Name: cultivate primary nasal epithelial cells from children and reprogram into induced pluripotent stem cells
Uploaded: 2016-03-10T14:00:00
Description: Watch this Scientific Journal Video about Cultivate Primary Nasal Epithelial Cells from Children and Reprogram into Induced Pluripotent Stem Cells at JoVE.com

The authors would like to acknowledge the Pluripotent Stem Cell Facility and the Confocal Imaging Core at Cincinnati Children&#39;s Hospital. This work was supported by R21AI119236 (HJ), R21AI101375 (HJ), NIH/NCATS 8UL1TR000077-04 (HJ), U19 AI070412 (HJ) and 2U19AI70235 (GKKH).

The following protocol follows the guidelines of the institutions human research ethics committee.
1. Sampling the Nasal Mucosa
NOTE: Obtain samples from subjects who are free of signs of respiratory viral infection.
<ol>
	<li>Prepare a 15 ml conical fresh before the participant visit and add 2 ml BEGM (Bronchial Epithelial Cell Growth Medium) plus 20 &#181;l sterile Penn/Strep/Fungizone (P/S/F) (0.01%).</li>
	<li>Open cytology brush just before taking the sample,...

Nasal epithelial cells (NECs) are an accessible platform for studying airway disease, and NEC-iPSCs offer an exciting avenue to explore disease development, treatment and therapy 1,31,32. NECs can easily be obtained without stressful or potentially harmful procedures 6,23. In our experience, the sampling of the nasal mucosa as described in this protocol appears to be less stressful and better perceived than blood collection for children. Therefore, this method may be particularly useful in pediatric...

Induced pluripotent stem cells from human samples (hiPSCs) are a fast developing technology of stem cell research. They offer an alternative to embryonic stem cell (hESC) research with far fewer ethical and moral drawbacks 1,2. Although they are not epigenetically identical to hESCs 3-5, hiPSCs offer a unique way to model development and disease phenotypes, and they can be derived from tissues relevant to the disease state 5-8. New methods of generating hiPSCs are constantly being explored to identify optimal cell types to start with, as a way to prepare GMP-quality iPSCs suitable for transplantation, and also to increase the timeliness and efficiency of the reprogramming process 6,9-11.
Airway epithelial cells are critical in the development of allergic inflammation 12, and the epithelium is a major driver of allergic responses and airway remodeling through interaction with immune and stromal cells. The airway epithelium plays an essential role in the origin and persistence of lung diseases such as asthma. However, lower airway epithelial cells are difficult to obtain in a clinical setting, especially from healthy control patients and children. Data from several studies support the premise that epithelial cells from nasal mucosa are a valid and practical proxy for lower airway epithelial cells 13-20, especially when studying responses to air pollutants and allergens. The nasal mucosa consists of more than 90% ciliated airway epithelial cells and sampling these nasal epithelial cells (NECs) can be readily performed in children as young as age four or five, as it is less invasive than other cell/tissue sampling techniques and is associated with minimal risk of adverse events such as infection 20-23. It offers a rapid and simple way to sample both healthy and diseased children without long, unnecessary, and often painful bronchoscopy procedures that necessitate sedation. Previous studies have found that disease subtypes related to asthma severity can be distinguished in both the nasal mucosa as well as bronchial cell samples taken from asthmatic children, and gene expression between the two tissue types was similar in about 90% of non-ubiquitous genes 22,24. As a source for iPSCs, NECs offer advantages over other frequently utilized cell types. Fibroblasts are often used for iPSC generation, but although these cells can easily be cultured from a skin biopsy, this process typically requires local anesthesia, an incision, and sutures, and is associated with some risk of infection. Therefore, obtaining informed consent from patients for this type of biopsy can be difficult 25. One alternative to fibroblasts is peripheral blood mononuclear cells (PBMCs). However, it may be difficult to obtain sufficient blood for iPSC generation from pediatric patients. In addition, there are limitations of downstream applications for fibroblast and blood cell derived iPSCs, especially their differentiation capacity to certain cell types 5,26. Therefore, given the relative accessibility and the low risk of side effects following their collection, NECs represent an ideal cell source for iPSC generation from pediatric populations.
iPSCs have received a lot of attention recently as a platform for studying human development, generating novel disease models, and as a potential source of cells for personalized therapies. Before the full potential of this technology can be realized, the molecular underpinnings of the reprogramming process need to be elucidated, but for now this protocol and the procedures outlined within will elucidate the research studies focused on airway exposures, as well as provide a platform for studying the effects of personalized medicine involving iPSCs.
The collaborative work of several labs has led to the generation of a successful technique for not only sampling the nasal mucosa, but also culturing NECs, and reprogramming these cells to iPSCs 23. This article provides an outline of a protocol for optimal sampling, culturing, and reprogramming conditions.

<table border="0" cellpadding="0" cellspacing="0" width="655">
	<tbody>
		<tr height="20">
			<td height="20" style="height:20px;width:207px;">15mL conical</td>
			<td style="width:203px;">Fisher Scientific</td>
			<td style="width:108px;">14-959-49D</td>
			<td style="width:139px;">Protocol Step 1.1.</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:207px;">BEGM</td>
			<td>Lonza</td>
			<td>CC-3170</td>
			<td>Protocol Step 1.1.</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:207px;">Penn/Strep/Fungicide</td>
			<td>Life Technologies</td>
			<td>15240-062</td>
			<td>Protocol Step 1.1.</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:207px;">Penn/Strep</td>
			<td>Life Technologies</td>
			<td>15140-122</td>
			<td>Protocol Step 4.a.</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:207px;">cytosoft cytology brush</td>
			<td>Fisher Scientific</td>
			<td>22-263-357</td>
			<td>Protocol Step 1.2.</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:207px;">trypan blue</td>
			<td>Fisher Scientific</td>
			<td>MT-25-900-CI</td>
			<td>Protocol Step 2.1.</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:207px;">hemacytometer</td>
			<td>Fisher Scientific</td>
			<td>02-671-54</td>
			<td>Protocol Step 2.1.</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:207px;">PBS</td>
			<td>Fisher Scientific</td>
			<td>BP2438-4</td>
			<td>Protocol Step 2.2.</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:207px;">Cytology Funnel Clips</td>
			<td>Fisher Scientific</td>
			<td>10-357</td>
			<td>Protocol Step 2.2.</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:207px;">cytospin funnel</td>
			<td>Fisher Scientific</td>
			<td>23-640-320</td>
			<td>Protocol Step 2.2.</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:207px;">Cytospin 4</td>
			<td>Fisher Scientific</td>
			<td>A78300003</td>
			<td>Protocol Step 2.2.</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:207px;">blank slide</td>
			<td>Fisher Scientific</td>
			<td>S95933</td>
			<td>Protocol Step 2.2.</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:207px;">hema 3 stain kit</td>
			<td>Fisher Scientific</td>
			<td>22-122-911</td>
			<td>Protocol Step 2.2.</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:207px;">Bovine Dermal Colagen, type 1</td>
			<td>Life Technologies</td>
			<td>A1064401</td>
			<td>Protocol Step 3.2.</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:207px;">T25 flask</td>
			<td>Fisher Scientific</td>
			<td>08-772-45</td>
			<td>Protocol Step 3.3.</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:207px;">Trypsin</td>
			<td>Lonza</td>
			<td>CC-5012</td>
			<td>Protocol Step 5.2.</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:207px;">Trypsin Neutralizing Solution</td>
			<td>Lonza</td>
			<td>CC-5002</td>
			<td>Protocol Step 5.2.</td>
		</tr>
		<tr height="44">
			<td height="44" style="height:44px;width:207px;">Fetal Bovine Serum (FBS), heat sterilized at 65&#176;C for 30min</td>
			<td>Sigma-Aldrich</td>
			<td>F2442</td>
			<td>Protocol Step 5.5.</td>
		</tr>
		<tr height="82">
			<td height="82" style="height:82px;width:207px;">Dimethyl sulfoxide Hybri-Max&#8482;, sterile-filtered, BioReagent, suitable for hybridoma, &#8805;99.7% -&#160;</td>
			<td>Sigma-Aldrich</td>
			<td>D2650</td>
			<td>Protocol Step 5.5.</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:207px;">polycistonic lentivirus*</td>
			<td>e.g. Millipore</td>
			<td>SCR511</td>
			<td>Protocol Step 6.4.&#160; 
			A commercial source of reprogramming vector is listed. We routinely use the 4-in-1 plasmid reported by Voelkel et al (PMID: 20385817) to generate VSV-G-pseudotyped polycistronic reprogramming lentivirus in-house. This plasmid can be obtained by contacting&#160;</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:207px;">polybrene</td>
			<td>Santa Cruz Biotechnology</td>
			<td>sc-134220</td>
			<td>Protocol Step 6.4.</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:207px;">Irradiated CF1 MEFs</td>
			<td>GlobalStem&#160;</td>
			<td>GSC-6301G</td>
			<td>Protocol Step 6.4.</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:207px;">hESC media</td>
			<td>See recipe included in protocol</td>
			<td></td>
			<td>Protocol Step 6.11.</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:207px;">SB431542</td>
			<td>Stemgent</td>
			<td>04-0010</td>
			<td>Protocol Step 6.11.</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:207px;">PD0325901</td>
			<td>Stemgent</td>
			<td>04-0006</td>
			<td>Protocol Step 6.11.</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:207px;">Thiazovivin&#160;</td>
			<td>Stemgent</td>
			<td>04-0017</td>
			<td>Protocol Step 6.11.</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:207px;">hESC-qualified Matrigel</td>
			<td>BD Biosciences</td>
			<td>354277</td>
			<td>Protocol Step 6.13.</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:207px;">Corning plate, 6 well</td>
			<td>Fisher Scientific</td>
			<td>08-772-1B</td>
			<td>Protocol Step 6.13.</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:207px;">mTeSR1</td>
			<td>StemCell</td>
			<td>5850</td>
			<td>Protocol Step 6.13.</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:207px;">250mL disposable filter flask (0.22&#181;m)</td>
			<td>Fisher</td>
			<td>SCGP-U02-RE&#160;</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:207px;">dispase</td>
			<td>StemCell</td>
			<td>7923</td>
			<td>Protocol Step 7.3.</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:207px;">DMEM/F12</td>
			<td>Life Technologies</td>
			<td>11320-033</td>
			<td>Protocol Step 7.3.</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:207px;">cell lifter</td>
			<td>Fisher Scientific</td>
			<td>08-100-240</td>
			<td>Protocol Step 7.4.</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">hESC Media**</td>
			<td></td>
			<td></td>
			<td>Protocol Step 6.11. 
			components should be mixed and then filter sterilized. Media can be kept at 4&#176;C for up to two weeks. When warming media, do not leave at 37&#176;C longer than 15 min</td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:207px;">DMEM-F12 50/50 media</td>
			<td>Invitrogen</td>
			<td>&#160;11330-032</td>
			<td>Final Concentration</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:207px;">KO replacement serum (KO-SR)</td>
			<td>Invitrogen</td>
			<td>10828-028</td>
			<td align="right">0.2</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:207px;">200mM L-glutamine</td>
			<td>Invitrogen</td>
			<td>25030-081</td>
			<td>1mM</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:207px;">55mM &#223;-mercaptoethanol</td>
			<td>Invitrogen</td>
			<td>21985-023</td>
			<td>0.1mM</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:207px;">100x non-essential amino acids</td>
			<td>Invitrogen</td>
			<td>11140-050</td>
			<td>1x</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:207px;">2ug/mL Basic-Fibroblast Growth Factor (b-FGF)</td>
			<td>Invitrogen</td>
			<td>13256-029</td>
			<td>4ng/mL</td>
		</tr>
	</tbody>
</table>

cultivate primary nasal epithelial cells from children and reprogram into induced pluripotent stem cells

Nasal epithelial cells (NECs) are the part of the airways that respond to air pollutants and are the first cells infected with respiratory viruses. They are also involved in many airway diseases through their innate immune response and interaction with immune and airway stromal cells. NECs are of particular interest for studies in children due to their accessibility during clinical visits. Human induced pluripotent stem cells (iPSCs) have been generated from multiple cell types and are a powerful tool for modeling human development and disease, as well as for their potential applications in regenerative medicine. This is the first protocol to lay out methods for successful generation of iPSCs from NECs derived from pediatric participants for research purposes. It describes how to obtain nasal epithelial cells from children, how to generate primary NEC cultures from these samples, and how to reprogram primary NECs into well-characterized iPSCs. Nasal mucosa samples are useful in epidemiological studies related to the effects of air pollution in children, and provide an important tool for studying airway disease. Primary nasal cells and iPSCs derived from them can be a tool for providing unlimited material for patient-specific research in diverse areas of airway epithelial biology, including asthma and COPD research.

The initial part of the nose, called the nasal vestibule, is the area surrounded by cartilage 29. The brush needs to go smoothly past this area of the nose, beyond the nasal valve (ostium internum, or the &#34;black hole&#34; seen at the back of the nare), and the sample is obtained from the inferior turbinate (Figure 1). The nasal turbinates are bony structures that increase the surface area of the nose 29, making them an ideal location for sampling...

Watch this Scientific Journal Video about Cultivate Primary Nasal Epithelial Cells from Children and Reprogram into Induced Pluripotent Stem Cells at JoVE.com

Cultivate Primary Nasal Epithelial Cells from Children and Reprogram into Induced Pluripotent Stem Cells

This publication demonstrates methods for successful sampling and culture of nasal epithelial mucosa from children, and reprogramming these cells to induced Pluripotent Stem Cells (iPSCs).

Nasal epithelial cells (NECs) are the part of the airways that respond to air pollutants and are the first cells infected with respiratory viruses. They ...

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