Materials and Reagents

Pipette tips (MidSci, Avant low binding tips)
35-mm cell culture dish (Thermo Fisher Scientific, catalog number: 153066 )
6-well plates (Corning, Falcon^®, catalog number: 353934 )
100 mm cell culture dish (Thermo Fisher Scientific, Thermo Scientific^TM, catalog number: 150464 )
NIH-3T3 cells (ATCC, catalog number: CRL-1658 )
3-5 week old K12^rtTA/rtTA/TetO-Cre/Rosa^mTmG (see Notes)
Ketamine/HCl 100 mg/ml (KetaJect; Henry Schein Animal Health, catalog number: 010177 )
Xylazine AnaSed^® 100 mg/ml (Santa Cruz Biotechnology, catalog number: sc-362949Rx )
Collagenase A (Sigma-Aldrich, Roche Diagnostics, catalog number: 10103578001 )
Dispase II (Sigma-Aldrich, catalog number: 4942078001)
Manufacturer: Roche Diagnostics, catalog number: 04942078001 .
Mitomycin C (Sigma-Aldrich, catalog number: M7949-2MG )
Phosphate buffered saline (PBS)
Trypsin (2.5%) (Thermo Fisher Scientific, Gibco^TM, catalog number: 15090046 )
Versene (Thermo Fisher Scientific, Gibco^TM, catalog number: 15040066 )
Dulbecco’s modified Eagle medium (DMEM) without calcium and magnesium (Thermo Fisher Scientific, Gibco^TM, catalog number: 21068028 )
Ham’s F12 Nutrient Mix (Thermo Fisher Scientific, Gibco^TM, catalog number: 11765047 )
Fetal Bovine Serum (Thermo Fisher Scientific, Gibco^TM, catalog number: 10082147 )
Human recombinant epidermal growth factor (Merck, catalog number: GF144 )
L-Glutamine (Thermo Fisher Scientific, Gibco^TM, catalog number: 25030081 )
Calcium Chloride solution 1 M (Sigma-Aldrich, catalog number: 21115 )
Human corneal growth supplement (Thermo Fisher Scientific, Gibco^TM, catalog number: S0095 )
Penicillin-streptomycin (10,000 U/ml) (Thermo Fisher Scientific, Gibco^TM, catalog number: 15140148 )
Amphotericin B (Thermo Fisher Scientific, catalog number: 15290026 )
Dulbecco’s modified Eagle medium (DMEM) high glucose (Thermo Fisher Scientific, Gibco^TM, catalog number: 11960044 )
Stem Cell Media (see Recipes)
3T3 media (see Recipes)

Equipment

Pipettes
Microdissection scissors (Fine Science Tools, catalog number: 15000-00 )
Forceps (Fine Science Tools, catalog number: 11252-23 )
Scissors (Fine Science Tools, catalog number: 14060-09 )
Hemocytometer (Hausser Scientific, catalog number: 3200 )
Dissecting Scope (ZEISS, model: Stemi DV4 )
BSL2 Laminar flow hood (Thermo Fisher Scientific, Thermo Scientific^TM, model: 1300 Series A2 , catalog number: 1387)
CO₂ incubator (Thermo Fisher Scientific, Thermo Scientic^TM, model: NAPCO Series 8000 WJ )
Centrifuge (Hettich, model: Rotina 35 )
Inverted fluorescent microscope (Zeiss Observer Z1 with an apotome attachment) (ZEISS, model: AxioObserver Z1 )

Software

AxioVison 4.7
ImageJ

Procedure

Removal of vibrissae (Figure 1)
1. Sacrifice 3-5 weeks old K12^rtTA/rtTA/TetO-Cre/Rosa^mTmG mice with ketamine/xylazine injection followed by cervical dislocation.
2. Remove the lip pad containing vibrissae with scissors and place in Stem Cell Media.
3. Under a dissecting microscope, remove the subcutaneous fat and connective tissue with forceps and scissors to expose the rows of vibrissae.
4. Remove individual vibrissae by pulling away from the pad using fine forceps.
  
  Figure 1. Isolation of the hair follicle bulge region from K12^rtTA/rtTA/TetO-Cre/Rosa^mTmG reporter mice. A. Diagram of the triple transgenic mouse model showing the inducible, tissue-specific nature of the system. B. Scheme showing isolation of the hair follicle bulge region. bg–bulge region. Parts Reprinted with permission from Blazejewska et al., 2009.
Isolation of hair follicle-derived stem cells
1. Expose the epithelial cores of the vibrissae by cutting the collagen capsule, loosening it from the core and pulling it down along the hair shaft.
2. Section the epithelial cores into three portions (Figure 2). Place the middle portion containing the hair follicle bulge region into a 35-mm dish containing 2 ml of collagenase (2 mg/ml) and incubate at 37 °C with 5% CO₂ for 1 h in order to remove any residual mesenchymal remnants of the capsule.
3. Transfer the partially digested hair follicle bulge region to another 35-mm dish containing 5 ml dispase/trypsin (2.4 U/0.05%) solution and digest for 1.5 h at 37 °C with 5% CO₂ in a humidified chamber to obtain a single-cell suspension of epithelial cells.
  
  Figure 2. Scheme for the clonal growth assay. The bulge region of the hair follicle is separated via mechanical dissection and enzymatically dissociated. The cells are plated on a 3T3 feeder layer and allowed to form holoclones (white circle at 2 weeks). The red channel depicts the membrane-bound tomato red fluorescence as these cells were derived from the reporter mice described in Figure 1.
Preparing feeder layer
1. Add Mitomycin C (40 μg/ml) to a 70% confluent dish of NIH3T3 cells and incubate in a humidified chamber at 37 °C with 5% CO₂ for 2 h.
2. Replace the Mitomycin C with 3T3 media.
3. Trypsinize and seed at 2 x 10⁵ cells per well of a 6-well culture plate.
Expansion/clonal growth assay
1. Enrich stem and progenitor cells by seeding at 1 x 10³ cells/cm² onto a Mitomycin C inactivated NIH 3T3 feeder layer in a 6-well culture dish (see above).
2. Cultivate for 14-21 days in a humidified chamber at 37 °C with 5% CO₂ to obtain holoclones (Figure 2). Change the medium every 2 days. Cultivate until holoclones are obtained. These will be large colonies containing tightly packed cells.
Subcultivation of hair follicle stem cells
1. Remove the 3T3 feeder layer with Versene for 60 sec at room temperature.
2. Wash 2 times with phosphate buffered saline (PBS).
3. Remove the attached holoclones with trypsin (0.25% Trypsin-EDTA) for 15 min at 37 °C with 5% CO₂.
4. Centrifuge at 170 x g for 5 min.
5. Cells are ready for application of choice (Note 6).

Data analysis

The conditions provided in this protocol have been optimized to obtain holoclones, which were assessed based on the size of the colony and colony forming efficiency. A detailed analysis of the isolation and clonal expansion of the hair follicle stem cells can be found at Blazejewska et al., 2009. (Stem Cells 2009 27(3):642-652)

Notes

Perform all cell culture work in a class II biological safety cabinet.
All animal protocols were approved by the Institutional Animal Care and Use Committee at the University of Cincinnati. Genetically modified mouse lines Krt12rtTA (Chikama et al., 2005), TetO-cre (Perl et al., 2002) and Gt(ROSA)26Sortm4(ACTB-tdTomato,-EGFP)Luo/J(ROSAmTmG) (Muzumdar et al., 2007) have been previously described. Compound transgenic mice were generated by breeding individual mouse lines to create K12^rtTA/rtTA/TetO-Cre/Rosa^mTmG. This dual reporter mouse model uses the keratin 12 promoter (corneal epithelium specific) to drive the expression of the Tet-on system. In conjunction with doxycycline and cre, the membrane tomato red hair follicle stem cells will turn green if they have differentiated into corneal epithelial cells.
Take care when removing the hair follicle and exposing the epithelial cores as to not damage the bulge region stem cells with the forceps.
All cell counts were performed using a hemocytometer.
The purity of the hair follicle stem cell cultures could be assessed in a parallel experiment by examining the expression of Krt15.
Bio-protocol title “Murine Hair Follicle Derived Stem Cell Transplantation onto the Cornea Using a Fibrin Carrier” (Call et al., 2018) demonstrates the use of the bulge derived hair follicle stem cells to treat a mouse model of limbal stem cell deficiency.

Recipes

Stem cell media
3 parts DMEM/High glucose without Ca²⁺ or Mg²⁺1 part Ham’s F12
10% FBS
10 ng/ml EGF
500 mg/L L-glutamine
0.4 mM calcium chloride
1x human corneal growth supplement
10,000 U/ml penicillin
10,000 μg/ml streptomycin
25 μg/ml amphotericin B
3T3 media
DMEM-high glucose
10% FBS
10,000 U/ml penicillin
10,000 U/ml streptomycin
25 μg/ml amphotericin B

Acknowledgments

This study was supported in part by grants from the NIH/NEI EY011845, Ohio Lions Eye Research Foundation to W.W.K. This work was adapted from Blazejewska et al., 2009 in Stem Cells. Authors do not have any conflicts of interest.

References

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