 medium (E8) by thawing one bottle of E8 supplement at 4 °C overnight. Mix the supplement with 500 mL of E8 medium and antibiotics if needed. NOTE: Working E8 solution should be used up within 2 weeks. Prepare the hPSC freezing medium by mixing 90 mL of complete E8 medium with 10 mL of DMSO for a total volume of 100 mL. Filter sterilize. Prepare the day 0 to day 1 differentiation medium by mixing 100 mL of essential 6 (E6) medium with 10 µM SB431542, 1 ng/mL BMP4, 300 nM CHIR99021, and 10 µM Y27632 for a total volume of 100 mL. Prepare the day 2 to day 10 differentiation medium by mixing 100 mL of E6 medium with 10 µM SB and 0.75 µM CHIR99021 for a total volume of 100 mL. Prepare the day 10 to day 14 spheroid medium by mixing neurobasal medium with 2 mL of B27 (50x), 1 mL of N2 (100x), 2 mM L-Glutamate, 3 µM CHIR99021, and 10 ng/mL FGF2 for a total volume of 100 mL. Prepare the day 14 to day 28 medium for spheroid long term maintenance by adding 0.5 µM of fresh RA to the day 10 to day 14 spheroid medium for every feeding. NOTE: Always keep RA at -80 °C. Prepare the SymN maturation medium by mixing neurobasal medium with 2 mL of B27 (50x), 1 mL of N2 (100x), 2 mM L-glutamate, 25 ng/mL GDNF, 25 ng/mL BDNF, 25 ng/mL NGF, 200 µM ascorbic acid, and 0.2 mM dbcAMP for a total volume of 100 mL. The solution should be used within 2 weeks. Before each feeding, add 0.125 µM fresh RA. This solution is used from day 14 (option 1) or day 28 (option 2). Prepare the FACS buffer by mixing DMEM with 2% FBS, 2 mM L-glutamate and antibiotics if needed for a total volume of 100 mL. hPSC maintenance Thawing and keeping hPSCs Prepare one VTN coated 100 mm dish. To thaw a vial of hPSCs directly from liquid nitrogen, put the vial into a 37 °C water bath, carefully swinging the tube in the water until it thaws. Transfer the thawed hPSCs to a 15 mL tube containing 10 mL of 1x PBS, and centrifuge at 200 x g for 4 min. Discard the supernatant and add 1 mL of E8 medium to the tube. Pipette a few times to fully resuspend the pellet and then add another 9 mL of E8 medium to reach 10 mL total. Aspirate the VTN solution from the 100 mm dish. Transfer the hPSCs to a 100 mm dish, shake gently (up-down and left-right, not in circles) to make sure cells are distributed evenly in the dish Incubate at 37 °C, in 5% CO2. On the following day, aspirate all medium and feed with 10 mL of E8. Feed this way every day for the next 3–4 days and then prepare to split. Splitting hPSCs NOTE: hPSCs at the point of splitting should be 80%–90% confluent. Big colonies with smooth and bright edges should be observed. However, contact between each colony should be avoided (Figure 2B, day 0 and Figure 6B). Prepare VTN-coated 100 mm dishes as needed. Aspirate the E8 and wash the dish that needs to be split 1x with 1x PBS. Aspirate the 1x PBS and add 4 mL of 0.25 M EDTA. Incubate for 2 min at 37 °C, 5% CO2. NOTE: The hPSCs should be split/replated as small colonies. Do not treat with EDTA longer than 2 min to prevent separation into single cells. The cells should be still attached to the dish surface after the 2 min treatment. Aspirate the EDTA, detach the colonies by strongly pipetting 10 mL of E8 medium onto the dish surface, and collect all the medium and cells in a 15 mL tube. With hPSCs at 80%–90% confluency, split colonies by 1:15-1:20. For example, to split hPSCs by 1:20 into one 100 mm dish, take 500 µL of E8/hPSCs solution and mix with 9.5 mL of fresh E8 medium. Plate hPSCs in VTN-coated 100 mm dishes. NOTE: It is advised to establish the ideal split ratio for each researcher and cell line independently. Freezing hPSCs For one 100 mm dish of hPSCs that is ready to be split, prepare three cryovials and 3.5 mL of freezing medium. NOTE: Media and vials should be kept in 4 °C or on ice until usage. Aspirate E8 and wash the dish 2x with 1x PBS. Aspirate 1x PBS and add 4 mL of 0.25 M EDTA, incubate for 2 min at 37 °C, in 5% CO2. NOTE: hPSCs should be frozen as small colonies at the time that they would be split. Do not treat the cells with EDTA longer than 2 min to prevent separation into single cells. Cells should be still attached on the dish’s surface after the 2 min treatment. Aspirate the EDTA, detach the colonies by strongly pipetting 10 mL of 1x PBS on the dish’s surface, and collect all medium and cells in a 50 mL tube. Add 20 mL of 1x PBS and centrifuge at 200 x g for 4 min to wash out the remaining EDTA. Discard the supernatant and resuspend the pellet in 3 mL of freezing medium. Distribute hPSCs evenly into the three cryovials, 1 mL each. Store at -80 °C overnight in a controlled freezing box or a styrofoam sandwich to ensure a slow temperature drop, and then transfer to a liquid nitrogen tank for long term storage. 2. Seeding hPSCs to start the differentiation (day 0) NOTE: hPSCs should be ready for differentiation after being stabilized (i.e., being split 2–3x after thawing). Be sure that the colonies are healthy, with smooth, shiny edges, and minimal differentiation (Figure 2B). Prepare basement membrane matrix-coated dishes (24 well or 6 well dishes) one day before day 0 as needed. Bring the dishes to RT at the start of differentiation. Make the day 0–1 differentiation medium as needed. Aspirate the E8 from the confluent, ready to split hPCSs, and wash the dish 2x with 1x PBS. Add 7 mL of 0.25 M EDTA per 100 mm dish, incubate at 37 °C, 5% CO2, for 15 min. NOTE: At this point, EDTA treatment is prolonged to disperse into single cells. Pipet off all the hPSCs (they should be floating) and transfer to a 50 mL tube. Add the same amount or more of 1x PBS as EDTA solution to dilute out the EDTA. Centrifuge at 200 x g for 4 min. Discard the supernatant, add 1 mL of day 0–1 differentiation medium and pipet to homogenize the cells. Follow by adding more medium and then mix to dilute the cell solution to a concentration ideal to count the cells. NOTE: Do not overdilute the cell solution. Cells from one full 100 mm dish should be diluted in 5 mL of medium to start. Count the cell number using an automated cell counter or hemocytometer. Dilute the cell solution as needed to reach 125,000 cells/cm2 in a low final volume (e.g., 2 mL per well for a 6 well dish or 500 µL per well for a 24 well dish). NOTE: A low volume helps the cells attach faster. Aspirate all of the basement membrane matrix solution from the coated dishes and plate the cell solution into the wells. Incubate at 37 °C, in 5% CO2. 3. Neural crest cell induction (day 1 to day 10, Figure 2A) On day 1 feed the cells with day 0–1 differentiation medium (3 mL per well for 6 well dishes and 1 mL per well for 24 well dishes). On day 2 feed the cells with day 2–day 10 differentiation medium (3 mL per well for 6 well dishes and 1 mL per well for 24 well dishes). From now on, the cells should be fed every other day until day 10 (i.e., the next feeding day will be day 4). NOTE: From day 6 on, NC ridges should be detected (Figure 2B). To check if differentiation is taking place, it is advised to carry a parallel differentiation culture in smaller wells (i.e., 24 wells), that can be stained for SOX10/AP2a and used for marker gene expression along the time of differentiation (Figure 2B,C). If sorting cells, proceed to section 4. Otherwise, proceed to section 5. 4. Fluorescence activated cell sorting (FACS) for neural crest marker CD49D and aggregating NC cells in spheroids NOTE: For FACS sorting, the samples should be kept on ice and not be exposed to light after staining until sorting. Prepare FACS buffer if the cells are sorted. Prepare day 10–14 spheroid medium. On day 10, remove the medium, and wash 1x with 1x PBS. Add dissociation solution (see Table of Materials) at 2 mL per well for a 6 well dish or 1 mL per well for a 24 well dish, and incubate at 37 °C, 5% CO2 for 20 min. Pipet off all the hPSCs and transfer to a 50 mL tube. Fill up the rest of the tube with FACS buffer and centrifuge at 200 x g for 4 min. NOTE: Each 50 mL tube can accommodate up to 20 mL or less of cell solution. The volume of the FACS buffer should be high enough to neutralize the dissociation solution. Discard the supernatant, resuspend the cells with the appropriate amount of FACS buffer (~2 mL per well of a 6 well plate), and count to determine the cell number. Prepare the following samples. Sample 1 (unstained control): 1 x 106 cells in 400 µL of FACS buffer. Filter the cells through a 20 µm strainer cap and keep the tube on ice. Sample 2 (DAPI only control): 1 x 106 cells in 400 µL of FACS buffer containing 0.5 ug/mL DAPI. Filter the cells through a FACS tube with a strainer cap and keep the tube on ice. Sample 3 (CD49d-labeled): Suspend the rest of the cells with FACS buffer containing PE/Cy7-conjugated CD49D antibody (5 µL for 1 x 106 cells per 100 µL of FACS buffer) in a 15 mL tube and incubate on ice for 20 min. Fill up the tubes with FACS buffer and centrifuge at 200 x g for 4 min. Discard the supernatant and resuspend every 5–10 x 106 cells in 1 mL of FACS buffer containing 0.5 ug/mL DAPI according to the manufacturer’s instructions. Filter the cells through the FACS tube with the strainer cap and keep the tube on ice. Prepare collection FACS tubes containing 2 mL of FACS buffer. Sort through the FACS machine with lasers that can detect DAPI and PE-Cy7 to isolate the CD49D+ population. After sorting, count the sorted cells. Centrifuge all sorted cells and resuspend in day 10–14 spheroid medium to a final concentration of 0.5 x 106 cells per 500 µL of medium. Plate 0.5 x 106 cells per well into ultra-low attachment 24 well plates. Incubate the cells at 37 °C, in 5% CO2. 5. Aggregating NC cells in spheroids If not using FACS to isolate the NC cells and instead aggregating them into spheroids directly, first prepare cells as described in steps 4.2–4.5. Fill up the rest of the tube with 1x PBS, and centrifuge at 200 x g for 4 min. Discard the supernatant, resuspend the cells with an appropriate amount of day 10–14 spheroid medium (e.g., ~2 mL of medium per well for a 6 well plate), and count to determine the cell number. Dilute the cells in day 10–14 spheroid medium to 0.5 x 106 cells per 500 µL of medium. Plate 500 µL of the cell suspension per well in ultra-low attachment 24 well plates. Incubate the cells at 37 °C, in 5% CO2. 6. NC spheroid maintenance and sympathetic progenitor induction (day 10 to day 14, Figure 4A) Option 1: Minimal spheroid culture On day 11, add 500 µL of day 10–14 spheroid medium to the NC spheroids without aspirating existing medium from day 10. Incubate at 37 °C, in 5% CO2. On day 12, tilt the plate to accumulate the NC spheroids on one side of the wells. Carefully aspirate and discard as much medium as possible, and feed with 1 mL of day 10–14 spheroid medium. Keep feeding the cells every day until day 14. Optional: If the spheroids aggregate and generate a large clump, use a pipette to break the spheroid clumps up. This also ensures that individual spheroids do not get too large. NOTE: The ideal spheroid size range should be around 100–500 μm. Within that range, the size of individual spheroids is not critical. However, the morphology, such as a smooth and clear edges (Figure 3 and Figure 6) is important for further success. At day 14, each 24 well plate ideally contains about 50–60 spheroids of different sizes within the abovementioned size range. Option 2: Expanded spheroid culture On day 15, to keep NC spheroids, feed with 1.5 mL of day 10–14 spheroid medium containing 0.5 µM RA. Incubate at 37 °C, 5% CO2. NOTE: RA should be added fresh for every feeding and always be stored at -80 °C. From now on, feed every other day up to day 28 and then continue with plating of the spheroids (section 7.1). NOTE: The growing spheroids are split approximately 1x per week by pipetting them with a 1 mL pipette to break them up. They are split at an approximate ratio of 1:2–1:4. Within the 2 week expansion period, the cells should roughly quadruple in number. 7. SymN differentiation and maturation (Option 1: after day 14; Option 2: after day 28) Plating spheroids in regular dishes Prepare PO/LM/FM-coated 24 well plates. Prepare symN medium containing 0.125 µM RA (add fresh every feed) and 10 µM Y27632 (day 14 only). On day 14, tilt the plate to accumulate NC spheroids on one side of the wells. Carefully aspirate and discard as much medium as possible, and feed with 1 mL of symN medium. Remove LM/FN from the coated plates. Split and plate each well from the 24 well plate into 4 separate wells of the new, coated 24 well plate. Each original well will have 1 mL, containing ~50–60 spheroids. This yields 250 µL, containing approximately 10–15 spheroids for each well on the new plate. Add 250 µL of additional medium per well. NOTE: This is a split of 1:4; make sure that the spheroids are distributed properly within the solution so that the split is relatively even. The number of spheroids is not counted because the final number does not affect the success of generating symNs. Incubate at 37 °C, 5% CO2. On day 15 (or day 29 for option 2), feed by replacing all medium with 1 mL of symN medium containing 0.125 µM RA. From now on, the neurons should be fed every 2 days until day 20 (or day 35 for option 2). After day 20 (or day 35 for option 2), the neurons should be fed by carefully replacing only half of the existing medium (500 µL). From now on, feed every week unless the medium quickly turns yellow. Keep feeding weekly until the desired time point. NOTE: symNs tend to aggregate in ganglia-like structures and are prone to detach from the culture dishes. To prevent this, half-feedings and minimal handling is recommended. Plating cells for electrophysiological recording Prepare PO/LM/FM-coated 96 well electrophysiology plates. Prepare symN medium containing 0.125 µM RA (add fresh every feed) and 10 µM Y27632 (day 14 only). On day 14, collect all spheroids, then centrifuge them at 200 x g for 4 min. Discard the supernatant, add 2 mL of dissociation solution, and transfer the mixture back to one of the wells of the ultra-low attachment plate. Incubate at 37 °C, in 5% CO2 for 20–45 min. NOTE: Depending on the size of the spheroids, the dissociation period can be longer than 20 min. Check the cell’s dissociation every 10 min up to 45 min. Optionally, 0.1 mg/mL of DNase can be added with dissociation solution to prevent free DNA from dead cells making the solution sticky. This is optional in this protocol because the spheroids will not aggregate once they are fully dissociated. Pipet to fully dissociate the spheroids, then centrifuge at 200 x g for 4 min. Discard the supernatant, resuspend the cells with appropriate amount of symN medium, and count the cell number. Plate the cells at 100,000/cm2 in PO/LM/FN-coated electrophysiology wells in 200 µL total volume per well. On day 15 (or day 29 for option 2), follow the same feeding processes as in section 7.1. The total volume after day 15 (or day 29 for option 2) should be 300 µL per well. Measure the electrical signals using a multielectrode array machine after day 20 (or day 35 for option 2). NOTE: In option 2, spheroids can be plated anytime between day 14–day 28. The first electrical signals measurements can be conducted 1 week after plating the spheroids.

Representative Results

In this protocol, we give instructions on how to generate symNs from hPSCs. The culture conditions demonstrated here were improved from an earlier published protocol23,24 (Figure 1A) to feeder-free and chemically defined conditions (Figure 1B). Two options are provided, one where symNs are made within 20 days, and another where the NCCs can be expanded for 2 weeks to generate more cells that can then be …

Discussion

We recently published two reviews, one discussing the use of hPSC-derived symNs for disease modeling³¹ as well as an in-depth comparison of available differentiation protocols²². Thus, here we focus on troubleshooting the current protocol to help the interested researcher succeed in making symNs. During the entire differentiation process, in order to gain consistent data as well as healthy differentiated cells, contamination at all stages should be carefully controlled. In …

Materials

100 mm cell culture dishes	Falcon	353003
15 mL conical tissue culture tubes	VWR/Corning	89039-664
24-well tissue culture plates	Falcon	353047
24-well ultra-low-attachment plates	Corning	07 200 601 and 07 200 602
5% CO2/20% O2 tissue culture incubator	Thermo Fisher/Life Technologies	Heracell VIOS 160i
50 ml conical tissue culture tubes	VWR/Corning	89039-656
6-well tissue culture plates	Costar	3516
Accutase	Innovation Cell Technologies	AT104500	Cell dissociation solution
Anti-AP2a antibody	Abcam	ab108311	Host: Rabbit; 1:400 dilution
Anti-Ascl1 antibody	BD Pharmingen	556604	Host: Mouse IgG1; 1:200 dilution
Anti-CD49D antibody	BioLegend	304313	Host: Mouse IgG1; 5 μl/million cells in 100 μl volume
Anti-CD49D antibody (isotype)	BioLegend	400125	Host: Mouse IgG1; 5 μl/million cells in 100 μl volume
Anti-DAPI antibody	Sigma	D9542	1:1000 dilution
Anti-DBH antibody	Immunostar	22806	Host: Rabbit; 1:500 dilution
Anti-GFP antibody	Abcam	ab13970	Host: Chicken; 1:1000 dilution
Anti-HOXC9 antibody	Santa Cruz Biotechnology	sc-365692	Host: Mouse IgG1; 1:100 dilution
Anti-NET1 antibody	Mab	NET17-1	Host: Mouse; 1:1000 dilution
Anti-PRPH antibody	Santa Cruz Biotechnology	SC-377093/H0112	Host: Mouse IgG2a; 1:200 dilution
Anti-SOX10 antibody	Abcam	ab50839	Host: Mouse; 1:100 dilution
Anti-TH antibody	Pel-Freez	P40101- 150	Host: Rabbit; 1:500 dilution
Ascorbic acid	Sigma	A8960-5G	Stock concentration: 100 mM
B27 supplement	Thermo Fisher/Life Technologies	12587-010	Stock concentration: 50x
BDNF	R&D Systems	248-BD	Stock concentration: 10 μg/mL
BMP4	R&D Systems	314-BP	Stock concentration: 6 mM
Cell counter	Thermo Fisher/Life Technologies	Countess II
Cell counting chamber slides	Invitrogen	C10312
Centrifuge	Eppendorf	57021&5424R
CHIR99021	R&D Systems	4423	Stock concentration: 6 mM
Cryo-vial	Thermo Fisher/Life Technologies	375353
dbcAMP	Sigma	D0627	Stock concentration: 100 mM
DMEM	Thermo Fisher/Life Technologies	10829-018	Stock concentration: 1x
DMEM/F12	Thermo Fisher/Life Technologies	11330-057	Stock concentration: 1x
DMSO	Thermo Fisher/Life Technologies	BP231-100
E6 medium	gibco	A15165-01
E8 medium	gibco	A15169-01	Stock concentration: 1x
E8 supplement	gibco	A15171-01	Stock concentration: 50x
EDTA	Sigma	ED2SS	Stock concentration: 0.5 M
Electrophysiology plates (AXION cytoview MEA96)	Axion BioSystems	M768-tMEA-96W
FACS machine	Beckman Coulter	CytoFLEX (for FACS)
FACS machine	Beckman Coulter	MoFlo Astrios EQ (for sorting)
FACS tubes (blue filter cap)	Falcon	352235
FACS tubes (white cap)	Falcon	352063
Fetal bovine serum (FBS)	Atlanta Biologicals	S11150
GDNF	PeproTech	450	Stock concentration: 10 μg/mL
Geltrex	Invitrogen	A1413202	Basement membrane matrix; Stock concentration: 100x
hPSCs	Thomson et al., (1998)	WA09
hPSCs	Oh et al. (2016)	H9-PHOX2B::eGFP
Human fibronectin (FN)	VWR/Corning	47743-654	Stock concentration: 1 mg/mL
L-glutamine	Thermo Fisher/Gibco	25030-081	Stock concentration: 200 mM
LN tank	Custom Biogenic Systems	V-1500AB
MEA reader	Axion BioSystems	Maestro Pro
Mouse laminin I (LM)	R&D Systems	3400-010-01	Stock concentration: 1 mg/mL
N2 supplement	Thermo Fisher/Life Technologies	17502-048	Stock concentration: 100x
Neurobasal medium	gibco	21103-049	Stock concentration: 1x
NGF	PeproTech	450-01	Stock concentration: 25 μg/mL
Phosphate-buffered saline (PBS)	Gibco	14190-136	Stock concentration: 1x
Poly-L-ornithine hydrobromide (PO)	Sigma	P3655	Stock concentration: 15 mg/mL
Primocin (antibiotics)	InvivoGen	ANTPM1	Stock concentration: 50 mg/mL
qPCR machine	Bio-Rad Laboratories	C1000 Touch
qPCR plates	Bio-Rad Laboratories	HSP9601
recombinant FGF2	R&D Systems	233-FB/CF	Stock concentration: 10 μg/mL
Retinoic acid	Sigma	R2625	Stock concentration: 1 mM
SB431542	Tocris/R&D Systems	1614	Stock concentration: 10 mM
Trypan blue	Corning	MT-25-900-CI
Vitronectin (VTN)	Thermo Fisher/Life Technologies	A14700	Stock concentration: 0.5 mg/mL
Water bath	VWR/Corning	706308
Y27632	R&D Systems	1254	Stock concentration: 10 mM