Differentiating Immortalized Multipotent Otic Progenitors into Spiral Ganglion Neurons and Evaluating the Differentiation

Abstract

Source: Azadeh, J., et al. Initiating Differentiation in Immortalized Multipotent Otic Progenitor Cells. J. Vis. Exp. (2016).

The video demonstrates the differentiation of immortalized multipotent otic progenitors (iMOPs) into spiral ganglion neurons (SGNs) and immunofluorescence-based confirmation of the differentiation. The iMOPs are plated onto culture substrate-coated coverslips in a neuronal differentiation medium for differentiation into SGNs. The differentiated cells are labeled with antibodies specific for neuronal differentiation markers and analyzed under a microscope to confirm differentiation.

Protocol

1. Differentiating iMOP-Derived Neurons

1. Make 50 ml of neuronal differentiation media: Neurobasal media, 1X B27 supplement, 2 mM L-Glutamine. Thaw bottle of 50X B27 and 200 mM L-Glutamine in 37 °C water bath for 5 min. Add 1ml 50X B27 and 0.5 ml 200 mM L-Glutamine to 48.5 ml of Neurobasal media.
2. Coat coverglass by placing 12 mm round 1.5 glass coverslips in a sterile 10 cm plate and add 70% ethanol (EtOH) to the plate to sterilized and clean the coverslips.
3. Gently agitate the coverslips to ensure they are covered in ethanol. Leave the plate for 10 min at RT. Rinse the coverslips 3 times with sterile 1X phosphate-buffered saline (PBS) to wash out the remaining ethanol. Rinse the coverslip once with sterile H₂O to wash out the remaining 1X PBS.
4. Aspirate the H₂O using a 2 ml aspirating pipette and let the coverslips dry. Expose the coverslips to UV light in the tissue culture hood for 15 min. Store coverslips in a sterile environment if not immediately used.
5. Place one 12 mm round coverslip in each well of a 24 well dish. Shake plate gently to ensure that the coverslips lie flat on the bottom of the well.
6. Thaw 2 mg/ml poly-D-lysine stock solution at room temperature (RT) and dilute to a 10 µg/ml poly-D-lysine concentration in 1X PBS. Add 0.25 ml of 10 ug/ml poly-D-lysine to the wells. Leave the plate in the 37 °C incubator for 1 hr.
7. Wash the wells 3 times with sterile 1X PBS. Thaw 10 mg/ml laminin stock solution at RT and dilute to 10 µg/ml in 1X PBS. Add 0.25 ml of 10 µg/ml laminin working solution into a single well of a 24 multi-well dish. Incubate the plate at 37 °C incubator. Aspirate out the laminin solution using a 2 ml aspirating pipette.
8. Wash the coverslip 3 times with 1X PBS by adding in 1 ml of 1X PBS with a P1000 pipette and removing the 1X PBS by aspirating with a 2 ml aspirating pipette. Leave 1X PBS from last wash in the well until cells are ready to be plated. Initiate neuronal differentiation by harvesting, dissociating and counting cells. Plate 1 x 10⁶ cells in a 6 cm dish at Day -3.
9. On Day 0, harvest, dissociate and count cells. Seed 1 x 10⁵ - 1.5 x 10⁵ iMOP cells into 0.5 ml pre-warmed neuronal differentiation media per well in a 24 multi-well dish. Aspirate and add pre-warmed neuronal differentiation media to cultures every other day.
10. Fix iMOP-derived neurons in 4% formaldehyde for 15 min. at RT on Day 7. Remove formaldehyde solution, wash iMOP-derived neurons with wash buffer (1X PBS containing 0.1% TritonX-100) before incubating in blocking buffer (1X PBS containing 10% normal goat serum and 0.1% Triton X-100) for 1 hr.
    1. Replace buffer and incubate samples in blocking buffer with diluted antibody. Incubate at 4 °C. Wash samples with 1X PBS containing 0.1% Triton X-100 subject cells to immunostaining. Wash the coverglass with 1X PBS before placing onto mounting media. Allow the mounting media to dry at 4 °C before acquiring epifluorescence images.

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
1.5 Thickness Glass Coverslip (Round 12 mm)	Electron Microscopy Sciences 7	72230-01
DMEM/F12	Life Technologies	11320-082
Neurobasal Medium	Life Technologies	21103
Phosphate Buffered Saline (PBS) pH 7.4	Life Technologies	10010-023
B27 Supplement (50X) Serum Free	Life Technologies	17504-044	Stored as 1 ml aliquots
L-Glutamine(200 mM)	Life Technologies	25030-081	Stored as 5 ml aliquots
Natural Mouse Laminin	Life Technologies	23017-015	Stored as 1 mg/ml aliquots
Prolong Gold Antifade Mountant	Life Technologies	47743-736	Stored as 10 mg/ml 100 µl aliquots
Moxi Z Mini Automated Cell Counter	Orflo	MXZ001
Moxi Z Cassette Type S	Orflo	MXC002
Poly-D-Lysine	Sigma	P7886	Resuspended in 1X PBS and stored as 10 mg/ml 100 µl aliquots
5 ml pipet individually wrapped paperback (200/case)	Thermo Fisher Scientific	1367811D
10 ml pipet individually wrapped paperback (200/case)	Thermo Fisher Scientific	1367811E
Tissue Culture Treated Biolite 24- Well Plate	Thermo Fisher Scientific	130188
Tissue Culture Treated Biolite 6- Well Plate	Thermo Fisher Scientific	130184
Tissue Culture Treated 6 cm Dish	Thermo Fisher Scientific	130181
EMD Millipore Millex Sterile Syringe PVDF Filter Pore size: 0.22 μm	Thermo Fisher Scientific	SLGV033RS
TipOne filter pipet tips 0.1 - 10 μl elongated filter tip	USA Scientific	1120-3810
TipOne filter pipet tips 1 - 20 μl filter tip	USA Scientific	1120-1810
TipOne filter pipet tips 1 - 200 μl filter tip	USA Scientific	1120-8810
TipOne filter pipet tips 101 - 1000 μl filter tip	USA Scientific	1126-7810
15 ml conical tubes sterile 20 bags of 25 tubes (500 tubes)	USA Scientific	1475-0511
50 ml conical tubes sterile 20 bags of 25 tubes (500 tubes)	USA Scientific	1500-1211