differentiating immortalized multipotent otic progenitors into spiral ganglion neurons and evaluating the differentiation

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

First, clean round glass coverslips by placing them in a sterile 100-millimeter plate, and submerging them in 70% ethanol. Gently agitate the coverslips to ensure they are covered and leave the dish at room temperature for 10 minutes. Next, rinse the coverslips three times in sterile 1x PBS and then once with sterile water.
Then, let the coverslips dry by exposing them to UV light in a tissue culture hood for 15 minutes. For immediate use, place one sterile coverslip into each well of a 24-well plate, and gently shake the plate to assure that all coverslips lie flat. Then, coat the coverslips by adding 0.25 milliliters of 1x PBS containing 10 micrograms per milliliter of poly-D-lysine to each well.
After the plate has incubated for one hour at 37 degrees Celsius, remove the poly-D-lysine solution and wash the wells three times with sterile 1x PBS. Once the liquid from the last wash has been aspirated, introduce 0.25 milliliters of 1x PBS containing 10 micrograms per milliliter laminin into each of the 24 wells. After incubating the plate overnight at 37 degrees Celsius, aspirate the laminin solution. Then, wash each well with 1 milliliter of 1x PBS three times, but leave the PBS from the last wash in the wells until the cells are ready to be plated.
To begin neuronal differentiation, count the cells from dissociated otospheres as previously described and then resuspend them in prewarmed neuronal differentiation medium. Proceed by seeding between 1 and 1.5 x 105 iMOP cells into each well of the plate containing the coated coverslips. Incubate the plate at 37 degrees Celsius with 5% carbon dioxide, replacing the neuronal differentiation medium every other day.
After seven days, aspirate the medium and fix the cells, which should be attached to the coverslips, by adding 4% formaldehyde into each well and leaving them for 15 minutes at room temperature. Then, remove the formaldehyde and wash the cells once in 1x PBS containing 0.1% Triton X-100. Next, remove the wash buffer and add blocking buffer consisting of 1x PBS containing 10% normal goat serum and 0.1% Triton X-100 to each well.
Leave the cells for one hour at room temperature and then remove the liquid and replace it with blocking buffer containing the appropriate dilution of Cdkn1b or Tubb3 antibody. Following an overnight incubation at 4 degrees Celsius, wash the cells once with 1x PBS containing 1% Triton X-100 and then proceed with immunostaining as previously described. After staining has been completed, perform a final wash by adding 1x PBS to the wells.
To prepare them for analysis, remove the coverslips to which stain cells are attached from the 24-well plate and place them onto mounting medium on a glass slide. After letting the mounting medium dry overnight at 4 degrees Celsius, acquire epifluorescence images as previously described.

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1. 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% ethanol (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 phosphate-buffered saline (PBS) to wash out the remaining ethanol. Rinse the coverslip once with sterile H2O to wash out the remaining 1X PBS.</li>
	<li>Aspirate the H2O using a 2 ml aspirating pipette and let the coverslips dry. Expose the coverslips to UV light in the tissue culture hood for 15 min. Store coverslips in a sterile environment if not immediately used.</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 room temperature (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. Plate 1 x 106 cells in a 6 cm dish at Day -3.</li>
	<li>On Day 0, harvest, dissociate and count cells. Seed 1 x 105 - 1.5 x 105 iMOP cells into 0.5 ml pre-warmed neuronal differentiation media per well in a 24 multi-well dish. Aspirate and add pre-warmed neuronal differentiation media to cultures every other day.</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 immunostaining. 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.</li>
	</ol>
	</li>
</ol>

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>1.5 Thickness Glass Coverslip (Round 12 mm)</td>
			<td>Electron Microscopy Sciences 7</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>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)</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>Prolong Gold Antifade Mountant</td>
			<td>Life Technologies</td>
			<td>47743-736</td>
			<td>Stored as 10 mg/ml 100 &#181;l aliquots</td>
		</tr>
		<tr>
			<td>Moxi Z Mini Automated Cell Counter</td>
			<td>Orflo</td>
			<td>MXZ001</td>
			<td></td>
		</tr>
		<tr>
			<td>Moxi Z Cassette Type S</td>
			<td>Orflo</td>
			<td>MXC002</td>
			<td></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 &#181;l 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</td>
			<td></td>
		</tr>
		<tr>
			<td>Tissue Culture Treated 6 cm Dish</td>
			<td>Thermo Fisher Scientific</td>
			<td>130181</td>
			<td></td>
		</tr>
		<tr>
			<td>EMD Millipore Millex Sterile Syringe PVDF Filter Pore size: 0.22 &#956;m</td>
			<td>Thermo Fisher Scientific</td>
			<td>SLGV033RS</td>
			<td></td>
		</tr>
		<tr>
			<td>TipOne filter pipet tips 0.1 - 10 &#956;l elongated filter tip</td>
			<td>USA Scientific</td>
			<td>1120-3810</td>
			<td></td>
		</tr>
		<tr>
			<td>TipOne filter pipet tips 1 - 20 &#956;l filter tip</td>
			<td>USA Scientific</td>
			<td>1120-1810</td>
			<td></td>
		</tr>
		<tr>
			<td>TipOne filter pipet tips 1 - 200 &#956;l filter tip</td>
			<td>USA Scientific</td>
			<td>1120-8810</td>
			<td></td>
		</tr>
		<tr>
			<td>TipOne filter pipet tips 101 - 1000 &#956;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>

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 ...

Take a multi-well plate containing sterile coverslips coated with a cell culture substrate.
Introduce immortalized multipotent otic progenitors, or iMOPs, in a neuronal differentiation medium.
iMOPs are precursors to various inner ear cells, including spiral ganglion neurons, or SGNs, that transmit auditory information to the brain.
Incubate, allowing the cells to adhere to the substrate. The medium induces differentiation of the iMOPs into SGNs.
Add a fixative to induce protein crosslinking, preserving cell morphology, and a detergent solution to permeabilize cellular membranes.
Introduce a blocking reagent to prevent non-specific labeling.
Add fluorophore-conjugated antibodies specific for neuronal differentiation markers and incubate, allowing the antibodies to label the SGNs.
Wash to remove unbound antibodies, and mount the coverslips with a mounting medium containing a fluorescent nuclear stain.
Using fluorescence microscopy, detect the antibody signals to confirm the differentiation of iMOPs into SGNs.

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비디오: 불멸화된 다능성 귀 전구세포를 나선형 신경절 뉴런으로 분화시키고 분화 평가 - 실험

Neuronal Differentiation from Mouse Embryonic Stem Cells In vitro

The neural differentiation of mouse embryonic stem cells (mESCs) is a potential tool for elucidating the key mechanisms involved in neurogenesis and potentially aid in regenerative medicine. Here, we established an efficient and low cost method for neuronal differentiation from mESCs in vitro, using the strategy of combinatorial screening. Under the conditions defined here, the 2-day embryoid body formation + 6-day retinoic acid induction protocol permits fast and efficient differentiation from mESCs into neural precursor cells (NPCs), as seen by the formation of well-stacked and neurite-like A2lox and 129 derivatives that are Nestin positive. The healthy state of embryoid bodies and the timepoint at which retinoic acid (RA) is applied, as well as the RA concentrations, are critical in the process. In the subsequent differentiation from NPCs into neurons, N2B27 medium II (supplemented by Neurobasal medium) could better support the long term maintenance and maturation of neuronal cells. The presented method is highly efficiency, low cost and easy to operate, and can be a powerful tool for neurobiology and developmental biology research.

Here, we established a low cost and easy to operate method that directs fast and efficient differentiation from embryonic stem cells into neurons. This method is suitable for popularization among laboratories and can be a useful tool for neurological research.

시험관 내 마우스 배아 줄기 세포로부터의 신경 분화

The neural differentiation of mouse embryonic stem cells (mESCs) is a potential tool for elucidating the key mechanisms involved in neurogenesis and ...

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JoVE Journal

신경과학

In vitro Modeling for Neurological Diseases using Direct Conversion from Fibroblasts to Neuronal Progenitor Cells and Differentiation into Astrocytes

Research on neurological disorders focuses primarily on the impact of neurons on disease mechanisms. Limited availability of animal models severely impacts the study of cell type specific contributions to disease. Moreover, animal models usually do not reflect variability in mutations and disease courses seen in human patients. Reprogramming methods for generation of induced pluripotent stem cells (iPSCs) have revolutionized patient specific research and created valuable tools for studying disease mechanisms. However, iPSC technology has disadvantages such as time, labor commitment, clonal selectivity and loss of epigenetic markers. Recent modifications of these methods allow more direct generation of cell lineages or specific cell types, bypassing clonal isolation or a pluripotent stem cell state. We have developed a rapid direct conversion method to generate induced Neuronal Progenitor Cells (iNPCs) from skin fibroblasts utilizing retroviral vectors in combination with neuralizing media. The iNPCs can be differentiated into neurons (iNs) oligodendrocytes (iOs) and astrocytes (iAs). iAs production facilitates rapid drug and disease mechanism testing as differentiation from iNPCs only takes 5 days. Moreover, iAs are easy to work with and are generated in pure populations at large numbers. We developed a highly reproducible co-culture assay using mouse GFP+ neurons and patient derived iAs to evaluate potential therapeutic strategies for numerous neurological and neurodegenerative disorders. Importantly, the iA assays are scalable to 384-well format facilitating the evaluation of multiple small molecules in one plate. This approach allows simultaneous therapeutic evaluation of multiple patient cell lines with diverse genetic background. Easy production and storage of iAs and capacity to screen multiple compounds in one assay renders this methodology adaptable for personalized medicine.

We describe a protocol to reprogram human skin-derived fibroblasts into induced Neuronal Progenitor Cells (iNPCs), and their subsequent differentiation into induced Astrocytes (iAs). This method leads to fast and reproducible generation of iNPCs and iAs in large quantities.

섬유아세포에서 신경 전구세포로의 직접 변환을 이용한 신경질환 시험관 모델링 및 성상세포로의 분화

Research on neurological disorders focuses primarily on the impact of neurons on disease mechanisms. Limited availability of animal models severely ...

Mouse Dorsal Root Ganglion Cryosectioning

A Procedure for Mouse Dorsal Root Ganglion Cryosectioning

High-quality mouse dorsal root ganglion (DRG) cryostat sections are crucial for proper immunochemistry staining and RNAscope studies in the research of inflammatory and neuropathic pain, itch, as well as other peripheral neurological conditions. However, it remains a challenge to consistently obtain high-quality, intact, and flat cryostat sections onto glass slides because of the tiny sample size of the DRG tissue. So far, there is no article describing an optimal protocol for DRG cryosectioning. This protocol presents a step-by-step method to resolve the frequently encountered difficulties associated with DRG cryosectioning. The presented article explains how to remove the surrounding liquid from the DRG tissue samples, place the DRG sections on the slide facing the same orientation, and flatten the sections on the glass slide without curving up. Although this protocol has been developed for cryosectioning the DRG samples, it can be applied for the cryosectioning of many other tissues with a small sample size.

저자 스포트라이트: A Procedure for Mouse Dorsal Root Ganglion Cryosectioning  

Presented here is the development for consistently acquiring high-quality dorsal root ganglion cryostat sections.

마우스 등쪽 뿌리 신경절 냉동 절제술을 위한 절차

High-quality mouse dorsal root ganglion (DRG) cryostat sections are crucial for proper immunochemistry staining and RNAscope studies in the research of ...

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

내레이션 대본

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