Method Article

Initiating Differentiation in Immortalized Multipotent Otic Progenitor Cells

DOI:

10.3791/53692

⸱

January 2nd, 2016

 ,  ,  ,  , 
1Cell Biology & Neuroscience, Rutgers University

* These authors contributed equally

In This Article

Summary

$$\rightleftharpoonup{xx}$$ $$\longleftharp{xx}$$, $$\longrightharp{xx}$$,

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

$$\rightleftharpoonup{xx}$$ $$\longleftharp{xx}$$, $$\longrightharp{xx}$$,

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 (p27KIP) 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

$$\rightleftharpoonup{xx}$$ $$\longleftharp{xx}$$, $$\longrightharp{xx}$$,

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 loss1-4. 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 therapies5-8. A flurry of progress has shown that pluripotent stem cells and inner ear derived progenitors can differentiate into hair cells and SGNs at various stages of maturity. Mammalian embryonic stem cells (ESC) and induced pluripotent stem cells (iPSC) can be used to generate functional hair cells and SGNs9-11. Stem cells and progenitor cells derived from the mammalian inner ear have also been shown to form hair cells and neurons with properties of their in vivo cellular counterparts12-16.

Use of iPSC or ESC-derived otic progenitors to replace lost hair cells and SGNs requires efficient differentiation. Improper differentiation or continued proliferation of engrafted stem-derived progenitors in the inner ear can exacerbate inner ear function and pose a tumorigenic risk such as teratomas formation in the inner ear17. There is a clear need for developing culture conditions and understanding differentiation of otic progenitors. One strategy in developing these methods is to recapitulate cell fate decisions made by neurosensory progenitors during inner ear development. Protocols that prevent proliferation and direct otic progenitors into hair cells or SGNs will help improve safety as well as efficacy of replacement therapies.

During development, the inner ear begins with the thickening of surface ectoderm in a restricted region between rhombomeres 5 and 6 to become the otic placode. As the otic placode invaginates to form an otic cup, a collection of cells in the anterior region of the otic cup gives rise to the neural-sensory-competent domain (NSD), which contains precursors of hair cells and neurons of the inner ear18. Fate mapping studies from mouse, chicken and zebrafish developing inner ear suggest multiple populations of neurosensory progenitors that give rise to the sensory hair cells, surrounding supporting cells and otic neurons19-22. The high mobility group transcription factor, Sox2, has been implicated in sensory cell specification and used as a marker for inner ear progenitors23,24. Hypomorphic mutations that decrease Sox2 expression levels in the inner ear result in the loss of the hair cells, supporting cells and SGNs in the cochlea25,26.

To study otic progenitor cells undergoing cell fate decisions, a fate restricted immortalized multipotent otic progenitor (iMOP) cell line from Sox2 expressing cochlear progenitors was previously established. iMOP cells were originally derived from embryonic E12.5-13.5 cochlea and infected with a c-Myc retrovirus27. iMOP cells can continually proliferate as colony forming cells known as otospheres and have the capacity to differentiate into hair cells, supporting cells and SGNs27. Understanding the capacity of iMOP cells to differentiate into distinct otic lineages allows application of these findings to efficiently generate iPSC or ESC-derived hair cells and SGNs. Efficient differentiation protocols will open new avenues for cell replacement therapies of inner ear diseases that are recalcitrant to conventional treatments. A crucial issue in generating otic cells by in vitro cell culture is to have differentiation markers that help determine if cells are undergoing differentiating. Cdkn1b (p27KIP) has been extensively used as an early marker for differentiation in developing inner ear, however, expression of Cdkn1b in iMOP cells and how it correlates to differentiation has not been addressed. In this study, the current culture conditions and how Cdkn1b expression correlates to other markers of iMOP differentiation are described.

Access restricted. Please log in or start a trial to view this content.

Protocol

$$\rightleftharpoonup{xx}$$ $$\longleftharp{xx}$$, $$\longrightharp{xx}$$,

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 fresh media to cultures every other day by doubling the volume of media. Ensure that concentration of bFGF does not drop below 5 ng/ml.
    1. Culture iMOP cells at 37 °C with 5% CO2. Passage cells after 5 - 7 days in culture as listed in the steps below. Transfer culture to a 15 ml conical using a 10 ml pipette tip.
  3. Make 1 mM EDTA in HBSS by diluting 0.1 ml of 0.5 M EDTA pH 8.0 into 50 ml of HBSS. Pre-warm 1 mM EDTA made in HBSS in a 37 °C water bath.
  4. Harvest cells by gravity sedimentation or centrifugation at 200 x g for 5 min. at RT. For gravity sedimentation, place the 15 ml conical containing the cultures in the 37 °C incubator for 5 - 10 min. After that time, observe the otospheres collect at the bottom of the 15 ml conical.
    NOTE: Excessive centrifugal force can cause cell damage.
  5. Carefully aspirate spent media using a 2 ml aspirating pipette without disturbing the cell pellet. Add 0.5 ml of pre-warmed 1 mM EDTA HBSS solution to cell pellet. Using a P1000 pipette, gently pipet up and down 2 - 3 times. Place conical at 37 °C and incubate for <5 min to facilitate dissociation into single cells.
  6. Gently swirl the cell solution to determine if otospheres are dissociated. If no otospheres sediment to the bottom of the conical, the cells are dissociated. The time of incubation may vary.
    NOTE: Prolonged incubation with EDTA will result in excessive cell death.
  7. Remove cells from 37 °C, add 2 ml of media to neutralize and dilute the EDTA. Collect the cells by centrifugation at 200 x g for 5 min at RT. Aspirate out diluted EDTA using a 2 ml aspirating pipette and add 5 ml of 1X PBS to wash the cells.
  8. Spin cells down at 200 x g for 5 min at RT and aspirate out 1X PBS using a 2 ml aspirating pipette. Resuspend the cells using a P1000 pipette in 0.5 ml of iMOP culture media by gently pipetting up and down 2 - 3 times.
  9. Count cells using a microfluidic particle counter with the appropriate cassettes28. A confluent plate of iMOP cells will contain ~6 X106 cells. Dilute cells 1:100 in media and add 75 ul of cell solution into the cassette for counting. Plate 1 x 106 cells in a 6 cm dish in iMOP culture media (~1:10 dilution). Passage iMOPs every 5 - 7 days.
    NOTE: Cells that form large otospheres or attach to the bottom of the tissue culture dish will differentiate. Cells will also die if the cultures are over confluent.

2. Freezing and Thawing IMOP Cells

  1. Prepare synthetic freezing medium by thawing and equilibrating the solution to 4 °C prior to freezing cells.
  2. Collect cells from a confluent 6 cm dish of iMOP cells. Use a 10 ml pipette and transfer cells (~6 - 8 x 106 cells) into a 15 ml conical.
  3. Harvest cells by gravity sedimentation or centrifugation at 200 x g for 5 min at RT. If the otospheres are small, cells will not settle by gravity sedimentation. Optional: Count cells using step 1.9 to determine total cell numbers.
  4. Aspirate spent media using a 2ml aspirating pipette while leaving the loose cell pellet behind.
  5. Add 0.25 ml of synthetic freezing media to resuspend cells at a density of ~5 x 105 to 3 x 106 cells/ml. Gently pipette cells with a P1000 pipette. Transfer the cell suspension to cryogenic vials using a P1000 pipette with 1 ml filtered tip.
  6. Place the vials in an alcohol free freezing container and place the freezing container at -80 °C to reduce the temperature 1 °C per minute until the temperature reaches -80 °C.
  7. For long-term storage of cells, transfer the vials to the vapor phase of a liquid nitrogen storage tank. To thaw out cells for culture, equilibrate cryogenic vial containing frozen cells at -80 °C.
  8. Pre-warm iMOP culture media in a 37 °C water bath.
  9. Thaw the frozen vial quickly by swirling the bottom of the vial in a 37 °C water bath. Add 1 ml of pre-warmed iMOP culture media to thawed cells once the last ice crystal disappears. Transfer cells into a 15 ml conical and add an additional 4 ml of iMOP culture media
  10. Spin the 15 ml conical for 5 min. at 200 x g and aspirate the spent media. Resuspend the cells in 2 ml of iMOP culture media and plate cells in a 6 cm dish. Incubate cultures at 37 °C with 5% CO2 for expansion.

3. Differentiating IMOP Cells into Sensory Epithelia

  1. Prepare 50 ml of iMOP sensory epithelia differentiation media: DMEM/F12, 1X B27 supplement, 25 µg/ml carbenecillin. Make 50 ml of iMOP sensory epithelia differentiation media. Warm up 49 ml of DMEM/F12 in a 50 ml conical in a 37 °C water bath. Thaw 50X B27 supplement and 100 mg/ml carbenecillin aliquots for 5 min. in a 37 °C water bath. Add 1 ml 50X B27 and 12.5 µl of 100 mg/ml carbenecillin into DMEM/F12.
  2. To harvest, dissociate, resuspend and count cells repeat steps 1.4-1.9
  3. Plate 1 x 106 cells in a 6 cm dish at Day -3 using iMOP culture media. On Day 0, transfer cultures using a 10 ml pipette into a 15 ml conical. Collect the otospheres by gravity sedimentation as stated in step 1.6. Aspirate out spent media using a 2 ml aspirating pipette and leave the otospheres on the bottom of the conical.
  4. Gently add in 2 ml of sensory epithelia differentiation media. Transfer otospheres to a 6 cm dish using a large bore 10 ml pipette.
    NOTE: Mechanical shearing from harsh pipetting can dissociate cells from the otospheres.
  5. Add 2 ml of fresh sensory epithelial differentiation media to cultures every other day. If necessary, cells can be collected by gravity sedimentation and media replaced.
  6. Collect otospheres at Day 10 by transferring to a 15 ml conical and allowing the otospheres to sediment as stated in step 1.6.Aspirate the spent media using a 2 ml aspirating pipette and leave the otospheres undisturbed.
  7. Fix otospheres by incubating in 4% formaldehyde in 1X PBS for 15 min at RT. Remove formaldehyde solution, wash otospheres 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 the otosphere to immunostaining27. Transfer otospheres into 1X PBS and place otospheres into mounting media on a glass slide using a P1000 pipette. Put a coverslip over the sample and allow mounting media to dry at 4 °C.
  8. Acquire epifluorescence images using an inverted microscope setup equipped with a 16 bit CCD camera and either a 20X 0.75 air or a 40X 1.3 NA oil immersion objective. Collect fluorescence from different color channels using the listed (excitation and emission) wavelengths: blue (377 ± 25 nm/447 ± 30 nm), green (475 ± 25 nm/540 ± 25 nm), red (562 ± 20nm/ and 625 ± 20nm) and infrared (628 ± 20nm/ and 692 ± 20 nm).

4. Assaying EdU Incorporation

  1. On Day -3, plate 1 x 106 iMOP cells in a 6 cm dish. Three days later on Day 0, harvest, dissociate, resuspend and count cells by repeating steps 1.4-1.9.
  2. Plate 2.5 x 105 cells in iMOP culture media and 5 x105 cells in sensory epithelia differentiation media in different wells of a 6 well dish.
  3. Determine the percentage of cells in S phase by EdU incorporation on day 3 by using an EdU incorporation assay
    1. Add EdU stock directly to the iMOP cultures to obtain a final concentration of 1 µM EdU in the culture media.
    2. Incubate EdU in iMOP cultures for 2 hr and incubate at 37 °C with 5% CO2. Harvest, dissociate and collect cells repeat steps 1.4-1.9. Add in 4% formaldehyde in 1X PBS for 15 min. at RT to fix cells. Harvest cells by centrifugation at 200 x g for 5 min at RT. Remove formaldehyde solution and properly dispose.
    3. Label nuclei of cells with Hoechst and incorporated EdU with green fluorescence dye-azide according to manufacturer's protocol.
      NOTE: All washes are done with 1X PBS, 3% BSA and 0.1% Tween 20. Wash cells with 1X PBS twice.
    4. Mount cells on a slide using antifade reagent and place a 1.5 cover glass over the sample. Acquire fluorescent images of labeled cells using an epifluorescence microscopy.

5. 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% 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 PBS to wash out the remaining ethanol. Rinse the coverslip once with sterile H20 to wash out remaining 1X PBS.
  4. Aspirate the H20 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 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 in steps 1.4-1.9.Plate 1 x 106 cells in a 6 cm dish at Day -3.
  9. On Day 0, harvest, dissociate and count cells by repeating 1.4-1.9. 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 immunostaining27. Wash the coverglass with 1X PBS before placing onto mounting media. Allow the mounting media to dry at 4 °C before acquiring epifluorescence images as listed in step 3.8.

Access restricted. Please log in or start a trial to view this content.

Results

$$\rightleftharpoonup{xx}$$ $$\longleftharp{xx}$$, $$\longrightharp{xx}$$,

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...

Access restricted. Please log in or start a trial to view this content.

Discussion

$$\rightleftharpoonup{xx}$$ $$\longleftharp{xx}$$, $$\longrightharp{xx}$$,

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...

Access restricted. Please log in or start a trial to view this content.

Disclosures

$$\rightleftharpoonup{xx}$$ $$\longleftharp{xx}$$, $$\longrightharp{xx}$$,

The authors have nothing to disclose.

Acknowledgements

$$\rightleftharpoonup{xx}$$ $$\longleftharp{xx}$$, $$\longrightharp{xx}$$,

The work was supported in part by the Duncan and Nancy MacMillan Faculty Development Chair Endowment Fund (K.Y.K.), Busch Biomedical Research Grant (K.Y.K.) and the Rutgers Faculty Development Grant (K.Y.K.).

Access restricted. Please log in or start a trial to view this content.

Materials

List of materials used in this article
NameCompanyCatalog NumberComments
CoolCell LX Alcohol-Free Cell Freezing ContainersBioCisionBCS-405
Cryogenic Vials (2 ml) Corning430654
1.5 Thickness Glass Coverslip (Round 12 mm)Electron Microscopy Sciences72230-01
DMEM/F12Life Technologies11320-082
Neurobasal MediumLife Technologies21103
Phosphate Buffered Saline (PBS) pH 7.4Life Technologies10010-023
Hank's Balanced Salt Solution (HBSS)Life Technologies14025-092
B27 Supplement (50X) Serum FreeLife Technologies17504-044Stored as 1 ml aliquots
L-Glutamine(200 mM) Life Technologies25030-081Stored as 5 ml aliquots
Natural Mouse LamininLife Technologies23017-015Stored as 1 mg/ml aliquots
Click-iT EdU Alexa Fluor 488 Life TechnologiesC10337
Synth-A-Freeze Cryopreservation MediaLife TechnologiesA12542-01
Prolong Gold Antifade MountantLife Technologies47743-736Stored as 10 mg/ml 100 µl aliquots
Moxi Z Mini Automated Cell CounterOrflo MXZ001
Moxi Z Cassette Type SOrflo MXC002
Recombinant Murine Fibroblast Growth Factor, basic (bFGF)Peprotech450-33Resuspended in 0.1% BSA in H20 and stored as 20 mg/ml aliquots
Poly-D-LysineSigmaP7886Resuspended in 1X PBS and stored as 10 mg/ml 100 µl aliquots
Carbenicillin, Disodium SaltThermo Fisher ScientificBP2648-1Resuspended in 10 mM Hepes pH 7.4 and stored as 100 mg/ml aliquots
5 ml pipet individually wrapped paperback (200/case)Thermo Fisher Scientific1367811D
10 ml pipet individually wrapped paperback (200/case)Thermo Fisher Scientific1367811E
Tissue Culture Treated Biolite 24-Well PlateThermo Fisher Scientific130188
Tissue Culture Treated Biolite 6-Well PlateThermo Fisher Scientific130184 
Tissue Culture Treated 6 cm DishThermo Fisher Scientific130181 
EMD Millipore Millex Sterile Syringe PVDF Filter Pore size: 0.22 μmThermo Fisher ScientificSLGV033RS
TipOne filter pipet tips 0.1 - 10 μl elongated filter tipUSA Scientific1120-3810
TipOne filter pipet tips 1 - 20 μl filter tipUSA Scientific1120-1810
TipOne filter pipet tips 1 - 200 μl  filter tipUSA Scientific1120-8810
TipOne filter pipet tips 101 - 1000 μl filter tipUSA Scientific1126-7810
15 ml conical tubes sterile 20 bags of 25 tubes (500 tubes)USA Scientific1475-0511
50 ml conical tubes sterile 20 bags of 25 tubes (500 tubes)USA Scientific1500-1211

References

$$\rightleftharpoonup{xx}$$ $$\longleftharp{xx}$$, $$\longrightharp{xx}$$,
  1. Petit, C., Richardson, G. P. Linking genes underlying deafness to hair-bundle development and function. Nat Neurosci. 12 (6), 703-710 (2009).
  2. Kujawa, S. G., Liberman, M. C. Adding insult to injury: cochlear nerve degeneration after 'temporary' noise-induced hearing loss. J Neurosci. 29 (45), 14077-14085 (2009).
  3. Kujawa, S. G., Liberman, M. C. Acceleration of age-related hearing loss by early noise exposure: evidence of a misspent youth. J Neurosci. 26 (7), 2115-2123 (2006).
  4. Huth, M. E., Ricci, A. J., Cheng, A. G. Mechanisms of aminoglycoside ototoxicity and targets of hair cell protection. Int J Otolaryngol. 2011, 937861-93 (2011).
  5. Brigande, J. V., Heller, S. Quo vadis, hair cell regeneration? Nat Neurosci. 12 (6), 679-685 (2009).
  6. Groves, A. K. The challenge of hair cell regeneration. Exp Biol Med (Maywood). 235 (4), 434-446 (2010).
  7. Okano, T., Kelley, M. W. Stem cell therapy for the inner ear: recent advances and future directions. Trends Amplif. 16 (1), 4-18 (2012).
  8. Ronaghi, M., Nasr, M., Heller, S. Concise review: Inner ear stem cells--an oxymoron, but why? Stem Cells. 30 (1), 69-74 (2012).
  9. Chen, W., et al. Restoration of auditory evoked responses by human ES-cell-derived otic progenitors. Nature. 490 (7419), 278-282 (2012).
  10. Koehler, K. R., Mikosz, A. M., Molosh, A. I., Patel, D., Hashino, E. Generation of inner ear sensory epithelia from pluripotent stem cells in 3D culture. Nature. , (2013).
  11. Oshima, K., et al. Mechanosensitive hair cell-like cells from embryonic and induced pluripotent stem cells. Cell. 141 (4), 704-716 (2010).
  12. Diensthuber, M., Oshima, K., Heller, S. Stem/progenitor cells derived from the cochlear sensory epithelium give rise to spheres with distinct morphologies and features. J Assoc Res Otolaryngol. 10 (2), 173-190 (2009).
  13. Doetzlhofer, A., White, P., Lee, Y. S., Groves, A., Segil, N. Prospective identification and purification of hair cell and supporting cell progenitors from the embryonic cochlea. Brain Res. 1091 (1), 282-288 (2006).
  14. Doetzlhofer, A., White, P. M., Johnson, J. E., Segil, N., Groves, A. K. In vitro growth and differentiation of mammalian sensory hair cell progenitors: a requirement for EGF and periotic mesenchyme. Dev Biol. 272 (2), 432-447 (2004).
  15. Martinez-Monedero, R., Yi, E., Oshima, K., Glowatzki, E., Edge, A. S. Differentiation of inner ear stem cells to functional sensory neurons. Dev Neurobiol. 68 (5), 669-684 (2008).
  16. Oshima, K., Senn, P., Heller, S. Isolation of sphere-forming stem cells from the mouse inner ear. Methods Mol Biol. 493, 141-162 (2009).
  17. Nishimura, K., Nakagawa, T., Sakamoto, T., Ito, J. Fates of murine pluripotent stem cell-derived neural progenitors following transplantation into mouse cochleae. Cell Transplant. 21 (4), 763-771 (2012).
  18. Wu, D. K., Kelley, M. W. Molecular mechanisms of inner ear development. Cold Spring Harb Perspect Biol. 4 (8), a008409(2012).
  19. Jiang, H., et al. Lineage analysis of the late otocyst stage mouse inner ear by transuterine microinjection of a retroviral vector encoding alkaline phosphatase and an oligonucleotide library. PLoS One. 8 (7), e69314(2013).
  20. Sapede, D., Dyballa, S., Pujades, C. Cell lineage analysis reveals three different progenitor pools for neurosensory elements in the otic vesicle. J Neurosci. 32 (46), 16424-16434 (2012).
  21. Satoh, T., Fekete, D. M. Clonal analysis of the relationships between mechanosensory cells and the neurons that innervate them in the chicken ear. Development. 132 (7), 1687-1697 (2005).
  22. Raft, S., et al. Cross-regulation of Ngn1 and Math1 coordinates the production of neurons and sensory hair cells during inner ear development. Development. 134 (24), 4405-4415 (2007).
  23. Neves, J., Parada, C., Chamizo, M., Giraldez, F. Jagged 1 regulates the restriction of Sox2 expression in the developing chicken inner ear: a mechanism for sensory organ specification. Development. 138 (4), 735-744 (2011).
  24. Mak, A. C., Szeto, I. Y., Fritzsch, B., Cheah, K. S. Differential and overlapping expression pattern of SOX2 and SOX9 in inner ear development. Gene Expr Patterns. 9 (6), 444-453 (2009).
  25. Kiernan, A. E., et al. Sox2 is required for sensory organ development in the mammalian inner ear. Nature. 434 (7036), 1031-1035 (2005).
  26. Puligilla, C., Dabdoub, A., Brenowitz, S. D., Kelley, M. W. Sox2 induces neuronal formation in the developing mammalian cochlea. J Neurosci. 30 (2), 714-722 (2010).
  27. Kwan, K. Y., Shen, J., Corey, D. P. C-MYC transcriptionally amplifies SOX2 target genes to regulate self-renewal in multipotent otic progenitor cells. Stem Cell Reports. 4 (1), 47-60 (2015).
  28. Dittami, G. M., Sethi, M., Rabbitt, R. D., Ayliffe, H. E. Determination of mammalian cell counts, cell size and cell health using the Moxi Z mini automated cell counter. J Vis Exp. (64), (2012).
  29. Levenstein, M. E., et al. Basic fibroblast growth factor support of human embryonic stem cell self-renewal. Stem Cells. 24 (3), 568-574 (2006).
  30. Furue, M. K., et al. Heparin promotes the growth of human embryonic stem cells in a defined serum-free medium. Proc Natl Acad Sci U S A. 105 (36), 13409-13414 (2008).
  31. Lotz, S., et al. Sustained levels of FGF2 maintain undifferentiated stem cell cultures with biweekly feeding. PLoS One. 8 (2), e56289(2013).
  32. Brewer, G. J., Torricelli, J. R., Evege, E. K., Price, P. J. Optimized survival of hippocampal neurons in B27-supplemented Neurobasal, a new serum-free medium combination. J Neurosci Res. 35 (5), 567-576 (1993).
  33. Watanabe, K., et al. A ROCK inhibitor permits survival of dissociated human embryonic stem cells. Nat Biotechnol. 25 (6), 681-686 (2007).
  34. Ruben, R. J. Development of the inner ear of the mouse: a radioautographic study of terminal mitoses. Acta Otolaryngol. , Suppl 220. 1-44 (1967).
  35. Matei, V., et al. Smaller inner ear sensory epithelia in Neurog 1 null mice are related to earlier hair cell cycle exit. Dev Dyn. 234 (3), 633-650 (2005).
  36. Lee, Y. S., Liu, F., Segil, N. A morphogenetic wave of p27Kip1 transcription directs cell cycle exit during organ of Corti development. Development. 133 (15), 2817-2826 (2006).
  37. Chen, P., Segil, N. p27(Kip1) links cell proliferation to morphogenesis in the developing organ of Corti. Development. 126 (8), 1581-1590 (1999).
  38. Hasson, T., et al. Unconventional myosins in inner-ear sensory epithelia. J Cell Biol. 137 (6), 1287-1307 (1997).
  39. Barclay, M., Ryan, A. F., Housley, G. D. Type I vs type II spiral ganglion neurons exhibit differential survival and neuritogenesis during cochlear development. Neural Dev. 6, 33(2011).
  40. Bermingham, N. A., et al. Math1: an essential gene for the generation of inner ear hair cells. Science. 284 (5421), 1837-1841 (1999).
  41. Izumikawa, M., et al. Auditory hair cell replacement and hearing improvement by Atoh1 gene therapy in deaf mammals. Nat Med. 11 (3), 271-276 (2005).
  42. Kim, W. Y., et al. NeuroD-null mice are deaf due to a severe loss of the inner ear sensory neurons during development. Development. 128 (3), 417-426 (2001).
  43. Liu, M., et al. Essential role of BETA2/NeuroD1 in development of the vestibular and auditory systems. Genes Dev. 14 (22), 2839-2854 (2000).
  44. Ma, Q., Anderson, D. J., Fritzsch, B. Neurogenin 1 null mutant ears develop fewer, morphologically normal hair cells in smaller sensory epithelia devoid of innervation. J Assoc Res Otolaryngol. 1 (2), 129-143 (2000).
  45. Pang, Z. P., et al. Induction of human neuronal cells by defined transcription factors. Nature. 476 (7359), 220-223 (2011).
  46. Vierbuchen, T., et al. Direct conversion of fibroblasts to functional neurons by defined factors. Nature. 463 (7284), 1035-1041 (2010).
  47. Zhang, Y., et al. Rapid single-step induction of functional neurons from human pluripotent stem cells. Neuron. 78 (5), 785-798 (2013).
  48. Blanchard, J. W., et al. Selective conversion of fibroblasts into peripheral sensory neurons. Nat Neurosci. 18 (1), 25-35 (2015).

Access restricted. Please log in or start a trial to view this content.

Reprints and Permissions

Request permission to reuse the text or figures of this JoVE article

Request Permission

Tags

iMOP Cell DifferentiationbFGF Withdrawal ProtocolEdU Cell Proliferation AssayImmunostaining Cdkn1b Cdh1Immunostaining Cdkn1b Tubb3Otosphere Formation Gravity SedimentationNeuronal Differentiation MediumSensory Epithelial DifferentiationInner Ear Cell Replacement

Related Articles