Normal human scalp specimens were taken from volunteers without systemic disease, who were undergoing plastic surgery and all subjects gave written informed consent. Biopsies were uniformly taken from the occipital scalp region of men who did not suffer from male-pattern alopecia or from any other diseases affecting the growth of hair and scalp. All tissue donors (ten patients) were male with average of 33 years. (range 26∼41 years). The tissue sample was transported to the laboratory in the media consisting of Dulbecco’s Modified Eagle Medium (DMEM) (Gibco Life Technologies, Gaithersburg, MD, USA) containing antimycotic agents to yield final concentrations of 15 units /ml penicillin, 15 mg/ml streptomycin, 30 ug/ml neomycin (Sigma, St Louis, MO, USA) and 100 ug/ml Normosin™ (Invivogen, San diego, CA, USA).

The sample tissue was rinsed in Hanks Bss to remove blood and debris and was placed in a 100-mm plastic tissue culture dish (Nunc, Rochester, NY, USA). The entire procedure was performed under a sterile laminar flow hood. Fat and connective tissue was cleared from the sample tissue. The rest of the tissue was minced with sterile scalpels into 1∼2 mm fragments and digested with Accumax™ (2%; Chemicon international Inc., CA, USA) and Dispase (0.4 mg/ml; Sigma.) in DMEM medium (Gibco Life Technologies) which consisted with 10% fetal bovine serum (Gibco Life Technologies) plus antibiotic and antimycotic agents (as above) for 30min at 37°C on a shaking incubator. And then the tissue washed with sterile PBS was digested with collagenase type IA (2 mg/ml; Sigma) in DMEM medium (Gibco Life Technologies) which consisted with 10% fetal bovine serum (Gibco Life Technologies) plus antibiotic and antimycotic agents (as above) for 30 min at 37°C on a shaking incubator. The tissue digest was vigorously pipetted. After gentle centrifugation (2,500 g, 5 min), the supernatant was removed, and the pellet was resuspended in Medium 199/Ham’s F12 (M199/F12; 1:1) medium which suplemented with 10 ng/ml human EGF, 20 ng/ml FGF, 5 mg/ml insulin, 0.5 mg/ml hydrocortisone, 2 mg/ml human transferin, 125 ug/ml bovine serum albumin, 10% fetal bovine serum (FBS) and antibiotic and antimycotic agents (as above) and distributed on a 25 cm² flask (Nunc). After 3 days, Culture medium of the hair follicle cells attached on the flask was changed to serum free medium (without FBS) and suspended cells on the flask were transfer to 75 cm² flask (Nunc) with complete culture medium (as above). As a same manner, the complete culture medium was changed to serum free medium (without FBS) after 3days and placed for 14 days at 37°C in a humidified atmosphere containing 5% CO₂. Each culture medium was carefully removed every 3 days and replaced by fresh culture medium. After 7 days in culture, all culture medium was changed to Defined keratinocytes serum free medium (Gibco Life Technologies). Atfter 2 weeks in primary culture, cells were collected by incubation with 0.5 mg/mL trypsin, 0.2 mg/mL EDTA (ethylenediamine tetraacetic acid) solution (Gibco Life Technologies), for 5 min at 37°C. After this incubation period, clusters of cells were released from the hair follicles. These were harvested by centrifugation at 300 g at 4°C for 10 min.

To remove cell clump before isolation, The cells were vigorously pipetted and added to a stacked sterile mesh sieve assembly with 50 Pre-Separation Filters (30 um size; Miltenyi Biotech, Bergish-Gladbach, Germany). After the human hair follicle cells collected were added to the top of the sieve assembly, cell aggragates or large particles from the samples were retained in mesh sieve while single cells passed through to the receptacle below. The single human hair follicle cells were resuspended cold phosphate buffered saline containing 0.5% bovine serum albumin (BSA) and 5 mm EDTA (MACS buffer), pH 7.4. and uniform, magnetic beads (Miltenyi Biotech), which were coated with a monoclonal antibody specific for human CD34, were used to separate CD34+ cells. The human hair follicle cells were resuspended to a concentration of 1×10⁸ cells in 300 ul PBS, 5 mM EDTA. These cells were incubated with 300 ul MACS buffer, 100 ul beads and blocking reagent (human IgG to prevent non-specific binding) per 10⁸ cells for 30 minutes at 4°C. After washing with MACS buffer, cells were loaded onto a MiniMACS column (Miltenyi Biotec Ltd, Bisley, Germany) held within a high-density magnetic field to retain CD34+ cells. Unbound cells (CD34− cells) were eluted with MACS buffer and retained CD34+ cells were eluted by washing with buffer after the column had been removed from the magnet. Cells in the MACS positive fraction were applied to a second column and the purification step repeated. Human hair follicle cells and cells from the MACS positive and negative fractions were assessed by cell counting and flow cytometry. Selected CD34+ cells were detached from the magnetic beads using the plunger. CD34+ and CD34− cells were resuspended in Iscove’s modified Dulbecco’s medium (IMDM) (Gibco Life Technologies).

To confirm the CD34+ and CD34− populations by FACS analysis, Each 2×10⁴ cells of the CD34+ and CD34− cells isolated as described above seeded in each well of 96 well plate. Cells were incubated for 40 min at room temperature with 1:10 final fluorescein isothiocyanate (FITC)-, phycoerythrin (PE)-conjugated mAb in PBS, 2% FCS. After washing, Cells were fixed with 1% paraformaldehyde in PBS before analysis on a flow-cytometry (FACSCalibur, BD Immunocytometry Systems, CA, USA). The mAbs were: CD34-FITC, CD45-FITC, CD24-PE, CD44-PE, CD133-PE, CD29-PE (BD Phar-Mingen, CA, USA). Controls did not include any mAb.

A total of 20 female BALB/c AnNCrjbji-nuBR mice (mean body weight 25.2±2.0 g) were obtained at 6 wks of age from Charles River (Boston, MA, USA) and used in accordance with the laboratory animal guidelines of Seoul National University. Mice were kept in protective cages (five mice in each) with unrestricted access to water and diet at standard laboratory conditions and were kept under 12h light/dark cycles. The animals were divided into four experimental groups as follows: group I (n=5), no injection; group II (n=5), Saline injection alone, as a control; group III (n=5), intradermal transplantation of isolated CD34− hair follicle cells; group IV (n=5), intradermal transplantation of isolated CD34+ hair follicle cells. Before transplantation, the hair of each group mice was removed by the topical application of calcium thioglycolate. The next day, approximately 1×10⁵ isolated CD34+ and CD34− hair follicle cells suspended in 50 uL of physiologic saline were injected into the parietal area of each group mice head using a 30-gauge Venocath (Becton Dickinson Medical systems, Sandy, UT) and 50 uL of physiologic saline was injected into same area of group I, II mice. In the in vivo studies, hair-regrowth activities when injecting cells was measured by observing hair of each mouse every day. All experiments lasted for 30 days. Fig. 3 shows the scale for evaluating hair growth in this mouse.

After 30 days from injection of the cells, biopsies were obtained from the parietal area of the mouse head. Samples of parietal skin from all group homogenized in lysis buffer (150 mM NaCl, 50 mM Tris-HCl, pH 7.4, 2 mM ethylenediamine tetraacetic acid (EDTA), 1% NP-40) containing protease inhibitors (Complete, Roche, Mannheim, Germany) on ice. Homogenate was centrifuged at 12,000 g for 1 h at 4°C. The supernatant was collected and subjected to determination of protein concentration using commercial protein assay kit (Bio-Rad, Richmond, CA, USA). Equal amounts of protein samples (25μg/lane) were subjected to 12% SDS-PAGE and transferred to nitrocellulose membranes. The membranes were subsequently incubated with the corresponding primary antibodies, as indicated: mouse anti-Bcl-2 monoclonal antibody (Zymed Laboratories Inc., CA, USA); rabbit anti-MAPK polyclonal antibodies (Zymed Laboratories Inc); rabbit anti-p38 monoclonal antibodies, anti-Bax polyclonal antibody (Santa Cruz Biotechnology, CA, USA); rabbit anti-active MAPK, anti-active p38 MAPK polyclonal antibodies (Promega Corporation, WI, USA); rabbit anti-Akt polyclonal antibodies (Cell Signaling Technology Inc. Beverly, MA, USA); (Sigma). Antibody recognition was detected with the respective secondary antibody, ether anti-mouse IgG, or anti-rabbit IgG antibodies linked to horse-radish peroxidase (Zymed Laboratories Inc.). Antibody-bound proteins were detected by the ECL western blotting analysis system (Amersham Pharmacia Biotech U.K. Limited).

After 30 days from injection of the cells, biopsies were obtained from the parietal area of the mouse head after anesthesia with dimethyl ether. Samples of parietal skin from control group and experiment group were fixed in 10% (wt/vol) neutral buffered formalin for 24∼48 hours, dehydrated in an alcohol-xylene series, and embedded in paraffin wax. From each block, sections with 3μm thickness were prepared and stained with haematoxylin and eosin (HE) for histological examination and it was serially sectioned at 3μm, floated on a water bath containing diethylpyrocarbonate-treated water, and mounted on positively charged slides (Superfrost/Plus slide, Erie Scientific Co., Portsmouth, NH).

The specific primers for human Alu sequences were Alu-sense, 5’ ACG CCT GTA ATC CCA GCA CTT 3’, and Alu-antisense, 5’ TCG CCC AGG CTG GAG TGC A 3’, which produced a band of 224 bp. The primers were positioned in the most conserved area of the Alu sequence (25). The PCR amplifications were performed by using the following settings: 94°C for 30 s, 58°C for 30 s and 72°C for 45 s, with a total 30 cycles. The PCR products were analysed in 1% agarose.

Polymerase chain reaction (PCR) products were purified using a 30-kd cutoff membrane ultrafiltration filter. The nucleotide sequences of the purified PCR products were determined by use of Big Dye chemistry with the ABI Prism Sequencer (Applied Biosystems, Foster City, CA, USA). Sequencing was performed of the purified PCR products before PCR products were labeled by random priming with digoxigenin-dUTP (Roche, Grenzacherstrasse, Switzerland), according to the manufacturer’s instructions.

Sections were deparaffinized in xylene and rehydrated in phosphate-buffered saline (PBS) (pH 7.4, 0.01 M) for 5 minutes. Deproteinization was carried out in 0.2 N HCl for 20 minutes at room temperature. Tissues were then digested at 37 C for 20 minutes in 100μg/ml proteinase K (GIBCO-BRL) in PBS. After digestion, tissues were fixed in 4% paraformaldehyde in PBS for 10 minutes. After rinsing twice with PBS, the slides were acetylated in 300 ml of 0.1 mM triethanolamine-HCl buffer (pH 8.0) to which 0.75 ml of acetic anhydride (0.25%) had been added. After 5 minutes, an additional 0.75 ml of acetic anhydride was added, and 5 minutes later the slides were rinsed in 2× saline sodium citrate (SSC) (1× SSC contains 50 mM NaCl and 15 mM sodium citrate, pH 7.0). The slides were allowed to equilibrate for 60 minutes in a standard hybridization buffer that consisted of 5× SSC with 50% deionized formamide, 10× 2% buffered blocking solution (Roche), 0.1% N-lauroylsarcosine, and 0.02% sodium dodecyl sulfate. Hybridization was done overnight at 45°C. The digoxigenin-labeled probe (0.1 ng/μl) was diluted in 300μl of the standard hybridization buffer, heated for 10 minutes at 95°C on a heating block, and quenched on ice before being applied to the tissue sections. Approximately, 50 ng of the digoxigenin-labeled probe was added to the standard hybridization buffer (50 μl), heated for 10 minutes at 95°C on a heating block, and quenched on ice before being layered over the section again. Fluid was held in place by a coverslip, and the edges were sealed with rubber cement. After overnight hybridization, sections were thoroughly washed twice in 4× SSC for 5 minutes at room temperature, twice in 2× SSC for 10 minutes at 37 C, twice in 0.2× SSC for 5 minutes at room temperature, and once in maleic acid buffer (100 mM maleic acid and 150 mM NaCl, pH 7.5) for 5 minutes at room temperature. For detection of hybridization, sections were incubated with anti-digoxigenin conjugated with alkaline phosphatase (Roche) diluted 1:250 in 0.1 M Tris-HCl (pH 7.4) and 0.15 M NaCl with 1% blocking reagent (Roche). After three washes in buffer, substrate consisting of nitroblue tetrazolium and 5-bromocresyl-3-indolylphosphate was layered over the sections. Color was allowed to develop for 5∼8 hours in the dark, and the reaction was stopped by dipping slides briefly in Triethylenediaminetetraacetic acid buffer (10 mM Tris-HCl and 1 mM ethylenediaminetetraacetic acid, pH 8.0). Sections were allowed to dry completely, dipped into the absolute xylene, and coverslipped with Canada balsam mounting medium (Hayashi Pure Chemical Industries Ltd., Osaka, Japan).

All experiments were conducted at least in triplicate (n=3), and the results are expressed as the mean±SD. Statistical analysis was conducted via analysis of variance (ANOVA), followed by Duncan’s multiple range test.

To confirm the CD34+ and CD34− population isolated by MACS, we performed FACS analysis of each population. CD34+ population was stained with antibodies to CD34 (FITC conjugate) and then subjected to FACS analysis gating out debris and dead cells, a distinct CD34+ population was found, which comprised 98% of isolated cells. Most of hair follicle CD34+ cells were CD133+ (approximately 99%), CD24+ (approximately 98%), CD29+ (approximately 99%), CD44+ (approximately 98%), but, a small population (approximately 1%) of human hair follicle CD34+ cells was also found to CD45+ (Fig. 1A). As might be expected, distinct CD34− population was found, which comprised very small percentage (approximately 1%) when were stained with antibodies to CD34 (FITC conjugate). Most of hair follicle CD34− cells were also negative for CD133 (approximately 17%). However, the intensity of expression of CD24 (approximately 96%), CD29 (approximately 99%), CD44 (approximately 92%) of CD34 negative population was also very high (approximately 96%) and the intensity of expression of CD45 was very low (approximately 2%), same as CD34+ population (Fig. 1B).

New hairs were observed to head and dorsal area of mouse skin which had transplanted hair follicle CD34+ cells (Fig. 2B). But, CD34+ transplanted mice have more hairs and whiskers compared with CD34− transplanted mice (Data not shown). All mice of cell transplanted group (Experiment group) successfully produced new hair at 3 weeks after injection of the cells but physiologic saline-injected mice (Control group) fail to produce hair in the skin (Fig. 2A).

In comparison with the transplantation of vehicle only as the vehicle control and the no transplantation of as the negative control, CD34 positive and negative cells isolated from human hair follicle were transplanted to evaluate their efficacy on the promotion of hair growth using immunodeficient nude mice. Fig. 4 shows these results of quantified hair growth after transplantation using the scale illustrated in Fig. 3 and demonstrates hair growth for the negative control and vehicle control (Saline), respectively. In negative control mice, hair growth showed only a faint appearance 25 days after transplantation. After the transplantation of saline, it took about 25 days for the hair of mice to grow, indicating no effect of vehicle in the proliferative rate of hair growth. However, after transplantation of CD34+ and Cd34− hair follicle cells, it was evident that both were able to facilitate hair growth compare to the control.CD34+ transplanted mice have more hairs, whiskers and earlier compared with CD34− transplanted mice.

To examine Histological sections of skin in control group and experiment group, biopsies were obtained from the head parietal skin of each group 30 days after injection of human CD34+ hair follicle cells and haematoxylin and eosin (HE) staining was carried out. Very Few hair follicle was observed in the head parietal skin of a control mouse (Fig. 5A, B) but new hair follicles were observed in the head parietal skin of human CD34+ hair follicle cell-injected mouse (Fig. 5C, D).

In situ hybridization of skin samples from each group with human cell specific probe, revealed the presence of transplanted human hair follicle CD34+ cells in the ORS keratinocytes of the nude mouse skin. The human specific DNA was identified around ORS of new hair matrix in CD34+ hair follicle-injected mouse (Fig 6B, C; arrows). This result indicated that human hair follicle cells transplanted were migrated and localized in ORS of new hair follicle: Control mouse was not detected (Fig 6A).

Western blotting methods were used to investigate the expression pattern of several cell signaling proteins in control mouse and transplanted mouse. The results indicate that control mouse and transplanted mouse have the same expression level of total p38, pp38, ERK and AKT. But pERK expression levels were significant increased in transplantated mouse. The apoptosis-suppressing Bcl-2 was significant increased and the apoptosis-promoting Bax was significant decreased in CD34+ cell-transplantated mouse compared with control mouse (Fig. 7).