This work was financially supported by ANR (Agence nationale de la Recherche), AFM (Association Fran&ccedil;aise contre les Myopathies), FEDER in PACA and IRME (Institut de Recherche sur la Moelle &eacute;pini&egrave;re et l'Enc&eacute;phale). We greatfully thank Marie Pierre Blanchard (Jean Roche Institute) for her efficient help during time lapse recording.

1. Collection of Olfactory Mucosa in Rats
<ol>
 <li>Begin by preparing three 35 mm Petri dishes filled with DMEM/HAM F12 culture medium in a clean culture hood.</li>
 <li>Any method of euthanasia must be approved in advance by the institution's animal care and use committee and carried out by qualified personnel. After the rat has entered deep anesthesia with sodium pentobarbital or other injectable forms of anesthesia such as ketamine/xylazine, decapitate and remove the skin. Inhalant anesthetics should be avoided. Adequacy of anesthesia will be assessed by toe pinch prior to decapitation. Remove the lower jaw with scissors and with the help of a rongeur, eliminate the facial muscles on both sides.</li>
 <li>Starting from the back of the incisors, remove with a rongeur the bone covering the nasal cavity, one side at a time. The olfactory turbinates come into sight as orange/brown organs located in the back of the nose.</li>
 <li>Delicately discard the turbinates with a forceps. Using a 26 gauge needle, isolate the olfactory mucosa lying on the septum by cutting the tissue along three lines: the arc of the perpendicular plate, the cribriform plate and the ceiling of the nasal cavity.</li>
 <li>Collect biopsies on both sides and transfer them in a DMEM/HAM F12-filled Petri dish. This procedure should not take longer than 10 minutes from the onset euthanasia.</li>
 <li>Now, in order to remove the mucus, transfer the biopsies two times in medium-filled Petri dishes.</li>
</ol>
2. Collection of Olfactory Mucosa in Humans
<ol>
 <li>This procedure should be carried out by an Ear Nose and Throat (ENT) surgeon, in accordance with the relevant local ethical committee(s), and every outpatient should sign an informed consent form.</li>
 <li>Using a 0&deg; or 30&deg; rigid endoscope (4 mm diameter), inspect both nasal cavities and assess the putative presence of polyps or any inflammatory lesion. Choose the best nasal cavity, taking into account the deviation of the septum. </li>
 <li>Using a cotton applicator, apply a local anesthetic, such as lidocaine with epinephrine, for 10 minutes.</li>
 <li>With a throughcut ethmoid forceps, collect a two square millimeter biopsy either at the root of the medial aspect of the middle turbinate or on the septum in the dorsomedial area. </li>
 <li>The olfactory biopsy is then transferred, using a sterile needle, into a sterile 2 ml tube filled with 1 ml of DMEM/HAM F12. Tip the tube upside down to make sure that the biopsy is immersed in the culture medium.</li>
 <li>Insert the tube in a refrigerated container and transport it to the research laboratory. At this stage, the biopsy can be used per se for comparative molecular studies focused on specific brain diseases or processed for generating stem cells.</li>
</ol>
3. Isolation of Olfactory Stem Cells from Human and Rat Mucosa
<ol>
 <li>Wash the biopsies in DMEM/HAM F12. Incubate the biopsies in a Petri dish filled with 1 ml of dispase II solution (2.4 IU/ml), for 1 hour at 37&deg;C.</li>
 <li>Next, under a dissecting microscope with a diffracted inverted light, the olfactory epithelium is removed from the underlying lamina propria using a micro spatula. </li>
 <li>The olfactory epithelium is thinner and looks translucent over a black background compared to the lamina propria which is striped orange/brown. Over a white background, the epithelium looks gray and the lamina propria, brown.</li>
 <li>Once purified, transfer the lamina propria into a Petri dish filled with DMEM/HAM F12. </li>
 <li>If the tissue is from a rodent, then cut the lamina propria into small pieces with two 25 gauge needles. Then, transfer the pieces to a 15 ml tube filled with 1 ml of collagenase IA.</li>
 <li>In the tube, using a sterile plastic pipette, dissociate the tissue. Then, incubate the tube for 10 minutes at 37&deg;C.</li>
 <li>To terminate the dissociation, gently rock the tube and add 9 ml of Ca-free and Mg-free PBS and centrifuge at 200 g for 5 minutes.</li>
 <li>Resuspend the cell pellet in DMEM/HAM F12 culture medium supplemented with 10% fetal calf serum, antibiotics and plate on plastic culture dishes.</li>
 <li>Now, if the tissue is human, then slice the lamina propria into 3 to 4 pieces with a thickness ranging from 200 to 500 &mu;m. </li>
 <li>Insert each strip into its own 2 cm diameter culture dish and cover the tissue with sterile 1.3 cm diameter glass cover slips. </li>
 <li>Then, add 500 &mu;l of culture medium (DMEM/HAM F12 supplemented with 10% fetal calf serum and antibiotics) to each culture dish.</li>
 <li>For either tissue type, renew the culture medium every 2 to 3 days.</li>
 <li>Five to seven days after, stem cells will begin to invade the culture dish and after two weeks they should be confluent. When confluency is reached, passage and transfer the cells to culture flasks.</li>
</ol>
4. Sphere Formation and Neuronal Differentiation of Olfactory Stem Cells
<ol>
 <li>To generate stem cell spheres, incubate the flasks for two hours at 37&deg;C with poly-L-lysine. (TEXT: 5 g/cm2).</li>
 <li>Plate the cells at a density of 16,000 cells per square centimeter in the treated flasks.</li>
 <li>Every two days, feed the cells with 0.2ml per square centimeter of supplemented medium (DMEM/HAM F12 supplemented with insulin, transferrin, selenium (ITS-X, 1%), EGF (50 ng/ml) and FGF2 (50 ng/ml)).</li>
 <li>Two to five days later, collect floating cell spheres and either re-plate or dissociate them before grafting in animal models of cell therapy. </li>
 <li>To differentiate olfactory stem cells into neuron-like cells, cultivate them for 21 days in Neurobasal medium containing B-27, penicillin, streptomycin, glutamine and glutamate.</li>
 <li>Then make medium changes every 3 days. Neuron-like cells should appear after two to three weeks.</li>
</ol>
5. Representative Results: 
Nasal human explant-outgrowing stem cells (Figure 2A) are dividing rapidly and confluency can be reached within one to two weeks. One key feature of stemness, nestin expression, was evaluated (Figure 2B). When grown on poly-L-lysine with a serum-free culture medium supplemented with EGF (50 ng/ml) and FGF2 (50 ng/ml), olfactory stem cells give rise to spheres (figure 2C). When grown in serum-containing culture medium newly plated spheres give rise to GFAP-expressing cells (&tilde;50%), tubulin-expressing cells (&tilde;10-15%) and O4-expressing cells (&tilde;2-5%)9 (Figures 2D-F). However, the fate of the sphere-derived cells can be modified. For example, when grown in a Neurobasal culture medium supplemented with B27 and glutamate, most of the nasal olfactory stem cells into neuron-like cells expressing &beta;-III tubulin (Figure 2G) and MAP2 (Figure 2H). 
<img src="/files/ftp_upload/2762/2762fig1.jpg" alt="Figure 1"/> 
Figure 1. Overall scheme of the experiment. Olfactory mucosa biopsies are excised from rat or human nasal cavity. Explants can be used per se for comparative molecular studies aiming to identify biomarkers in brain diseases. For isolating olfactory stem cells, the interactions between the lamina propria and the neuro-epithelium are disrupted with the dispase II enzyme and, after 45 minutes, the epithelium is removed with a micro-spatula. Rodent olfactory stem cells are further selected by dissociating the lamina propria with collagenase IA. For human tissue, pieces of olfactory lamina propria are cultured under glass coverslip until outgrowing stem cells invade the whole well. After proliferation, using an appropriate culture medium, olfactory stem cells can generate spheres or differentiate into neuron-like cells. Olfactory stem cells can be used to i) repair brain diseases or trauma or ii) identify molecular markers of central nervous system diseases. Illustrations are made with the help of Servier Medical Art.
<img src="/files/ftp_upload/2762/2762fig2.jpg" alt="Figure 2"/> 
Figure 2. Culture and differentiation of nasal human olfactory stem cells. Human stem cells growing out of the lamina propria explant (A) are dividing rapidly, when cultivated in a serum-containing medium. Stem cells express the stemness marker nestin (B). When plated on poly-L-lysine-coated plastic and cultivated in a serum-free culture medium supplemented with EGF and FGF2, olfactory stem cells generate spheres (C). Sphere-derived cells, when plated in serum-containing culture medium, give rise to GFAP-expressing cells (&tilde;50%), tubulin-expressing cells (&tilde;10-15%) and O4-expressing cells (&tilde;2-5%)9 (D-F). When grown in a Neurobasal culture medium supplemented with B27 and glutamate, they differentiate into neuron-like cells expressing &beta;-III tubulin (G) and MAP2 (H).

<ol>
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H., McGrath, J., Mackay-Sim, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Altered+adhesion,+proliferation+and+death+in+neural+cultures+from+adults+with+schizophrenia.">Altered adhesion, proliferation and death in neural cultures from adults with schizophrenia.</a> Schizophr Res. 40, 211-218 (1999).</li><li>Matigian, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Disease-specific,+neurosphere-derived+cells+as+models+for+brain+disorders.">Disease-specific, neurosphere-derived cells as models for brain disorders.</a> Dis Model Mech. 3, 11-12 (2010).</li><li>Arnold, S. 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D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell+cycle+alterations+in+biopsied+olfactory+neuroepithelium+in+schizophrenia+and+bipolar+I+disorder+using+cell+culture+and+gene+expression+analyses.">Cell cycle alterations in biopsied olfactory neuroepithelium in schizophrenia and bipolar I disorder using cell culture and gene expression analyses.</a> Schizophr Res. 82, 163-173 (2006).</li><li>Roisen, F. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Adult+human+olfactory+stem+cells.">Adult human olfactory stem cells.</a> Brain Res. 890, 11-22 (2001).</li><li>Murrell, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multipotent+stem+cells+from+adult+olfactory+mucosa.">Multipotent stem cells from adult olfactory mucosa.</a> Dev Dyn. 233, 496-515 (2005).</li><li>Tome, M., Lindsay, S. L., Riddell, J. S., Barnett, S. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+of+nonepithelial+multipotent+cells+in+the+embryonic+olfactory+mucosa.">Identification of nonepithelial multipotent cells in the embryonic olfactory mucosa.</a> Stem Cells. 27, 2196-2208 (2009).</li><li>Delorme, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+human+nose+harbors+a+niche+of+olfactory+ectomesenchymal+stem+cells+displaying+neurogenic+and+osteogenic+properties.">The human nose harbors a niche of olfactory ectomesenchymal stem cells displaying neurogenic and osteogenic properties.</a> Stem Cells Dev. 19, 853-866 (2010).</li><li>Xiao, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+adult+olfactory+neural+progenitors+rescue+axotomized+rodent+rubrospinal+neurons+and+promote+functional+recovery.">Human adult olfactory neural progenitors rescue axotomized rodent rubrospinal neurons and promote functional recovery.</a> Exp Neurol. 194, 12-30 (2005).</li><li>Xiao, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+adult+olfactory+neural+progenitors+promote+axotomized+rubrospinal+tract+axonal+reinnervation+and+locomotor+recovery.">Human adult olfactory neural progenitors promote axotomized rubrospinal tract axonal reinnervation and locomotor recovery.</a> Neurobiol Dis. 26, 363-374 (2007).</li><li>Pandit, S. R., Sullivan, J. M., Egger, V., Borecki, A. 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No conflicts of interest declared.

The techniques presented here make the rodent and human olfactory mucosa a useful model for clinical research into the causes of neurodevelopmental and neurodegenerative diseases as well as a tool for repairing the pathological or traumatized brain. The protocol is relatively straightforward and can be easily carried out by an experienced cell biologist. The success rate for the biopsy and culture techniques is high.
Critical steps 
<ul>
	<li>For the collection of rodent olfactory mucosa, it is recommended not to exceed a time limit of 10 minutes between euthanasia and the final excision of the olfactory tissue. </li>
	<li>The dissociation of the rodent olfactory lamina propria is usually achieved after 10 min. incubation in collagenase IA. If not, we recommend to collect the day after the supernatant in which undissociated bits of lamina propria float, centrifuge it at 200 g and mechanically dissociate the floating lamina using a Pasteur pipette before replating the cells in a new well. The first well containing the already attached cells is filled with fresh culture medium.</li>
	<li>For the human lamina propria, which is more compact than the rodent tissue, we do not recommend an enzymatic dissociation. The tissue is sliced and each explant is inserted between the bottom of the plastic dish and a glass coverslip. Successful cultures include explants whose thickness range from 200 to 500 &mu;m.</li>
</ul>
Possible modifications
<ul>
	<li>The current protocol can be slightly modified in order to generate olfactory neurons in vitro. For that purpose, the neuro-epithelium is not removed and the whole olfactory mucosa is sliced with a McIlwain chopper (200 &mu;m thickness). Each explant is plated in a dish, partially dried for one hour and then rehydrated with FCS-containing culture medium. During the first days post plating, epithelial and mesenchymal cells grow out of the explant. Then, neuron progenitors will migrate on the top of this cell layer and differentiate into neurons. </li>
	<li>Purification of olfactory stem cells can be achieved using flow cytometry. Specific surface markers can be retrieved from the list published in the characterization paper11.</li>
	<li>For transplantation experiments, it is possible to use a strain of rats whose olfactory stem cells are GFP-positive. This rat strain (Sprague Dawley, eGFP driven by the PGK promoter) is available at ITERT (Nantes, France). For inserting the GFP gene in human olfactory stem cells, we use a method based on lentivirus infection.</li>
	<li>This paper is focused on olfactory ecto-mesenchymal stem cells. However, another cell type of interest, the olfactory ensheathing cells, can be purified from the same tissue. We described a method for their collection and purification1,17 and we used human nasal ensheathing cells for a phase I/IIa clinical trial in paraplegic patients18.</li>
	<li>As a member of the mesenchymal stem cell superfamily, OE-MSCs are able, under appropriate culture conditions, to differentiate into adipocytes, osteocytes and myocytes11.</li>
</ul>
Future applications
<ul>
	<li>We have already used human olfactory biopsies to study cellular and molecular anomalies in patients with autism, bipolar disorder, familial dysautonomia, Parkinson's disease, Alzheimer's disease and schizophrenia2-7. Theoretically, all brain diseases can be studied using nasal biopsies or peripheral olfactory stem cells.</li>
	<li>Rodent and human nasal olfactory stem cells have already been grafted in animal models of amnesia, Parkinson's disease, cochlear damage and spinal cord trauma12-16. It is conceivable to transplant these cells into animal models of Alzheimer's disease, brain ischemia, Multiple sclerosis.</li>
</ul>

<table border="1">
	<tbody>
		<tr>
			<td>Name of the reagent</td>
			<td>Company</td>
			<td>Catalogue number</td>
		</tr>
		<tr>
			<td colspan="3">Collection of olfactory mucosa in rats</td>
		</tr>
		<tr>
			<td>DMEM/HAM F12</td>
			<td>Invitrogen</td>
			<td>31331-028</td>
		</tr>
		<tr>
			<td>Sodium Pentobarbital</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Rongeur</td>
			<td>FST</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>26 gauge needle</td>
			<td>Terumo</td>
			<td>NN-2613R</td>
		</tr>
		<tr>
			<td>Forceps</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td colspan="3">Collection of olfactory mucosa in humans</td>
		</tr>
		<tr>
			<td>Rigid endoscope</td>
			<td>Karl Storz or Richard Wolf Medical</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Lidocaine</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Epinephrine</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Throughcut ethmoid forceps</td>
			<td>Karl Storz or Richard Wolf Medical</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td colspan="3">Isolation of olfactory stem cells</td>
		</tr>
		<tr>
			<td>Dispase II</td>
			<td>Roche</td>
			<td>10 295 825 001</td>
		</tr>
		<tr>
			<td>Dissecting microscope</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Micro spatula</td>
			<td>FST</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Collagenase IA</td>
			<td>Sigma-Aldrich</td>
			<td>C9891</td>
		</tr>
		<tr>
			<td>Ca-free/Mg-free PBS</td>
			<td>Invitrogen</td>
			<td>14190-250</td>
		</tr>
		<tr>
			<td>Fetal calf serum</td>
			<td>Invitrogen</td>
			<td>10270098</td>
		</tr>
		<tr>
			<td>Glass coverslip</td>
			<td>Knittel Glaser</td>
			<td>001/35</td>
		</tr>
		<tr>
			<td colspan="3">Sphere formation and neuronal differentiation</td>
		</tr>
		<tr>
			<td>Poly-L-lysine</td>
			<td>Sigma-Aldrich</td>
			<td>P-1274</td>
		</tr>
		<tr>
			<td>Insulin transferrin selenium (ITS)</td>
			<td>Invitrogen</td>
			<td>51500056</td>
		</tr>
		<tr>
			<td>EGF</td>
			<td>R&amp;D Systems</td>
			<td>236-EG</td>
		</tr>
		<tr>
			<td>FGF2</td>
			<td>R&amp;D Systems</td>
			<td>233-FB</td>
		</tr>
		<tr>
			<td>Neurobasal medium</td>
			<td>Invitrogen</td>
			<td>21103-049</td>
		</tr>
		<tr>
			<td>B-27 Serum-Free Supplement</td>
			<td>Invitrogen</td>
			<td>17504-044</td>
		</tr>
		<tr>
			<td>Penicillin/streptomycin</td>
			<td>Invitrogen</td>
			<td>15140122</td>
		</tr>
		<tr>
			<td>Glutamine </td>
			<td>Invitrogen</td>
			<td>25030024</td>
		</tr>
		<tr>
			<td>Glutamate</td>
			<td>Sigma-Aldrich</td>
			<td>&nbsp;</td>
		</tr>
	</tbody>
</table>

isolating nasal olfactory stem cells from rodents or humans

The olfactory mucosa, located in the nasal cavity, is in charge of detecting odours. It is also the only nervous tissue that is exposed to the external environment and easily accessible in every living individual. As a result, this tissue is unique for anyone aiming to identify molecular anomalies in the pathological brain or isolate adult stem cells for cell therapy. 
Molecular abnormalities in brain diseases are often studied using nervous tissue samples collected post-mortem. However, this material has numerous limitations. In contrast, the olfactory mucosa is readily accessible and can be biopsied safely without any loss of sense of smell1. Accordingly, the olfactory mucosa provides an "open window" in the adult human through which one can study developmental (e.g. autism, schizophrenia)2-4 or neurodegenerative (e.g. Parkinson, Alzheimer) diseases4,5. Olfactory mucosa can be used for either comparative molecular studies4,6 or in vitro experiments on neurogenesis3,7.
The olfactory epithelium is also a nervous tissue that produces new neurons every day to replace those that are damaged by pollution, bacterial of viral infections. This permanent neurogenesis is sustained by progenitors but also stem cells residing within both compartments of the mucosa, namely the neuroepithelium and the underlying lamina propria8-10. We recently developed a method to purify the adult stem cells located in the lamina propria and, after having demonstrated that they are closely related to bone marrow mesenchymal stem cells (BM-MSC), we named them olfactory ecto-mesenchymal stem cells (OE-MSC)11.
Interestingly, when compared to BM-MSCs, OE-MSCs display a high proliferation rate, an elevated clonogenicity and an inclination to differentiate into neural cells. We took advantage of these characteristics to perform studies dedicated to unveil new candidate genes in schizophrenia and Parkinson's disease4. We and others have also shown that OE-MSCs are promising candidates for cell therapy, after a spinal cord trauma12,13, a cochlear damage14 or in an animal models of Parkinson's disease15 or amnesia16.
In this study, we present methods to biopsy olfactory mucosa in rats and humans. After collection, the lamina propria is enzymatically separated from the epithelium and stem cells are purified using an enzymatic or a non-enzymatic method. Purified olfactory stem cells can then be either grown in large numbers and banked in liquid nitrogen or induced to form spheres or differentiated into neural cells. These stem cells can also be used for comparative omics (genomic, transcriptomic, epigenomic, proteomic) studies.

Watch this Scientific Journal Video about Isolating Nasal Olfactory Stem Cells from Rodents or Humans at JoVE.com

Isolating Nasal Olfactory Stem Cells from Rodents or Humans

We describe here a method for biopsying olfactory mucosa from rat and human nasal cavities. These biopsies can be used for either identifying molecular anomalies in brain diseases or isolating multipotent adult stem cells that can be utilized for cell transplantation in animal models of brain trauma/disease.

The olfactory mucosa, located in the nasal cavity, is in charge of detecting odours. It is also the only nervous tissue that is exposed to the external ...

isolating-nasal-olfactory-stem-cells-from-rodents-or-humans

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JoVE Journal

Neuroscience

30.6K vistas.  Aix Marseille University. Se describe aquí un método para la biopsia de mucosa olfatoria de ratas y humanos cavidades nasales. Estas biopsias se pueden utilizar tanto para la identificación de anomalías moleculares en las enfermedades cerebrales o el aislamiento de células madre adultas multipotentes que pueden ser utilizados para el trasplante de células en modelos animales de trauma cerebral / enfermedad

Video: Isolating Nasal Olfactory Stem Cells from Rodents or Humans

Electrophysiological Measurements from a Moth Olfactory System

Insect olfactory systems provide unique opportunities for recording odorant-induced responses in the forms of electroantennograms (EAG) and single sensillum recordings (SSR), which are summed responses from all odorant receptor neurons (ORNs) located on the antenna and from those housed in individual sensilla, respectively. These approaches have been exploited for getting a better understanding of insect chemical communication. The identified stimuli can then be used as either attractants or repellents in management strategies for insect pests.

Insect olfactory systems provide unique opportunities for recording odorant-induced responses in the forms of electroantennograms (EAG) and single sensillum recordings (SSR), which are summed responses from all odorant receptor neurons (ORNs) located on the antenna and from those housed in individual sensilla, respectively.

Insect olfactory systems provide unique opportunities for recording odorant-induced responses in the forms of electroantennograms (EAG) and single ...

Isolating LacZ-expressing Cells from Mouse Inner Ear Tissues using Flow Cytometry

Isolation of specific cell types allows one to analyze rare cell populations such as stem/progenitor cells. Such an approach to studying inner ear tissues presents a unique challenge because of the paucity of cells of interest and few transgenic reporter mouse models. Here, we describe a protocol using fluorescence-conjugated probes to selectively label LacZ-positive cells from the neonatal cochleae.
The most common underlying pathology of sensorineural hearing loss is the irreversible damage and loss of cochlear sensory hair cells, which are required to transduce sound waves to neural impulses. Recent evidence suggests that the murine auditory and vestibular organs harbor stem/progenitor cells that may have regenerative potential1,2. These findings warrant further investigation, including identifying specific cell types with stem/progenitor cell characteristics. The Wnt signaling pathway has been demonstrated to play a critical role in maintaining stem/progenitor cell populations in several organ systems3-7. We have recently identified Wnt-responsive Axin2-expressing cells in the neonatal cochlea, but their function is largely unknown8.
To better understand the behavior of these Wnt-responsive cells in vitro, we have developed a method of isolating Axin2-expressing cells from cochleae of Axin2-LacZ reporter mice9. Using flow cytometry to isolate Axin2-LacZ positive cells from the neonatal cochleae, we could in turn execute a variety of experiments on live cells to interrogate their behavior as stem/progenitor cells. Here, we describe in detail the steps for the microdissection of neonatal cochlea, dissociation of these tissues, labeling of the LacZ-positive cells using a fluorogenic substrate, and cell sorting. Techniques for dissociating cochleae into single cells and isolating cochlear cells via flow cytometry have been described2,10-12. We have made modifications to these techniques to establish a novel protocol to isolate LacZ-expressing cells from the neonatal cochlea.

Flow cytometry is a powerful tool allowing for the isolation and study of specific cell populations. This protocol describes steps for isolating LacZ-expressing cells from cochlear tissues from neonatal transgenic mice. Dissociated cochlear cells were labeled using fluorescent-conjugated substrates of &#946;-galactosidase prior to separation via flow cytometry.

Isolation of specific cell types allows one to analyze rare cell populations such as stem/progenitor cells. Such an approach to studying inner ear tissues ...

Expansion of Embryonic and Adult Neural Stem Cells by In Utero Electroporation or Viral Stereotaxic Injection

Somatic stem cells can divide to generate additional stem cells (expansion) or more differentiated cell types (differentiation), which is fundamental for tissue formation during embryonic development and tissue homeostasis during adulthood 1. Currently, great efforts are invested towards controlling the switch of somatic stem cells from expansion to differentiation because this is thought to be fundamental for developing novel strategies for regenerative medicine 1,2. However, a major challenge in the study and use of somatic stem cell is that their expansion has been proven very difficult to control.
Here we describe a system that allows the control of neural stem/progenitor cell (altogether referred to as NSC) expansion in the mouse embryonic cortex or the adult hippocampus by manipulating the expression of the cdk4/cyclinD1 complex, a major regulator of the G1 phase of the cell cycle and somatic stem cell differentiation 3,4. Specifically, two different approaches are described by which the cdk4/cyclinD1 complex is overexpressed in NSC in vivo. By the first approach, overexpression of the cell cycle regulators is obtained by injecting plasmids encoding for cdk4/cyclinD1 in the lumen of the mouse telencephalon followed by in utero electroporation to deliver them to NSC of the lateral cortex, thus, triggering episomal expression of the transgenes 5-8. By the second approach, highly concentrated HIV-derived viruses are stereotaxically injected in the dentate gyrus of the adult mouse hippocampus, thus, triggering constitutive expression of the cell cycle regulators after integration of the viral construct in the genome of infected cells 9. Both approaches, whose basic principles were already described by other video protocols 10-14, were here optimized to i) reduce tissue damage, ii) target wide portions of very specific brain regions, iii) obtain high numbers of manipulated cells within each region, and iv) trigger high expression levels of the transgenes within each cell. Transient overexpression of the transgenes using the two approaches is obtained by different means i.e. by natural dilution of the electroporated plasmids due to cell division or tamoxifen administration in Cre-expressing NSC infected with viruses carrying cdk4/cyclinD1 flanked by loxP sites, respectively 9,15.
These methods provide a very powerful platform to acutely and tissue-specifically manipulate the expression of any gene in the mouse brain. In particular, by manipulating the expression of the cdk4/cyclinD1 complex, our system allows the temporal control of NSC expansion and their switch to differentiation, thus, ultimately increasing the number of neurons generated in the mammalian brain. Our approach may be critically important for basic research and using somatic stem cells for therapy of the mammalian central nervous system while providing a better understanding of i) stem cell contribution to tissue formation during development, ii) tissue homeostasis during adulthood, iii) the role of adult neurogenesis in cognitive functions, and perhaps, iv) better using somatic stem cells in models of neurodegenerative diseases.

Expansion of Embryonic and Adult Neural Stem Cells by In Utero Electroporation or Viral Stereotaxic Injection

Controlling the expansion of somatic stem cells is a major factor hampering their study and use in therapy. Here we describe a system to temporally control neural stem cells expansion during development and adulthood, which can be used to increase the number of neurons generated in the mouse brain.

Somatic stem cells can divide to generate additional stem cells (expansion) or more differentiated cell types (differentiation), which is fundamental for ...

Isolation and Culture of Neural Crest Cells from Embryonic Murine Neural Tube

The embryonic neural crest (NC) is a multipotent progenitor population that originates at the dorsal aspect of the neural tube, undergoes an epithelial to mesenchymal transition (EMT) and migrates throughout the embryo, giving rise to diverse cell types 1-3. NC also has the unique ability to influence the differentiation and maturation of target organs4-6. When explanted in vitro, NC progenitors undergo self-renewal, migrate and differentiate into a variety of tissue types including neurons, glia, smooth muscle cells, cartilage and bone. 
NC multipotency was first described from explants of the avian neural tube7-9. In vitro isolation of NC cells facilitates the study of NC dynamics including proliferation, migration, and multipotency. Further work in the avian and rat systems demonstrated that explanted NC cells retain their NC potential when transplanted back into the embryo10-13. Because these inherent cellular properties are preserved in explanted NC progenitors, the neural tube explant assay provides an attractive option for studying the NC in vitro. 
To attain a better understanding of the mammalian NC, many methods have been employed to isolate NC populations. NC-derived progenitors can be cultured from post-migratory locations in both the embryo and adult to study the dynamics of post-migratory NC progenitors11,14-20, however isolation of NC progenitors as they emigrate from the neural tube provides optimal preservation of NC cell potential and migratory properties13,21,22. Some protocols employ fluorescence activated cell sorting (FACS) to isolate a NC population enriched for particular progenitors11,13,14,17. However, when starting with early stage embryos, cell numbers adequate for analyses are difficult to obtain with FACS, complicating the isolation of early NC populations from individual embryos. Here, we describe an approach that does not rely on FACS and results in an approximately 96% pure NC population based on a Wnt1-Cre activated lineage reporter23. 
The method presented here is adapted from protocols optimized for the culture of rat NC11,13. The advantages of this protocol compared to previous methods are that 1) the cells are not grown on a feeder layer, 2) FACS is not required to obtain a relatively pure NC population, 3) premigratory NC cells are isolated and 4) results are easily quantified. Furthermore, this protocol can be used for isolation of NC from any mutant mouse model, facilitating the study of NC characteristics with different genetic manipulations. The limitation of this approach is that the NC is removed from the context of the embryo, which is known to influence the survival, migration and differentiation of the NC2,24-28.

Isolation of embryonic neural crest from the neural tube facilitates the use of in vitro methods for studying migration, self-renewal, and multipotency of neural crest.

The embryonic neural crest (NC) is a multipotent progenitor population that originates at the dorsal aspect of the neural tube, undergoes an epithelial to ...

A cGMP-applicable Expansion Method for Aggregates of Human Neural Stem and Progenitor Cells Derived From Pluripotent Stem Cells or Fetal Brain Tissue

A cell expansion technique to amass large numbers of cells from a single specimen for research experiments and clinical trials would greatly benefit the stem cell community. Many current expansion methods are laborious and costly, and those involving complete dissociation may cause several stem and progenitor cell types to undergo differentiation or early senescence. To overcome these problems, we have developed an automated mechanical passaging method referred to as &#8220;chopping&#8221; that is simple and inexpensive. This technique avoids chemical or enzymatic dissociation into single cells and instead allows for the large-scale expansion of suspended, spheroid cultures that maintain constant cell/cell contact. The chopping method has primarily been used for fetal brain-derived neural progenitor cells or neurospheres, and has recently been published for use with neural stem cells derived from embryonic and induced pluripotent stem cells. The procedure involves seeding neurospheres onto a tissue culture Petri dish and subsequently passing a sharp, sterile blade through the cells effectively automating the tedious process of manually mechanically dissociating each sphere. Suspending cells in culture provides a favorable surface area-to-volume ratio; as over 500,000 cells can be grown within a single neurosphere of less than 0.5 mm in diameter. In one T175 flask, over 50 million cells can grow in suspension cultures compared to only 15 million in adherent cultures. Importantly, the chopping procedure has been used under current good manufacturing practice (cGMP), permitting mass quantity production of clinical-grade cell products.

This protocol describes a novel mechanical chopping method that allows the expansion of spherical neural stem and progenitor cell aggregates without dissociation to a single cell suspension.&#160; Maintaining cell/cell contact allows rapid and stable growth for over 40 passages.

A cell expansion technique to amass large numbers of cells from a single specimen for research experiments and clinical trials would greatly benefit the ...

Feeder-free Derivation of Neural Crest Progenitor Cells from Human Pluripotent Stem Cells

Human pluripotent stem cells (hPSCs) have great potential for studying human embryonic development, for modeling human diseases in the dish and as a source of transplantable cells for regenerative applications after disease or accidents. Neural crest (NC) cells are the precursors for a large variety of adult somatic cells, such as cells from the peripheral nervous system and glia, melanocytes and mesenchymal cells. They are a valuable source of cells to study aspects of human embryonic development, including cell fate specification and migration. Further differentiation of NC progenitor cells into terminally differentiated cell types offers the possibility to model human diseases in vitro, investigate disease mechanisms and generate cells for regenerative medicine. This article presents the adaptation of a currently available in vitro differentiation protocol for the derivation of NC cells from hPSCs. This new protocol requires 18 days of differentiation, is feeder-free, easily scalable and highly reproducible among human embryonic stem cell (hESC) lines as well as human induced pluripotent stem cell (hiPSC) lines. Both old and new protocols yield NC cells of equal identity.

Neural crest (NC) cells derived from human pluripotent stem cells (hPSC) have great potential for modeling human development and disease and for cell replacement therapies. Here, a feeder-free adaptation of the currently widely used in vitro differentiation protocol for the derivation of NC cells from hPSCs is presented.

Human pluripotent stem cells (hPSCs) have great potential for studying human embryonic development, for modeling human diseases in the dish and as a ...

Directed Dopaminergic Neuron Differentiation from Human Pluripotent Stem Cells

Dopaminergic (DA) neurons in the substantia nigra pars compacta (also known as A9 DA neurons) are the specific cell type that is lost in Parkinson&#8217;s disease (PD). There is great interest in deriving A9 DA neurons from human pluripotent stem cells (hPSCs) for regenerative cell replacement therapy for PD. During neural development, A9 DA neurons originate from the floor plate (FP) precursors located at the ventral midline of the central nervous system. Here, we optimized the culture conditions for the stepwise differentiation of hPSCs to A9 DA neurons, which mimics embryonic DA neuron development. In our protocol, we first describe the efficient generation of FP precursor cells from hPSCs using a small molecule method, and then convert the FP cells to A9 DA neurons, which could be maintained in vitro for several months. This efficient, repeatable and controllable protocol works well in human embryonic stem cells (hESCs) and human induced pluripotent stem cells (hiPSCs) from normal persons and PD patients, in which one could derive A9 DA neurons to perform in vitro disease modeling and drug screening and in vivo cell transplantation therapy for PD.

We, based on knowledge from developmental biology and published research, developed an optimized protocol to efficiently generate A9 midbrain dopaminergic neurons from both human embryonic stem cells and human induced pluripotent stem cells, which would be useful for disease modeling and cell replacement therapy for Parkinson&#8217;s disease.

Dopaminergic (DA) neurons in the substantia nigra pars compacta  (also known as A9 DA neurons) are the specific cell type that is lost in ...

Modeling Astrocytoma Pathogenesis In Vitro and In Vivo Using Cortical Astrocytes or Neural Stem Cells from Conditional, Genetically Engineered Mice

Current astrocytoma models are limited in their ability to define the roles of oncogenic mutations in specific brain cell types during disease pathogenesis and their utility for preclinical drug development. In order to design a better model system for these applications, phenotypically wild-type cortical astrocytes and neural stem cells (NSC) from conditional, genetically engineered mice (GEM) that harbor various combinations of floxed oncogenic alleles were harvested and grown in culture. Genetic recombination was induced in vitro using adenoviral Cre-mediated recombination, resulting in expression of mutated oncogenes and deletion of tumor suppressor genes. The phenotypic consequences of these mutations were defined by measuring proliferation, transformation, and drug response in vitro. Orthotopic allograft models, whereby transformed cells are stereotactically injected into the brains of immune-competent, syngeneic littermates, were developed to define the role of oncogenic mutations and cell type on tumorigenesis in vivo. Unlike most established human glioblastoma cell line xenografts, injection of transformed GEM-derived cortical astrocytes into the brains of immune-competent littermates produced astrocytomas, including the most aggressive subtype, glioblastoma, that recapitulated the histopathological hallmarks of human astrocytomas, including diffuse invasion of normal brain parenchyma. Bioluminescence imaging of orthotopic allografts from transformed astrocytes engineered to express luciferase was utilized to monitor in vivo tumor growth over time. Thus, astrocytoma models using astrocytes and NSC harvested from GEM with conditional oncogenic alleles provide an integrated system to study the genetics and cell biology of astrocytoma pathogenesis in vitro and in vivo and may be useful in preclinical drug development for these devastating diseases.

Modeling Astrocytoma Pathogenesis In Vitro and In Vivo Using Cortical Astrocytes or Neural Stem Cells from Conditional, Genetically Engineered Mice

Phenotypically wild-type astrocytes and neural stem cells harvested from mice engineered with floxed, conditional oncogenic alleles and transformed via viral Cre-mediated recombination can be used to model astrocytoma pathogenesis in vitro and in vivo by orthotopic injection of transformed cells into brains of syngeneic, immune-competent littermates.

Current astrocytoma models are limited in their ability to define the roles of oncogenic mutations in specific brain cell types during disease ...

Olfactory Neurons Obtained through Nasal Biopsy Combined with Laser-Capture Microdissection: A Potential Approach to Study Treatment Response in Mental Disorders

Bipolar disorder (BD) is a severe neuropsychiatric disorder with poorly understood pathophysiology and typically treated with the mood stabilizer, lithium carbonate. Animal studies as well as human genetic studies indicate that lithium affects molecular targets that are involved in neuronal growth, survival and maturation, and notably molecules involved in Wnt signaling. Given the ethical challenge to obtaining brain biopsies for investigating dynamic molecular changes associated with lithium-response in the central nervous system (CNS), one may consider the use of neurons obtained from olfactory tissues to achieve this goal.The olfactory epithelium contains olfactory receptor neurons at different stages of development and glial-like supporting cells. This provides a unique opportunity to study dynamic changes in the CNS of patients with neuropsychiatric diseases, using olfactory tissue safely obtained from nasal biopsies. To overcome the drawback posed by substantial contamination of biopsied olfactory tissue with non-neuronal cells, a novel approach to obtain enriched neuronal cell populations was developed by combining nasal biopsies with laser-capture microdissection. In this study, a system for investigating treatment-associated dynamic molecular changes in neuronal tissue was developed and validated, using a small pilot sample of BD patients recruited for the study of the molecular mechanisms of lithium treatment response.

In this study, a novel platform to investigate intraneuronal molecular signatures of treatment response in bipolar disorder (BD) was developed and validated. Olfactory epithelium from BD patients was obtained through nasal biopsies. Then laser-capture microdissection was combined with Real Time RT PCR to investigate the molecular signature of lithium response in BD.

Bipolar disorder (BD) is a severe neuropsychiatric disorder with poorly understood pathophysiology and typically treated with the mood stabilizer, lithium ...

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.

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