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, and be sure to keep brush sterile and do not touch it to any surfaces besides the nasal surfaces.</li>
	<li>Ask participant to sit still and tilt head up towards ceiling. Have smaller or more restless children sit on their hands and/or lying down to avoid swatting brush away.</li>
	<li>Aim brush at the back of the nose where the passage narrows (Looks like a small black hole). Slide brush down and twist wrists as the brush is removed from nostril.</li>
	<li>Place brush in conical and submerge in BEGM. Cut off excess brush and replace cap on tube. DO NOT VORTEX. 
	NOTE: If the participant is willing, obtain a second brush in the same manner but in the second nostril. Obtaining a brushing of both nostrils will result in a higher likelihood that the sampling will be successful. Sampling the same nostril may result in bleeding and will not likely improve the sample.</li>
	<li>Keep samples as close to 37 &#176;C as possible during transport using a beaker with warm water.</li>
</ol>
2. Cell Count and Cytospin
<ol>
	<li>Gently agitate the brush in BEGM and take 10 &#181;l of the sample for a cell count using a hemacytometer. Add 10 &#181;l of a live/dead stain and count only live cells under a microscope.</li>
	<li>Take 50 &#181;l of the sample and dilute to 200 &#181;l with 1x PBS. Add to cytospin funnel attached to glass slide and spin at 300 rpm for 2 min. Let slide dry O/N and stain slide following manufacturer&#39;s instructions. Allow to dry O/N, preferably in the dark.</li>
	<li>Stain Slide with Hema 3 Stain
	<ol>
		<li>Transfer each solution (Hema fixative, light blue; Hema 3 Solution I, pink; and Hema 3 Solution II, deep blue) into a staining dish; Keep covered when not in use. Insert slides into slide staining boat</li>
		<li>Dip slides continuously in fixative for 30 sec, then allow excess to drain. Dip slides continuously in Solution I for 30 sec, then allow excess to drain. Dip slides continuously in Solution II for 30 sec, then allow excess to drain</li>
		<li>Rinse with deionized water until water runs clear. Allow to dry O/N, preferably in the dark</li>
	</ol>
	</li>
	<li>Look at slide under microscope and count cells, determine percentage of epithelial cells. Should be 90% or greater if sampling is good.</li>
</ol>
3. Seeding Cells
NOTE: Carry out all cell culture procedures in a proper and certified tissue culture hood using sterile technique.
<ol>
	<li>Coat plates with Bovine Dermal Collagen (BDC), type 1. Add 0.5 ml of 3 mg/ml BDC diluted in 49.5 ml 1x PBS. Prepare less if only a few plates will be used. Coat a T25 flask with 2 ml BDC and incubate in sterile hood O/N or at 37 &#176;C for 2 hr, if necessary.</li>
	<li>After incubation, aspirate any remaining liquid. Expose flasks to UV light for 30min in sterile hood. Store flasks for up to one month at 4 &#176;C, but bring them to RT or 37 &#176;C before seeding cells.</li>
	<li>Keep brush and cells in BEGM at 37 &#176;C until ready to seed. Gently swish brush inside conical, but do not vortex.
	<ol>
		<li>Plate 7 x 105 cells in a T25 flask. If there is less, use a smaller container, such as one well of a 6-well plate. This size plate requires about 3 x 105 cells. 
		NOTE: If there are not this many cells, it is not advisable to continue.</li>
	</ol>
	</li>
	<li>Under a sterile hood, take brush out of tube and gently rotate brush on the surface of the coated flask, being careful not to scratch the coating. Discard brush in an appropriate biohazard container.</li>
	<li>Slowly pipette the remaining cells in BEGM out of the conical and into the T25 flask. Observe the cells under microscope. A number of floating cells will be observed and there may be some debris from the nose, which is acceptable at this stage (Figure 3A).</li>
</ol>
4. Cell Culture
<ol>
	<li>After seeding cells, let them settle for 48 hr without disruption (Figure 3). After 48 hr, slowly and gently add 2 ml warm BEGM and 20&#160;&#181;l sterile Penicilin/Streptomycin/Fungizone (P/S/F). 
	NOTE: DO NOT aspirate the original 2 ml of media.</li>
	<li>On the 4th day after seeding, carefully aspirate all liquid, and replace with 4 ml fresh, warmed BEGM plus 1x Pen/Strep (fungizone is no longer necessary). Change media every two days and assess cells for attachment, growth, and confluence. When cells reach ~80% confluence, usually in 2-3 weeks, they are ready to be passaged.</li>
	<li>One T25 flask (about 2.5 x 106 cells when confluent) can be passaged into three T25 flasks or one T75 flask. After the first passage, cells should be robust and healthy, reaching confluence about every three days.</li>
</ol>
5. Passaging Cells
<ol>
	<li>Gently aspirate the media from the plate. Add 1 ml 0.05% Trypsin solution per T25 flask to the plate and place in the incubator for about 4min, or until cells detach from plate. Check the level of detachment of cells every 2 min and do not leave Trypsin on longer than 10 min.</li>
	<li>Add 5 ml Trypsin Neutralizing Solution (TNS), or serum-containing media to neutralize the enzyme. Remove all the liquid and cells from the flask and place in a 15 ml conical tube.</li>
	<li>Centrifuge at 200 x g (acceleration 0 deceleration 0) for 5 min to obtain a cell pellet. Aspirate supernatant and resuspend cells in BEGM + P/S in the volume required for the new flasks (4 ml for a T25 flask, 10 ml for a T75 flask)</li>
	<li>Perform cell count with a live/dead stain as described above (2.1). Add appropriate amount of BEGM and cells to the coated flasks and return to the incubator. Maintain as detailed above or cryopreserve in freezing medium (70% BEGM, 20% FBS and 10% DMSO) at a concentration of 0.5-2 x 106 cells per ml.</li>
</ol>
6. Reprogramming to iPSCs
NOTE: Before generating iPSCs, ensure adherence to all institutional regulations governing the generation and use of human iPSCs. Sterile technique is especially important for iPSC cultures, as culture medium does not routinely contain antibiotics. It is critical that NECs appear healthy and are robustly proliferative for successful reprogramming.
<ol>
	<li>Plate 5 x 105 NECs per well of a 6 well tissue culture plate. After 24 hr, add polybrene to BEGM to a final concentration of 8 &#181;g/ml and transduce NECs by adding lentiviral particles expressing Oct4, Sox2, Klf4, c-Myc to the media. Aim to achieve a multiplicity of infection (MOI) of ~527.
	<ol>
		<li>To determining the MOI, prepare serial dilutions of the concentrated-lentiviral stock and transduce HT1080 cells with these dilutions. After first unequivocally detection of dTomato expression in the HT1080 cells, calculate the number of &#34;transducing units/ml&#34; by scoring the number of dTomato-positive cells/small clumps of cells in each dilution. 
		NOTE: Typically, there will be dilutions that are too high (everything is dTomato positive) and too low (none or only a couple of positive cells).</li>
		<li>Perform scoring from the dilutions in which discrete positive cells/small clumps of cells is identified. Determine the titer by correcting the number of transducing units/ml with the appropriate dilution factor. MOI is then the ratio of the number of transduced cells to the number of virus particles 27. 
		NOTE: Each dTomato positive cell/small clump is assumed to arise from a single virus particle.</li>
	</ol>
	</li>
	<li>About 3 hr post-transduction, remove media and replace with fresh BEGM. Discard lentivirus-containing media using institutionally-approved procedures. Return transduced NECs to the incubator for 3 days.</li>
	<li>On day 3, replace media with fresh BEGM, and then incubate for another 3 days. Aspirate spent media, wash cells with 1x PBS and add 1 ml 0.05% Trypsin solution to the well. Passage cells with Trypsin as written above (section 5). Carefully aspirate supernatant and resuspend cells in 4 ml BEGM + P/S. Cells can be passaged to a new well of a 6 well plate coated with BDC, or added to MEF cultures, if ready (see next step).</li>
	<li>Prepare MEF coated plates. On day 4 add 0.1% gelatin solution (1 ml/well) to 2 wells of a 6 well (to perform +/- Thiazovivin (SPT) see step 6.6). On the next day, thaw a vial of inactivated MEFs 28.</li>
	<li>Place MEFs into 10 ml of MEF media (DMEM + 1x NEAA + 10% dFCS). Spin at 200 x g for 5 min to pellet. Resuspend in MEF media. Aspirate gelatin from 6 well plate and add 1.87 x 105 MEFs per well of the gelatin-coated 6 well plate. Incubate O/N at 37 &#176;C, or for a minimum of 24 hr.</li>
	<li>Transfer 2 ml of BEGM containing transduced cells per well into 2 wells of a previously coated 6 well plate and incubate O/N. On the next day, replace BEGM with 2.5 ml per well of standard hESC media containing 4 ng/ml basic fibroblast growth factor. Feed cells with fresh hESC media +/- SPT daily for 10 days 
	NOTE: It is possible to add a cocktail of small molecules (SB431542, PD0325901, and Thiazovivin (SPT) cocktail) to enhance the efficiency and kinetics of reprogramming for NECs 23.</li>
	<li>After ten days, continue to feed daily using hESC media without SPT. Feed cultures daily until hESC-like colonies appear (Figure 5A, P0 colony). Identify colonies harboring putative iPSCs by their high nucleus:cytoplasmic ratio and prominent nucleoli.</li>
	<li>Manually excise and transfer colonies with hESC-like morphology to separate wells for culture and expansion as separate lines in a feeder-free system. Excised colonies should adapt rapidly to these conditions.
	<ol>
		<li>After plating excised colonies these discrete putative iPSC lines are now considered passage 1 (p1). Feed cultures daily with mTeSR1. Passage and expand iPSC lines using standard procedures. It may take several days to a week for p1 colonies to reach the size at which they should be passaged.</li>
	</ol>
	</li>
</ol>
7. Maintaining iPSCs in Culture
<ol>
	<li>Feed and evaluate cultures daily. Manually excise areas exhibiting overt differentiation by scraping with a sterile glass pipette or micro pipette tip. Change media after daily picking.</li>
	<li>Passage cells after approximately every four days: Gently aspirate media, add 1 ml warm Dispase (1 mg/ml) per well of a 6-well plate (half the feeding volume), then incubate at 37 &#176;C for about 4 min. The edges of the colonies will begin to lift, which looks like a white outline around the colony. Gently aspirate the Dispase and wash 3x with warmed DMEM/F12.</li>
	<li>Add 2 ml mTeSR1 and use a cell lifter to lift the colonies from the plate. Use a 5 ml serological pipette or P1000 micropipette to gently triturate the cells until the colonies are broken into smaller clusters.
	<ol>
		<li>Avoid producing very small clusters or single cells, as survival and subsequent proliferation will be sub-optimal. When re-plating colonies, a 1:4 to 1:6 split will maintain optimal colony density. Gently remove colonies and media from the plate and add to new, matrigel-coated wells.</li>
	</ol>
	</li>
</ol>

The authors have no conflicts of interest to disclose.

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...

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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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Developmental Biology

11.1K Views.  Cincinnati Children's Hospital. 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).

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

Large-Scale Production of Cardiomyocytes from Human Pluripotent Stem Cells Using a Highly Reproducible Small Molecule-Based Differentiation Protocol

Maximizing the benefit of human pluripotent stem cells (hPSCs) for research, disease modeling, pharmaceutical and clinical applications requires robust methods for the large-scale production of functional cell types, including cardiomyocytes. Here we demonstrate that the temporal manipulation of WNT, TGF-&#946;, and SHH signaling pathways leads to highly efficient cardiomyocyte differentiation of single-cell passaged hPSC lines in both static suspension and stirred suspension bioreactor systems. Employing this strategy resulted in ~ 100% beating spheroids, consistently containing &#62; 80% cardiac troponin T-positive cells after 15 days of culture, validated in multiple hPSC lines. We also report on a variation of this protocol for use with cell lines not currently adapted to single-cell passaging, the success of which has been verified in 42 hPSC lines. Cardiomyocytes generated using these protocols express lineage-specific markers and show expected electrophysiological functionalities. Our protocol presents a simple, efficient and robust platform for the large-scale production of human cardiomyocytes.

Here, we present a robust, fast and scalable cardiomyocyte differentiation protocol for human pluripotent stem cells (hPSCs). Cardiomyocytes derived using this large-scale method can provide sufficient cell numbers for their effective use in human cardiovascular disease modeling, high-throughput drug screening, and potentially clinical applications.

Maximizing the benefit of human pluripotent stem cells (hPSCs) for research, disease modeling, pharmaceutical and clinical applications requires robust ...

The Production of Pluripotent Stem Cells from Mouse Amniotic Fluid Cells Using a Transposon System

Induced pluripotent stem (iPS) cells are generated from mouse and human somatic cells by forced expression of defined transcription factors using different methods. Here, we produced iPS cells from mouse amniotic fluid cells, using a non-viral-based transposon system. All obtained iPS cell lines exhibited characteristics of pluripotent cells, including the ability to differentiate toward derivatives of all three germ layers in vitro and in vivo. This strategy opens up the possibility of using cells from diseased fetuses to develop new therapies for birth defects.

In this study, we generate induced pluripotent stem cells from mouse amniotic fluid cells, using a non-viral-based transposon system.

Induced pluripotent stem (iPS) cells are generated from mouse and human somatic cells by forced expression of defined transcription factors using ...

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 ...

Differentiating Chondrocytes from Peripheral Blood-derived Human Induced Pluripotent Stem Cells

In this study, we used peripheral blood cells (PBCs) as seed cells to produce chondrocytes via induced pluripotent stem cells (iPSCs) in an integration-free method. Following embryoid body (EB) formation and fibroblastic cell expansion, the iPSCs are induced for chondrogenic differentiation for 21 days under serum-free and xeno-free conditions. After chondrocyte induction, the phenotypes of the cells are evaluated by morphological, immunohistochemical, and biochemical analyses, as well as by the quantitative real-time PCR examination of chondrogenic differentiation markers. The chondrogenic pellets show positive alcian blue and toluidine blue staining. The immunohistochemistry of collagen II and X staining is also positive. The sulfated glycosaminoglycan (sGAG) content and the chondrogenic differentiation markers COLLAGEN 2 (COL2), COLLAGEN 10 (COL10), SOX9, and AGGRECAN are significantly upregulated in chondrogenic pellets compared to hiPSCs and fibroblastic cells. These results suggest that PBCs can be used as seed cells to generate iPSCs for cartilage repair, which is patient-specific and cost-effective.

We present a protocol to generate a chondrogenic lineage from human peripheral blood (PB) via induced pluripotent stem cells (iPSCs) using an integration-free method, which includes embryoid body (EB) formation, fibroblastic cells expansion, and chondrogenic induction.

In this study, we used peripheral blood cells (PBCs) as seed cells to produce chondrocytes via induced pluripotent stem cells (iPSCs) in an ...

Rapid, Directed Differentiation of Retinal Pigment Epithelial Cells from Human Embryonic or Induced Pluripotent Stem Cells

We describe a robust method to direct the differentiation of pluripotent stem cells into retinal pigment epithelial cells (RPE). The purpose of providing a detailed and thorough protocol is to clearly demonstrate each step and to make this readily available to researchers in the field. This protocol results in a homogenous layer of RPE with minimal or no manual dissection needed. The method presented here has been shown to be effective for induced pluripotent stem cells (iPSC) and human embryonic stem cells. Additionally, we describe methods for cryopreservation of intermediate cell banks that allow long-term storage. RPE generated using this protocol might be useful for iPSC disease-in-a-dish modeling or clinical application.

This protocol describes how to produce retinal pigment epithelial cells (RPE) from pluripotent stem cells. The method uses a combination of growth factors and small molecules to direct the differentiation of stem cells into immature RPE in fourteen days and mature, functional RPE after three months.

We describe a robust method to direct the differentiation of pluripotent stem cells into retinal pigment epithelial cells (RPE). The purpose of providing ...

Computational Analysis of the Caenorhabditis elegans Germline to Study the Distribution of Nuclei, Proteins, and the Cytoskeleton

The Caenorhabditis elegans (C. elegans) germline is used to study several biologically important processes including stem cell development, apoptosis, and chromosome dynamics. While the germline is an excellent model, the analysis is often two dimensional due to the time and labor required for three-dimensional analysis. Major readouts in such studies are the number/position of nuclei and protein distribution within the germline. Here, we present a method to perform automated analysis of the germline using confocal microscopy and computational approaches to determine the number and position of nuclei in each region of the germline. Our method also analyzes germline protein distribution that enables the three-dimensional examination of protein expression in different genetic backgrounds. Further, our study shows variations in cytoskeletal architecture in distinct regions of the germline that may accommodate specific spatial developmental requirements. Finally, our method enables automated counting of the sperm in the spermatheca of each germline. Taken together, our method enables rapid and reproducible phenotypic analysis of the C. elegans germline.

Computational Analysis of the Caenorhabditis elegans Germline to Study the Distribution of Nuclei, Proteins, and the Cytoskeleton

We present an automated method for three-dimensional reconstruction of the Caenorhabditis elegans germline. Our method determines the number and position of each nucleus within the germline and analyses germline protein distribution and cytoskeletal structure.

The Caenorhabditis elegans (C. elegans) germline is used to study several biologically important processes including stem cell development, apoptosis, and ...

Efficient and Scalable Directed Differentiation of Clinically Compatible Corneal Limbal Epithelial Stem Cells from Human Pluripotent Stem Cells

Corneal limbal epithelial stem cells (LESCs) are responsible for continuously renewing the corneal epithelium, and thus maintaining corneal homeostasis and visual clarity. Human pluripotent stem cell (hPSC)-derived LESCs provide a promising cell source for corneal cell replacement therapy. Undefined, xenogeneic culture and differentiation conditions cause variation in research results and impede the clinical translation of hPSC-derived therapeutics. This protocol provides a reproducible and efficient method for hPSC-LESC differentiation under xeno- and feeder cell-free conditions. Firstly, monolayer culture of undifferentiated hPSC on recombinant laminin-521 (LN-521) and defined hPSC medium serves as a foundation for robust production of high-quality starting material for differentiations. Secondly, a rapid and simple hPSC-LESC differentiation method yields LESC populations in only 24 days. This method includes a four-day surface ectodermal induction in suspension with small molecules, followed by adherent culture phase on LN-521/collagen IV combination matrix in defined corneal epithelial differentiation medium. Cryostoring and extended differentiation further purifies the cell population and enables banking of the cells in large quantities for cell therapy products. The resulting high-quality hPSC-LESCs provide a potential novel treatment strategy for corneal surface reconstruction to treat limbal stem cell deficiency (LSCD).

This protocol introduces a simple two-step method for differentiating corneal limbal epithelial stem cells from human pluripotent stem cells under xeno- and feeder cell-free culture conditions. The cell culture methods presented here enable cost-efficient, large-scale production of clinical quality cells applicable to corneal cell therapy use.

Corneal limbal epithelial stem cells (LESCs) are responsible for continuously renewing the corneal epithelium, and thus maintaining corneal homeostasis ...

RNA-based Reprogramming of Human Primary Fibroblasts into Induced Pluripotent Stem Cells

Induced pluripotent stem cells (iPSCs) have proven to be a valuable tool to study human development and disease. Further advancing iPSCs as a regenerative therapeutic requires a safe, robust, and expedient reprogramming protocol. Here, we present a clinically relevant, step-by-step protocol for the extremely high-efficiency reprogramming of human dermal fibroblasts into iPSCs using a non-integrating approach. The core of the protocol consists of expressing pluripotency factors (SOX2, KLF4, cMYC, LIN28A, NANOG, OCT4-MyoD fusion) from in vitro transcribed messenger RNAs synthesized with modified nucleotides (modified mRNAs). The reprogramming modified mRNAs are transfected into primary fibroblasts every 48 h together with mature embryonic stem cell-specific microRNA-367/302 mimics for two weeks. The resulting iPSC colonies can then be isolated and directly expanded in feeder-free conditions. To maximize efficiency and consistency of our reprogramming protocol across fibroblast samples, we have optimized various parameters including the RNA transfection regimen, timing of transfections, culture conditions, and seeding densities. Importantly, our method generates high-quality iPSCs from most fibroblast sources, including difficult-to-reprogram diseased, aged, and/or senescent samples.

Here we describe a clinically relevant, high-efficiency, feeder-free method to reprogram human primary fibroblasts into induced pluripotent stem cells using modified mRNAs encoding reprogramming factors and mature microRNA-367/302 mimics. Also included are methods to assess reprogramming efficiency, expand clonal iPSC colonies, and confirm expression of the pluripotency marker TRA-1-60.

Induced pluripotent stem cells (iPSCs) have proven to be a valuable tool to study human development and disease. Further advancing iPSCs as a regenerative ...

Efficient Differentiation of Human Pluripotent Stem Cells into Liver Cells

The liver detoxifies harmful substances, secretes vital proteins, and executes key metabolic activities, thus sustaining life. Consequently, liver failure&#8212;which can be caused by chronic alcohol intake, hepatitis, acute poisoning, or other insults&#8212;is a severe condition that can culminate in bleeding, jaundice, coma, and eventually death. However, approaches to treat liver failure, as well as studies of liver function and disease, have been stymied in part by the lack of a plentiful supply of human liver cells. To this end, this protocol details the efficient differentiation of human pluripotent stem cells (hPSCs) into hepatocyte-like cells, guided by a developmental roadmap that describes how liver fate is specified across six consecutive differentiation steps. By manipulating developmental signaling pathways to promote liver differentiation and to explicitly suppress the formation of unwanted cell fates, this method efficiently generates populations of human liver bud progenitors and hepatocyte-like cells by days 6 and 18 of PSC differentiation, respectively. This is achieved through the temporally-precise control of developmental signaling pathways, exerted by small molecules and growth factors in a serum-free culture medium. Differentiation in this system occurs in monolayers and yields hepatocyte-like cells that express characteristic hepatocyte enzymes and have the ability to engraft a mouse model of chronic liver failure. The ability to efficiently generate large numbers of human liver cells in vitro has ramifications for treatment of liver failure, for drug screening, and for mechanistic studies of liver disease.

This protocol details a monolayer, serum-free method to efficiently generate hepatocyte-like cells from human pluripotent stem cells (hPSCs) in 18 days. This entails six steps as hPSCs sequentially differentiate into intermediate cell-types such as the primitive streak, definitive endoderm, posterior foregut and liver bud progenitors before forming hepatocyte-like cells.

The liver detoxifies harmful substances, secretes vital proteins, and executes key metabolic activities, thus sustaining life. Consequently, liver ...

Generation of 3D Skin Organoid from Cord Blood-derived Induced Pluripotent Stem Cells

The skin is the body&#8217;s largest organ and has many functions. The skin acts as a physical barrier and protector of the body and regulates bodily functions. Biomimetics is the imitation of the models, systems, and elements of nature for the purpose of solving complex human problems1. Skin biomimetics is a useful tool for in vitro disease research and in vivo regenerative medicine. Human induced pluripotent stem cells (iPSCs) have the characteristic of unlimited proliferation and the ability of differentiation to three germ layers. Human iPSCs are generated from various primary cells, such as blood cells, keratinocytes, fibroblasts, and more. Among them, cord blood mononuclear cells (CBMCs) have emerged as an alternative cell source from the perspective of allogeneic regenerative medicine. CBMCs are useful in regenerative medicine because human leukocyte antigen (HLA) typing is essential to the cell banking system. We provide a method for the differentiation of CBMC-iPSCs into keratinocytes and fibroblasts and for generation of a 3D skin organoid. CBMC-iPSC-derived keratinocytes and fibroblasts have characteristics similar to a primary cell line. The 3D skin organoids are generated by overlaying an epidermal layer onto a dermal layer. By transplanting this 3D skin organoid, a humanized mice model is generated. This study shows that a 3D human iPSC-derived skin organoid may be a novel, alternative tool for dermatologic research in vitro and in vivo.

We propose a protocol that shows how to differentiate induced pluripotent stem cell-derived keratinocytes and fibroblasts and generate a 3D skin organoid, using these keratinocytes and fibroblasts. This protocol contains an additional step of generating a humanized mice model. The technique presented here will improve dermatologic research.

The skin is the body&#8217;s largest organ and has many functions. The skin acts as a physical barrier and protector of the body and regulates bodily ...