Human iPSC-Derived Neuron and Oligodendrocyte Co-culture as a Small-Molecule Screening Assay for Myelination
人 iPSC 衍生神经元与少突胶质细胞共培养用于髓鞘形成的小分子筛选分析
发布: 2025年05月05日第15卷第9期 DOI: 10.21769/BioProtoc.5227 浏览次数: 1262
评审: Xiaokang WuShanmugaPriyaa MadhukaranKrishna Murthy NakuluriMithun Santra
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Q&A
Abstract
Neurons and oligodendrocytes are the building blocks of the brain. Neurons form synaptic connections and transmit signals, while oligodendrocytes, including oligodendrocyte precursor cells (OPCs) and their derivatives, are vital for central nervous system maintenance and myelination. The demand for human-specific neuron-oligodendrocyte model systems to study these interactions has grown, yet co-culture protocols remain limited. Recent advancements in the field provide methods for deriving co-cultures of neurons and OPCs from human induced pluripotent stem cells (hiPSC), each with distinct benefits and challenges. This study presents a time-efficient, reproducible method to derive neurons and O4-expressing oligodendrocytes, followed by a straightforward co-culture system that minimizes astrocyte differentiation and ensures robust neuron and oligodendrocyte populations.
Key features
• Reliable, stable generation of neurons and O4-expressing oligodendrocytes within a practical timeframe.
• Co-culture system utilizing hIPSC-derived neurons and O4-expressing oligodendrocytes.
• Maturation of neurons and oligodendrocytes achieved within 10 days of co-culturing.
Keywords: hiPSC (hiPSC)
Human NPCs (人神经前体细胞（NPCs）)
Neurons (神经元)
O4-expressing oligodendrocyte (O4 表达的少突胶质细胞)
Co-culture (共培养)
Myelin (髓鞘)
Screening assay (筛选分析)
Background
Neurons and oligodendrocytes are pivotal cell types in the brain, supporting the central nervous system and participating in white matter formation [1–4]. This is critical for higher brain functions such as social and cognitive learning [5–8]. Neurons establish synaptic connections and transmit signals across the brain [9,10], while oligodendrocyte lineage cells—including oligodendrocyte precursor cells (OPCs), myelinating oligodendrocytes, and transitional cell types—perform essential functions for central nervous system (CNS) maintenance and myelination of axons [3,11,12]. Beyond myelination, oligodendrocytes support neuronal energy metabolism, help buffer potassium [13], and promote synaptic plasticity through the secretion of neurotrophic factors such as BDNF [14,15]. Early in life, the disruption of the oligodendrocyte-neuron unit results in axonal dysfunction and impairs neurodevelopment [16,17]; later, it can be observed in traumatic injuries, Alzheimer’s disease, demyelinating diseases such as multiple sclerosis [18–20], and psychiatric disorders such as bipolar disorders and schizophrenia [21].
Consequently, there is an increasing demand for the development of human-based neuron-oligodendrocyte model systems for studying these interactions in both healthy and diseased brains. Compared to equivalent models derived from mice, primary cultures, or animal-derived co-cultures [22,23], the induced pluripotent stem cell (iPSC) technology allows the generation of several cell types that can respond to the full spectrum of human nutritional and hormonal stimuli.
Although numerous methods exist for deriving neurons or OPCs individually from iPSCs [24,25], protocols for their co-culture are limited. Over the past five years, there has been a rise in neuron-oligodendrocyte co-culture studies. Notably, Dooves et al. [26], Assetta et al. [27], and von der Bey et al. [28] have developed comprehensive protocols for deriving neurons and OPCs from human iPSCs. Each protocol offers distinct advantages and drawbacks. Dooves et al. [26] presented a straightforward but time-consuming method involving small molecule-directed differentiation, achieving mature neurons by day 37 and mature oligodendrocytes by day 67, followed by a 28-day co-culture period. In contrast, Assetta et al. and von der Bey et al. achieved neuron and oligodendrocyte maturation and subsequent co-culture in a shorter timeframe, utilizing pro-myelinating compounds (small molecules that promote myelination by the direct interaction with the oligodendrocytes) or complex media changes [27,28].
In this study, we generate well-characterized populations of neurons and O4-expressing oligodendrocytes separately from the same iPSC line and from neural progenitor cells (NPCs) in a time-efficient, robust, and reproducible manner. Additionally, we aimed to establish a straightforward co-culture system using these defined cell types. Neurons were derived using a standardized small molecule-driven differentiation protocol, incorporating the CultureOneTM Supplement (Gibco) to minimize astrocyte production. This method yielded stable neuronal cultures after 29 days of differentiation. O4-expressing oligodendrocytes, cells that are considered pre-myelinating oligodendrocytes, were generated by integrating methodologies from Ehrlich et al. [25] and Dooves et al. [26]. A tetracycline-inducible lentivirus transduction method [25,29] was combined with simplified media changes [26], achieving stable O4-expressing oligodendrocytes after 28 days of differentiation. Compared to other protocols, this approach ensures that most of the cell population differentiates into a high percentage of neurons or oligodendrocyte lineage cells. In addition, the experimenter can control the percentage of astrocytes in co-culture. While the presence of astrocytes maintains neurons in culture for longer [30], they also decrease the percentage of neurons after differentiation and might represent a confounding factor in studying the exclusive interaction between neurons and oligodendrocytes [31].
This co-culture demonstrates the potential for future studies investigating neuron-oligodendrocyte crosstalk in various human myelin disorders [32]. The co-culture also offers high translational potential to humans and broad scientific impact. Since neurons and oligodendrocytes retain the genome of the donor [33], they could be used to develop drug screening platforms for targeted drug discovery and personalized medicine in a reasonable time.
Materials and reagents
Biological materials
1. Lentiviral vector (provided by Prof. Dr. Kuhlman [25,29])
Reagents
1. 4’,6-diamidino-2-phenylindole (DAPI) (Sigma-Aldrich, catalog number: D9542)
2. Accutase (Sigma-Aldrich, catalog number: A6964-500ML)
3. Antibiotic antimycotic (Merck, Sigma-Aldrich, catalog number: A5955-20ML)
4. Anti-forkhead box protein G1 (FoxG1) (dilution 1:400; working concentration: 2.25 μg/mL) (Abcam, catalog number: Ab18259)
5. Anti-galactosylceramidase (GalC) (dilution 1:250; working concentration: 4 μg/mL) (Millipore, catalog number: MAB342)
6. Anti-glial fibrillary acidic protein (GFAP) (dilution 1:3,500; working concentration: 0.5 μg/mL) (Novusbio, catalog number: NBP1-05198)
7. Anti-myelin-associated protein (MAG) (dilution 1:400; working concentration: 5 μg/mL) (Merck, catalog number: MAB1567)
8. Anti-microtubule-associated protein 2 (MAP2) (dilution 1:900; working concentration: 0.84 μg/mL) (Abcam, catalog number: Ab183830)
9. Anti-myelin basic protein (MBP) (dilution 1:1,500; initial concentration not disclosed from the manufacturer) (Invitrogen, catalog number: PA1-10008)
10. Anti-nestin (dilution 1:250; working concentration: 4 μg/mL) (Merck, Sigma-Aldrich, catalog number: MAB5326)
11. Anti-neuronalNuclei (NeuN) (dilution 1:500; working concentration: 2 μg/mL) (Merck, catalog number: ABN78)
12. Anti-neurofilament heavy (NFH) (dilution 1:400; working concentration: 20 μg/mL) (Merck, catalog number: N4142)
13. Anti-neural/glial antigen 2 (NG2) (dilution 1:100; working concentration: 10 μg/mL) (Invitrogen, catalog number: 14-6504-82)
14. Anti-oligodendrocyte marker O4 (O4) (dilution 1:400; working concentration: 1.25 μg/mL) (Merck, catalog number: MAB1326)
15. Anti-oligodendrocyte transcription factor 2 (Olig2) (dilution 1:250; initial concentration not disclosed from the manufacturer) (Merck, catalog number: AB9610)
16. Anti-paired box protein (Pax6) (dilution 1:100; working concentration: 20 μg/mL) (BioLegend, catalog number: 901301)
17. Anti-Sox10 (dilution 1:250; working concentration: 4 μg/mL) (Millipore, catalog number: AB5727)
18. Anti-class III-beta tubulin (Tuj1) (dilution 1:750; working concentration: 1.32 μg/mL) (BioLegend, catalog number: MMS-435P)
19. Ascorbic acid (AA) (Sigma-Aldrich, catalog number: A4403-100MG)
20. B27 with vitamin A (Thermo Fisher Scientific, catalog number: 17504044)
21. B27 without vitamin A (Thermo Fisher Scientific, catalog number: 12587010)
22. Basic fibroblast growth factor (bFGF or FGF) (Sigma-Aldrich, catalog number: SRP4037-50UG)
23. Biotin (Sigma-Aldrich, catalog number: B4501)
24. Brain-derived neurotrophic factor (BDNF) (PeproTech, catalog number: AF-450-02-10UG)
25. CHIR99021 (Cayman Chemicals, catalog number: CAY13122-10mg)
26. Cholest-4-en-3-one (Sigma-Aldrich, catalog number: 188174)
27. Clemastine fumarate salt (Sigma-Aldrich, catalog number: SML0445)
28. CultureOneTM supplement (100×) (Thermo Fisher Scientific, catalog number: A3320201)
29. Cyclic adenosine monophosphate (Merck, catalog number: D0260-25UG)
30. DMEM/F12 – L-glutamine (Thermo Fisher Scientific, Gibco, catalog number: 21331-020)
31. DMEM/F12 +/+ L-glutamine/sodium bicarbonate (Thermo Fisher Scientific, Gibco, catalog number: 11320-033)
32. DMEM/F12 + GlutaMAX (Thermo Fisher Scientific, Gibco, catalog number: 31331-093)
33. Dimethyl sulfoxide (DMSO) (Sigma-Aldrich, catalog number: D2438)
34. Donkey-anti-rabbit (Alexa 488) (Jackson Immuno Research, catalog number: 711-545-152)
35. Donkey-anti-chicken (Alexa 647) (Jackson Immuno Research, catalog number: 703-175-155)
36. Donkey-anti-mouse (Cy3) (Jackson Immuno Research, catalog number: 715-165-150)
37. Dorsomorphin (Cayman Chemicals, catalog number: 11967-5mg)
38. Doxycycline (Sigma-Aldrich, catalog number: D3447-500MG)
39. DPBS (-/- Ca2+ and Mg2+) (Gibco, catalog number: 14190-094)
40. DPBS (+/+ Ca2+ and Mg2+) (Gibco, catalog number: 14040-133)
41. Epidermal growth factor (EGF) (Sigma-Aldrich, catalog number: SRP3027-500UG)
42. Fluoromount G (Invitrogen, catalog number: 00-4958-02)
43. Geltrex (Thermo Fisher Scientific, catalog number: A1413202)
44. Glial cell line–derived neurotrophic factor (GDNF) (PeproTech, catalog number: 450-10)
45. GlutaMAX (Thermo Fisher Scientific, catalog number: 35050-038)
46. Hydrochloric acid (HCl) (Sigma-Aldrich, catalog number: 258148)
47. Human leukemia inhibitory factor (hLIF) (PeproTech, catalog number: AF-300-05-25UG)
48. Insulin (Sigma-Aldrich, catalog number: I9278-5mL)
49. BioLaminin (LN521) (Biolaminin, catalog number: LN521-02)
50. MEM non-essential amino acids (Thermo Fisher Scientific, catalog number: 11140-035)
51. Miconazole nitrate salt (Sigma-Aldrich, catalog number: M3512)
52. Mouse laminin (L2020) (Sigma-Aldrich, catalog number: L2020-1MG)
53. N1 supplement (Sigma-Aldrich, catalog number: N6530-5ml)
54. N2 supplement (Thermo Fisher Scientific, catalog number: 17052048)
55. Neurobasal media (Thermo Fisher Scientific, Gibco, catalog number: 21103-049)
56. Noggin (PeproTech, catalog number: 120-10C-50UG)
57. Normal donkey serum (Merck, catalog number: S30-100ml)
58. NSC freezing media (Merck, catalog number: SCM007)
59. NT3 (Merck, catalog number: CS204470)
60. Papain (Worthington Biochemistry, catalog number: LK003178)
61. Paraformaldehyde (PFA) (Sigma-Aldrich, catalog number: 441244)
62. PBS (Gibco, catalog number: 10010-015)
63.Poly-L-ornithine solution (PLO) (Sigma-Aldrich, catalog number: P4957-50mL)
64. Potassium phosphate (Sigma-Aldrich, catalog number: P5655-500G)
65. Protamine sulfate (Sigma-Aldrich, catalog number: 110123)
66. Purmorphamine (Calbiochem, catalog number: 540220-5mg)
67. Puromycin (Thermo Fisher Scientific, catalog number: J67236)
68. Rock inhibitor (also known as Y-27632) (Lubioscience, catalog number: HB2297-5MG)
69. SB431542 (Sigma-Aldrich, catalog number: S4317)
70. Smoothened agonist (SAG) (Calbiochem, catalog number: 566660)
71. Sodium hydroxide solution (NaOH) (Fuka, catalog number: 35255)
72. Sodium phosphate (ACROS, catalog number: 204855000)
73. Triiodothyronine (T3) (Merck, catalog number: T6397-100MG)
74. Triton X-100 (Sigma-Aldrich, catalog number: 93443-100ml)
75. β-Mercaptoethanol (Sigma-Aldrich, catalog number: M3148-25ml)
Solutions
1. Neuronal differentiation media with CultureOneTM supplement for neurons (NDMC) (see Recipes)
2. Neural maintenance media for oligodendrocyte progenitor cells (OPC) and oligodendrocytes (NMM) (see Recipes)
3. Neural stem cell maintenance media for NPCs maintenance (NSMM) (see Recipes)
4. Neuroglia co-culture media (see Recipes)
5. NPC transduction media for transduction of NPCs to obtain (OPC) (NTM) (see Recipes)
6. Clemastine or miconazole treatment
7. Sodium phosphate buffer (see Recipes)
8. 4% PFA (see Recipes)
9. Blocking buffer (PBS+) (see Recipes)
10. Permeabilization buffer (see Recipes)
11. Poly-L-ornithine/BioLaminin (PLO/LN521) coating (see Recipes)
12. Poly-L-ornithine/mouse Laminin (PLO/L2020) coating (see Recipes)
13. Geltrex coating (see Recipes)
Recipes
Media recipes
1. Neuronal differentiation media with CultureOneTM supplement for neurons (NDMC)
| Reagent | Final concentration | Amount |
|---|---|---|
| Neurobasal media | N/A | 474 mL |
| GlutaMAX (100×) | 1× | 5 mL |
| B27 supplemented with vitamin A (50×) | 1× | 10 mL |
| Antibiotic antimycotic (100×) | 1× | 5 mL |
| Ascorbic acid (100 mM) | 200 μM | 1 mL |
| CultureOneTM supplement (100×) | 1× | 5 mL |
| Total | N/A | 500 mL |
To prepare a 100 mM stock solution of ascorbic acid, dissolve 100 mg of the powder in 5.67 mL of sterile ddH2O.
Mix reagents and filter (pore size 0.4 μm or smaller) media. Media can be stored for 2 weeks at 4 °C.
Note: The CultureOneTM supplement minimizes astrocyte differentiation. It can be introduced during a later phase of differentiation to obtain the desired number of astrocytes.
2. Neural maintenance media for oligodendrocyte progenitor cells and oligodendrocytes (NMM)
| Reagent | Final concentration | Amount |
|---|---|---|
| Neurobasal media | N/A | 241 mL |
| DMEM/F12 + GlutaMAX | N/A | 241 mL |
| GlutaMAX (100×) | 0.5× | 2.5 mL |
| MEM non-essential amino acids (100×) | 0.5× | 2.5 mL |
| B27 supplement without vitamin A (50×) | 0.5× | 5 mL |
| N2 supplement (100×) | 1× | 2.5 mL |
| Antibiotic antimycotic (100×) | 1× | 5 mL |
| Insulin (10.5 mg/mL) | 5 μg/mL | 238 μL |
| β-Mercaptoethanol (143 mM) | 10 μM | 35 μL |
| Total | N/A | 500 mL |
Mix reagents and filter (pore size 0.4 μm or smaller) media. Media can be stored for 2 weeks at 4 °C.
3. Neural stem cell maintenance media for NPC maintenance (NSMM)
| Reagent | Final concentration | Amount |
|---|---|---|
| Neurobasal media | N/A | 237.5 mL |
| DMEM/F12 +/+ L-glutamine/sodium bicarbonate | N/A | 237.5 mL |
| GlutaMAX (100×) | 1× | 5 mL |
| B27 supplemented with vitamin A (50×) | 1× | 10 mL |
| N2 supplement (100×) | 1× | 5 mL |
| Antibiotic antimycotic (100×) | 1× | 5 mL |
| Total | N/A | 500 mL |
Mix reagents and filter (pore size 0.4 μm or smaller) media. Media can be stored for 2 weeks at 4 °C.
4. Neuroglia co-culture media
| Reagent | Final concentration | Amount |
|---|---|---|
| Neurobasal media | N/A | 237.5 mL |
| DMEM/F12 – L-glutamine | N/A | 237.5 mL |
| MEM non -essential amino acids (100×) | 1× | 5 mL |
| B27 supplemented with vitamin A (50×) | 1× | 10 mL |
| N1 supplement (100×) | 1× | 5 mL |
| Antibiotic antimycotic (100×) | 1× | 5 mL |
| Total | N/A | 500 mL |
Mix reagents and filter (pore size 0.4 μm or smaller) media. Media can be stored for 2 weeks at 4 °C.
5. NPC transduction media for transduction of NPCs to obtain oligodendrocyte progenitor cells (NTM)
| Reagent | Final concentration | Amount |
|---|---|---|
| Neurobasal media | N/A | 241.25 mL |
| DMEM/F12 +/+ L-glutamine/sodium bicarbonate | N/A | 241.25 mL |
| GlutaMAX (100×) | 1× | 5 mL |
| B27 supplement without vitamin A (50×) | 0.5× | 5 mL |
| N2 supplement (100×) | 0.5× | 2.5 mL |
| Antibiotic antimycotic (100×) | 1× | 5 mL |
| Total | N/A | 500 mL |
Mix reagents and filter (pore size 0.4 μm or smaller) media. Media can be stored for 2 weeks at 4 °C.
6. Clemastine or miconazole treatment
Clemastine: Prepare a 100 mM stock solution dissolving 45.9 mg of clemastine in 1 mL of DMSO. Store at -20 °C.
Miconazole: Prepare a 100 mM stock solution by dissolving 47.914 mg of miconazole in 1 mL of DMSO. Store at -20 °C.
Immunostaining recipes
7. Sodium phosphate buffer, 0.4 M stock, pH 7.4–7.6
| Reagent | Final concentration | Amount |
|---|---|---|
| Sodium phosphate | N/A | 0.53 g |
| Potassium phosphate | N/A | 0.28 g |
| Deionized water | N/A | ~50 mL |
| HCl | 1 M | As needed |
| NaOH | 1 M | As needed |
| Total | N/A | 50 mL |
Dissolve in ~90% of deionized water, adjust pH to 7.4–7.6 with 1 M HCl or 1 M NaOH, and then bring to the final volume.
8. 4% Paraformaldehyde fixative (PFA), pH 7–7.4
| Reagent | Final concentration | Amount |
|---|---|---|
| Paraformaldehyde | N/A | 2 g |
| Deionized water | N/A | ~37.5 mL |
| 0.4 M sodium phosphate buffer | 0.4 M | 12.5 mL |
| HCl | 1 M | As needed |
| Total | N/A | 50 mL |
a. Add 2/3 deionized water to a flask and heat to 60 °C, keeping below 70 °C.
b. Add the paraformaldehyde with constant stirring for 10 min.
c. Slowly add a few drops of 10 M NaOH to the cloudy mixture, stirring until the solution clears.
d. Stir a few minutes longer, adding more NaOH if necessary to clear.
e. Remove from heat and cool to room temperature.
f. Filter with #3 Whatman® filter paper.
g. Add the 0.4 M sodium phosphate buffer.
h. Adjust pH to 7–7.4 with 1 M HCL and check with a pH meter.
i. Bring to the final volume with deionized water.
Notes:
1. Paraformaldehyde will never appear to be dissolving until the NaOH is added.
2. Be patient, allow time for the NaOH to cause the paraformaldehyde to dissolve before adding more.
3. There is always a little colloidal “fluff’ in an unfiltered state.
4. Do not weigh paraformaldehyde in plastic weigh boats; instead, use a glass beaker to minimize static dispersion of paraformaldehyde dust.
5. Wash out the filter paper with tap water before discarding, rinse everything that was in contact with paraformaldehyde, and make sure you collect it in a special container.
9. Blocking buffer (PBS+)
| Reagent | Final concentration | Amount |
|---|---|---|
| PBS | N/A | 45 mL |
| Normal donkey serum (100%) | 10% | 5 mL |
| Total | N/A | 50 mL |
Mix reagents and filter (pore size 0.2 μm). Blocking buffer can be stored at 4 °C for 48 h.
10. Permeabilization buffer
| Reagent | Final concentration | Amount |
|---|---|---|
| Blocking buffer (PBS+) | N/A | 49 mL |
| Triton X-100 (10%) | 0.2 % | 1 mL |
| Total | N/A | 50 mL |
Use the filtered PBS+. Permeabilization buffer can be stored at 4 °C for 48 h.
Coating of plates
11. Poly-L-ornithine/BioLaminin (PLO/LN521) coating
a. Place coverslips into a 24-well plate.
b. Dilute PLO in PBS (500 μL/well = 75 μL PLO + 425 μL PBS).
c. Incubate at room temperature for 2 h.
d. Remove PLO and wash three times with PBS.
e. Allow coverslips to dry slightly before the next step.
f. Add 10 μg/mL of LN521 + cold DPBS ++ [50 μL BioLaminin + 450 μL DPBS+ Ca2+ and Mg2+(DPBS++)].
g. Cover with parafilm.
h. Incubate at 37 °C overnight or at room temperature for 48 h.
i. Remove BioLaminin and immediately add medium with the cells.
Notes:
1. PLO should be stored at -20 °C in smaller aliquots upon arrival.
2. Thaw LN521 on ice, aliquot, and store at -20 °C.
3. PLO/LN521 coverslips can be prepared in advance. To store, add cold DPBS to the wells, wrap the plate with parafilm, and keep it at 4 °C for up to two weeks. Before use, place the plate in the incubator for 30 minutes. Ensure that there is sufficient liquid to cover the wells to prevent them from drying out.
12. Poly-L-ornithine/mouse Laminin (PLO/L2020) coating
a. Place coverslips into a 24-well plate.
b. Dilute PLO in PBS (500 μL/well = 75 μL PLO + 425 μL PBS).
c. Incubate at room temperature for 2 h.
d. Remove PLO and wash three times with PBS.
e. Allow coverslips to dry slightly before the next step.
f. Add L2020 + cold DPBS ++ (50 μL L2020 + 4.95 mL DPBS++).
g. Cover with parafilm.
h. Incubate at 37 °C overnight or at room temperature for 48 h.
i. Remove L2020 and immediately add medium with the cells.
Notes:
1. Thaw L2020 on ice, aliquot, and store at -20 °C.
2. PLO/L2020 coverslips can be prepared in advance. To store, add cold DPBS to the wells, wrap the plate with parafilm, and keep it at 4 °C for up to two weeks. Before use, place the plate in the incubator for 30 min. Ensure that there is sufficient liquid to cover the wells to prevent them from drying out.
13. Geltrex coating
a. Dilute Geltrex 1:1 with cold DMEM/F12 – L-glutamine and aliquot in appropriate sizes.
b. Store aliquots at -20 °C.
c. Dilute 1:100 with cold DMEM/F12 – L-glutamine.
d. Add the appropriate amount to flask or well (5 mL per T75 flask; 1 mL per 6-well plate).
e. Incubate at 37 °C for 1 h.
f. Remove Geltrex and immediately add medium with the cells.
CRITICAL: Geltrex must be handled at low temperatures to prevent polymerization. It is recommended to pre-cool laboratory consumables, such as tubes for aliquots, pipette tips, and Falcon tubes, prior to diluting and aliquoting. Ideally, perform this work on a cooling block or on ice.
Laboratory supplies
1. 0.2 μm filters (Sarstedt, catalog number: 83.1826.001)
2. 1.5 mL Eppendorf tubes (Eppendorf, catalog number: 0030120.086)
3. 10 μL pipette tips (Tip One, catalog number: S1111-3700)
4. 1,000 μL pipette tips (Tip One, catalog number: S1111-6001)
5. L graduated pipette (Greiner Bio-One, catalog number: 607180)
6. 15 mL Falcon (Cellstar Tubes, catalog number: 188271N)
7. 200 μL pipette tips (Tip One, catalog number: S1111-0006)
8. 24-well plate (Falcon, catalog number: 353047)
9. 50 mL Falcon (Cellstar Tubes, catalog number: 227261)
10. 5 mL graduated pipette (Greiner Bio-One, catalog number: 606180)
11. 6-well plates (Falcon, catalog number: 353046)
12. Whatman® qualitative filterpapier (Merck, catalog number: WHA1001090)
13. Coverslips (VWR, catalog number: 631-1578)
14. Microscope slides (25 mm × 75 mm × 1.0 mm, super frost) (Epredia, catalog number: J1800AMNZ)
15. One-way 20 mL syringes (Injekt, catalog number: 4606205V
16. 500 mL vacuum filter, 0.45 μm (Sarstedt, catalog number: 83.3941)
17. T75 flask (Sarstedt, catalog number: 833911002)
Equipment
1. Pipet boy (Integra, catalog number: 155000)
2. Incubator (Binder, catalog number: 9040-0131)
3. Benchtop centrifuge 420R (Hettich, model: Rotina 420R)
4. Benchtop centrifuge (Eppendorf, model: 5430/5430R)
5. 4 °C refrigerated storage unit (Liebherr, model: K3130Index20C/001)
6. -20 °C ProFiline freezer (Liebherr, model: GG5210Index40A/006)
7. 37 °C water bath (Sun Lab, model: SU1811)
8. Widefield microscope Zeiss, Axio Observer 1 (Center for Microscopy and Image Analysis, University of Zurich)
Software and datasets
1. Fiji ImageJ-win64
2. GraphPad Prism V10
3. ZEN V3.1 (blue edition)
Procedure
文章信息
稿件历史记录
提交日期: Oct 29, 2024
接收日期: Jan 26, 2025
在线发布日期: Feb 20, 2025
出版日期: May 5, 2025
版权信息
© 2025 The Author(s); This is an open access article under the CC BY-NC license (https://creativecommons.org/licenses/by-nc/4.0/).
如何引用
Chie, S. E., Szentpetery, Z., Generali, M., Kuhlmann, T., Natalucci, G. and Miletta, M. C. (2025). Human iPSC-Derived Neuron and Oligodendrocyte Co-culture as a Small-Molecule Screening Assay for Myelination. Bio-protocol 15(9): e5227. DOI: 10.21769/BioProtoc.5227.
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分类
干细胞 > 多能干细胞 > 细胞分化
细胞生物学 > 细胞成像 > 荧光
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