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INTRODUCTION skin was cut into 4 mm × 2 mm pieces, placed in a centrifuge
tube containing 0.25% Dispase (Shanghai Biological Technical
The discovery of hair follicle stem cells (HFSCs) and the Company, China), and digested overnight at 4 °C. The hair
development of methods to culture these cells in vitro have follicles were then gently pulled out in the direction of hair
led to the possibility of developing new tissue engineering growth. The dissociated adipose tissue was then washed
treatments for cutaneous damage caused by burns. HFSCs with PBS and hair follicles were added to a 60-mm petri
[1]
are highly proliferative multipotent stem cells with multi-lineage dish containing 10% fetal bovine serum (FBS) in Dulbecco’s
potential. They can differentiate into a range of distinct cell types Modified Eagle Media (DMEM; Hyclone, USA). Bulge areas
including epidermal cells, hair follicle cells, and sebaceous were isolated from the hair follicles by incubation with 2.5
cells. Both the proliferation rate and the differentiation g/L trypsin and 0.04% EDTA for 10 min at 37 °C, followed by
[2]
capacity of HFSCs are influenced by autologous genes and centrifugation and resuspension in K-SFM medium (Shanghai
external signals. The Wnt/β-catenin signaling pathway plays Biological Technical Company, Shanghai, China) at a density
7
a crucial role in the development of follicles and hair. [3,4] of 5 × 10 cells/L. Cells were incubated at 37 °C under 5% CO 2
During both embryonic development and tumorigenesis in a humidified incubator. The culture medium was replaced
Wnt proteins bind to their receptors and activate Wnt/β- every 3-4 days, and HFSCs were cultured and passaged three
catenin signaling, leading to β-catenin accumulation in the times prior to being used for all experiments.
cytoplasm and subsequent nuclear translocation. β-catenin
binding to lymphoid enhancer factor-1 (LEF1) activates Measuring the proliferation rate of HFSCs
target genes and accelerates the directional differentiation To measure their rates of proliferation, HFSCs were seeded at the
of HFSCs. [3,4] Other factors, including chemicals (e.g. LiCl) following four different densities: 1 × 10 , 1 × 10 , 1 × 10 , and
3
5
4
6
and growth factors, influence cytoplasmic β-catenin levels in 1 × 10 cells/mL of 24-well plate. Growth curves of the HFSCs
[1]
5
HFSCs, leading to HFSC differentiation. However, details of were obtained by seeding 1 × 10 logarithmically growing cells
the mechanism require further investigation. The purpose of in 1mL medium into each well of a 24-well culture plate. A total
this study was to investigate how keratinocyte growth factor of 36 wells were used. Hoechst 33258 DNA quantitation was
(KGF) and LiCl influence the function of the Wnt/β-catenin performed every day after 7 days in culture.
signaling pathway and its interaction with other signaling
components during human HFSC differentiation into dermal Immunofluorescence assay
papilla and epidermal cells. Immunofluorescence analysis was performed as previously
[5]
described. Briefly, sterile glass coverslips were placed into
METHODS 60 mm dishes, and approximately 2 × 10 cells/mL of 24-well
4
plate were seeded into each dish. After 7 days in culture,
Tissue harvest and cell isolation adherent cells were stained for pan-cytokeratin, K19-Cy3, and
Fresh scalp specimens were obtained under aseptic condition β1-integrin and analyzed by immunofluorescence microscopy.
and HFSCs were isolated using previously established
[1]
methods. Briefly, scalp skin from patients undergoing Treatment with LiCl and KGF
surgery was washed repeatedly with phosphate buffered To investigate the effects of specific compounds on HFSC
solution (PBS) containing 5% penicillin and streptomycin differentiation, HFSCs were treated with LiCl (0.5, 1.5, 10,
(North China Pharmaceutical Corporation, China). After or 25 mmol/L) or KGF (10, 25, 50, or 100 µg/L). After 7 days,
removing subcutaneous adipose tissue and impurities, the Hoechst 33258 DNA quantitation was performed to compare
Table 1: Primer sequences and amplified fragments
Primer pairs Sequences (5ʹ to 3ʹ) Denaturation temperature Fragment size (bp)
(°C)
β-catenin forward TACCTCCCAAGTCCTGTATGAG 56.0 180
β-catenin reverse TGAGCAGCATCAAACTGTGTAG

APC forward TTAAAGCAAGTTGAGGCACTG 55.5 270
APC reverse ACCGGCTTCCATAAGAACGGA
Axin forward GCGGGACAGATTGATTCACTT 57.0 190
Axin reverse TGTGGACACCAGTTCTCCCT
GSK-3β forward ATTTCCAGGGGATAGTGGTGT 56.0 154
GSK-3β reverse TCCTGACGAATCCTTAGTCCAAG

LEF1 forward AATGAGAGCGAATGTCGTTGC 56.5 137
LEF1 reverse GCTGTCTTTCTTTCCGTGCTA

GAPDH forward TGTTGCCATCAATGACCCCTT 57.0 202
GAPDH reverse CTCCACGACGTACTCAGCG

APC: adenomatous polyposis coli; GSK-3β: glycogen synthase kinase-3β; LEF1: lymphoid enhancer factor-1; GAPDH: glyceraldehyde-3-phospha
40 Plast Aesthet Res || Vol 3 || Issue 2 || Feb 29, 2016