Name: Cultivate Primary Nasal Epithelial Cells from Children and Reprogram into Induced Pluripotent Stem Cells (Video) | JoVE Journal
Uploaded: 2016-03-10T14:00:00.000+00:00
Duration: 12 min 8 s
Description: Learn to cultivate primary nasal epithelial cells from children and reprogram them into induced pluripotent stem cells using non-invasive sampling. This method enables asthma research and disease modeling without stressful procedures.

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5 minutes

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>

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<li>Lai, P. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Alternate+methods+of+nasal+epithelial+cell+sampling+for+airway+genomic+studies%5BTitle%5D)+AND+%22J+Allergy+Clin+Immunol%22%5BJournal%5D)">Alternate methods of nasal epithelial cell sampling for airway genomic studies.</a> J Allergy Clin Immunol. , (2015).</li>
<li>Shi, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+combined+chemical+and+genetic+approach+for+the+generation+of+induced+pluripotent+stem+cells%5BTitle%5D)+AND+%22Cell+Stem+Cell%22%5BJournal%5D)">A combined chemical and genetic approach for the generation of induced pluripotent stem cells.</a> Cell Stem Cell. 2 (6), 525-528 (2008).</li>
<li>Okita, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+more+efficient+method+to+generate+integration-free+human+iPS+cells%5BTitle%5D)+AND+%22Nature%22%5BJournal%5D)">A more efficient method to generate integration-free human iPS cells.</a> Nature. 8 (5), 409-412 (2011).</li>
<li>Dye, B. R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((In+vitro+generation+of+human+pluripotent+stem+cell+derived+lung+organoids%5BTitle%5D)+AND+%22Elife%22%5BJournal%5D)">In vitro generation of human pluripotent stem cell derived lung organoids.</a> Elife. 4, (2015).</li>
<li>Huang, S. X., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Efficient+generation+of+lung+and+airway+epithelial+cells+from+human+pluripotent+stem+cells%5BTitle%5D)+AND+%22Nat+Biotechnol%22%5BJournal%5D)">Efficient generation of lung and airway epithelial cells from human pluripotent stem cells.</a> Nat Biotechnol. 32 (1), 84-91 (2014).</li>
<li>Wong, A. P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Directed+differentiation+of+human+pluripotent+stem+cells+into+mature+airway+epithelia+expressing+functional+CFTR+protein%5BTitle%5D)+AND+%22Nature+biotechnology%22%5BJournal%5D)">Directed differentiation of human pluripotent stem cells into mature airway epithelia expressing functional CFTR protein.</a> Nature biotechnology. 30 (9), 876-882 (2012).</li>
<li>Marchetto, M. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Transcriptional+signature+and+memory+retention+of+human-induced+pluripotent+stem+cells%5BTitle%5D)+AND+%22PloS+one%22%5BJournal%5D)">Transcriptional signature and memory retention of human-induced pluripotent stem cells.</a> PloS one. 4 (9), e7076(2009).</li>
<li>Nukaya, D., Minami, K., Hoshikawa, R., Yokoi, N., Seino, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Preferential+gene+expression+and+epigenetic+memory+of+induced+pluripotent+stem+cells+derived+from+mouse+pancreas%5BTitle%5D)+AND+%22Genes+Cells%22%5BJournal%5D)">Preferential gene expression and epigenetic memory of induced pluripotent stem cells derived from mouse pancreas.</a> Genes Cells. 20 (5), 367-381 (2015).</li>
<li>Vaskova, E. A., Stekleneva, A. E., Medvedev, S. P., Zakian, S. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((%27Epigenetic+memory%27+phenomenon+in+induced+pluripotent+stem+cells%5BTitle%5D)+AND+%22Acta+Naturae%22%5BJournal%5D)">'Epigenetic memory' phenomenon in induced pluripotent stem cells.</a> Acta Naturae. 5 (4), 15-21 (2013).</li>
<li>Bar-Nur, O., Russ, H. A., Efrat, S., Benvenisty, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Epigenetic+memory+and+preferential+lineage-specific+differentiation+in+induced+pluripotent+stem+cells+derived+from+human+pancreatic+islet+beta+cells%5BTitle%5D)+AND+%22Cell+Stem+Cell%22%5BJournal%5D)">Epigenetic memory and preferential lineage-specific differentiation in induced pluripotent stem cells derived from human pancreatic islet beta cells.</a> Cell Stem Cell. 9 (1), 17-23 (2011).</li>
<li>Jandial, R., Levy, M. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Cellular+alchemy%3A+induced+pluripotent+stem+cells+retain+epigenetic+memory%5BTitle%5D)+AND+%22World+Neurosurg%22%5BJournal%5D)">Cellular alchemy: induced pluripotent stem cells retain epigenetic memory.</a> World Neurosurg. 75 (1), 5-6 (2011).</li>
<li>Kim, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Epigenetic+memory+in+induced+pluripotent+stem+cells%5BTitle%5D)+AND+%22Nature%22%5BJournal%5D)">Epigenetic memory in induced pluripotent stem cells.</a> Nature. 467 (7313), 285-290 (2010).</li>
<li>Ohi, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Incomplete+DNA+methylation+underlies+a+transcriptional+memory+of+somatic+cells+in+human+iPS+cells%5BTitle%5D)+AND+%22Nat+Cell+Biol%22%5BJournal%5D)">Incomplete DNA methylation underlies a transcriptional memory of somatic cells in human iPS cells.</a> Nat Cell Biol. 13 (5), 541-549 (2011).</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><tbody><tr><td>15 ml conical</td><td>Fisher Scientific</td><td>14-959-49D</td><td>Protocol Step 1.1.</td></tr><tr><td>BEGM</td><td>Lonza</td><td>CC-3170</td><td>Protocol Step 1.1.</td></tr><tr><td>Penn/Strep/Fungicide</td><td>Life Technologies</td><td>15240-062</td><td>Protocol Step 1.1.</td></tr><tr><td>Penn/Strep</td><td>Life Technologies</td><td>15140-122</td><td>Protocol Step 4.a.</td></tr><tr><td>cytosoft cytology brush</td><td>Fisher Scientific</td><td>22-263-357</td><td>Protocol Step 1.2.</td></tr><tr><td>trypan blue</td><td>Fisher Scientific</td><td>MT-25-900-CI</td><td>Protocol Step 2.1.</td></tr><tr><td>hemacytometer</td><td>Fisher Scientific</td><td>02-671-54</td><td>Protocol Step 2.1.</td></tr><tr><td>PBS</td><td>Fisher Scientific</td><td>BP2438-4</td><td>Protocol Step 2.2.</td></tr><tr><td>Cytology Funnel Clips</td><td>Fisher Scientific</td><td>10-357</td><td>Protocol Step 2.2.</td></tr><tr><td>cytospin funnel</td><td>Fisher Scientific</td><td>23-640-320</td><td>Protocol Step 2.2.</td></tr><tr><td>Cytospin 4</td><td>Fisher Scientific</td><td>A78300003</td><td>Protocol Step 2.2.</td></tr><tr><td>blank slide</td><td>Fisher Scientific</td><td>S95933</td><td>Protocol Step 2.2.</td></tr><tr><td>hema 3 stain kit</td><td>Fisher Scientific</td><td>22-122-911</td><td>Protocol Step 2.2.</td></tr><tr><td>Bovine Dermal Colagen, type 1</td><td>Life Technologies</td><td>A1064401</td><td>Protocol Step 3.2.</td></tr><tr><td>T25 flask</td><td>Fisher Scientific</td><td>08-772-45</td><td>Protocol Step 3.3.</td></tr><tr><td>Trypsin</td><td>Lonza</td><td>CC-5012</td><td>Protocol Step 5.2.</td></tr><tr><td>Trypsin Neutralizing Solution</td><td>Lonza</td><td>CC-5002</td><td>Protocol Step 5.2.</td></tr><tr><td>Fetal Bovine Serum (FBS), heat sterilized at 65 &amp;deg;C for 30 min</td><td>Sigma-Aldrich</td><td>F2442</td><td>Protocol Step 5.5.</td></tr><tr><td>Dimethyl sulfoxide Hybri-Max&amp;trade;, sterile-filtered, BioReagent, suitable for hybridoma, &amp;ge;99.7%</td><td>Sigma-Aldrich</td><td>D2650</td><td>Protocol Step 5.5.</td></tr><tr><td>polycistonic lentivirus*</td><td>&lt;em&gt;e.g.&lt;/em&gt; Millipore</td><td>SCR511</td><td>Protocol Step 6.4.&amp;nbsp;&lt;br/&gt; 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&amp;nbsp;</td></tr><tr><td>polybrene</td><td>Santa Cruz Biotechnology</td><td>sc-134220</td><td>Protocol Step 6.4.</td></tr><tr><td>Irradiated CF1 MEFs</td><td>GlobalStem&amp;nbsp;</td><td>GSC-6301G</td><td>Protocol Step 6.4.</td></tr><tr><td>hESC media</td><td>See recipe included in protocol</td><td></td><td>Protocol Step 6.11.</td></tr><tr><td>SB431542</td><td>Stemgent</td><td>04-0010</td><td>Protocol Step 6.11.</td></tr><tr><td>PD0325901</td><td>Stemgent</td><td>04-0006</td><td>Protocol Step 6.11.</td></tr><tr><td>Thiazovivin&amp;nbsp;</td><td>Stemgent</td><td>04-0017</td><td>Protocol Step 6.11.</td></tr><tr><td>hESC-qualified Matrigel</td><td>BD Biosciences</td><td>354277</td><td>Protocol Step 6.13.</td></tr><tr><td>Corning plate, 6 well</td><td>Fisher Scientific</td><td>08-772-1B</td><td>Protocol Step 6.13.</td></tr><tr><td>mTeSR1</td><td>StemCell</td><td>5850</td><td>Protocol Step 6.13.</td></tr><tr><td>250 ml disposable filter flask (0.22 &amp;micro;m)</td><td>Fisher</td><td>SCGP-U02-RE&amp;nbsp;</td><td></td></tr><tr><td>dispase</td><td>StemCell</td><td>7923</td><td>Protocol Step 7.3.</td></tr><tr><td>DMEM/F12</td><td>Life Technologies</td><td>11320-033</td><td>Protocol Step 7.3.</td></tr><tr><td>cell lifter</td><td>Fisher Scientific</td><td>08-100-240</td><td>Protocol Step 7.4.</td></tr><tr><td>hESC Media**</td><td></td><td></td><td>Protocol Step 6.11.&lt;br/&gt; components should be mixed and then filter sterilized. Media can be kept at 4 &amp;deg;C for up to two weeks. When warming media, do not leave at 37 &amp;deg;C longer than 15 min</td></tr><tr><td>DMEM-F12 50/50 media</td><td>Invitrogen</td><td>&amp;nbsp;11330-032</td><td>Final Concentration</td></tr><tr><td>KO replacement serum (KO-SR)</td><td>Invitrogen</td><td>10828-028</td><td>0.2</td></tr><tr><td>200 mM L-glutamine</td><td>Invitrogen</td><td>25030-081</td><td>1 mM</td></tr><tr><td>55 mM &amp;szlig;-mercaptoethanol</td><td>Invitrogen</td><td>21985-023</td><td>0.1 mM</td></tr><tr><td>100x non-essential amino acids</td><td>Invitrogen</td><td>11140-050</td><td>1x</td></tr><tr><td>2 &amp;micro;g/ml Basic-Fibroblast Growth Factor (b-FGF)</td><td>Invitrogen</td><td>13256-029</td><td>4 ng/ml</td></tr></tbody></table>

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

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 "black hole" 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. The ar...

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 ...

The overall goal of this procedure is to grow nasal epithelial cells obtained from children and reprogram them into induced pluripotent stem cells. This method can help answer key questions in the field of aerated disease such as how asthma develops. The main advantage of this technique is that it collects airway epithelial cells from children without stressful procedures.In culture, these are in a homogeneous population, and serves as an excellent source to generate induced pluripotent stem cells or iPSCs. We first had the idea for this study when we talked to Drs. Ji and Hershey about their study to collect meso-epithelial cells from children to investigate epigenetic variation in asthma and air pollution.Visual demonstration of these techniques is critical because nasal sampling and reprogramming to iPSCs of nasal epithelial cells are difficult techniques to learn since they're based on personal experience and not established protocols. Prior to the participant's visit, transfer two milliliters of fresh bronchial epithelial growth medium, abbreviated BEGM, to a 15 milliliter conical tube. Then, to this medium, add 20 microliters of a mixture of penicillin, streptomycin, and amphotericin b to a final concentration of 0.01%When the participant arrives, direct them to a chair to sit.Then open a cytology brush immediately before taking the sample, and do not touch it to any surfaces to ensure that it remains sterile. Proceed to instruct the participant to sit still, and tilt their head upwards towards the ceiling. If they are restless, have them sit on their hands so that they do not attempt to swat the brush away.Next, aim and insert the brush to the back of the nose where the passage narrows. Afterwards, remove the brush from the nostril by sliding it down with a twisting motion of the wrist. Continue by placing the brush in the previously prepared conical tube, and ensure that it is submerged in BEGM.Then cut off the excess portion of the brush and fasten the cap on the tube. After the sample has been collected, immediately place it in a beaker filled with warm water so that it is kept as close to 37 degrees Celsius as possible. Then transport it to the laboratory.Once in the laboratory, gently agitate the brush in the BEGM. Next, remove 10 microliters of the sample and to this, add 10 microliters of a live/dead cell stain. Proceed to introduce this mixture onto a hemocytometer.And count only the live cells under a microscope. Following cell counting, take 50 microliters of the sample and proceed to dilute it to 200 microliters with 1X PBS. Transfer the remainder of the undiluted cell mixture, along with the brush, to a regulated 37 degree Celsius waterbath until cell seeding.Proceed to add the PBS-diluted sample to a cytospin funnel attached to a glass slide. And spin the entire aparatus at 400 rpm for two minutes. Afterwards, remove the funnel and allow the slide to dry overnight in the dark.After staining the slide with Hema 3, as discussed in the text protocol, observe it under a microscope and count the cells. Identify epithelial cells by their unique characteristics. A columnar shape, cilia, and one round nucleus, and determine the percentage of these cells in the preparation.To begin seeding, gently swirl the brush inside the conical within a certified tissue culture hood, and then remove it from the tube. Afterwards, gently rotate the brush on the inner surface of a T25 flask, previously coated with bovine dermal collagen, taking care not to scratch the coating. Then discard the brush in an appropriate biohazard container.Slowly pipet the remaining cells in BEGM from the conical into the T25 flask, at least 7 times 10 to the fifth cells. Proceed to observe the cells under an inverted microscope. After culturing and passaging cells, as described in the text, proceed to plate 5 times 10 to the fifth nasal epithelial cells in two milliliters of BEGM per well of a six well tissue culture plate.Then incubate the cells for 24 hours at 37 degrees Celsius and 5%carbon dioxide. Following incubation, assure that cells are robustly proliferating. Then add Polybrene to each BEGM-containing well to a final concentration of eight micrograms per milliliter under a fume hood.Proceed to transduce the nasal epithelial cells by adding lentiviral particles, expressing Oct4, Sox2, Klf4, and c-Myc to the medium in each well as discussed in the text protocol. After incubating the transduced cells for approximately three hours, remove the virus-containing medium and discard it, using institutionally approved procedures. Then replace this medium with BEGM, and return the transduced nasal epithelial cells to the incubator.After three days, replace the medium with fresh BEGM. And then incubate the transduced cells under the same conditions. The next day, add 0.1%gelatin solution to two wells of a six-well plate in order to begin preparing mouse embryonic fibroblast-coated plates.Then cover the plate with its corresponding lid, and incubate it overnight at 37 degrees Celsius. Proceed on day five, to thaw a vial containing approximately 5 times 10 to the fifth inactivated mouse embryonic fibroblasts, abbreviated as MEFs. Then place these cells into 10 milliliters of MEF medium.And proceed to spin them at 200 x g for five minutes. Afterwards, aspirate the supernatant and resuspend the MEFs in the same medium. Once these cells have been counted, aspirate the gelatin solution from the previously prepared six-well plate.Next, add 1.87 times 10 to fifth MEFs into each gelatin-coated well of the plate, and incubate it at 37 degrees Celsius for a minimum of 24 hours. Then on day six of culture, aspirate the spent medium from the transduced cells and wash them in 1X PBS. Next, add one milliliter of 0.05%trypsin solution to each well of the plate.After the cells are detached, proceed to neutralize with trypsin neutralizing solution or TNS, then collect and centrifuge the cells as previously discussed. Following centrifugation, aspirate the supernatant, and resuspend the cells in four milliliters of BEGM, supplemented with penicillin and streptomycin. Proceed to introduce two milliliters of this cell suspension into each of gelatin-and MEF-coated wells of the previously prepared plate.After incubating the cells overnight, replace the BEGM with 2.5 milliliters of standard human embryonic stem cell, abbreviated as hESC, medium, containing four nanograms per milliliter basic fibroblast growth factor. Proceed to culture the cells and feed them daily with fresh hESC medium until hESC-like colonies, harboring putative induced pluripotent stem cells, or iPSCs, are observed in the cultures. Manually excise, and transfer these colonies to separate wells of a 24-well plate in order to culture and expand them as separate lines in a feeder-free system.Initially, nasal samples are a mix of cell types and debris. However, following culture and medium changes, epithelial cells become predominent in these preparations, as shown in this picture take one day after cell passaging. Here, epithelital cells, growing to confluence, demonstrate large nuclei and stretched cytoplasm.Due to the properties of the lentiviral particles used in this study, when such nasal epithelial cells are transduced, they express the tdTomato fluorescent marker, as demonstrated by the upper right image of cells, four days post-transduction. A comparison with the corresponding bright-field picture reveals that not every cell demonstrates tdTomato signal as evidenced by the merged image on the bottom. Thus, not every cell is transduced.However, transduction efficiency is approximately 90%When transduced nasal epithelial cells are cultured as described in the protocol, they give rise to iPSC colonies. Following iPSC colony harvest, and culture in standard feeder-free conditions, individual cells in such colonies demonstrated typical hESC-like morphology, such as a round shape, large nucleus, and high nucleus to cytoplasm ratio as demonstrated by this magnified image. Once mastered, this technique can be done in one and a half hours, if performed properly.When attempting this procedure, it's important to remember to take the sample correctly, keep the cells at 37 degrees Celsius, and plate them as quickly as possible at the correct density. Following this procedure, other methods like setting up an air-liquid interface using primary nasal epithelial cells can be performed in order to answer additional questions like how the air we respond to allergens and pathogens. After its development, this technique paved the way for researchers to model diseases such as asthma, COPD, and cystic fibrosis.This technique also helps study environmental effects on lung development in the in vitro human system. After watching this video, you should have a good idea of how to cultivate nasal epithelial cells from children and generate iPSCs from this population. Since we work with human embryonic stem cells and induced pluripotent stem cells, remember to always adhere to institutional and national guidelines.

cultivate-primary-nasal-epithelial-cells-from-children-reprogram-into

Research

JoVE Journal

Developmental Biology

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.

Isolating Nasal Olfactory Stem Cells from Rodents or Humans

Nasal epithelial cells, obtained through superficial scrape biopsy of human volunteers, are expanded and transferred onto tissue culture inserts. Upon reaching confluency, cells are grown at air liquid interface, yielding cultures of ciliated and non-ciliated cells. Differentiated nasal epithelial cell cultures provide viable experimental models for studying the respiratory mucosa.

Culturing of Human Nasal Epithelial Cells at the Air Liquid Interface

Generation of induced pluripotent stem cells provides fascinating prospects for the derivation of autologous transplants. However, progression through a pluripotent state and laborious re-differentiation still hinders clinical translation. Here we describe the derivation of adult human fibroblasts and their direct conversion into induced neural progenitor cells and the subsequent differentiation into neural lineages.

Derivation of Adult Human Fibroblasts and their Direct Conversion into Expandable Neural Progenitor Cells

Expansion of human pediatric esophageal epithelial cells utilizing conditional reprogramming provides investigators with a patient-specific population of cells that can be utilized for engineering esophageal constructs for autologous implantation to treat defects or injury and serve as a reservoir for therapeutic screening assays.

Conditional Reprogramming of Pediatric Human Esophageal Epithelial Cells for Use in Tissue Engineering and Disease Investigation

This protocol describes the reprogramming of primary amniotic fluid and membrane mesenchymal stem cells into induced pluripotent stem cells using a non-integrating episomal approach in fully chemically defined conditions. Procedures of extraction, culture, reprogramming, and characterization of the resulting induced pluripotent stem cells by stringent methods are detailed.

Reprogramming Primary Amniotic Fluid and Membrane Cells to Pluripotency in Xeno-free Conditions

Direct neuronal reprogramming generates neurons that maintain the age of the starting somatic cell. Here, we describe a single vector-based method to generate induced neurons from dermal fibroblasts obtained from adult human donors.

Simple Generation of a High Yield Culture of Induced Neurons from Human Adult Skin Fibroblasts

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.

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

Cell reprogramming requires the introduction of key genes, which regulate and maintain the pluripotent cell state. The protocol described enables the formation of induced pluripotent stem cells (iPSCs) colonies from human dermal fibroblasts without viral/integrating methods but using non-modified RNAs (NM-RNAs) combined with immune evasion factors reducing cellular defense mechanisms.

Isolation of Adult Human Dermal Fibroblasts from Abdominal Skin and Generation of Induced Pluripotent Stem Cells Using a Non-Integrating Method

Here we describe the isolation, amplification, and differentiation of primary human nasal epithelial (HNE) cells at the air-liquid interface and a biobanking protocol allowing to successfully freeze and then thaw amplified HNE. The protocol analyzes electrophysiological properties of differentiated HNE cells and CFTR-related chloride secretion correction upon different modulator treatments.

Primary Human Nasal Epithelial Cells: Biobanking in the Context of Precision Medicine

Recent advances in human induced pluripotent stem cell differentiation protocols allow for the stepwise derivation of organ-specific cell types. Here, we provide detailed steps for the maintenance and expansion of iPSC-derived airway basal cells and their differentiation into a mucociliary epithelium in air-liquid interface cultures.

Generation of Airway Epithelial Cell Air-Liquid Interface Cultures from Human Pluripotent Stem Cells

Learn to cultivate primary nasal epithelial cells from children and reprogram them into induced pluripotent stem cells using non-invasive sampling. This method enables asthma research and disease modeling without stressful procedures.

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

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