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 border="0" cellpadding="0" cellspacing="0" width="1075">
	<tbody>
		<tr height="20">
			<td height="20" style="height:20px;width:455px;">CoolCell LX Alcohol-Free Cell Freezing Containers</td>
			<td style="width:196px;">BioCision</td>
			<td style="width:145px;">BCS-405</td>
			<td style="width:280px;"></td>
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		<tr height="20">
			<td height="20" style="height:20px;width:455px;">Cryogenic Vials (2 ml)&#160;</td>
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			<td style="width:145px;">430654</td>
			<td style="width:280px;"></td>
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			<td height="20" style="height:20px;width:455px;">1.5 Thickness Glass Coverslip (Round 12 mm)</td>
			<td style="width:196px;">Electron Microscopy Sciences</td>
			<td style="width:145px;">72230-01</td>
			<td style="width:280px;"></td>
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			<td height="20" style="height:20px;width:455px;">DMEM/F12</td>
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			<td style="width:145px;">11320-082</td>
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			<td height="20" style="height:20px;width:455px;">Neurobasal Medium</td>
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			<td style="width:145px;">21103</td>
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			<td height="20" style="height:20px;width:455px;">Phosphate Buffered Saline (PBS) pH 7.4</td>
			<td style="width:196px;">Life Technologies</td>
			<td style="width:145px;">10010-023</td>
			<td style="width:280px;"></td>
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			<td height="20" style="height:20px;width:455px;">Hank&#39;s Balanced Salt Solution (HBSS)</td>
			<td style="width:196px;">Life Technologies</td>
			<td style="width:145px;">14025-092</td>
			<td style="width:280px;"></td>
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			<td height="20" style="height:20px;width:455px;">B27 Supplement (50X) Serum Free</td>
			<td style="width:196px;">Life Technologies</td>
			<td style="width:145px;">17504-044</td>
			<td style="width:280px;">Stored as 1 ml aliquots</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:455px;">L-Glutamine(200 mM)&#160;</td>
			<td style="width:196px;">Life Technologies</td>
			<td style="width:145px;">25030-081</td>
			<td style="width:280px;">Stored as 5 ml aliquots</td>
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		<tr height="20">
			<td height="20" style="height:20px;width:455px;">Natural Mouse Laminin</td>
			<td style="width:196px;">Life Technologies</td>
			<td style="width:145px;">23017-015</td>
			<td style="width:280px;">Stored as 1 mg/ml aliquots</td>
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			<td height="20" style="height:20px;width:455px;">Click-iT EdU Alexa Fluor 488&#160;</td>
			<td style="width:196px;">Life Technologies</td>
			<td style="width:145px;">C10337</td>
			<td style="width:280px;"></td>
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			<td style="width:145px;">A12542-01</td>
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			<td style="width:196px;">Life Technologies</td>
			<td style="width:145px;">47743-736</td>
			<td style="width:280px;">Stored as 10 mg/ml 100 &#181;l aliquots</td>
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			<td height="20" style="height:20px;width:455px;">Moxi Z Mini Automated Cell Counter</td>
			<td style="width:196px;">Orflo&#160;</td>
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			<td height="20" style="height:20px;width:455px;">Moxi Z Cassette Type S</td>
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			<td height="40" style="height:40px;width:455px;">Recombinant Murine Fibroblast Growth Factor, basic (bFGF)</td>
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			<td style="width:145px;">450-33</td>
			<td style="width:280px;">Resuspended in 0.1% BSA in H20 and stored as 20 mg/ml aliquots</td>
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		<tr height="40">
			<td height="40" style="height:40px;width:455px;">Poly-D-Lysine</td>
			<td style="width:196px;">Sigma</td>
			<td style="width:145px;">P7886</td>
			<td style="width:280px;">Resuspended in 1X PBS and stored as 10 mg/ml 100 &#181;l aliquots</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:455px;">Carbenicillin, Disodium Salt</td>
			<td style="width:196px;">Thermo Fisher Scientific</td>
			<td style="width:145px;">BP2648-1</td>
			<td style="width:280px;">Resuspended in 10mM Hepes pH 7.4 and stored as 100 mg/ml aliquots</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:455px;">5 ml pipet individually wrapped paperback (200/case)</td>
			<td style="width:196px;">Thermo Fisher Scientific</td>
			<td>1367811D</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:455px;">10 ml pipet individually wrapped paperback (200/case)</td>
			<td style="width:196px;">Thermo Fisher Scientific</td>
			<td>1367811E</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:455px;">Tissue Culture Treated Biolite 24 -Well Plate</td>
			<td style="width:196px;">Thermo Fisher Scientific</td>
			<td style="width:145px;">130188</td>
			<td style="width:280px;"></td>
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			<td height="20" style="height:20px;width:455px;">Tissue Culture Treated Biolite 6 -Well Plate</td>
			<td style="width:196px;">Thermo Fisher Scientific</td>
			<td style="width:145px;">130184&#160;</td>
			<td style="width:280px;"></td>
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			<td height="20" style="height:20px;width:455px;">Tissue Culture Treated 6 cm Dish</td>
			<td style="width:196px;">Thermo Fisher Scientific</td>
			<td style="width:145px;">130181&#160;</td>
			<td style="width:280px;"></td>
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		<tr height="20">
			<td height="20" style="height:20px;">EMD Millipore Millex Sterile Syringe PVDF Filter Pore size: 0.22&#956;m</td>
			<td style="width:196px;">Thermo Fisher Scientific</td>
			<td>SLGV033RS</td>
			<td></td>
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		<tr height="20">
			<td height="20" style="height:20px;width:455px;">TipOne filter pipet tips 0.1-10 ul elongated filter tip</td>
			<td>USA Scientific</td>
			<td>1120-3810</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:455px;">TipOne filter pipet tips 1-20 ul filter tip</td>
			<td>USA Scientific</td>
			<td>1120-1810</td>
			<td></td>
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		<tr height="20">
			<td height="20" style="height:20px;width:455px;">TipOne filter pipet tips 1-200 ul&#160; filter tip</td>
			<td>USA Scientific</td>
			<td>1120-8810</td>
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			<td height="20" style="height:20px;width:455px;">TipOne filter pipet tips 101-1000 ul filter tip</td>
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			<td>1126-7810</td>
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		<tr height="20">
			<td height="20" style="height:20px;width:455px;">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 height="20">
			<td height="20" style="height:20px;width:455px;">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...

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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 ...

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Universidad de Cartagena UDEC

Research

JoVE Journal

Developmental Biology

8.4K 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

Derivation of Cardiac Progenitor Cells from Embryonic Stem Cells

Cardiac progenitor cells (CPCs) have the capacity to differentiate into cardiomyocytes, smooth muscle cells (SMC), and endothelial cells and hold great promise in cell therapy against heart disease. Among various methods to isolate CPCs, differentiation of embryonic stem cell (ESC) into CPCs attracts great attention in the field since ESCs can provide unlimited cell source. As a result, numerous strategies have been developed to derive CPCs from ESCs. In this protocol, differentiation and purification of embryonic CPCs from both mouse and human ESCs is described. Due to the difficulty of using cell surface markers to isolate embryonic CPCs, ESCs are engineered with fluorescent reporters activated by CPC-specific cre recombinase expression. Thus, CPCs can be enriched by fluorescence-activated cell sorting (FACS). This protocol illustrates procedures to form embryoid bodies (EBs) from ESCs for CPC specification and enrichment. The isolated CPCs can be subsequently cultured for cardiac lineage differentiation and other biological assays. This protocol is optimized for robust and efficient derivation of CPCs from both mouse and human ESCs.

In this protocol, derivation of cardiac progenitor cells from both mouse and human embryonic stem cells will be illustrated. A major strategy in this protocol is to enrich cardiac progenitor cells with flow cytometry using fluorescent reporters engineered into the embryonic stem cell lines.

Cardiac progenitor cells (CPCs) have the capacity to differentiate into cardiomyocytes, smooth muscle cells (SMC), and endothelial cells and hold great ...

Direct Induction of Human Neural Stem Cells from Peripheral Blood Hematopoietic Progenitor Cells

Human disease specific neuronal cultures are essential for generating in vitro models for human neurological diseases. However, the lack of access to primary human adult neural cultures raises unique challenges. Recent developments in induced pluripotent stem cells (iPSC) provides an alternative approach to derive neural cultures from skin fibroblasts through patient specific iPSC, but this process is labor intensive, requires special expertise and large amounts of resources, and can take several months. This prevents the wide application of this technology to the study of neurological diseases. To overcome some of these issues, we have developed a method to derive neural stem cells directly from human adult peripheral blood, bypassing the iPSC derivation process. Hematopoietic progenitor cells enriched from human adult peripheral blood were cultured in vitro and transfected with Sendai virus vectors containing transcriptional factors Sox2, Oct3/4, Klf4, and c-Myc. The transfection results in morphological changes in the cells which are further selected by using human neural progenitor medium containing basic fibroblast growth factor (bFGF) and vascular endothelial growth factor (VEGF). The resulting cells are characterized by the expression for neural stem cell markers, such as nestin and SOX2. These neural stem cells could be further differentiated to neurons, astroglia and oligodendrocytes in specified differentiation media. Using easily accessible human peripheral blood samples, this method could be used to derive neural stem cells for further differentiation to neural cells for in vitro modeling of neurological disorders and may advance studies related to the pathogenesis and treatment of those diseases.

A method was developed to directly derive human neural stem cells from hematopoietic progenitor cells enriched from peripheral blood cells.

Human disease specific neuronal cultures are essential for generating in vitro models for human neurological diseases. However, the lack of access to ...

Analysis of Zebrafish Larvae Skeletal Muscle Integrity with Evans Blue Dye

The zebrafish model is an emerging system for the study of neuromuscular disorders. In the study of neuromuscular diseases, the integrity of the muscle membrane is a critical disease determinant. To date, numerous neuromuscular conditions display degenerating muscle fibers with abnormal membrane integrity; this is most commonly observed in muscular dystrophies. Evans Blue Dye (EBD) is a vital, cell permeable dye that is rapidly taken into degenerating, damaged, or apoptotic cells; in contrast, it is not taken up by cells with an intact membrane. EBD injection is commonly employed to ascertain muscle integrity in mouse models of neuromuscular diseases. However, such EBD experiments require muscle dissection and/or sectioning prior to analysis. In contrast, EBD uptake in zebrafish is visualized in live, intact preparations. Here, we demonstrate a simple and straightforward methodology for performing EBD injections and analysis in live zebrafish. In addition, we demonstrate a co-injection strategy to increase efficacy of EBD analysis. Overall, this video article provides an outline to perform EBD injection and characterization in zebrafish models of neuromuscular disease.

In this study, we describe a straightforward method to perform Evans Blue Dye (EBD) analysis on zebrafish larvae. This technique is a powerful tool for the characterization of skeletal muscle integrity and delineation of zebrafish models of muscular dystrophy, and is a valuable method for the development of novel therapeutics.

The zebrafish model is an emerging system for the study of neuromuscular disorders.  In the study of neuromuscular diseases, the integrity of the muscle ...

Isolating Mesangiogenic Progenitor Cells (MPCs) from Human Bone Marrow

In a research study aimed to isolate human bone marrow (hBM)-derived Mesenchymal Stromal Cells (MSCs) for clinical applications, we identified a novel cell population specifically selected for growth in human serum supplemented medium. These cells are characterized by morphological, phenotypic, and molecular features distinct from MSCs and we named them Mesodermal Progenitor Cells (MPCs). MPCs are round, with a thick highly refringent core region; they show strong, trypsin resistant adherence to plastic. Failure to expand MPCs directly revealed that they are slow in cycling. This is as also suggested by Ki-67 negativity. On the other hand, culturing MPCs in standard medium designed for MSC expansion, gave rise to a population of exponentially growing MSC-like cells. Besides showing mesenchymal differentiation capacity MPCs retained angiogenic potential, confirming their multiple lineage progenitor nature. Here we describe an optimized highly reproducible protocol to isolate and characterize hBM-MPCs by flow cytometry (CD73, CD90, CD31, and CD45), nestin expression, and F-actin organization. Protocols for mesengenic and angiogenic differentiation of MPCs are also provided. Here we also suggest a more appropriate nomenclature for these cells, which has been re-named as "Mesangiogenic Progenitor Cells".

Isolating Mesangiogenic Progenitor Cells MPCs from Human Bone Marrow

Here we describe an optimized, highly reproducible protocol to isolate Mesodermal Progenitor Cells (MPCs) from human bone marrow (hBM). MPCs were characterized by flow cytometry and nestin expression. They showed the ability to give rise to exponentially growing MSC-like cell cultures while retaining their angiogenic potential.

In a research study aimed to isolate human bone marrow (hBM)-derived Mesenchymal Stromal Cells (MSCs) for clinical applications, we identified a novel ...

Directed Differentiation of Primitive and Definitive Hematopoietic Progenitors from Human Pluripotent Stem Cells

One of the major goals for regenerative medicine is the generation and maintenance of hematopoietic stem cells (HSCs) derived from human pluripotent stem cells (hPSCs). Until recently, efforts to differentiate hPSCs into HSCs have predominantly generated hematopoietic progenitors that lack HSC potential, and instead resemble yolk sac hematopoiesis.&#160;These resulting hematopoietic progenitors may have limited utility for in vitro disease modeling of various adult hematopoietic disorders, particularly those of the lymphoid lineages. However, we have recently described methods to generate erythro-myelo-lymphoid multilineage definitive hematopoietic progenitors from hPSCs using a stage-specific directed differentiation protocol, which we outline here. Through enzymatic dissociation of hPSCs on basement membrane matrix-coated plasticware, embryoid bodies (EBs) are formed. EBs are differentiated to mesoderm by recombinant BMP4, which is subsequently specified to the definitive hematopoietic program by the GSK3&#946; inhibitor, CHIR99021. Alternatively, primitive hematopoiesis is specified by the PORCN inhibitor, IWP2. Hematopoiesis is further driven through the addition of recombinant VEGF and supportive hematopoietic cytokines. The resulting hematopoietic progenitors generated using this method have the potential to be used for disease and developmental modeling, in vitro.

Here, we present human pluripotent stem cell (hPSC) culture protocols, used to differentiate hPSCs into CD34+ hematopoietic progenitors. This method uses stage-specific manipulation of canonical WNT signaling to specify cells exclusively to either the definitive or primitive hematopoietic program.

One of the major goals for regenerative medicine is the generation and maintenance of hematopoietic stem cells (HSCs) derived from human pluripotent stem ...

In Vitro Differentiation of Human Pluripotent Stem Cells into Trophoblastic Cells

The placenta is the first organ to develop during embryogenesis and is required for the survival of the developing embryo. The placenta is comprised of various trophoblastic cells that differentiate from the extra-embryonic trophectoderm cells of the preimplantation blastocyst. As such, our understanding of the early differentiation events of the human placenta is limited because of ethical and legal restrictions on the isolation and manipulation of human embryogenesis. Human pluripotent stem cells (hPSCs) are a robust model system for investigating human development and can also be differentiated in vitro into trophoblastic cells that express markers of the various trophoblast cell types. Here, we present a detailed protocol for differentiating hPSCs into trophoblastic cells using bone morphogenic protein 4 and inhibitors of the Activin/Nodal signaling pathways. This protocol generates various trophoblast cell types that can be transfected with siRNAs for investigating loss-of-function phenotypes or can be infected with pathogens. Additionally, hPSCs can be genetically modified and then differentiated into trophoblast progenitors for gain-of-function analyses. This in vitro differentiation method for generating human trophoblasts starting from hPSCs overcomes the ethical and legal restrictions of working with early human embryos, and this system can be used for a variety of applications, including drug discovery and stem cell research.

In Vitro Differentiation of Human Pluripotent Stem Cells into Trophoblastic Cells

Here, we present a protocol to efficiently generate human trophoblastic cells from human pluripotent stem cells using bone morphogenic protein 4 and inhibitors of the Activin/Nodal pathways. This method is suitable for the efficient differentiation of human pluripotent stem cells and can generate large quantities of cells for genetic manipulation.

The placenta is the first organ to develop during embryogenesis and is required for the survival of the developing embryo. The placenta is comprised of ...

In Vitro Differentiation of Human Mesenchymal Stem Cells into Functional Cardiomyocyte-like Cells

Myocardial infarction and the subsequent ischemic cascade result in the extensive loss of cardiomyocytes, leading to congestive heart failure, the leading cause of mortality worldwide. Mesenchymal stem cells (MSCs) are a promising option for cell-based therapies to replace current, invasive techniques. MSCs can differentiate into mesenchymal lineages, including cardiac cell types, but complete differentiation into functional cells has not yet been achieved. Previous methods of differentiation were based on pharmacological agents or growth factors. However, more physiologically relevant strategies can also enable MSCs to undergo cardiomyogenic transformation. Here, we present a differentiation method using MSC aggregates on cardiomyocyte feeder layers to produce cardiomyocyte-like contracting cells.
Human umbilical cord perivascular cells (HUCPVCs) have been shown to have a greater differentiation potential than commonly investigated MSC types, such as bone marrow MSCs (BMSCs). As an ontogenetically younger source, we investigated the cardiomyogenic potential of first-trimester (FTM) HUCPVCs compared to older sources. FTM HUCPVCs are a novel, rich source of MSCs that retain their in utero immunoprivileged properties when cultured in vitro. Using this differentiation protocol, FTM and term HUCPVCs achieved significantly increased cardiomyogenic differentiation compared to BMSCs, as indicated by the increased expression of cardiomyocyte markers (i.e., myocyte enhancer factor 2C, cardiac troponin T, heavy chain cardiac myosin, signal regulatory protein &#945;, and connexin 43). They also maintained significantly lower immunogenicity, as demonstrated by their lower HLA-A expression and higher HLA-G expression. Applying aggregate-based differentiation, FTM HUCPVCs showed increased aggregate formation potential and generated contracting cells clusters within 1 week of co-culture on cardiac feeder layers, becoming the first MSC type to do so. 
Our results demonstrate that this differentiation strategy can effectively harness the cardiomyogenic potential of young MSCs, such as FTM HUCPVCs, and suggests that in vitro pre-differentiation could be a potential strategy to increase their regenerative efficacy in vivo.

In Vitro Differentiation of Human Mesenchymal Stem Cells into Functional Cardiomyocyte-like Cells

Here, we present a method to efficiently harness the cardiac differentiation potential of young sources of human mesenchymal stem cells in order to generate functional, contracting, cardiomyocyte-like cells in vitro.

Myocardial infarction and the subsequent ischemic cascade result in the extensive loss of cardiomyocytes, leading to congestive heart failure, the leading ...

Single-cell Photoconversion in Living Intact Zebrafish

Animal and plant tissue is composed of distinct populations of cells. These cells interact over time to build and maintain the tissue and can cause disease when disrupted. Scientists have developed clever techniques to investigate characteristics and natural dynamics of these cells within intact tissue by expressing fluorescent proteins in subsets of cells. However, at times, experiments require more selected visualization of cells within the tissue, sometimes at the single-cell or population-of-cells manner. To achieve this and visualize single cells within a population of cells, scientists have utilized single-cell photoconversion of fluorescent proteins. To demonstrate this technique, we show here how to direct UV light to an Eos-expressing cell of interest in an intact, living zebrafish. We then image those photoconverted Eos+ cells 24 h later to determine how they changed in the tissue. We describe two techniques: single cell photoconversion and photoconversions of populations of cell. These techniques can be used to visualize cell-cell interactions, cell-fate and differentiation, and cell migrations, making it a technique that is applicable in numerous biological questions.

Here, we present a protocol to show how cell photoconversion is achieved through UV exposure to specific areas expressing the fluorescent protein, Eos, in living animals.

Animal and plant tissue is composed of distinct populations of cells. These cells interact over time to build and maintain the tissue and can cause ...

Induction of Endothelial Differentiation in Cardiac Progenitor Cells Under Low Serum Conditions

Cardiac progenitor cells (CPCs) may have therapeutic potential for cardiac regeneration after injury. In the adult mammalian heart, intrinsic CPCs are extremely scarce, but expanded CPCs could be useful for cell therapy. A prerequisite for their use is their ability to differentiate in a controlled manner into the various cardiac lineages using defined and efficient protocols. In addition, upon in vitro expansion, CPCs isolated from patients or preclinical disease models may offer fruitful research tools for the investigation of disease mechanisms.
Current studies use different markers to identify CPCs. However, not all of them are expressed in humans, which limits the translational impact of some preclinical studies. Differentiation protocols that are applicable irrespective of the isolation technique and marker expression will allow for the standardized expansion and priming of CPCs for cell therapy purpose. Here we describe that the priming of CPCs under a low fetal bovine serum (FBS) concentration and low cell density conditions facilitates the endothelial differentiation of CPCs. Using two different subpopulations of mouse and rat CPCs, we show that laminin is a more suitable substrate than fibronectin for this purpose under the following protocol: after culturing for 2 - 3 days in medium including supplements that maintain multipotency and with 3.5% FBS, CPCs are seeded on laminin at &#60;60% confluence and cultured in supplement-free medium with low concentrations of FBS (0.1%) for 20 - 24 hours before differentiation in endothelial differentiation medium. Because CPCs are a heterogeneous population, serum concentrations and incubation times may need to be adjusted depending on the properties of the respective CPC subpopulation. Considering this, the technique can be applied to other types of CPCs as well and provides a useful method to investigate the potential and mechanisms of differentiation and how they are affected by disease when using CPCs isolated from respective disease models.

This protocol describes an endothelial differentiation technique for cardiac progenitor cells. It particularly focuses on how serum concentration and cell-seeding density affect the endothelial differentiation potential.

Cardiac progenitor cells (CPCs) may have therapeutic potential for cardiac regeneration after injury. In the adult mammalian heart, intrinsic CPCs are ...

In Vitro Generation of Somite Derivatives from Human Induced Pluripotent Stem Cells

In response to signals such as WNTs, bone morphogenetic proteins (BMPs), and sonic hedgehog (SHH) secreted from surrounding tissues, somites (SMs) give rise to multiple cell types, including the myotome (MYO), sclerotome (SCL), dermatome (D), and syndetome (SYN), which in turn develop into skeletal muscle, axial skeleton, dorsal dermis, and axial tendon/ligament, respectively. Therefore, the generation of SMs and their derivatives from human induced pluripotent stem cells (iPSCs) is critical to obtain pluripotent stem cells (PSCs) for application in regenerative medicine and for disease research in the field of orthopedic surgery. Although the induction protocols for MYO and SCL from PSCs have been previously reported by several researchers, no study has yet demonstrated the induction of SYN and D from iPSCs. Therefore, efficient induction of fully competent SMs remains a major challenge. Here, we recapitulate human SM patterning with human iPSCs in vitro by mimicking the signaling environment during chick/mouse SM development, and report on methods of systematic induction of SM derivatives (MYO, SCL, D, and SYN) from human iPSCs under chemically defined conditions through the presomitic mesoderm (PSM) and SM states. Knowledge regarding chick/mouse&#160;SM development was successfully applied to the induction of SMs with human iPSCs. This method could be a novel tool for studying human somitogenesis and patterning without the use of embryos and for cell-based therapy and disease modeling.

We present here a protocol for the differentiation of human induced pluripotent stem cells into each somite derivative (myotome, sclerotome, dermatome, and syndetome) in chemically defined conditions, which has applications in future disease modeling and cell-based therapies in orthopedic surgery.

In response to signals such as WNTs, bone morphogenetic proteins (BMPs), and sonic hedgehog (SHH) secreted from surrounding tissues, somites (SMs) give ...

Initiating Differentiation in Immortalized Multipotent Otic Progenitor Cells

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Derivation of Cardiac Progenitor Cells from Embryonic Stem Cells

Direct Induction of Human Neural Stem Cells from Peripheral Blood Hematopoietic Progenitor Cells

Analysis of Zebrafish Larvae Skeletal Muscle Integrity with Evans Blue Dye

Isolating Mesangiogenic Progenitor Cells (MPCs) from Human Bone Marrow

Directed Differentiation of Primitive and Definitive Hematopoietic Progenitors from Human Pluripotent Stem Cells

In Vitro Differentiation of Human Pluripotent Stem Cells into Trophoblastic Cells

In Vitro Differentiation of Human Mesenchymal Stem Cells into Functional Cardiomyocyte-like Cells

Single-cell Photoconversion in Living Intact Zebrafish

Induction of Endothelial Differentiation in Cardiac Progenitor Cells Under Low Serum Conditions

In Vitro Generation of Somite Derivatives from Human Induced Pluripotent Stem Cells