hHFSCs were obtained from human scalp tissues from healthy adult patients (average age, 30 years) undergoing cosmetic plastic surgery. All protocols of human tissue handling were approved by the Research Ethical Committee of the Shanghai 9th People’s Hospital (Shanghai, China) and written informed consent was obtained from the patients. Briefly, two to three pieces of scalp debris (4–9 mm) were postoperatively collected and washed two to three times with phosphate-buffered saline (PBS). The fat tissue was carefully removed and the scalp was cut into smaller sections. The scalp sections were then transferred to a 15-ml centrifuge tube and washed three times to remove any remaining hair or blood clots. Washed tissue pieces were digested with 1 mg/ml collagenase type I (Invitrogen Life Technologies, Carlsbad, CA, USA) at 37°C with occasional agitation. After 2 h of digestion, single-hair follicles were released from the full-thickness skin, filtered through a 40-mm cell strainer (BD Biosciences, San Jose, CA, USA) and washed extensively in PBS. Subsequent to this, each hair follicle was placed in one well of a 96-well plate (BD Biosciences), to allow for cell migration onto the tissue culture plastic, and was cultured in 100 μl low-glucose Dulbecco’s modified Eagle’s medium (LG-DMEM; Gibco, Grand Island, NY, USA) containing 10% foetal bovine serum (FBS; HyClone, Logan, UT, USA). Cells that originated from the bulge region were visually identified as epidermal keratinocytes, while cells migrating from the dermal sheath or papilla exhibited the morphological appearance of mesenchymal cells. The wells populated with cells originating from the dermal sheath or papilla were selected, pooled and expanded. The cells were then plated in 100-mm culture dishes (Falcon, Oxnard, CA, USA) at a density of 4×10⁴ cells/cm² and cultured in LG-DMEM (Gibco) supplemented with 10% FBS, 100 U/ml penicillin (Sigma-Aldrich, St. Louis, MO, USA), and 100 mg/ml streptomycin (Sigma-Aldrich) (defined as growth medium). The medium was changed twice a week. Cells were passaged when they had reached 70–80% confluence and second passage hHFSCs were used in the subsequent experiments. The characterisation of hHFSCs was determined by the CD marker profile and the ability to differentiate into osteogenic, adipogenic and chondrogenic lineages, as previously described (data not shown) (15,16).

To evaluate the effects of TGF-β1 and PDGF-BB on the differentiation of hHFSCs into SMCs, hHFSCs reaching subconfluence were cultured in DMEM supplemented with 5 ng/ml recombinant human TGF-β1 (R&D Systems, Minneapolis, MN, USA) and 10 ng/ml recombinant human PDGF-BB with 1% FBS. DMEM supplemented with 1% FBS was defined as the basal medium (BM). Human umbilical artery SMCs (hUASMCs) served as a positive control. The culture media were changed every two days. Cell characterisation and functional evaluation were performed subsequent to eight days of culture.

The hHFSCs were harvested, resuspended in PBS, fixed with 4% paraformaldehyde (Sigma-Aldrich) for 15 min and permeabilised with 0.1% Triton X-100 (Sigma-Aldrich) for 10 min. Subsequent to washing with PBS, the cells were blocked with 3% bovine serum albumin (BSA, Sigma-Aldrich) for 30 min and incubated with the following primary antibodies: Mouse monoclonal anti-α-smooth muscle actin (α-SMA, C6198, Sigma-Aldrich), rabbit monoclonal anti-α-calponin (ab46794; Abcam, Cambridge, UK) and mouse monoclonal anti-smooth muscle myosin heavy chain (SM-MHC, M7786, Sigma-Aldrich). Following incubation with the primary antibodies for 60 min at room temperature (RT), the cultures were washed with PBS three times. Fluorescein isothiocyanate (FITC)-conjugated goat anti-rabbit secondary antibody (Millipore, Billerica, MA, USA) was used to detect the localisation of anti-α-calponin antibodies. FITC-conjugated goat anti-mouse secondary antibody (Millipore) was used to detect the localisation of anti-α-SMA and anti-SM-MHC antibodies. In addition, cell nuclei were stained with propidium iodide. Control samples consisted of cells without primary antibody and were used to assess background fluorescence. The images were viewed by a fluorescence microscope (Eclipse E400 epi-fluorescence microscope; Nikon, Tokyo, Japan).

Cells were trypsinised, centrifuged at 500 × g for 5 min (Allegra 64R; Beckman Coulter, Brea, CA, USA), resuspended in PBS/1% BSA and incubated with anti-α-SMA (dilution, 1:100; Sigma-Aldrich), anti-α-calponin (dilution, 1:100; Abcam) and anti-SM-MHC (dilution 1:100; Sigma-Aldrich) for 30 min at RT on a shaking plate (Lab Rotors; Thermo Scientific, Logan UT, USA). The cells were then washed, resuspended in PBS/1% BSA with FITC-conjugated secondary antibody and incubated for 30 min at RT on a shaking plate. Subsequent to this, cells were washed and fixed. FITC-conjugated isotype-matching immunoglobulins were used to determine non-specific staining. Fluorescence was determined using a flow cytometer (FACSCalibur; Becton-Dickinson, San Jose, CA, USA) and the data were analysed using CellQuest software (Becton-Dickinson).

Expression levels of SMC-specific markers (α-SMA, α-calponin and SM-MHC) were identified by isolating the total RNA from cells using the RNeasy total RNA isolation kit (Qiagen, Inc., Valencia, CA, USA) and cDNA was synthesised using the SuperScript First-strand Synthesis system (Life Technologies, Barcelona, Spain). Specific genes were amplified by PCR using the Fast-Run Taq Master kit (Protech Technologies Inc., Taipei, Taiwan). The primer sequences designed using the Primer Express software are listed in Table I. The cDNA product was amplified by PCR using standard methods (35 cycles of 95°C for 30 sec, 56°C for 30 sec and 72°C for 60 sec). Gel electrophoresis was then performed on a 2% agarose gel treated with ethidium bromide and bands were visualised using a UV light box. hUASMCs and human chondrocyte cells (hCs) served as positive and negative controls, respectively.

The contractility of cells was measured as previously described (28). Briefly, cells were trypsinised by treatment with trypsin-ethylenediaminetetraacetic acid, counted and resuspended in serum-free DMEM at a density of 1×10⁶ cells/ml. The prepared cell suspension was added to a collagen gel solution to achieve a final concentration of 3 mg collagen/ml and a density of 4×10⁵ cells/ml. The cell collagen mixture was poured into 12-well culture plates for 1 h to polymerise the collagen cell lattices. The lattices were mechanically released from the bottom of the tissue culture dishes by gently pipetting medium at the lattice-dish interface to initiate collagen gel contraction. The extent of gel contraction of each group at different time points was calculated by measuring the dimensions of the gel lattices viewed by a digital camera. The area of the gel lattices was analysed using NIH ImageJ software (National Institutes of Health, Bethesda, MD, USA). The relative lattice area was obtained by dividing the area at each time point by the initial area of the lattice.

Each experiment was repeated at least three times. The results are presented as the mean ± SD. Comparisons between groups were performed by a paired Student’s t-test. P<0.05 was considered to indicate a statistically significant difference.

The total quantity of cells isolated from each scalp tissue sample ranged from 5×10⁴ to 1×10⁵ cells. Approximately 0.5–2% of the isolated tissue cells were identified to be the adherent stem cells. Subsequent to 1–2 days of culture, the hHFSCs were observed to be elongated (Fig. 1A). Within another 3–4 days, the cells reached confluency and were subsequently passaged onto a novel plate.

At the second passage, 5 ng/ml TGF-β1 and 10 ng/ml PDGF-BB were selected to induce the differentiation of hHFSCs into the SMC lineage. hHFSCs treated with TGF-β1 and PDGF-BB for eight days acquired a spindle morphology and grew in a hill and valley pattern similar to that previously observed in primary isolated hUASMCs. No obvious change was identified in the hHFSCs cultured in BM (Fig. 1B–D). Fifth passage hHFSCs appeared to be a relatively homogenous population that exhibited a fibroblast-like spindle morphology.

To determine whether TGF-β1 and PDGF-BB induced the differentiation of hHFSCs into the SMC phenotype, SM-specific proteins (α-SMA, α-calponin and SM-MHC) were detected by immunofluorescence staining. In parallel, the three markers in hUASMCs were also analysed as a positive control. As shown in Fig. 2, there was a baseline expression of α-SMA, but marginal expression of α-calponin and SM-MHC in undifferentiated hHFSCs cultured in BM. When cultured in DMEM supplemented with TGF-β1 and PDGF-BB, expression levels of α-SMA and α-calponin were identified to be enhanced. Furthermore, expression of SM-MHC was markedly increased, reaching a similar level to that in the hUASMCs.

To determine the percentage of SM differentiated cells in the hHFSCs, the expression levels of α-SMA, α-calponin and SM-MHC were analysed by flow cytometry. As shown in Fig. 3, the α-SMA was detected in 17.41±0.78%, 45.32±1.21% and 89.46±0.83% of undifferentiated hHFSCs, induced hHFSCs and hUASMCs, respectively. By comparison, marginal expression of α-calponin (3.47±0.82%) and SM-MHC (2.78±0.63%) was observed in the undifferentiated hHFSCs, while their expression levels reached 57.24±2.08% and 70.71±1.78%, respectively, in the induced hHFSCs, which was closer to the expression observed in hUASMCs.

The gene expression profile analysed by RT-PCR further confirmed the differentiation of hHFSCs into SMCs. As shown in Fig. 4, undifferentiated hHFSCs expressed low levels of α-SMA mRNA and did not express α-calponin or SM-MHC, which was similar to that observed in the hCs. By contrast, these factors were upregulated in induced hHFSCs at a similar level to that in hUASMCs. These data support the hypothesis that hHFSCs are capable of differentiating into SMCs when induced by TGF-β1 and PDGF-BB.

To evaluate whether SMCs, differentiated from hHFSCs by TGF-β1 and PDGF-BB induction, exhibit the ability to contract and generate force, the contractility of the smooth muscle-like cells was analysed using a collagen lattice gel contraction assay. The shrinkage of gels embedded with differentiated SMCs occurred in a time-dependent manner. After 48 h, the area of gel lattice was markedly decreased. However, collagen gel matrix containing undifferentiated hHFSCs contracted to a lesser extent. Compared with the gel containing undifferentiated cells, the area of the collagen lattice with differentiated hHFSCs was significantly diminished after 6 h (Fig. 5). Thus, these results showed that with the combined induction by TGF-β1 and PDGF-BB, the hHFSCs differentiated into the SMC phenotype and acquired the contractile ability observed in hUASMCs.