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.).

1. Maintaining Self-renewal in IMOP Cells
<ol>
	<li>Prepare iMOP culture media: DMEM/F12, 1X B27 supplement, 25 &#181;g/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 &#176;C water bath.
	<ol>
		<li>Thaw 50X B27 supplement and filter-sterilized 100 mg/ml carbenecillin aliquots for 5 min in a 37 &#176;C water bath. Thaw out 100 &#181;g/ml bFGF aliquot at RT. Add 1 ml 50X B27, 10 &#181;l of 100 &#181;g/ml bFGF and 12.5 &#181;l of 100 mg/ml carbenecillin into DMEM/F12.</li>
	</ol>
	</li>
	<li>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.
	<ol>
		<li>Culture iMOP cells at 37 &#176;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.</li>
	</ol>
	</li>
	<li>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 &#176;C water bath.</li>
	<li>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 &#176;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.</li>
	<li>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 &#176;C and incubate for &#60;5 min to facilitate dissociation into single cells.</li>
	<li>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.</li>
	<li>Remove cells from 37 &#176;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.</li>
	<li>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.</li>
	<li>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.</li>
</ol>
2. Freezing and Thawing IMOP Cells
<ol>
	<li>Prepare synthetic freezing medium by thawing and equilibrating the solution to 4 &#176;C prior to freezing cells.</li>
	<li>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.</li>
	<li>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.</li>
	<li>Aspirate spent media using a 2ml aspirating pipette while leaving the loose cell pellet behind.</li>
	<li>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.</li>
	<li>Place the vials in an alcohol free freezing container and place the freezing container at -80 &#176;C to reduce the temperature 1 &#176;C per minute until the temperature reaches -80 &#176;C.</li>
	<li>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 &#176;C.</li>
	<li>Pre-warm iMOP culture media in a 37 &#176;C water bath.</li>
	<li>Thaw the frozen vial quickly by swirling the bottom of the vial in a 37 &#176;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</li>
	<li>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 &#176;C with 5% CO2 for expansion.</li>
</ol>
3. Differentiating IMOP Cells into Sensory Epithelia
<ol>
	<li>Prepare 50 ml of iMOP sensory epithelia differentiation media: DMEM/F12, 1X B27 supplement, 25 &#181;g/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 &#176;C water bath. Thaw 50X B27 supplement and 100 mg/ml carbenecillin aliquots for 5 min. in a 37 &#176;C water bath. Add 1 ml 50X B27 and 12.5 &#181;l of 100 mg/ml carbenecillin into DMEM/F12.</li>
	<li>To harvest, dissociate, resuspend and count cells repeat steps 1.4-1.9</li>
	<li>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.</li>
	<li>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.</li>
	<li>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.</li>
	<li>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.</li>
	<li>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.
	<ol>
		<li>Replace buffer and incubate samples in blocking buffer with diluted antibody. Incubate at 4 &#176;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 &#176;C.</li>
	</ol>
	</li>
	<li>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 &#177; 25 nm/447 &#177; 30 nm), green (475 &#177; 25 nm/540 &#177; 25 nm), red (562 &#177; 20nm/ and 625 &#177; 20nm) and infrared (628 &#177; 20nm/ and 692 &#177; 20 nm).</li>
</ol>
4. Assaying EdU Incorporation
<ol>
	<li>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.</li>
	<li>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.</li>
	<li>Determine the percentage of cells in S phase by EdU incorporation on day 3 by using an EdU incorporation assay
	<ol>
		<li>Add EdU stock directly to the iMOP cultures to obtain a final concentration of 1 &#181;M EdU in the culture media.</li>
		<li>Incubate EdU in iMOP cultures for 2 hr&#160;and incubate at 37 &#176;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.</li>
		<li>Label nuclei of cells with Hoechst and incorporated EdU with green fluorescence dye-azide according to manufacturer&#39;s protocol. 
		NOTE: All washes are done with 1X PBS, 3% BSA and 0.1% Tween 20. Wash cells with 1X PBS twice.</li>
		<li>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.</li>
	</ol>
	</li>
</ol>
5. Differentiating IMOP-Derived Neurons
<ol>
	<li>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 &#176;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.</li>
	<li>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.</li>
	<li>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.</li>
	<li>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.</li>
	<li>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.</li>
	<li>Thaw 2 mg/ml poly-D-lysine stock solution at RT and dilute to a 10 &#181;g/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 &#176;C incubator for 1 hr.</li>
	<li>Wash the wells 3 times with sterile 1X PBS. Thaw 10 mg/ml laminin stock solution at RT and dilute to 10 &#181;g/ml in 1X PBS. Add 0.25 ml of 10 &#181;g/ml laminin working solution into a single well of a 24 multi-well dish. Incubate the plate at 37 &#176;C incubator. Aspirate out the laminin solution using a 2 ml aspirating pipette.</li>
	<li>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.</li>
	<li>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.</li>
	<li>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.
	<ol>
		<li>Replace buffer and incubate samples in blocking buffer with diluted antibody. Incubate at 4 &#176;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 &#176;C before acquiring epifluorescence images as listed in step 3.8.</li>
	</ol>
	</li>
</ol>

The authors have nothing to disclose.

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...

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.

<table><tbody><tr><td>CoolCell LX Alcohol-Free Cell Freezing Containers</td><td>BioCision</td><td>BCS-405</td><td></td></tr><tr><td>Cryogenic Vials (2 ml)&amp;nbsp;</td><td>Corning</td><td>430654</td><td></td></tr><tr><td>1.5 Thickness Glass Coverslip (Round 12 mm)</td><td>Electron Microscopy Sciences</td><td>72230-01</td><td></td></tr><tr><td>DMEM/F12</td><td>Life Technologies</td><td>11320-082</td><td></td></tr><tr><td>Neurobasal Medium</td><td>Life Technologies</td><td>21103</td><td></td></tr><tr><td>Phosphate Buffered Saline (PBS) pH 7.4</td><td>Life Technologies</td><td>10010-023</td><td></td></tr><tr><td>Hank&#039;s Balanced Salt Solution (HBSS)</td><td>Life Technologies</td><td>14025-092</td><td></td></tr><tr><td>B27 Supplement (50X) Serum Free</td><td>Life Technologies</td><td>17504-044</td><td>Stored as 1 ml aliquots</td></tr><tr><td>L-Glutamine(200 mM)&amp;nbsp;</td><td>Life Technologies</td><td>25030-081</td><td>Stored as 5 ml aliquots</td></tr><tr><td>Natural Mouse Laminin</td><td>Life Technologies</td><td>23017-015</td><td>Stored as 1 mg/ml aliquots</td></tr><tr><td>Click-iT EdU Alexa Fluor 488&amp;nbsp;</td><td>Life Technologies</td><td>C10337</td><td></td></tr><tr><td>Synth-A-Freeze Cryopreservation Media</td><td>Life Technologies</td><td>A12542-01</td><td></td></tr><tr><td>Prolong Gold Antifade Mountant</td><td>Life Technologies</td><td>47743-736</td><td>Stored as 10 mg/ml 100 &amp;micro;l aliquots</td></tr><tr><td>Moxi Z Mini Automated Cell Counter</td><td>Orflo&amp;nbsp;</td><td>MXZ001</td><td></td></tr><tr><td>Moxi Z Cassette Type S</td><td>Orflo&amp;nbsp;</td><td>MXC002</td><td></td></tr><tr><td>Recombinant Murine Fibroblast Growth Factor, basic (bFGF)</td><td>Peprotech</td><td>450-33</td><td>Resuspended in 0.1% BSA in H20 and stored as 20 mg/ml aliquots</td></tr><tr><td>Poly-D-Lysine</td><td>Sigma</td><td>P7886</td><td>Resuspended in 1X PBS and stored as 10 mg/ml 100 &amp;micro;l aliquots</td></tr><tr><td>Carbenicillin, Disodium Salt</td><td>Thermo Fisher Scientific</td><td>BP2648-1</td><td>Resuspended in 10 mM Hepes pH 7.4 and stored as 100 mg/ml aliquots</td></tr><tr><td>5 ml pipet individually wrapped paperback (200/case)</td><td>Thermo Fisher Scientific</td><td>1367811D</td><td></td></tr><tr><td>10 ml pipet individually wrapped paperback (200/case)</td><td>Thermo Fisher Scientific</td><td>1367811E</td><td></td></tr><tr><td>Tissue Culture Treated Biolite 24-Well Plate</td><td>Thermo Fisher Scientific</td><td>130188</td><td></td></tr><tr><td>Tissue Culture Treated Biolite 6-Well Plate</td><td>Thermo Fisher Scientific</td><td>130184&amp;nbsp;</td><td></td></tr><tr><td>Tissue Culture Treated 6 cm Dish</td><td>Thermo Fisher Scientific</td><td>130181&amp;nbsp;</td><td></td></tr><tr><td>EMD Millipore Millex Sterile Syringe PVDF Filter Pore size: 0.22 &amp;mu;m</td><td>Thermo Fisher Scientific</td><td>SLGV033RS</td><td></td></tr><tr><td>TipOne filter pipet tips 0.1 - 10 &amp;mu;l elongated filter tip</td><td>USA Scientific</td><td>1120-3810</td><td></td></tr><tr><td>TipOne filter pipet tips 1 - 20 &amp;mu;l filter tip</td><td>USA Scientific</td><td>1120-1810</td><td></td></tr><tr><td>TipOne filter pipet tips 1 - 200 &amp;mu;l&amp;nbsp; filter tip</td><td>USA Scientific</td><td>1120-8810</td><td></td></tr><tr><td>TipOne filter pipet tips 101 - 1000 &amp;mu;l filter tip</td><td>USA Scientific</td><td>1126-7810</td><td></td></tr><tr><td>15 ml conical tubes sterile 20 bags of 25 tubes (500 tubes)</td><td>USA Scientific</td><td>1475-0511</td><td></td></tr><tr><td>50 ml conical tubes sterile 20 bags of 25 tubes (500 tubes)</td><td>USA Scientific</td><td>1500-1211</td><td></td></tr></tbody></table>

initiating differentiation in immortalized multipotent otic progenitor cells

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 &#946;-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.

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...

Watch this Scientific Journal Video about Initiating Differentiation in Immortalized Multipotent Otic Progenitor Cells at JoVE.com

Initiating Differentiation in Immortalized Multipotent Otic Progenitor Cells

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.

Use of human induced pluripotent stem cells (iPSC) or embryonic stem cells (ESC) for cell replacement therapies holds great promise. Several limitations ...

initiating-differentiation-immortalized-multipotent-otic-progenitor

Research

JoVE Journal

Developmental Biology

8.5K Views.  Rutgers University. 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.

Video: Initiating Differentiation in Immortalized Multipotent Otic Progenitor Cells

Progenitor-derived Oligodendrocyte Culture System from Human Fetal Brain

Differentiation of human neural progenitors into neuronal and glial cell types offers a model to study and compare molecular regulation of neural cell lineage development. In vitro expansion of neural progenitors from fetal CNS tissue has been well characterized. Despite the identification and isolation of glial progenitors from adult human sub-cortical white matter and development of various culture conditions to direct differentiation of fetal neural progenitors into myelin producing oligodendrocytes, acquiring sufficient human oligodendrocytes for in vitro experimentation remains difficult. Differentiation of galactocerebroside+ (GalC) and O4+ oligodendrocyte precursor or progenitor cells (OPC) from neural precursor cells has been reported using second trimester fetal brain. However, these cells do not proliferate in the absence of support cells including astrocytes and neurons, and are lost quickly over time in culture. The need remains for a culture system to produce cells of the oligodendrocyte lineage suitable for in vitro experimentation.
Culture of primary human oligodendrocytes could, for example, be a useful model to study the pathogenesis of neurotropic infectious agents like the human polyomavirus, JCV, that in vivo infects those cells. These cultured cells could also provide models of other demyelinating diseases of the central nervous system (CNS). Primary, human fetal brain-derived, multipotential neural progenitor cells proliferate in vitro while maintaining the capacity to differentiate into neurons (progenitor-derived neurons, PDN) and astrocytes (progenitor-derived astrocytes, PDA) This study shows that neural progenitors can be induced to differentiate through many of the stages of oligodendrocytic lineage development (progenitor-derived oligodendrocytes, PDO). We culture neural progenitor cells in DMEM-F12 serum-free media supplemented with basic fibroblast growth factor (bFGF), platelet derived growth factor (PDGF-AA), Sonic hedgehog (Shh), neurotrophic factor 3 (NT-3), N-2 and triiodothyronine (T3). The cultured cells are passaged at 2.5e6 cells per 75cm flasks approximately every seven days. Using these conditions, the majority of the cells in culture maintain a morphology characterized by few processes and express markers of pre-oligodendrocyte cells, such as A2B5 and O-4. When we remove the four growth factors (GF) (bFGF, PDGF-AA, Shh, NT-3) and add conditioned media from PDN, the cells start to acquire more processes and express markers specific of oligodendrocyte differentiation, such as GalC and myelin basic protein (MBP). We performed phenotypic characterization using multicolor flow cytometry to identify unique markers of oligodendrocyte.

Primary, human fetal brain-derived, multipotential progenitor cells proliferate in vitro while maintaining the capacity to differentiate into neurons and astrocytes. This work shows that neural progenitors can be induced to differentiate through stages of the oligodendrocytic lineage by conditioning with select growth factors.

Differentiation of human neural progenitors into neuronal and glial cell types offers a model to study and compare molecular regulation of neural cell ...

Neuroscience

Differentiation of Embryonic Stem Cells into Oligodendrocyte Precursors

Oligodendrocytes are the myelinating cells of the central nervous system. For regenerative cell therapy in demyelinating diseases, there is significant interest in deriving a pure population of lineage-committed oligodendrocyte precursor cells (OPCs) for transplantation. OPCs are characterized by the activity of the transcription factor Olig2 and surface expression of a proteoglycan NG2. Using the GFP-Olig2 (G-Olig2) mouse embryonic stem cell (mESC) reporter line, we optimized conditions for the differentiation of mESCs into GFP+Olig2+NG2+ OPCs. In our protocol, we first describe the generation of embryoid bodies (EBs) from mESCs. Second, we describe treatment of mESC-derived EBs with small molecules: (1) retinoic acid (RA) and (2) a sonic hedgehog (Shh) agonist purmorphamine (Pur) under defined culture conditions to direct EB differentiation into the oligodendroglial lineage. By this approach, OPCs can be obtained with high efficiency (&gt;80%) in a time period of 30 days. Cells derived from mESCs in this protocol are phenotypically similar to OPCs derived from primary tissue culture. The mESC-derived OPCs do not show the spiking property described for a subpopulation of brain OPCs in situ. To study this electrophysiological property, we describe the generation of spiking mESC-derived OPCs by ectopically expressing NaV1.2 subunit. The spiking and nonspiking cells obtained from this protocol will help advance functional studies on the two subpopulations of OPCs.

We describe a small molecule-based protocol for differentiation of mouse embryonic stem cells into oligodendrocyte precursor cells (OPCs). This protocol generates Olig2+NG2+ OPCs with high efficiency by 30 days of differentiation. We also describe a method to generate "spiking" OPCs that can fire action potentials.

Oligodendrocytes are the myelinating cells of the central nervous system. For regenerative cell therapy in demyelinating diseases, there is significant ...

Biology

Differentiation and Characterization of Osteoclasts from Human Induced Pluripotent Stem Cells

This protocol details the propagation and passaging of human iPSCs and their differentiation into osteoclasts. First, iPSCs are dissociated into a single-cell suspension for further use in embryoid body induction. Following mesodermal induction, embryoid bodies undergo hematopoietic differentiation, producing a floating hematopoietic cell population. Subsequently, the harvested hematopoietic cells undergo a macrophage colony-stimulating factor maturation step and, finally, osteoclast differentiation. After osteoclast differentiation, osteoclasts are characterized by staining for TRAP in conjunction with a methyl green nuclear stain. Osteoclasts are observed as multinucleated, TRAP+ polykaryons. Their identification can be further supported by Cathepsin K staining. Bone and mineral resorption assays allow for functional characterization, confirming the identity of bona fide osteoclasts. This protocol demonstrates a robust and versatile method to differentiate human osteoclasts from iPSCs and allows for easy adoption in applications requiring large quantities of functional human osteoclasts. Applications in the areas of bone research, cancer research, tissue engineering, and endoprosthesis research could be envisioned.

This protocol presents the differentiation of human osteoclasts from induced pluripotent stem cells (iPSCs) and describes methods for the characterization of osteoclasts and osteoclast precursors.

This protocol details the propagation and passaging of human iPSCs and their differentiation into osteoclasts. First, iPSCs are dissociated into a ...

Differentiation of Newborn Mouse Skin Derived Stem Cells into Germ-like Cells In vitro

Studying germ cell formation and differentiation has traditionally been very difficult due to low cell numbers and their location deep within developing embryos. The availability of a "closed" in vitro based system could prove invaluable for our understanding of gametogenesis. The formation of oocyte-like cells (OLCs) from somatic stem cells, isolated from newborn mouse skin, has been demonstrated and can be visualized in this video protocol. The resulting OLCs express various markers consistent with oocytes such as Oct4 , Vasa , Bmp15, and Scp3. However, they remain unable to undergo maturation or fertilization due to a failure to complete meiosis. This protocol will provide a system that is useful for studying the early stage formation and differentiation of germ cells into more mature gametes. During early differentiation the number of cells expressing Oct4 (potential germ-like cells) reaches ~5%, however currently the formation of OLCs remains relatively inefficient. The protocol is relatively straight forward though special care should be taken to ensure the starting cell population is healthy and at an early passage.

Differentiation of Newborn Mouse Skin Derived Stem Cells into Germ-like Cells In vitro

In recent years the formation of germ cells using in vitro culture methods has been demonstrated using several different types of somatic stem cells. This article will visually present the differentiation of newborn mouse derived stem cells to early oocyte-like cells.

Studying  germ cell formation and differentiation has traditionally been very difficult  due to low cell numbers and their location deep within developing ...

Generation of Human Neurons and Oligodendrocytes from Pluripotent Stem Cells for Modeling Neuron-Oligodendrocyte Interactions

In Alzheimer&#8217;s disease (AD) and other neurodegenerative disorders, oligodendroglial failure is a common early pathological feature, but how it contributes to disease development and progression, particularly in the gray matter of the brain, remains largely unknown. The dysfunction of oligodendrocyte lineage cells is hallmarked by deficiencies in myelination and impaired self-renewal of oligodendrocyte precursor cells (OPCs). These two defects are caused at least in part by the disruption of interactions between neuron and oligodendrocytes along the buildup of pathology. OPCs give rise to myelinating oligodendrocytes during CNS development. In the mature brain cortex, OPCs are the major proliferative cells (comprising ~5% of total brain cells) and control new myelin formation in a neural activity-dependent manner. Such neuron-to-oligodendrocyte communications are significantly understudied, especially in the context of neurodegenerative conditions such as AD, due to the lack of appropriate tools. In recent years, our group and others have made significant progress to improve currently available protocols to generate functional neurons and oligodendrocytes individually from human pluripotent stem cells. In this manuscript, we describe our optimized procedures, including the establishment of a co-culture system to model the neuron-oligodendrocyte connections. Our illustrative results suggest an unexpected contribution from OPCs/oligodendrocytes to the brain amyloidosis and synapse integrity and highlight the utility of this methodology for AD research. This reductionist approach is a powerful tool to dissect the specific hetero-cellular interactions out of the inherent complexity inside the brain. The protocols we describe here are expected to facilitate future studies on oligodendroglial defects in the pathogenesis of neurodegeneration.

The neuron-glial interactions in neurodegeneration are not well understood due to inadequate tools and methods. Here, we describe optimized protocols to obtain induced neurons, oligodendrocyte precursor cells, and oligodendrocytes from human pluripotent stem cells and provide examples of the values of these methods in understanding cell-type-specific contributions in Alzheimer&#8217;s disease.

In Alzheimer&#8217;s disease (AD) and other neurodegenerative disorders, oligodendroglial failure is a common early pathological feature, but how it ...

Differentiating Human Embryonic Stem Cells into Neural Progenitors

Source: Jeon, K. et al., <a href="https://app.jove.com/v/60571">Efficient Neural Differentiation using Single-Cell Culture of Human Embryonic Stem Cells.</a> J. Vis. Exp.&#160;(2020)
This video demonstrates the differentiation of human embryonic stem cells, or hESCs, directly into neural progenitor cells without using feeder cells or any intermediate steps. The selective inhibition of bone morphogenetic protein or BMP and transforming growth factor-beta, or TGF-&#946;, signaling pathways leads to hESCs differentiation into neural progenitor cells, or NPCs.

1. Preparation of human embryonic stem cells (hESC)-qualified Basement Membrane Matrix-coated Plates

	Slowly thaw the hESC-qualified basement membrane ...

JoVE Encyclopedia of Experiments

Neuronal Culture Techniques

Advanced Neuronal Models

A Co-culture Method to Model Neuron-Oligodendrocyte Interactions

Source: Assetta, B.,et al. <a href="https://app.jove.com/v/61778">Generation of Human Neurons and Oligodendrocytes from Pluripotent Stem Cells for Modeling Neuron-Oligodendrocyte Interactions.</a> J. Vis. Exp. (2020).
This video demonstrates the establishment of a co-culture of human induced neurons (iNs) and induced oligodendrocyte precursor cells (iOPCs). iNs are mixed with iOPCs in a co-culture and then incubated to support the maturation of both iNs into neurons and iOPCs into oligodendrocytes. Neurons form synaptic connections with other neurons and oligodendrocytes, while oligodendrocytes develop myelin sheaths around the neurons&#39; axons.

1. Human neuron induction from human pluripotent stem cells

	Lentivirus preparation (~5 days)
	
		Plate ~1 million Human embryonic kidney 293T (HEK293T) ...

Co-culture and Interaction Studies

Differentiation of Neural Progenitor Cells into Neurons

Source: Mao, X., et al. <a href="https://app.jove.com/v/61190">Neuronal Differentiation from Mouse Embryonic Stem Cells In vitro.</a> J. Vis. Exp. (2020).
The video showcased a cell culture method for differentiating neural progenitor cells into neurons. By incubating cells in a nutrient-rich medium supplemented with growth factors, the progenitor cells matured into neurons with developed axons and dendrites. A stabilizing agent ensured cell viability and protected against oxidative damage throughout the differentiation process.

1. Differentiation from NPCs to neurons (Phase II)

	Seed about 5 x 105 mouse embryonic stem cells or mESC derivatives within 2 mL of basal ...

Differentiating Otic Progenitor Cells Into Sensory Epithelial Cells Followed by Immunostaining

Source: Azadeh, J. et al., <a href="https://app.jove.com/v/53692">Initiating Differentiation in Immortalized Multipotent Otic Progenitor Cells.</a>&#160;J. Vis. Exp. (2016).
This video demonstrates the differentiation of immortalized multipotent otic progenitor (iMOP) cells into sensory epithelial cells. It outlines the steps involved in culturing iMOP cells, forming otospheres, inducing differentiation, and visualizing cellular components and differentiation markers through fluorescence microscopy to confirm successful cell differentiation.

1. Maintaining Self-renewal in IMOP Cells

	Prepare iMOP culture media: DMEM/F12, 1X B27 supplement, 25 &#181;g/ml carbenicillin and 20 ng/ml basic ...

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

Source: Azadeh, J., et al. <a href="https://app.jove.com/v/53692">Initiating Differentiation in Immortalized Multipotent Otic Progenitor Cells.</a> 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.

1. Differentiating iMOP-Derived Neurons 

	Make 50 ml of neuronal differentiation media: Neurobasal media, 1X B27 supplement, 2 mM L-Glutamine. Thaw ...

Initiating Differentiation in Immortalized Multipotent Otic Progenitor Cells

Samenvatting

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