Plucked hairs from healthy donors were obtained from the occipital area of the scalp. The hairs were removed with depilation forceps. All human material was handled according to the Dutch Medical Treatment Agreement Act (Dutch Civil Code, Book 7, Section 7.7.5, article 7:467; http://www.dutchcivillaw.com/legislation/dcctitle7777.htm). Intact hair follicles (HFs) in the anagen phase were selected under a dissection microscope, and placed in DMEM/Ham’s F-12 1:1 (Biochrom AG, Berlin, Germany) containing 1 % GlutaMAX (100x; Life Technologies, Carlsbad, CA, USA) and 1 % Antibiotic Antimycotic Solution (100x; Sigma-Aldrich, St. Louis, MO, USA) (Fig. 1a). The HFs were processed the next morning.
Isolation and cultivation of HFBSCs
Isolation of HF stem cells was according to Sieber-Blum et al. (2004) with minor changes. Briefly, connective tissue (if present) was removed from the HF and the bulge-containing area was dissected out just below the sebaceous gland and well above the bulb (Fig. 1a). Then, a longitudinal section along the tissue of the bulge was made, to cause the tissue to unfold. During these procedures, care has to be taken to avoid dehydration of the HF. Before the start of the culture, tissue culture 12-well plates (TPP; Trasadingen, Switzerland) were coated with poly-d-lysine (PDL; Sigma-Aldrich) diluted in sterile demi water (1:10) at 37 °C and 5 % CO2 for 1 h. Then the PDL solution was removed and the wells air-dried under sterile conditions. Prior to usage, the PDL matrix was rehydrated with basic growth medium (BGM, 37 °C, 30 min). BGM consisted of DMEM/Ham’s F-12 1:1, containing 1 % GlutaMAX, 1 % Antibiotic Antimycotic Solution, supplemented with 10 % fetal bovine serum Gold (FBS; Life Technologies), 2 % B-27 Supplement without vitamin A (50x; Life Technologies), 1 % N-2 MAX Media Supplement (100x; R&D Systems, Minneapolis, MN, USA), recombinant human Fibroblast Growth Factor-basic (rhFGF-basic; 20 ng/ml; R&D Systems), and recombinant human Epidermal Growth Factor (rhEGF; 20 ng/ml; R&D Systems). After rehydration, the BGM was poured out of the wells, and one HF-bulge was placed in each well. The HFs were carefully pressed on the bottom of the well using a forceps. Subsequently, three incubation periods in a small drop of medium allowed the HF to attach to the matrix. Incubation was done at 37 °C and 5 % CO2 for 75 min. If necessary, some medium was added. Finally, 500 µl of freshly prepared BGM was added cautiously. The primary culture was established by the outgrowth of HF stem cells from the bulge, usually at 8–10 days after the start of the culturing. After 1 week of culturing, a complete medium change was performed, followed by replacement of half of the medium every other day. Three to four days after the start of outgrowth, the HF bulge was removed and some of the cultures were fixed with 1 % formaldehyde in PBS (FA) for immunohistochemical analysis of neural crest markers.
Expansion and cryopreservation
After removal of the bulge, cells were grown to 60–70 % confluence and enzymatically detached using pre-warmed 0.05 % trypsin–EDTA (Life Technologies) at 37 °C for precisely 2 min. Trypsinization was stopped by the addition of DMEM/HAM’s F-12 1:1 supplemented with 10 % FBS. The cells were centrifuged at 280×g for 10 min, and the cell pellet was suspended in 1 ml BGM. After cell counting (Logos Biosystems, Anyang-City, Korea), the cells were seeded at expansion density (2.5 × 103 cells per cm2) in a PDL-coated dish and allowed to expand until 60–70 % confluence. In general, cells were passaged three to four times. Each period of time prior to passaging was about 1 week. Doubling times were calculated at passages 2 and 3, using the site: Roth V. 2006 Doubling Time Computing, Available from: http://www.doubling-time.com/compute.php (Kim et al. 2011).
In addition, a portion of the cells was frozen at −80 °C at a concentration of 1 × 106 cells/ml in 90 % FBS with 10 % dimethyl sulfoxide (Sigma-Aldrich). After storage and thawing, the cells were suspended in 5 ml BGM, centrifuged, collected, suspended in BGM, carefully triturated, seeded at expansion density, and cultured at 37 °C and 5 % CO2.
Simulation of the transplantation procedure: ejection of cells
After cryopreservation, cells were cultured at 37 °C and 5 % CO2. After 1 week cells were enzymatically detached and centrifuged at 280×g for 10 min. They were suspended at a density of ~4.0 × 106 cells/ml in BGM medium and carefully triturated. Subsequently, 10 μl of the cell suspension was loaded into a 100 μl syringe with a 30 gauge needle and injected into a 1 ml Eppendorf tube using a programmable syringe pump (Prosense, Oosterhout, The Netherlands); settings: diameter 4.699 mm—rate 0.5 ml/min. Both cultured and cryopreserved cells were subjected to shear stress.
Viability was assessed using the trypan blue test. Trypan blue staining is based on the principle that live cells possess intact cell membranes that exclude the dye, whereas dead cells do not (Strober 2001). A 1:1 dilution of cell suspension and 0.4 % trypan blue was incubated for 2 min at room temperature. Next, the stained cells were counted using a Neubauer haemocytometer chamber and calculated using the following formula: vital cell rate (%) = number of vital cells/(number of vital cells + number of dead cells) × 100 %. Cells that had been cultured but not injected served as controls.
The paired, two-tailed Student’s t test was used to estimate the difference between the control and injected cells. The unpaired, two-tailed Student’s t test was used to estimate the difference between control cells and both cryopreserved and injected cryopreserved cells.
Neural differentiation of HFBSCs
Following outgrowth, expansion, and cryopreservation of HFBSCs, 2.5 × 105 cells in 500 µl of BGM were seeded per well of a 12-well plate. The cells were seeded via the side into PDL-coated wells containing PDL-coated cover glasses (Thermo Scientific, Waltham, MA, USA). It was essential in all the described procedures to prevent the cover glass sticking to the bottom of the well. Prior to PDL coating, the cover glasses were etched in 85 % phosphoric acid (Merck Millipore, Darmstadt, Germany) for 12 h (Beaudoin III et al. 2012). Subsequently, acid-treated cover glasses were rinsed extensively in ultrapure water and subjected to a graded series of 70, 90, and 96 % ethanol. Cover glasses were stored in 96 % ethanol. The 12-well plates and etched cover glasses were coated separately with PDL as described previously. After seeding, the cells were cultured at 37 °C and 5 % CO2, while their settlement underneath the cover glass was observed daily. When an appropriate density was achieved, i.e., 5 to 10 cells in one field of view (FOV, 10× magnification, an area of ~3.5 mm2), differentiation was induced by removal of 250 µl medium and replacement with 300 µl cAMP-containing induction medium (IM) (Jarmalavičiūtė et al. 2013). IM consisted of DMEM/Ham’s F-12 1:1 supplemented with 1.5 mM cAMP (Sigma-Aldrich), 1 % glutamax (Life Technologies), 10 ng/ml NGF, 10 ng/ml GDNF, 10 ng/ml BDNF (all from R&D Systems) and 2 % B27 + VitA (Life Technologies). If the appropriate density was not achieved, half of the medium was replaced with fresh BGM every other day. After IM was added, the cultures were allowed to differentiate for at least 60 h without disturbance due to opening of the incubator or observation of the cells. Subsequently, 250 µl medium was removed and again substituted with 300 µl IM. Thereafter, the medium was replenished with IM every other day. Cultures were observed for morphological changes on a daily basis. If no neuronal morphologies appeared after 7 days of differentiation, the culture underwent another period without disturbance in IM, and the above-mentioned differentiation procedure was followed again. After differentiation for 7–14 days, the cover glass was carefully removed, because cells were not only attached to the bottom of the well but sometimes also to the underside of the cover glass. The cells on the bottom of the well were fixed in 1 % FA for 15 min and processed for immunohistochemistry. Fixed cells were stored at 4 °C for a maximum period of 2 weeks.
Glial differentiation of HFBSCs
After expansion, a volume of 500 µl of BGM (without FBS) containing 1 × 105 cells was pipetted into each well of a 12-well plate. The cells were seeded via the side into uncoated wells which contained PDL-coated cover glasses. During culture, the cover glass should not stick to the bottom of the well. On the next day, half of the medium was replaced with serum-free BGM (Pacey et al. 2006). Cells were observed every other day to follow settlement and changes in morphology. The medium was exchanged with serum-free medium every other day until an average density of 10–20 cells per FOV underneath the cover glass was reached. Then half of the medium was replaced by IM. The cells underneath the cover glass usually adopt glial-like morphologies after 3–4 days of induction. After induction, the medium was exchanged once a week with IM. Cultures were maintained until networks of cells were observed. Because the networks were attached to the underside of the cover glass, the cover glasses were removed and placed upside down in another well of a 12-well dish. The cells were fixed in 1 % FA for 15 min.
Prior to immunohistochemistry, the cells were washed with 0.05 % Tween-20 in PBS for 5 min and permeabilized in 0.1 % Triton X-100 in PBS for 10 min. Cells were treated with blocking solution consisting of 5 % non-immune serum in 0.05 % Tween-20 in PBS for 30 min. Afterwards, the cells were incubated with the primary antibodies in blocking solution at 4 °C overnight (Petersen and Pedersen 2013). The primary antibodies used were: anti-Nestin (1:500, Biosensis, Thebarton, South Australia), anti-SLUG (1:125, Abcam (Cambridge, U.K.) ab 27568), anti-AP-2α (1:100, Santa Cruz Biotechnology (Santa Cruz, CA, USA) sc-53164), anti-SOX9 (1:500, Millipore (Billerica, MA, USA) AB5535), anti-SOX10 (1:200, Santa Cruz sc-17342), anti-β-III-tubulin (1:200, Abcam ab18207), anti-synapsin-1 (1:200, Abcam ab8), anti-myelin protein zero (MPZ; 1:200, Neuromics (Minneapolis, MN, USA) Ch23009), and anti-Krox20 (1:100, Covance, New York, NY, USA). The secondary fluorochrome-conjugated antibodies were diluted 1:500 in blocking buffer, and the cells were incubated at room temperature for 1 h. The secondary antibodies were conjugated with either Alexa Fluor 488 or Alexa Fluor 555 (Life Technologies). Nuclear counterstaining was performed with 1:1000 DAPI (Life Technologies, D3571) in PBS. The cells were covered with Vectashield (Vector Laboratories, Burlingame, CA, USA). Omission of the primary antibody served as a control for false cross-reactivity of the secondary antibody. Pertinent positive cell or tissue controls were used: RT4-D6P2T, a rat Schwann cell line (ATCC, Manassas, VA, USA) for anti-nestin, -SOX9, -Krox20, and -MPZ, the Melan-Ink4a cell line (Wellcome Trust Functional Genomics Cell Bank, London, UK) for anti-SOX10 and -β-III-tubulin (Locher et al. 2013), the SKBR3-breast cancer cell line for anti-SLUG and Ap-2α, and mouse brain for anti-synapsin-1.
Fluorescence imaging was performed using fluorescence microscopy (Olympus IX70) in combination with the LAS AF microscope software (Version 1.9.0 build 1633, Leica Microsystems). The data were corrected for background staining and normalized using the quantification method of the software. Only those immunostainings showing a peak maximum at emission of at least two times higher than the background were considered to be significantly positive. Pictures were processed using Adobe Photoshop CS6 Extended (Version: 13.0 x64, Adobe Systems Incorporated, San Jose, CA, USA).