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><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

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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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

Generation of Induced Pluripotent Stem Cells from Frozen Buffy Coats using Non-integrating Episomal Plasmids

Somatic cells can be reprogrammed into induced pluripotent stem cells (iPSCs) by forcing the expression of four transcription factors (Oct-4, Sox-2, Klf-4, and c-Myc), typically expressed by human embryonic stem cells (hESCs). Due to their similarity with hESCs, iPSCs have become an important tool for potential patient-specific regenerative medicine, avoiding ethical issues associated with hESCs. In order to obtain cells suitable for clinical application, transgene-free iPSCs need to be generated to avoid transgene reactivation, altered gene expression and misguided differentiation. Moreover, a highly efficient and inexpensive reprogramming method is necessary to derive sufficient iPSCs for therapeutic purposes. Given this need, an efficient non-integrating episomal plasmid approach is the preferable choice for iPSC derivation. Currently the most common cell type used for reprogramming purposes are fibroblasts, the isolation of which requires tissue biopsy, an invasive surgical procedure for the patient. Therefore, human peripheral blood represents the most accessible and least invasive tissue for iPSC generation.
In this study, a cost-effective and viral-free protocol using non-integrating episomal plasmids is reported for the generation of iPSCs from human peripheral blood mononuclear cells (PBMNCs) obtained from frozen buffy coats after whole blood centrifugation and without density gradient separation.

Induced pluripotent stem cells (iPSCs) represent a source of patient-specific tissues for clinical applications and basic research. Here, we present a detailed protocol to reprogram human peripheral blood mononuclear cells (PBMNCs) obtained from frozen buffy coats into viral-free iPSCs using non-integrating episomal plasmids.

Somatic cells can be reprogrammed into induced pluripotent stem cells (iPSCs) by forcing the expression of four transcription factors (Oct-4, Sox-2, ...

Generation of Integration-free Induced Pluripotent Stem Cells from Human Peripheral Blood Mononuclear Cells Using Episomal Vectors

Induced Pluripotent Stem Cells (iPSCs) hold great promise for disease modeling and regenerative therapies. We previously reported the use of Episomal Vectors (EV) to generate integration-free iPSCs from peripheral blood mononuclear cells (PB MNCs). The episomal vectors used are DNA plasmids incorporated with oriP and EBNA1 elements from the Epstein-Barr (EB) virus, which&#160;allow for replication and long-term retainment of plasmids in mammalian cells, respectively. With further optimization, thousands of iPSC colonies can be obtained from 1 mL of peripheral blood. Two critical factors for achieving high reprogramming efficiencies&#160;are: 1) the use of a 2A &#34;self-cleavage&#34; peptide to link OCT4 and SOX2, thus achieving equimolar expression of the two factors; 2) the use of two vectors to express MYC and KLF4 individually. Here we describe a step-by-step protocol for generating integration-free iPSCs from adult peripheral blood samples. The generated iPSCs are integration-free as residual episomal plasmids are undetectable after five passages. Although the reprogramming efficiency is comparable to that of Sendai Virus (SV) vectors, EV plasmids are considerably more economical than the commercially available SV vectors. This affordable EV reprogramming system holds potential for clinical applications in regenerative medicine and provides an approach for the direct reprogramming of PB MNCs to integration-free mesenchymal stem cells, neural stem cells, etc.

This protocol describes a detailed method for efficient generation of integration-free iPSCs from human adult peripheral blood cells. With the use of four oriP/EBNA-based episomal vectors to express the reprogramming factors, KLF4, MYC, BCL-XL, or OCT4 and SOX2, thousands of iPSC colonies can be obtained from 1 mL of peripheral blood.

Induced Pluripotent Stem Cells (iPSCs) hold great promise for disease modeling and regenerative therapies. We previously reported the use of Episomal ...

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

Identifying and expanding patient-specific cells in culture for use in tissue engineering and disease investigation can be very challenging. Utilizing various types of stem cells to derive cell types of interest is often costly, time consuming and highly inefficient. Furthermore, undesired cell types must be removed prior to using this cell source, which requires another step in the process. In order to obtain enough esophageal epithelial cells to engineer the lumen of an esophageal construct or to screen therapeutic approaches for treating esophageal disease, native esophageal epithelial cells must be expanded without altering their gene expression or phenotype. Conditional reprogramming of esophageal epithelial tissue offers a promising approach to expanding patient-specific esophageal epithelial cells. Furthermore, these cells do not need to be sorted or purified and will return to a mature epithelial state after removing them from conditional reprogramming culture. This technique has been described in many cancer screening studies and allows for indefinite expansion of these cells over multiple passages. The ability to perform esophageal screening assays would help revolutionize the treatment of pediatric esophageal diseases like eosinophilic esophagitis by identifying the trigger mechanism causing the patient's symptoms. For those patients who suffer from congenital defect, disease or injury of the esophagus, this cell source could be used as a means to seed a synthetic construct for implantation to repair or replace the affected region.

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.

Identifying and expanding patient-specific cells in culture for use in tissue engineering and disease investigation can be very challenging. Utilizing ...

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

Autologous cell-based therapies got a step closer to reality with the introduction of induced pluripotent stem cells. Fetal stem cells, such as amniotic fluid and membrane mesenchymal stem cells, represent a unique type of undifferentiated cells with promise in tissue engineering and for reprogramming into iPSC for future pediatric interventions and stem cell banking. The protocol presented here describes an optimized procedure for extracting and culturing primary amniotic fluid and membrane mesenchymal stem cells and generating episomal induced pluripotent stem cells from these cells in fully chemically defined culture conditions utilizing human recombinant vitronectin and the E8 medium. Characterization of the new lines by applying stringent methods &#8211; flow cytometry, confocal imaging, teratoma formation and transcriptional profiling &#8211; is also described. The newly generated lines express markers of embryonic stem cells &#8211; Oct3/4A, Nanog, Sox2, TRA-1-60, TRA-1-81, SSEA-4 &#8211; while being negative for the SSEA-1 marker. The stem cell lines form teratomas in scid-beige mice in 6-8 weeks and the teratomas contain tissues representative of all three germ layers. Transcriptional profiling of the lines by submitting global expression microarray data to a bioinformatic pluripotency assessment algorithm deemed all lines pluripotent and therefore, this approach is an attractive alternative to animal testing. The new iPSC lines can readily be used in downstream experiments involving the optimization of differentiation and tissue engineering.

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.

Autologous cell-based therapies got a step closer to reality with the introduction of induced pluripotent stem cells. Fetal stem cells, such as amniotic ...

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 ...

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

Generation of&#160;induced pluripotent stem cell (iPSCs) from adult skin fibroblasts and subsequent differentiation into somatic cells provides fascinating prospects for the derivation of autologous transplants that circumvent histocompatibility barriers. However, progression through a pluripotent state and subsequent complete differentiation into desired lineages remains a roadblock for the clinical translation of iPSC technology because of the associated neoplastic potential and genomic instability. Recently, we and others showed that somatic cells cannot only be converted into iPSCs but also into different types of multipotent somatic stem cells by using defined factors, thereby circumventing progression through the pluripotent state. In particular, the direct conversion of human fibroblasts into induced neural progenitor cells (iNPCs) heralds the possibility of a novel autologous cell source for various applications such as cell replacement, disease modeling and drug screening. Here, we describe the isolation of adult human primary fibroblasts by skin biopsy and their efficient direct conversion into iNPCs by timely restricted expression of Oct4, Sox2, Klf4, as well as c-Myc. Sox2-positive neuroepithelial colonies appear after 17 days of induction and iNPC lines can be established efficiently by monoclonal isolation and expansion. Precise adjustment of viral multiplicity of infection and supplementation of leukemia inhibitory factor during the induction phase represent critical factors to achieve conversion efficiencies of up to 0.2%. Thus far, patient-specific iNPC lines could be expanded for more than 12 passages and uniformly display morphological and molecular features of neural stem/progenitor cells, such as the expression of Nestin and Sox2. The iNPC lines can be differentiated into neurons and astrocytes as judged by staining against TUJ1 and GFAP, respectively. In conclusion, we report a robust protocol for the derivation and direct conversion of human fibroblasts into stably expandable neural progenitor cells that might provide a cellular source for biomedical applications such as autologous neural cell replacement and disease modeling.

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.

Generation of&#160;induced pluripotent stem cell (iPSCs) from adult skin fibroblasts and subsequent differentiation into somatic cells provides ...

Neuroscience

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

Diseases of the conducting airway such as asthma, cystic fibrosis (CF), primary ciliary dyskinesia (PCD), and viral respiratory infections are major causes of morbidity and mortality worldwide. In vitro platforms using human bronchial epithelial cells (HBECs) have been instrumental to our understanding of the airway epithelium in health and disease. Access to HBECs from individuals with rare genetic diseases or rare mutations is a bottleneck in lung research.
Induced pluripotent stem cells (iPSCs) are readily generated by "reprogramming" somatic cells and retain the unique genetic background of the individual donor. Recent advances allow for the directed differentiation of iPSCs to lung epithelial progenitor cells, alveolar type 2 cells, as well as the cells of the conducting airway epithelium via basal cells, the major airway stem cells.
Here we outline a protocol for the maintenance and expansion of iPSC-derived airway basal cells (hereafter iBCs) as well as their trilineage differentiation in air-liquid interface (ALI) cultures. iBCs are maintained and expanded as epithelial spheres suspended in droplets of extracellular matrix cultured in a primary basal cell medium supplemented with inhibitors of TGF-&#223; and BMP signaling pathways. iBCs within these epithelial spheres express key basal markers TP63 and NGFR, can be purified by fluorescence activated cell sorting (FACS), and when plated on porous membranes in standard ALI culture conditions, differentiate into a functional airway epithelium. ALI cultures derived from healthy donors are composed of basal, secretory and multiciliated cells and demonstrate epithelial barrier integrity, motile cilia, and mucus secretion. Cultures derived from individuals with CF or PCD recapitulate the dysfunctional CFTR-mediated chloride transport or immotile cilia, the respective disease-causing epithelial defects.
Here, we present a protocol for the generation of human cells that can be applied for modeling and understanding airway diseases.

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.

Diseases of the conducting airway such as asthma, cystic fibrosis (CF), primary ciliary dyskinesia (PCD), and viral respiratory infections are major ...

Selecting and Isolating Colonies of Human Induced Pluripotent Stem Cells Reprogrammed from Adult Fibroblasts

Herein we present a protocol of reprogramming human adult fibroblasts into human induced pluripotent stem cells (hiPSC) using retroviral vectors encoding Oct3/4, Sox2, Klf4 and c-myc (OSKM) in the presence of sodium butyrate 1-3. We used this method to reprogram late passage (&gt;p10) human adult fibroblasts derived from Friedreich's ataxia patient (GM03665, Coriell Repository). The reprogramming approach includes highly efficient transduction protocol using repetitive centrifugation of fibroblasts in the presence of virus-containing media. The reprogrammed hiPSC colonies were identified using live immunostaining for Tra-1-81, a surface marker of pluripotent cells, separated from non-reprogrammed fibroblasts and manually passaged 4,5. These hiPSC were then transferred to Matrigel plates and grown in feeder-free conditions, directly from the reprogramming plate. Starting from the first passage, hiPSC colonies demonstrate characteristic hES-like morphology. Using this protocol more than 70% of selected colonies can be successfully expanded and established into cell lines. The established hiPSC lines displayed characteristic pluripotency markers including surface markers TRA-1-60 and SSEA-4, as well as nuclear markers Oct3/4, Sox2 and Nanog. 
The protocol presented here has been established and tested using adult fibroblasts obtained from Friedreich's ataxia patients and control individuals 6, human newborn fibroblasts, as well as human keratinocytes.

We present a protocol for efficient reprogramming of human somatic cells into human induced pluripotent stem cells (hiPSC) using retroviral vectors encoding Oct3/4, Sox2, Klf4 and c-myc (OSKM) and identification of correctly reprogrammed hiPSC by live staining with Tra-1-81 antibody.

Herein we present a protocol of reprogramming human adult fibroblasts into human induced pluripotent stem cells (hiPSC) using retroviral vectors encoding ...

Biology

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

Induced pluripotent stem cells (iPSCs) could be considered, to date, a promising source of pluripotent cells for the management of currently untreatable diseases, for the reconstitution and regeneration of injured tissues and for the development of new drugs. Despite all the advantages related to the use of iPSCs, such as the low risk of rejection, the lessened ethical issues, and the possibility to obtain them from both young and old patients without any difference in their reprogramming potential, problems to overcome are still numerous. In fact, cell reprogramming conducted with viral and integrating viruses can cause infections and the introduction of required genes can induce a genomic instability of the recipient cell, impairing their use in clinic. In particular, there are many concerns about the use of c-Myc gene, well-known from several studies for its mutation-inducing activity. Fibroblasts have emerged as the suitable cell population for cellular reprogramming as they are easy to isolate and culture and are harvested by a minimally invasive skin punch biopsy. The protocol described here provides a detailed step-by-step description of the whole procedure, from sample processing to obtain cell cultures, choice of reagents and supplies, cleaning and preparation, to cell reprogramming by the means of a commercial non-modified RNAs (NM-RNAs)-based reprogramming kit. The chosen reprogramming kit allows an effective reprogramming of human dermal fibroblast to iPSCs and small colonies can be seen as early as 24 h after the first transfection, even with modifications with the respect to the standard datasheet. The reprogramming procedure used in this protocol offers the advantage of a safe reprogramming, without the risk of infections caused by viral vector-based methods, reduces the cellular defense mechanisms, and allows the generation of xeno-free iPSCs, all critical features that are mandatory for further clinical applications.

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.

Induced pluripotent stem cells (iPSCs) could be considered, to date, a promising source of pluripotent cells for the management of currently untreatable ...

Bioengineering

Culturing of Human Nasal Epithelial Cells at the Air Liquid Interface

In vitro models using human primary epithelial cells are essential in understanding key functions of the respiratory epithelium in the context of microbial infections or inhaled agents. Direct comparisons of cells obtained from diseased populations allow us to characterize different phenotypes and dissect the underlying mechanisms mediating changes in epithelial cell function. Culturing epithelial cells from the human tracheobronchial region has been well documented, but is limited by the availability of human lung tissue or invasiveness associated with obtaining the bronchial brushes biopsies. Nasal epithelial cells are obtained through much less invasive superficial nasal scrape biopsies and subjects can be biopsied multiple times with no significant side effects. Additionally, the nose is the entry point to the respiratory system and therefore one of the first sites to be exposed to any kind of air-borne stressor, such as microbial agents, pollutants, or allergens. 
Briefly, nasal epithelial cells obtained from human volunteers are expanded on coated tissue culture plates, and then transferred onto cell culture inserts. Upon reaching confluency, cells continue to be cultured at the air-liquid interface (ALI), for several weeks, which creates more physiologically relevant conditions. The ALI culture condition uses defined media leading to a differentiated epithelium that exhibits morphological and functional characteristics similar to the human nasal epithelium, with both ciliated and mucus producing cells. Tissue culture inserts with differentiated nasal epithelial cells can be manipulated in a variety of ways depending on the research questions (treatment with pharmacological agents, transduction with lentiviral vectors, exposure to gases, or infection with microbial agents) and analyzed for numerous different endpoints ranging from cellular and molecular pathways, functional changes, morphology, etc. 
In vitro models of differentiated human nasal epithelial cells will enable investigators to address novel and important research questions by using organotypic experimental models that largely mimic the nasal epithelium in vivo.

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.

In vitro models using human primary  epithelial cells are essential in understanding key functions of the respiratory  epithelium in the context of ...

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

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