Initiating Differentiation in Immortalized Multipotent Otic Progenitor Cells

Summary

The current protocols to maintain immortalized multipotent otic progenitor (iMOP) cells and otic differentiation are described. Culture conditions and molecular markers that indicate differentiation into sensory epithelia and spiral ganglion neurons (SGN) are highlighted.

Abstract

Use of human induced pluripotent stem cells (iPSC) or embryonic stem cells (ESC) for cell replacement therapies holds great promise. Several limitations including low yields and heterogeneous populations of differentiated cells hinder the progress of stem cell therapies. A fate restricted immortalized multipotent otic progenitor (iMOP) cell line was generated to facilitate efficient differentiation of large numbers of functional hair cells and spiral ganglion neurons (SGN) for inner ear cell replacement therapies. Starting from dissociated cultures of single iMOP cells, protocols that promote cell cycle exit and differentiation by basic fibroblast growth factor (bFGF) withdrawal were described. A significant decrease in proliferating cells after bFGF withdrawal was confirmed using an EdU cell proliferation assay. Concomitant with a decrease in proliferation, successful differentiation resulted in expression of molecular markers and morphological changes. Immunostaining of Cdkn1b (p27^KIP) and Cdh1 (E-cadherin) in iMOP-derived otospheres was used as an indicator for differentiation into inner ear sensory epithelia while immunostaining of Cdkn1b and Tubb3 (neuronal β-tubulin) was used to identify iMOP-derived neurons. Use of iMOP cells provides an important tool for understanding cell fate decisions made by inner ear neurosensory progenitors and will help develop protocols for generating large numbers of iPSC or ESC-derived hair cells and SGNs. These methods will accelerate efforts for generating otic cells for replacement therapies.

Introduction

The organs of the inner ear, the cochlea, utricle, saccule and three semicircular canals, mediate the ability to hear and balance. Within the cochlea, hair cells convert sounds into electrical signals that are relayed to the spiral ganglion neurons (SGN). The SGNs fire action potentials to propagate neural signals through the auditory circuit. Genetic mutations, ototoxic drugs and exposure to loud sounds contribute to hair cell and SGN death that result in hearing loss^1-4. Once lost, these cells are not replaced. Use of iPSC and ESC to generate nascent hair cells or SGNs holds great promise for inner ear cell replacement therapies^5-8. A flurry ....

Protocol

1. Maintaining Self-renewal in IMOP Cells

1. Prepare iMOP culture media: DMEM/F12, 1X B27 supplement, 25 µg/ml carbenecillin and 20 ng/ml bFGF. Make 50 ml of iMOP culture media using sterile reagents. Warm up 49 ml of DMEM/F12 in a 50 ml conical in a 37 °C water bath.
   1. Thaw 50X B27 supplement and filter-sterilized 100 mg/ml carbenecillin aliquots for 5 min in a 37 °C water bath. Thaw out 100 µg/ml bFGF aliquot at RT. Add 1 ml 50X B27, 10 µl of 100 µg/ml bFGF and 12.5 µl of 100 mg/ml carbenecillin into DMEM/F12.
2. Use 3 ml of media in a 60 mm tissue culture dish for culturing iMOP cells. Add f....

Results

bFGF Withdrawal Decreases Proliferation in IMOP Cells

To decrease the proliferative capacity of iMOP cells and initiate differentiation of iMOP cells, bFGF was withdrawn from the cultures. To confirm that growth factor withdrawal decreases proliferation, EdU incorporation was employed as a proliferation assay. The percentage of cells that incorporated EdU from otospheres cultured with iMOP culture media (contain.......

Discussion

Monitoring IMOP Cultures

A protocol for maintaining self-renewal and promoting differentiation of a novel iMOP cell line is described and additional plating formats are included (Table 1). Several critical steps that help with routine expansion and differentiation of iMOP cells are noted. Similar to pluripotent stem cell cultures, iMOP cells theoretically have an indefinite life-span. To ensure that cell lines are properly maintained, iMOP cultures are routine.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
CoolCell LX Alcohol-Free Cell Freezing Containers	BioCision	BCS-405
Cryogenic Vials (2 ml)	Corning	430654
1.5 Thickness Glass Coverslip (Round 12 mm)	Electron Microscopy Sciences	72230-01
DMEM/F12	Life Technologies	11320-082
Neurobasal Medium	Life Technologies	21103
Phosphate Buffered Saline (PBS) pH 7.4	Life Technologies	10010-023
Hank's Balanced Salt Solution (HBSS)	Life Technologies	14025-092
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
Click-iT EdU Alexa Fluor 488	Life Technologies	C10337
Synth-A-Freeze Cryopreservation Media	Life Technologies	A12542-01
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
Recombinant Murine Fibroblast Growth Factor, basic (bFGF)	Peprotech	450-33	Resuspended in 0.1% BSA in H20 and stored as 20 mg/ml aliquots
Poly-D-Lysine	Sigma	P7886	Resuspended in 1X PBS and stored as 10 mg/ml 100 µl aliquots
Carbenicillin, Disodium Salt	Thermo Fisher Scientific	BP2648-1	Resuspended in 10 mM Hepes pH 7.4 and stored as 100 mg/ml 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