TITLE: Derivation of Spinal Cord Organoids as a quantitative approach to study the mammalian Hedgehog Signalling
AUTHORS: Markus Holzner, Anton Wutz, Giulio Di Minin

[DESCRIPTION]
The Hedgehog (HH) pathway is crucial for embryonic development, and adult homeostasis. Its dysregulation is implicated in multiple diseases. Existing cellular models used to study HH signal regulation in mammals do not fully recapitulate the complexity of the pathway. Here we describe the procedure to derive Spinal Cord Organoids (SCOs) and we show how to apply them to quantitively study the activity of the HH pathway. During SCO formation, the specification of different categories of neural progenitors (NPC) depends on the intensity of the HH signal, mirroring the process that occurs during neural tube development. By assessing the number of NPCs within these distinct subgroups, we are able to categorize and quantify the activation level of the HH pathway. SCOs represent an accessible and reliable in-vitro tool to quantify HH signaling and investigate the contribution of genetic and chemical cues in the HH pathway regulation. 

[STEPS]
SECTION: mESC maintenance
1. Note: the procedure describes the necessary amounts for the expansion of mouse ESCs in a 3 cm dish. This dimension will provide enough cells to derive SCOs. Volumes can, however, easily be adapted according to the user’s needs.
 
 Pre-coat the necessary number of wells of a 3 cm dish with 1 mL of 0,2% gelatin. Leave for at least 10 min at room temperature (RT).

Note: Alternatively, ESCs can be grown on feeder cells (e.g., mouse embryonic fibroblasts). To improve ESC attachment in the absence of feeder cells 0,5% of FBS can be added to the gelatin solution.
 
split ES cells once colonies reach a confluency of 60-80%. 

Note: Proper ESC growth conditions are crucial for the generation of SCOs. ESCs should be passed every 2 days. Inspect ESC culture and check for colony size and morphology. An overly dark center indicates a necrotic area within the colony due to cell overgrowth and must be avoided. Adjust plated cell numbers to match the specific needs of your ESC line. Loss of compactness and loose borders are signs of differentiation. In this case, a co-culture on feeder cells is suggested to enhance the pluripotency state of the ESCs. 
 
Aspirate the culture media, wash cells once with 2 mL of PBS.
Incubate with an appropriate amount of Trypsin EDTA (200 µL per one well of a 3 cm dish) for 3-5 min at 37 °C. 
Once cells are detached from the plate, stop the reaction with 2 mL of ESC base media and transfer the cell suspension to a centrifuge tube.
Spin the cells at 900 rpm for 5 min and resuspend the cells in ESC maintenance media.
 Count cells and plate 400k ESCs on a gelatin pre-coated culture dish.

Note: For neuronal EB induction, resuspend the ES cells after centrifugation in the differentiation medium to avoid a carry-over of 2i and LIF.
SECTION: Derivation of Spinal Cord Organoids (SCOs)
2. Day 1. ESC plating in AggreWell plate for nEB formation
Timing: 1 to 2 hours, depending on the number of samples. 
 
Per each sample, prepare two wells of an AggreWell Plate. 
 Incline the AggreWell plate to an angle of 30-40° and slowly add 500 µL of differentiation medium to the bottom of each well. Make sure to evenly distribute the media and avoid bubble formation. Bubbles can be removed by pipetting or lightly shaking.
Plate 50k to 150k ESCs per well. Resuspend cells in 1 mL of differentiation media per sample, mix, and transfer cells into the AggreWell plate.

Note: The final dimension of nEBs is crucial for neuralization and pattering of later SCOs. The amount of ESCs plated in the AggreWell wells must therefore be adjusted and optimized according to the used ESCs.

 Distribute the cells by pipetting up and down slowly and avoid bubble formation. 
Seal the plate with Parafilm and spin for 5 min at 900 rpm. This step is optional, will however increase the size homogeneity of later formed nEBs.
Incubate plates at 37 °C and 5 % CO2 until Day 3. 
SECTION: Derivation of Spinal Cord Organoids (SCOs)
3. Day 3. Transfer of nEBs to a 10 cm culture dish 
Timing: 15 min per sample 
 
Transfer each sample (2 wells of the AggreWell plate) into one 10 cm dish. Use low-attachment plates to prevent nEBs from attaching to the plate.
Add 10 mL of differentiation media, supplemented with 100-200 nM retinoic acid to a 10 cm plate. 

Note: RA activity is strongly different between formulations and according to the storing conditions. We try to avoid freeze-thaw cycles. The optimal RA concentration should be defined by users.

With a cut tip and a P1000 pipet wash the nEBs out of their wells by slowly pipetting up and down, approximately 5 times. 

Note: When transferring the nEBs always use P1000 tips with the front cut off. The increased opening will help to maintain the nEBs structural integrity while handling.

Transfer the nEBs to their respective 10 cm plate. 
Evenly distribute nEBs in the plate by gently rocking the plates in circles, drawing an 8 when looked at from above. 
Carefully transfer the plates back to the incubator.
Incubate plates at 37 °C and 5 % CO2.

Note: From this point on we call the nEBs now Spinal Cord Organoids (SCOs)
SECTION: Derivation of Spinal Cord Organoids (SCOs)
4. Day 4. Patterning of the dorsal to ventral axis. 
Timing: 10 min
ES cells have now reached the stage of the neuronal plate. To trigger the dorsal to ventral axis patterning, samples can be treated with the SHH protein or hedgehog agonists like SAG or Purmorphamine (PMP). 

To induce specification, add SHH at 200 ng/mL or PMP at a final concentration of 1 to 2 µM. The negative control is maintained in only retinoic acid. 
Transfer plates back into the incubator and ensure an even distribution of SCOs among the whole plate.
Incubate plates at 37 °C and 5 % CO2.
SECTION: Derivation of Spinal Cord Organoids (SCOs)
5. Day 6. End of the Experiment and sample preparation for either qPCR analysis or IF staining.

SCOs are collected for downstream analysis

Note: Ventral markers are already detected after 24 h of SHH treatment and remain expressed up until 48 h of SHH treatment. Thereafter (day 8), NPCs start to differentiate and will express neuronal markers.
SECTION: Preparing SCOs for transcriptional analysis
6. Lysis of the samples

Prepare one microcentrifuge tube (2 mL) for each sample. Collect SCOs in the center of the 10 cm plate by slowly swirling the plate in small circles. 
Transfer the SCOs to the Eppendorf tube with a P1000 pipet (and a cut tip) and let them sediment by gravity. 
Remove supernatant and wash SCOs twice with each 2 mL of PBS, letting the SCOs sediment by gravity in between each washing step. Remove the supernatant.
SECTION: Preparing SCOs for transcriptional analysis
7. RNA extraction

Resuspend the SCOs in the lysis buffer of your preferred RNA extraction method. We use the Quiagen RNeasy kit with the following adjustments.
Add 500 µL of lysis buffer (RLT) per sample and dissociate SCOs by pipetting. 
Transfer samples to a spin column of the QIAshredder and collect the lysates by a short centrifugation (15 s, max speed). 
Follow the RNA extraction protocol as suggested by the manufacturer and include the optional on-column DNAse digestion. 
Extracted RNA is eluted with 40 µL of water. 

SECTION: Preparing SCOs for transcriptional analysis
8. cDNA preparation

cDNA is prepared with the Qiagen QuantiTect Reverse Transcription Kit following the manufacturer’s instructions. 
SECTION: Immunofluorescence analysis of SCO sections
9. Sample preparation for immunofluorescence analysis 
Timing: 4 h

Note: To maintain the SCOs structural integrity, avoid unnecessary pipetting as much as possible.

Transfer SCOs into microcentrifuge tubes by first collecting them in the center of the culture dish swirling the plate in small circles, and then taking them up with a cut, BSA-coated P1000 tip.

Note: Coat microcentrifuge tubes (2 mL Eppendorf tubes) with a BSA solution; discard BSA solution. Do the same for tips that are used to transfer the SCOs. This will prevent SCOs from sticking to the plastic of the tubes and tips.

Wash SCOs twice with ice-cold PBS. Let SCOs sink to the bottom of the microcentrifuge tube by gravity alone, remove the supernatant and add 1 mL of ice-cold PBS to each sample. Repeat this process once more.
Fixing SCOs. Remove PBS and add 1 mL of a freshly prepared 4 % PFA solution to each sample. Incubate for 30 min at 4 °C, shaking at about 100 rpm. 
Wash the fixed SCOs twice with each 1 mL of ice-cold PBS just as done before. After the second wash, remove almost all PBS but leave ca. 150 µL in each tube. 
Cryoprotect the SCOs in sucrose. For best results, the sucrose concentration is increased stepwise from 10 to 30%. Start by adding 1 mL of a 10% sucrose solution in PBS to each sample. You will notice, that the SCOs will now float at the interface between the sucrose solution and PBS. Incubate the samples again at 4 °C, shaking until the SCOs are saturated with sucrose and have sunk to the bottom of the microcentrifuge tube. This will take about 30 min. Repeat with 20% and 30% of sucrose solutions always leaving ca. 150 µL on top of the SCOs after each step.
Embedding and freezing. The tissue freezing medium is very viscous and is pipetted best with a cut P1000 tip. Add about 1 mL of the freezing medium to each sample. Swirl and resuspend SCOs stirring, not pipetting with a P200 tip inside the tube. Then transfer the SCOs into a Cryomold‱. Avoid air bubbles and evenly distribute SCOs over the whole area of the Cryomold‱. Top off with additional tissue freezing medium until the mold is filled. Transfer the container onto a cold metal block to freeze and then store samples at -80 °C.

Note: Freezing SCOs for later cryosections is best performed directionally, bottom to top. Therefore, add dry ice to a box and place a metal block inside. The block will cool down and serve as platform where the Cryomolds‱ can be placed for freezing.
SECTION: Immunofluorescence analysis of SCO sections
10. Preparing cryosections 
Timing 1h per sample 
Cryosections are prepared with a cryotome. Following the device specific instructions, cut 10-15 µm thick slices and collect them on SuperFrost‱ Plus slides. 
Let the sections dry at room temperature for 1 h. Cryosections can be stored at –80 °C.
SECTION: Immunofluorescence analysis of SCO sections
11. Immunofluorescence staining
Timing 2 Days

Let the slides warm up to RT. 
Rehydrate cryosections for 30 min in PBS. 
Permeabilization/Blocking. Incubate samples in PBS with 0,3% Triton X and 10% Donkey Serum for 30 min at RT.
Primary Antibody (AB) incubation. Prepare a master mix of 300 µL per slide. Primary antibodies are diluted in AB buffer and mixed well. The AB solution is added to each slide, and a piece of parafilm (equal size as the slide) is added carefully on top. The slides are incubated overnight at 4 °C in a humidified chamber. 
Samples are washed three times with PBS Triton X (0,1%) for 5 min each. 
Secondary AB incubation. Secondary ABs and Dapi are diluted in AB buffer. Again 300 µL of AB solution is added to each slide, protected with a piece of parafilm, and incubated at RT, in the dark, for 30 min. 
Samples are washed twice with PBS Triton X (0,1%) for each 5 min and once with PBS for 5 min.
Mount samples with Mowiol and leave to dry at RT, in the dark, overnight. Samples can now be analyzed with a fluorescence microscope. 

Note: We generally acquire SCOs picture using a 20X objective. For each condition, at least 10 independent SCOs are acquired and counted.