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Method Article

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

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DOI:

10.3791/53814

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March 10th, 2016

 ,  ,  ,  , 
1Pyrosequencing Core, Cincinnati Children's Hospital, 2Division of Developmental Biology, Cincinnati Children's Hospital, 3Division of Pulmonary Medicine, Seattle Children's Hospital, 4Division of Asthma Research, Cincinnati Children's Hospital

In This Article

Summary

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).

Abstract

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.

Introduction

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.

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Protocol

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.

  1. Prepare a 15 ml conical fresh before the participant visit and add 2 ml BEGM (Bronchial Epithelial Cell Growth Medium) plus 20 µl sterile Penn/Strep/Fungizone (P/S/F) (0.01%).
  2. 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.
  3. 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.
  4. 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.
  5. 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.
  6. Keep samples as close to 37 °C as possible during transport using a beaker with warm water.

2. Cell Count and Cytospin

  1. Gently agitate the brush in BEGM and take 10 µl of the sample for a cell count using a hemacytometer. Add 10 µl of a live/dead stain and count only live cells under a microscope.
  2. Take 50 µl of the sample and dilute to 200 µl 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's instructions. Allow to dry O/N, preferably in the dark.
  3. Stain Slide with Hema 3 Stain
    1. 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
    2. 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
    3. Rinse with deionized water until water runs clear. Allow to dry O/N, preferably in the dark
  4. Look at slide under microscope and count cells, determine percentage of epithelial cells. Should be 90% or greater if sampling is good.

3. Seeding Cells

NOTE: Carry out all cell culture procedures in a proper and certified tissue culture hood using sterile technique.

  1. 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 °C for 2 hr, if necessary.
  2. 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 °C, but bring them to RT or 37 °C before seeding cells.
  3. Keep brush and cells in BEGM at 37 °C until ready to seed. Gently swish brush inside conical, but do not vortex.
    1. 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.
  4. 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.
  5. 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).

4. Cell Culture

  1. 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 µl sterile Penicilin/Streptomycin/Fungizone (P/S/F).
    NOTE: DO NOT aspirate the original 2 ml of media.
  2. 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.
  3. 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.

5. Passaging Cells

  1. 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.
  2. 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.
  3. 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)
  4. 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.

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.

  1. 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 µg/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.
    1. 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 "transducing units/ml" 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).
    2. 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.
  2. 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.
  3. 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).
  4. 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.
  5. 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 °C, or for a minimum of 24 hr.
  6. 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.
  7. 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.
  8. 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.
    1. 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.

7. Maintaining iPSCs in Culture

  1. 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.
  2. 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 °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.
  3. 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.
    1. 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.

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Results

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...

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Discussion

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...

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Disclosures

The authors have no conflicts of interest to disclose.

Acknowledgements

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

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Materials

List of materials used in this article
NameCompanyCatalog NumberComments
15 ml conicalFisher Scientific14-959-49DProtocol Step 1.1.
BEGMLonzaCC-3170Protocol Step 1.1.
Penn/Strep/FungicideLife Technologies15240-062Protocol Step 1.1.
Penn/StrepLife Technologies15140-122Protocol Step 4.a.
cytosoft cytology brushFisher Scientific22-263-357Protocol Step 1.2.
trypan blueFisher ScientificMT-25-900-CIProtocol Step 2.1.
hemacytometerFisher Scientific02-671-54Protocol Step 2.1.
PBSFisher ScientificBP2438-4Protocol Step 2.2.
Cytology Funnel ClipsFisher Scientific10-357Protocol Step 2.2.
cytospin funnelFisher Scientific23-640-320Protocol Step 2.2.
Cytospin 4Fisher ScientificA78300003Protocol Step 2.2.
blank slideFisher ScientificS95933Protocol Step 2.2.
hema 3 stain kitFisher Scientific22-122-911Protocol Step 2.2.
Bovine Dermal Colagen, type 1Life TechnologiesA1064401Protocol Step 3.2.
T25 flaskFisher Scientific08-772-45Protocol Step 3.3.
TrypsinLonzaCC-5012Protocol Step 5.2.
Trypsin Neutralizing SolutionLonzaCC-5002Protocol Step 5.2.
Fetal Bovine Serum (FBS), heat sterilized at 65 °C for 30 minSigma-AldrichF2442Protocol Step 5.5.
Dimethyl sulfoxide Hybri-Max™, sterile-filtered, BioReagent, suitable for hybridoma, ≥99.7%Sigma-AldrichD2650Protocol Step 5.5.
polycistonic lentivirus*e.g. MilliporeSCR511Protocol Step 6.4. 
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 
polybreneSanta Cruz Biotechnologysc-134220Protocol Step 6.4.
Irradiated CF1 MEFsGlobalStem GSC-6301GProtocol Step 6.4.
hESC mediaSee recipe included in protocolProtocol Step 6.11.
SB431542Stemgent04-0010Protocol Step 6.11.
PD0325901Stemgent04-0006Protocol Step 6.11.
Thiazovivin Stemgent04-0017Protocol Step 6.11.
hESC-qualified MatrigelBD Biosciences354277Protocol Step 6.13.
Corning plate, 6 wellFisher Scientific08-772-1BProtocol Step 6.13.
mTeSR1StemCell5850Protocol Step 6.13.
250 ml disposable filter flask (0.22 µm)FisherSCGP-U02-RE 
dispaseStemCell7923Protocol Step 7.3.
DMEM/F12Life Technologies11320-033Protocol Step 7.3.
cell lifterFisher Scientific08-100-240Protocol Step 7.4.
hESC Media**Protocol Step 6.11.
components should be mixed and then filter sterilized. Media can be kept at 4 °C for up to two weeks. When warming media, do not leave at 37 °C longer than 15 min
DMEM-F12 50/50 mediaInvitrogen 11330-032Final Concentration
KO replacement serum (KO-SR)Invitrogen10828-0280.2
200 mM L-glutamineInvitrogen25030-0811 mM
55 mM ß-mercaptoethanolInvitrogen21985-0230.1 mM
100x non-essential amino acidsInvitrogen11140-0501x
2 µg/ml Basic-Fibroblast Growth Factor (b-FGF)Invitrogen13256-0294 ng/ml

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Primary Cell CultureLentiviral TransductionReprogramming ProtocolPediatric Airway SamplinghESC MediumMEF CoatingColony ExpansionFeeder Free System
Cultivate Primary Nasal Epithelial Cells from Children and Reprogram into Induced Pluripotent Stem Cells
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