Generation of Human Brain Organoids for Mitochondrial Disease Modeling

Podsumowanie

We describe a detailed protocol for the generation of human induced pluripotent stem cell-derived brain organoids and their use in modeling mitochondrial diseases.

Streszczenie

Mitochondrial diseases represent the largest class of inborn errors of metabolism and are currently incurable. These diseases cause neurodevelopmental defects whose underlying mechanisms remain to be elucidated. A major roadblock is the lack of effective models recapitulating the early-onset neuronal impairment seen in the patients. Advances in the technology of induced pluripotent stem cells (iPSCs) enable the generation of three-dimensional (3D) brain organoids that can be used to investigate the impact of diseases on the development and organization of the nervous system. Researchers, including these authors, have recently introduced human brain organoids to model mitochondrial disorders. This paper reports a detailed protocol for the robust generation of human iPSC-derived brain organoids and their use in mitochondrial bioenergetic profiling and imaging analyses. These experiments will allow the use of brain organoids to investigate metabolic and developmental dysfunctions and may provide crucial information to dissect the neuronal pathology of mitochondrial diseases.

Wprowadzenie

Mitochondrial diseases represent the largest class of inborn errors of metabolism¹. They are caused by genetic mutations disrupting different mitochondrial processes, including oxidative phosphorylation (OXPHOS)², respiratory chain assembly, mitochondrial dynamics, and mitochondrial DNA transcription or replication³. Tissues with energy requirements are particularly affected by mitochondrial dysfunction⁴. Accordingly, patients with mitochondrial diseases typically develop early-onset neurological manifestations.

There are currently no treatments available for children affected with mitochondrial diseases⁵. A major hindrance for drug development of mitochondrial diseases is the lack of effective models recapitulating the human disease course⁶. Several of the currently studied animal models do not exhibit the neurological defects present in the patients⁷. Hence, the mechanisms underlying the neuronal pathology of mitochondrial diseases are still not fully understood.

Recent studies generated iPSCs from patients affected by mitochondrial diseases and used these cells to obtain patient-specific neuronal cells. For example, genetic defects associated with the mitochondrial disease, Leigh syndrome, have been found to cause aberrations in cellular bioenergetics^8,9, protein synthesis¹⁰, and calcium homeostasis^9,11. These reports provided important mechanistic clues on the neuronal impairment occurring in mitochondrial diseases, paving the way for drug discovery for these incurable diseases¹².

Two-dimensional (2D) cultures, however, do not enable the investigation of the architectural complexity and regional organization of 3D organs¹³. To this end, the use of 3D brain organoids derived from patient-specific iPSCs¹⁴ may allow researchers to gain additional important information and thereby help to dissect how mitochondrial diseases impact the development and function of the nervous system¹⁵. Studies employing iPSC-derived brain organoids to investigate mitochondrial diseases are beginning to uncover the neurodevelopmental components of mitochondrial diseases.

Spinal cord organoids carrying mutations associated with the mitochondrial disease, mitochondrial encephalopathy, lactic acidosis, and stroke-like episodes syndrome (MELAS), showed defective neurogenesis and delayed motor neuron differentiation¹⁶. Cortical organoids derived from patients with the mitochondrial disease, Leigh syndrome, showed reduced size, defects in neural epithelial bud generation, and loss of cortical architecture¹⁷. Brain organoids from Leigh syndrome patients showed that the disease defects initiate at the level of neural progenitor cells, which cannot commit to mitochondrial metabolism, causing aberrant neuronal branching and morphogenesis¹⁸. Thus, neural progenitors may represent a cellular therapeutic target for mitochondrial diseases, and strategies promoting their mitochondrial function may support the functional development of the nervous system.

The use of brain organoids might help uncover the neurodevelopmental components of mitochondrial diseases. Mitochondrial diseases are mainly considered as early-onset neurodegeneration⁵. However, neurodevelopmental defects are also present in patients affected by mitochondrial diseases, including developmental delay and cognitive impairment¹⁹. Patient-specific brain organoids may help address these aspects and elucidate how mitochondrial diseases may impact human brain development. Mitochondrial dysfunction could also play a pathogenetic role in other more common neurological diseases, such as Alzheimer's disease, Parkinson's disease, and Huntington's disease⁴. Hence, elucidating the impact of mitochondrial defects in neurodevelopment using brain organoids might also be instrumental for the study of those diseases. This paper describes a detailed protocol for generating reproducible brain organoids that can be used for conducting disease modeling of mitochondrial diseases.

Protokół

NOTE: The use of human iPSCs may require an ethical approval. iPSCs used in this study were derived from healthy control individuals following local ethical approval (#2019-681). All cell culture procedures must be performed under a sterile cell culture hood, carefully disinfecting all reagents and consumables before transferring under the hood. Human iPSCs used for differentiation should have a passage number below 50 to avoid potential genomic aberrations that may occur upon extensive culture. The pluripotent state of the cells should be validated before organoid generation, for example, by monitoring the expression of pluripotency-associated markers such as NANOG or OCT4. Mycoplasma tests should be conducted weekly to ensure mycoplasma-free cultures.

1. Generation of brain organoids

1. Culture of human iPSCs
   1. Culture human iPSCs under feeder-free conditions in iPSC medium (see the Table of Materials) on coated 6-well plates and keep them in a humidified tissue culture incubator at 37 °C and 5% CO₂.
      ​NOTE: The carryover of feeder cells may hamper the organoid differentiation. Passage the cells at least once in feeder-free conditions.
   2. Passage the iPSCs at 80% confluency using enzyme-free detachment medium in ratios ranging from 1:4 to 1:12. To increase cell survival, add 10 µM Rho-associated protein kinase (ROCK) inhibitor (Y27632) after each splitting.
2. Dissociate the iPSCs (80% confluency)-Day 0.
   1. Prepare Cortical Differentiation Medium I (CDMI) (Table 1). Prewarm CDMI medium at room temperature (22-25 °C) before adding it to the cells.
   2. Wash the wells containing the iPSCs with phosphate-buffered saline (PBS) to remove dead cells and debris.
   3. Add 500 µL of prewarmed Reagent A (Table of Materials) to each well and incubate for 5 min at 37 °C. Check under the microscope to ensure cell detachment.
   4. Add 1 mL of iPSC medium to dilute reagent A to neutralize its activity.
   5. Use a 1000 µL pipette to dissociate the cells by pipetting up and down and transfer the cell suspension to a 15 mL centrifuge tube.
   6. Gently centrifuge the iPSCs at 125 x g for 5 min at room temperature (22-25 °C).
   7. Carefully aspirate the supernatant to avoid disturbing the cell pellet.
   8. Resuspend the pellet with 1 mL of CDMI to obtain a single-cell suspension, and count the cell number.
   9. Prepare the seeding medium with 9,000 iPSCs per 100 µL in CDMI supplemented with 20 µM ROCK inhibitor, 3 µM WNT-catenin inhibitor (IWR1), and 5 µM SB431542.
   10. Add 100 µL of seeding medium per well to a 96-well v-bottom plate.
   11. Keep the plate in a humidified tissue culture incubator at 37 °C and 5% CO₂.
3. Neurosphere generation
   1. On day 1, observe that round cell aggregates (neurospheres) with defined smooth borders are forming. Note the dead cells around the aggregates. Continue to culture in the incubator at 37 °C and 5% CO₂.
   2. On day 3, agitate the plate by tapping on the sides three times to detach dead cells.
   3. Add 100 µL of CDMI supplemented with 20 µM ROCK inhibitor, 3 µM IWR1, and 5 µM SB431542 to each well.
   4. Return the plate to the incubator at 37 °C and 5% CO₂.
   5. On day 6, carefully remove 80 µL of the supernatant medium from each well. Avoid touching the bottom of the well.
   6. Add 100 µL of CDMI supplemented with 3 µM IWR1 and 5 µM SB431542 to each well. Return the plate to the incubator at 37 °C and 5% CO₂.
   7. Repeat steps 5 and 6 every 3 days until day 18.
4. Transfer of neurospheres
   1. On day 18, prepare Cortical Differentiation Medium II (CDMII) (Table 1) and add 10 mL to a 100 mm ultra-low attachment cell culture plate.
   2. Use a 200 µL pipette with the tip cut off to transfer the round neurospheres from the 96-well plate to the 100 mm ultra-low attachment cell culture plate.
      NOTE: Be gentle to avoid damaging the neurospheres by making sure the opening of the tip is wide enough and that the aggregates are not aspirated too quickly.
   3. Remove 5 mL of medium from the plate containing the neurospheres and add 5 mL of fresh CDMII.
      NOTE: This procedure helps to reduce the amount of CDMI medium that may have carried over from the transfer of neurospheres.
   4. Place the plate on an orbital shaker at 70 rpm inside a humidified tissue culture incubator at 37 °C and 5% CO₂.
      NOTE: Visually inspect the neurospheres the next day. Increase the speed of the orbital shaker if the neurospheres are clumped together or attached to the bottom of the plate.
   5. Every 3 days, carefully aspirate the supernatant medium and replace it with fresh CDMII. Leave a small amount of the medium to prevent the neurospheres from drying out.
   6. On day 35, prepare Cortical Differentiation Medium III (CDMIII) (Table 1).
      NOTE: The matrix component should be dissolved in cold CDMIII.
   7. Aspirate the medium from the plate and add 10 mL of cold CDMIII.
      NOTE: It is more effective to use cold medium so that the matrix component can coat the organoids without forming clumps.
   8. After changing the medium, place the plate back on an orbital shaker at 70 rpm inside a humidified tissue culture incubator at 37 °C and 5% CO₂.
   9. Change the medium every 3-5 days depending on the rate of growth, as indicated by the color of the medium.
   10. On day 70, prepare Cortical Differentiation Medium IV (CDMIV) (Table 1). Use CDMIV medium until the desired age of organoids is reached. During this period, keep the plate on an orbital shaker set at 70 rpm inside a humidified tissue culture incubator (37 °C and 5% CO₂).
   11. Change the medium every 3-5 days, depending on the growth rate.

2. Immunostaining of brain organoids

1. Tissue preparation
   1. Prepare 4% paraformaldehyde (PFA) solution, and place it under a safety hood.
      NOTE: Wear personal safety equipment when handling PFA.
   2. Collect brain organoids and gently transfer them with a blunt-tipped 3 mL plastic Pasteur pipette to a 6-well plate filled with PFA.
      NOTE: Use organoids older than 40 days to allow the visualization of structures with higher cellular complexity.
   3. Keep the organoids in the PFA solution for 1 h at room temperature.
   4. Carefully remove the PFA with a 3 mL plastic Pasteur pipette, and wash the fixed organoids three times using PBS.
   5. Store the fixed organoids at 4 °C in PBS until further use.
2. Preparation of brain organoid slices
   1. Prepare a 3% agar solution and heat slowly until liquefied.
   2. Place the mold (the cut end of a 10 mL syringe) on a piece of absorbent filter paper (smooth side up). Place a droplet of agar onto it.
   3. Quickly take a single organoid out of the 6-well plate with a spatula and remove excessive PBS with filter paper.
      NOTE: Be careful not to touch the organoid directly with filter paper.
   4. Place the organoid onto the agar droplet.
   5. Repeat this procedure with up to three organoids.
      NOTE: Work fast to avoid solidification of the agar during this step.
   6. Refill the mold with agar until all the organoids are fully covered.
   7. Wait until the agar begins to solidify, and then gently transfer the entire mold containing the organoids, including the absorbent filter paper, onto a cooling element.
      NOTE: If a cooling element is unavailable, store the organoids for a few minutes in a refrigerator at 4 °C.
   8. In the meantime, prepare for the slicing procedure: place a razor blade (cleaned with acetone and washed with double-distilled water) into the holder of the vibratome, mount on the bath, and fill it with PBS.
   9. Remove the mold from (solidified) agar and use a scalpel to trim it to form a cube.
   10. Attach the agar cube containing the organoids on the carrier plate of the vibratome with adhesive gel (see the Table of Materials), and place it in the bath containing PBS.
   11. Adjust the vibratome (see the Table of Materials) to cut slices at a thickness of 150 µm.
       NOTE: Vibratome settings (proper angle, amplitude, frequency, and velocity of the blade) may be similar to those used for slicing fixed brain tissue derived from early postnatal animals. However, the ideal settings depend strongly on the type of the vibratome and must be determined in a first step to prevent distortion or even ripping of the tissue while cutting.
   12. Start the cutting procedure. Use a glass pipette or a spatula to gently transfer each freshly cut slice into a 24-well plate filled with PBS.
   13. Store the plate containing slices at 4 °C (for up to a few days) until further processing.
   14. Transfer the slices out of the plate with a glass pipette or a spatula onto microscope slides. Use a minimum of 2 slices per slide.
   15. Carefully remove the agar and excess PBS with a syringe.
   16. Allow the slices to dry until they adhere to the slides.
       NOTE: While microscope slides can be stored in plastic chambers filled with PBS at 4 °C, they should be stained as soon as possible after the slicing procedure.
3. Immunohistochemical staining
   1. Prepare the blocking solution (Table 1).
   2. Use a PAP pen to draw a hydrophobic border around the slices on the slide to help keep all the solutions on the slides.
   3. Carefully add the blocking solution on the slide, and incubate for 1 h at room temperature (22-25 °C). To avoid destroying the tissue, do not add the solution directly on top of the slices.
   4. Aspirate the blocking solution and apply the desired primary antibody diluted in the blocking solution.
   5. Incubate the slide overnight in a humidified chamber at 4 °C.
   6. Rinse the slide three times with 1x PBS for 10 min each.
   7. Incubate the slices with the specific secondary antibody diluted in the blocking solution and perform Hoechst staining (1:2,500) for 1 h at room temperature in the dark.
      NOTE: Remember to perform negative controls to confirm there are no non-specific binding or auto-fluorescence.
   8. Rinse three times with 1x PBS for 10 min each in the dark.
   9. Add one drop of mounting medium to the slice, place a coverslip on the edge of the drop, and slowly lay the coverslip down toward the slice to avoid air bubbles.
   10. Allow the slide to rest overnight at room temperature. Apply nail polish on the border of the coverslip to further seal the slide. For long-term storage, store at 4 °C.
4. Documentation of staining
   1. To scan large images for stitching, utilize a motorized upright wide-field microscope equipped with (see the Table of Materials for details) a high-quality objective; DAPI filter set (e.g., excitation (EX): 340-380 nm, dichroic mirror (DM): 400 nm, barrier filter (BA): 435-485 nm); fluorescein isothiocyanate filter set (e.g., EX: 465-495 nm, DM: 505 nm, BA: 515-555 nm); Alexa594 filter set (e.g., ET 575/40; T 600 LPXR; HC 623/24); digital camera; high-performance acquisition software allowing for automated stitches and stack operations.
   2. For image handling, use an image processing program capable of generating 8-bit tif-files, cropping stitches, adjusting contrast and brightness, merging the channels (e.g., blue, green, and red), and adding scale bars.
   3. To scan details, use a motorized confocal laser scanning microscope equipped with a high-quality objective, a UV laser (EX: 408 nm), an Argon laser (EX: 488 nm), a Helium-Neon laser (EX: 543), imaging software for a confocal microscope.
   4. For image handling of details, use an image processing program capable of generating maximum-intensity projections of confocal z-stacks (e.g., optical sections of 0.6 µm each), generating 8-bit tif-files, adjusting contrast and brightness, merging the channels (e.g., blue, green, and red), adding scale bars.
   5. Use a graphic editor to arrange the figures.

3. Bioenergetic profiling of brain organoids

1. Preparation of organoids for bioenergetic profiling
   1. Prepare the papain and DNase solution following the manufacturer's protocol.
   2. Transfer 3-5 organoids into a 6-well plate. Wash them two times with prewarmed PBS.
   3. Add 2 mL of prewarmed activated papain solution containing DNase. Using a blade, cut the organoids into small pieces.
   4. Place the plate onto an orbital shaker set at 27 rpm inside a cell culture incubator (at 37 °C, 5% CO₂), and incubate for 15-20 min.
      NOTE: The time of incubation depends on the organoid stage. Early-stage organoids can be used as they are. For organoids older than 3 months, it is recommended to cut the organoids into 2-3 pieces before dissociation and incubate the pieces at a rocking speed set at 27 rpm for 15-20 min at 37 °C. This procedure can help remove necrotic tissue that may be present in the later-stage organoids.
   5. Collect the digested tissues into a 15 mL tube and add 5 mL of organoid culture medium CDMIV (Table 1).
   6. Triturate the tissue with a 10 mL plastic pipette by pipetting up and down 10-15 times. Let the undissociated tissue settle down to the bottom of the tube.
   7. Carefully transfer the cell suspension to a 15 mL tube, avoiding any pieces of undissociated tissue. Filter the solution through a 40 µm cell strainer (e.g., polystyrene round-bottom tubes with cell strainer caps).
   8. Pellet the cells by centrifuging at 300 x g for 5 min at room temperature.
   9. Assess the cell number and quality using trypan blue.
   10. Plate the desired number (~20,000/well) of cells onto coated 96-well microplates. Change the medium 6-8 h after plating to Neuronal Medium (Table 1).
   11. Incubate the coated 96-well microplate in a CO₂ incubator (37 °C, 5% CO₂) for 4 days.
2. Bioenergetic profiling
   1. On day 3 after replating the dissociated cells, add 200 µL of calibration solution into each well of the bottom part of the 96-well microplate, and place the top green sensor cartridge onto the hydrated microplate.
      NOTE: Place the sensor cartridge on top of the microplate in the correct orientation, and ensure that the calibrant solution covers all the sensors.
   2. Incubate the hydrated 96-well microplate in a non-CO₂ incubator at 37 °C overnight.
   3. Turn on the analyzer to allow the instrument to stabilize at 37 °C overnight.
   4. On day 4 after replating, inspect the disassociated organoid culture on the 96-well microplate under the microscope to ensure that the cells appear as a confluent monolayer.
   5. Prepare Assay Medium (Table 1).
   6. Remove Neuronal Medium from all wells with a pipette without touching the bottom of the well to prevent cell damage. Alternatively, carefully invert the whole plate and then dry it on clean paper. Work quickly to avoid cell death.
   7. Wash the cells twice with prewarmed 200 µL of Assay Medium. Add Assay Medium to a final volume of 180 µL per well. Incubate the 96-well microplate in a non-CO₂ incubator at 37 °C for 1 h.
   8. Prepare 10 µM solutions of mitochondrial inhibitors in Assay medium. Note that the final concentration after injection is 1 µM.
   9. Load the sensor cartridge placed in the hydrated microplate with 10x solutions of the mitochondrial inhibitors.
      1. Add 18 µL of mitochondrial inhibitor 1 into port A.
      2. Add 19.8 µL of mitochondrial inhibitor 2 into port B.
      3. Add 21.6 µL of mitochondrial inhibitor 2 into port C.
      4. Add 23.4 µL of mitochondrial inhibitor 3 into port D.
   10. Place the loaded cartridge in the hydrated microplate in a non-CO₂ incubator at 37 °C until the start of the assay.
   11. Set up a running protocol in the instrument's software (Table 2).
   12. Press START. Take the loaded cartridge from the non-CO₂ incubator and place it into the analyzer for calibration.
       NOTE: Make sure the plate is inserted in the correct orientation and without the lid.
   13. Once the calibration step ends, remove the calibration plate. Take the 96-well microplate from the non-CO₂ incubator and place it into the analyzer. Click on CONTINUE to start the measurements.
   14. When the run is finished, remove the 96-well cell culture microplate from the analyzer and collect the medium from all the wells without disturbing the cells.
       NOTE: The medium can be stored at -20 °C and used later for measuring the amount of lactate released by the cells in the medium using an appropriate lactate assay kit.
   15. Wash the cells with 200 µL of 1x PBS in each well.
   16. After removing the PBS, freeze the plate at -20 °C.
       NOTE: The frozen plate can be used to quantify cells, proteins, or DNA in each well of the microplate. This quantification will be needed for normalizing the obtained bioenergetic rates. Follow the manufacturer´s instructions for cell, protein, or DNA quantification assays.

Wyniki

The protocol described here facilitates the robust generation of round organoids (Figure 1A). The generated organoids contain mature neurons that can be visualized using protein markers specific for axons (SMI312) and dendrites (microtubule-associated protein 2 (MAP2)) (Figure 1B). Mature organoids contain not only neuronal cells (MAP2-positive) but also glial cells (e.g., positive for the astrocyte marker ...

Dyskusje

This paper describes the reproducible generation of human iPSC-derived brain organoids and their use for mitochondrial disease modeling. The protocol described here is modified based on a previously published work²⁰. One major advantage of the present protocol is that it does not require the manual embedding of each organoid into a scaffolding matrix. In fact, the matrix solution is simply dissolved into the cell culture medium. Moreover, there is no need to employ expensive bioreactors, as organo...

Materiały

Name	Company	Catalog Number	Comments
2-mercaptoethanol	Gibco	31350-010
Affinity Designer	Serif (Europe) Ltd		Layout software; Vector graphics editor
Alexa Fluor 488 donkey anti-guinea pig	Sigma Aldrich	SAB4600033-250UL	1:300
Alexa Fluor 488 donkey anti-mouse	Thermo Fisher Scientific	A-31571	1:300
Antimycin A	Sigma Aldrich	1397-94-0
Anti-β-Tubulin III (TUJ-1)	Sigma Aldrich	T8578	1:2000
Argon Laser	Melles Griot		Any other Laser, e.g., diode lasers emitting 488 is fine, too
Ascorbic acid	Sigma	A92902
B-27 with Vitamin A	Gibco	17504044
Bacto Agar	Becton Dickinson		3% in PBS, store solution at -20 °C
BDNF	Miltenyi Biotec	130-093-0811
cAMP	Sigma	D0627
Cell Star cell culture 6 well plate	Greiner-Bio-One	657160
Chemically Defined Lipid Concentrate	Gibco	11905031
Confocal laser scanning microscope C1	Nikon Microscope Solutions		Modular confocal microscope system
Corning Matrigel Growth Factor Reduced (GFR) Basement membrane matrix, Phenol Red-free, LDEV-free	Corning	356231	Matrix component
CyQUANT Cell Proliferation Assay Kit	Thermo Fisher	C7026
DMEM/F12	ThermoFisher	31330038
DMSO	Sigma	D2660-100ML
Donkey anti-goat Cy3	Merck Millipore	AP180C	1:300
Donkey anti-mouse Cy3	Merck Millipore	AP192C	1:300
Donkey anti-rabbit Cy3	Merck Millipore	AP182C	1:300
DPBS	Gibco	14190250
DS-Q1Mc camera	Nikon Microscope Solutions
Eclipse 90i upright widefield microscope	Nikon Microscope Solutions
Eclipse E 600FN upright microscope	Nikon Microscope Solutions
Eclipse Ts2 Inverted Microscope	Nikon Microsope Solutions
EZ-C1 Silver Version 3.91	Nikon Microscope Solutions		Imaging software for confocal microscope
FCCP	Sigma Aldrich	370-86-5
Fetal Bovine Serum	Gibco	10270-106
GDNF	Miltenyi Biotec	130-096-291
Glasgow MEM	Gibco	11710-035
Glass Pasteur pipette	Brand	747715	Inverted
Glutamax	Gibco	35050-061
Helium-Neon Laser	Melles Griot		Every other Laser, e.g., diode lasers emitting 594 is fine, too
Heparin	Merck	H3149-25KU
HERACell 240i CO2 Incubator	Thermo Scientific	51026331
Hoechst 33342	Invitrogen	H3570	1:2500
Image J 1.53c	Wayne Rasband National Institute of Health		Image processing Software
Injekt Solo 10 mL/ Luer	Braun	4606108V
Knockout Serum Replacement	Gibco	10828010
Laser (407 nm)	Coherent		Any other Laser, e.g., diode lasers emitting 407 is fine, too
Map2	Synaptic Systems	No. 188004	1:1000
Maxisafe 2030i
MEM NEAA	Gibco	11140-050
mTeSR Plus	Stemcell Technology	85850	iPSC medium
Multifuge X3R Centrifuge	Thermo Scientific	10325804
MycoAlert Mycoplasma Detection Kit	Lonza	# LT07-218
N2 Supplement	Gibco	17502-048
Needle for single usage (23G x 1” TW)	Neoject	10016
NIS-Elements Aadvanced Research 3.2	Nikon		Imaging software
Oligomycin A	Sigma Aldrich	75351
Orbital Shaker Heidolph Unimax 1010	Heidolph	543-12310-00
PAP Pen	Sigma	Z377821-1EA	To draw hydrophobic barrier on slides.
Papain Dissociation System kit	Worthington	LK003150
Paraformaldehyde	Merck	818715	4% in PBS, store solution at -20 °C
Pasteur pipette 7mL	VWR	612-1681	Graduated up to 3 mL
Penicillin-Streptomycin	Gibco	15140-122
Plan Apo VC 20x / 0.75 air DIC N2  ∞/0.17 WD 1.0	Nikon Microscope Solutions		Dry Microscope Objective
Plan Apo VC 60x / 1.40 oil DIC N2 ∞/0.17 WD 0.13	Nikon Microscope Solutions		Oil Immersion Microscope Objective
Polystyrene Petri dish (100 mm)	Greiner Bio-One	664161
Polystyrene round-bottom tube with cell-strainer cap (5 mL)	Falcon	352235
Potassium chloride	Roth	6781.1
ProLong Glass Antifade Moutant	Invitrogen	P36980
Qualitative filter paper	VWR	516-0813
Rock Inhibitior	Merck	SCM075
Rotenone	Sigma	83-794
S100β	Abcam	Ab11178	1:600
SB-431542	Cayman Chemical Company	13031
Scalpel blades	Heinz Herenz Hamburq	1110918
SMI312	Biolegend	837904	1:500
Sodium bicarbonate	Merck/Sigma	31437-1kg-M
Sodium chloride	Roth	3957
Sodium dihydrogen phosphate	Applichem	131965
Sodium Pyruvate	Gibco	11360070
SOX2	Santa Cruz Biotechnology	Sc-17320	1:100
StemPro Accutase Cell Dissociation Reagent	Gibco/StemPro	A1110501	Reagent A
Super Glue Gel	UHU	63261	adhesive gel
SuperFrost Plus	VWR	631-0108
Syringe for single usage (1 mL)	BD Plastipak	300015
TB2 Thermoblock	Biometra
TC Plate 24 Well	Sarstedt	83.3922
TC Plate 6 Well	Sarstedt	83.392
TGFbeta3	Miltenyi Biotec	130-094-007
Tissue Culture Hood	ThermoFisher	51032711
TOM20	Santa Cruz Biotechnology	SC-11415	1:200
Triton-X	Merck	X100-5ML
UltraPure 0.5M EDTA	Invitrogen	15575020
Vibratome Microm HM 650 V	Thermo Scientific		Production terminated, any other adjustable microtome is fine, too.
Vibratome Wilkinson Classic Razor Blade	Wilkinson Sword	70517470
Whatman Benchkote	Merck/Sigma	28418852
Wnt Antagonist I	EMD Millipore Corp	3378738
XF 96 extracellular flux analyser	Seahorse Bioscience	100737-101
XF Assay DMEM Medium	Seahorse Bioscience	103680-100
XF Calibrant Solution	Seahorse Bioscience	100840-000
XFe96 FluxPak (96-well microplate)	Seahorse Bioscience	102416-100