Sonic hedgehog signalling regulates the self‐renewal and proliferation of skin‐derived precursor cells in mice

Abstract Objectives The sonic hedgehog (Shh) signalling pathway has an important role in the maintenance of various stem cells and organogenesis during development. However, the effect of Shh in skin‐derived precursors (SKPs), which have the capacity for multipotency and self‐renewal, is not yet clear. The present study investigated the effects of the Shh signalling pathway on the proliferation and self‐renewal of murine SKPs (mSKPs). Methods The Shh signalling pathway was activated by treatment with purmorphamine (Shh agonist) or recombinant Shh in mSKPs. Cyclopamine (Shh antagonist) or GANT‐61 (Gli inhibitor) was used to inhibit the pathway. Western blot, qPCR, and immunofluorescence were used to analyse the expression of genes related to self‐renewal, stemness, epithelial‐mesenchymal transition (EMT) and the Shh signalling pathway. In addition, cell proliferation and apoptosis were examined. Results Inhibiting the Shh signalling pathway reduced mSKP proliferation and sphere formation, but increased apoptosis. Activating this signalling pathway produced opposite results. The Shh signalling pathway also controlled the EMT phenotype in mSKPs. Moreover, purmorphamine recovered the self‐renewal and proliferation of aged mSKPs. Conclusion Our results suggest that the Shh signalling pathway has an important role in the proliferation, self‐renewal and apoptosis of mSKPs. These findings also provide a better understanding of the cellular mechanisms underlying SKP self‐renewal and apoptosis that allow more efficient expansion of SKPs.

self-renewal and differentiation potency. These two features in SKPs are regulated by intrinsic and extrinsic signals from various niches. 6,12 Sphere-type SKPs are generated using a suspension culture system. Dissociated single cells from primary spheres form secondary spheres expressing the SKP markers. Various studies have reported that SKP spheres can be obtained using 3D colony-forming systems (eg, methylcellulose or Matrigel), where the clonality of the spheres can also be confirmed. 1,2,13 The Hedgehog (Hh)-Gli signalling pathway participates in brain development, self-renewal of neural stem cells and proliferation of various precursor cells. [14][15][16] Recent reports also show that Hh-Gli signalling pathway controls the self-renewal of neural stem cells by regulating Nanog, p53 and Foxm1. 17 Patched, Gli1 and Gli2. 19 Furthermore, the Hh signalling pathway plays a critical role in endoderm and mesoderm development during embryogenesis. 20 Sonic hedgehog (Shh) knock-out mice are embryonic lethal because these mice have problems patterning vertebrate embryonic tissues (including the brain, spinal cord and axial skeleton). 21,22 Recent studies have demonstrated that Shh stimulates embryonic stem cell proliferation via Gli family activation and protein kinase C cooperation in mice. 23 The Hh signalling pathway also regulates the self-renewal of mammary stem cells via Bmi1, a Polycomb group protein. 24,25 Bmi1 participates in brain development and stem cell proliferation. It is also able to replace some reprogramming factors such as Sox2, Klf4 and N-myc when induced pluripotent stem (iPS) cells are generated from murine embryonic fibroblasts. 26,27 Synthetic or natural small molecules are widely used to understand and regulate stem cells. 20 Small molecules such as purmorphamine and oxysterol activate the Shh signalling pathway. They are able to replace Bmi1 to generate iPS cells. They also induce Bmi1, Sox2 and N-myc expression to promote the proliferation of neural precursor cells. 26 Both the epithelial-mesenchymal transition (EMT) and the mesenchymal-epithelial transition (MET) play important roles in embryonic development, fibrosis and cancer progression. [28][29][30] The EMT influences organ and tissue formation during embryogenesis, including the neural crest, heart, nervous system and craniofacial structure. 29 More recently, the effect of the EMT on the self-renewal and stemness of cancer stem cells (stem-like cells in tumours) was studied. 31 The EMT is characterized by cells losing their epithelial state and acquiring fibroblast-like properties. Cells produced by the EMT show decreased intercellular adhesion and elevated motility. 25 Specifically, they show decreased expression of E-cadherin and increased expression of mesenchymal cell markers (such as Ncadherin, fibronectin, vimentin and α-smooth muscle actin. 28,32,33 In the present study, the effects of the Shh signalling pathway on the generation and propagation of murine SKPs (mSKPs) were investigated. We also explored whether activation of the Shh signalling pathway contributes to the self-renewal and cell proliferation of SKPs. The sphere formation of SKPs in suspension culture revealed a correlation between the EMT and the Shh signalling pathway. Our study highlights the fact that the Shh pathway is important to the self-renewal and cell proliferation of SKPs.

| Chemicals
All inorganic and organic compounds were obtained from Sigma-Aldrich Korea (Yong-in, Korea). All liquid solutions were purchased from Thermo Fisher Scientific Korea (Seoul, Korea) unless otherwise stated.

| Propagation and isolation of mSKPs
The mSKPs were isolated by previously described protocols 1,2 , with a few modifications. Back skin obtained from E16.5-17.5 mouse embryos was washed three times in phosphate-buffered saline (PBS; WelGENE, Daegu, Korea) with 3X penicillin/streptomycin (Gibco, Grand Island, NY, USA) and then minced into small pieces using a blade. Small pieces of back skin were incubated for 40 minutes in a 37°C, 5% CO 2 cell culture incubator on a 60 mm culture dish containing 4 mL of 0.05% (w/v) trypsin solution (Gibco) or 1X TryPLE™ (Gibco). The incubated skin pieces were pipetted up and down 30 times for single cell dissociation. Dulbecco's modified Eagle medium (DMEM; WelGENE) with 10% foetal bovine serum (Atlas Biologicals, Fort Collins, CO, USA) was added onto the incubated skin pieces and dissociated single cells for enzyme neutralization. Skin and cell suspensions were strained through 100 and 40 μm nylon cell strainers (BD, Franklin Lakes, NJ, USA) for single cell isolation. Strained single cells were centrifuged at 250 g for 4 minutes. The cell pellet was resuspended in 5 mL of DMEM/F-12 (3:1 mixture, v/v; Gibco) containing 2% B27 supplement (B27; Gibco), 20 ng/mL epidermal growth factor (EGF; Peprotech, Rocky Hill, NJ, USA), and 40 ng/mL basic fibroblast growth factor (bFGF; Peprotech; called SKP medium). The single cells were counted and then cultured in a 90 mm Petri dish in a 37°C with 5% CO 2 atmosphere. Fresh SKP medium was replaced every 3 days.
The cells were passaged every 5-7 days. The formed spheres were single-cell dissociated by pipetting with Accutase™ (Gibco).

| Immunofluorescence staining
Immunofluorescence staining was performed according to standard protocols. Briefly, mSKPs or differentiated cells from mSKPs were fixed in 4% paraformaldehyde, permeabilized with 0.25% Triton X-100 (Sigma) and blocked with 1% goat serum in PBS.
The fixed cells were stained with antibodies against Table 1. The treated cells were covered with SlowFade antifade reagent with DAPI (SlowFade ™ Gold antifade with DAPI) for nuclear staining and covered with a glass coverslip. Images were captured with confocal microscopes (LSM800; Carl Zeiss, Oberkochen, Germany; FV-300, Olympus, Tokyo, Japan).

| Reverse transcription-PCR
For total RNA isolation from sphere-forming mSKPs, we followed the commercial protocol of the Ambion PureLink™ RNA Mini Kit (Thermo Fisher Scientific). For the synthesis of first-strand cDNA, reverse transcription was performed for 1 hour at 42°C in a final reaction volume of 25 μL using cDNA synthesis kit (Thermo Fisher Scientific). Synthetic cDNA from the RNA of mSKPs and differentiated cells was used for each PCR reaction. Each reaction contained 50 ng cDNA, 20 pmol each of specific primers, and AccuPower™ PCR Premix (Bioneer, Daejeon, Korea). Thermal cycle was repeated 34 times.

| Real-time quantitative PCR
The cDNA was analysed using real-time quantitative PCR (qPCR). For optimal quantification, primers were designed using Primer Express software (Applied Biosystems, Foster City, CA, USA). The qPCR reactions were performed using the ABI PRISM 7500 system and SYBR™ Premix Ex Taq II (Takara Bio Inc., Shiga, Japan). All samples were run in triplicate as technical replicates. The following amplification procedure was employed. Data were analysed using the 7500 System Sequence Detection software (Applied Biosystems). All samples had the same starting quantities of all candidate reference genes, based on the standard curves generated for those genes. All procedures and data analyses followed MIQE guidelines. 34 The specific primer sequences targeting genes for stemness, differentiation, EMT, Shh signalling and the neural crest are listed in Table 2.

| Protein extraction and Western blot
The mSKPs receiving various treatments were centrifuged and collected. Collected mSKPs were lysed by Passive lysis buffer (Promega, Madison, WI, USA). Protein was quantified using the Pierce BCA protein assay kit (Thermo Fisher Scientific). Equal amounts of protein (30-50 μg) from each treated group were analysed on a 12% sodium dodecyl sulphate polyacrylamide gel. After transfer to a nitrocellulose membrane, the membrane was incubated with primary and secondary antibodies on a shaker. Detection was performed using WesternBright™ Quantum (Advansta, Menlo Park, CA, USA), according to the manufacturer's recommended protocol. Western blot data were analysed using the GeneGnome XRQ System (Syngene, Cambridge, UK).

| Cell proliferation assays
The mSKPs were measured for cell proliferation using the WST-1 cell

| Apoptosis analysis by Annexin V assay using fluorescence-activated cell sorting
The mSKPs were plated at a density of 1.

| Statistical analyses
All numerical values in this study are expressed as the mean ± SD.
Statistical analyses were performed using a two-tailed Student's t test for comparison between two groups, or a one-way ANOVA for the comparison of three or more groups. Differences were considered statistically significant at P values <0.05.

| Isolation, culture and characterization of mSKPs from murine foetal back skin
The mSKPs formed spheres from single cells in suspension culture ( Figure 1A). After 5-7 days, the spheres grew larger, and some  Figure 1I).
These results demonstrate that cell spheres isolated from murine back skin have SKP properties.

| Activation of the Shh pathway by recombinant Shh (rShh) treatment in mSKPs
After treatment of 500 ng/mL rShh, mSKPs in the treated group formed larger spheres than the control group (Figure 2A,B). Several genes related to stemness, the neural crest and the Shh pathway were detected in the reverse transcription-PCR analysis ( Figure 2C).
These findings indicate that sphere formation and multipotency in mSKPs are influenced by activation of the Shh signalling pathway.

| Treatment with a Shh agonist promotes the proliferation of mSKPs and changes gene expression
The mSKPs treated with Pur (a Shh agonist) formed larger spheres, and their number also increased compared to the control group in all passages checked ( Figure 3A). The results of a WST-1 assay showed a significant increase in the proliferation rate after treatment with Pur at passages 1 and 2 ( Figure 3B). Although the total number of spheres with a diameter over 20 μm was not different from the control group ( Figure 3C-a), the number of spheres with a diameter over reprogramming-related genes via Shh signalling pathway activation.

| Treatment with a Smo inhibitor decreases proliferation in mSKPs and changes gene expression
The size and number of spheres decreased after treatment with CP (a Smo inhibitor), as observed using a stereomicroscope ( Figure 4A,S1).
A dose-dependent decrease in the proliferation of mSKPs due to CP was also demonstrated by a WST-1 assay ( Figure 4B). Cell morphology and proliferation in CP-and Pur-treated mSKPs were similar to the control group ( Figure 4A,B).
CP treatment decreased the total number of spheres, regardless of their diameter. The number of spheres after cotreatment with CP and Pur was similar to the controls, regardless of diameter. CPmediated effect was counteracted by Pur treatment (Figure 4C-a,b).
In addition, the effect of counteraction by cotreatment on the prolif- Ngfr (neural stem cell/precursor genes). However, the expression of pluripotency and reprogramming genes such as Oct4, Nanog, c-Myc and Klf4 was not significantly different between the CP-treated and control groups. In addition, Pur treatment elevated the expression of several key genes which had been decreased by CP treatment ( Figure 5). Therefore, it is possible that Pur treatment may recover a CP-inhibited Shh signalling pathway.

| The effect of a Gli inhibitor (GANT-61) on the self-renewal and proliferation of mSKPs
The influence of the Shh-Gli signalling pathway on mSKPs was investigated using the Gli inhibitor GANT-61. Treatment of mSKPs with GANT-61 led to reduced sphere formation compared to the control ( Figure 6A,S2A), and cell proliferation also decreased at passages 1 and 2 ( Figure 6B,S2B). Sphere size and number were also reduced by Gli1 inhibition ( Figure 6C). In addition, the expression of genes related to stemness and Shh signalling was decreased by the Gli1 inhibitor ( Figure 6D). These results demonstrated that the formation and proliferation of mSKP spheres are inhibited by a block of the Shh-Gli1 signalling pathway, caused by a Gli1 inhibitor.

| The Shh signalling pathway regulates the EMT in mSKPs
To examine the correlation between the Shh signalling pathway and the EMT in mSKPs, expression levels of EMT markers (such as α-Sma, Cdh2, Fn1, Vim and Tgf-β1) were compared between Pur-treated and nontreated mSKPs. The expression of EMT markers was increased by Shh activation, whereas expression was decreased by Shh inhibition. In addition, Pur recovered EMT markers that were inhibited by CP treatment ( Figure 8A). The Purpromoted activation of the Shh signalling pathway elevated EMT protein levels, as measured by Western blot and immunofluorescence. In contrast, inhibition of the Shh signalling pathway by CP decreased EMT protein expression. Among the EMT genes, α-Sma and Cdh2 were strongly affected by the Pur and CP treatments ( Figure 8B,C). The result shows that the Shh signalling pathway also regulates the EMT phenotype in mSKPs.

| Using Pur to promote activation of the Shh signalling pathway for the long-term culture of mSKPs
Although sphere formation in mSKPs was remarkably decreased after passage 3, proliferation and sphere formation in mSKPs were improved by Pur treatment (Figure 9A,B). These data suggest that activation of the Shh signalling pathway by Pur can revive the selfrenewal and proliferation of aged mSKPs.

| D ISCUSS I ON
The objective of this study was to investigate sphere formation and cell proliferation using Pur, a Shh agonist, to activate the Shh signalling pathway. We demonstrated that Pur increases the expression of stem cell genes (CD49f, Ngfr, nestin, Klf4 and Bmi1) and EMT genes (N-cad, α-Sma, fibronectin, vimentin and Tgf-β1) in mSKPs.

(B)
Our findings suggest that Pur promotes the proliferation of mSKPs in culture, and that the Shh signalling pathway regulates the selfrenewal of mSKPs.
The Hh signalling pathway is involved in the survival, proliferation and differentiation of cells in embryonic development. 20,21,35 Many other studies have shown that aberrant signalling in this pathway is related to a variety of human cancers. These include basal cell carcinomas, colorectal cancer, ovarian cancer and smallcell lung cancer. 14,29,30,36 Activation of the Shh signalling pathway has an essential role in controlling self-renewal and tumour initiation in melanoma. 37 In addition, the Shh signalling pathway increases the initial generation and self-renewal of neural cells. 16 To our knowledge, there is no available study as to whether the Shh signalling pathway influences the proliferation, self-renewal and apoptosis of mSKPs. We demonstrated that Pur treatment enhances the sphere formation capability of mSKPs, and this result shows that the Shh signalling pathway is related to the self-renewal and proliferation of mSKPs.
It has been suggested that Hh signalling plays a critical role in regulating the proliferation of various types of stem cells, including mammary, telencephalic and mesenchymal stem cells. Pur enhances cell proliferation and reduces apoptosis in human umbilical cord blood-derived mesenchymal stem cells. This is achieved through the RNA-binding protein Msi1, which regulates oncogenes, cell cycle genes and microRNAs. 38 We used Pur to activate the Shh signalling pathway because Pur showed a similar effect to rShh. After Pur treatment, mSKPs were evaluated according to sphere size and number to verify the capacity for sphere formation. It has been reported that the PI3K-AKT signalling pathway promotes self-renewal and inhibits senescence in human SKPs treated with small molecules. 13 We confirmed that the number of spheres with a diameter longer than 50 μm increased at passages 1 and 2 after Pur treatment. Our results suggest that Pur treatment activates the Shh signalling pathway to promote cell proliferation and self-renewal in SKPs. Although the expression of key pluripotency genes (Oct4 and Nanog) did  When human and mouse SKPs are cultured long-term, ageing and senescence occur. Sphere formation and cell proliferation are reduced, and these cells cannot maintain their self-renewal potency at late passages. 13 Our results show that activation of the Shh signalling pathway by Pur treatment improves the self-renewal of mSKPs during long-term culture in vitro.
In conclusion, the Shh-Gli signalling pathway plays an important role in the self-renewal, proliferation and inhibition of apoptosis in mSKPs. Pur is critical for the expansion of mSKPs since it enhances self-renewal and proliferation by activating the Shh signalling pathway. In the future, human SKPs could possibly be grown to sufficient numbers for therapy. The results of this study provide fruitful information that adds to our knowledge of stem cells and skin development.

ACKNOWLEDG EMENT
This study was supported by a grant from the National Research

CO N FLI C T O F I NTE R E S T
The authors declare no conflict of interest.