• Introduction
• Materials and Methods
• Chemicals
• Isolation and propagation of SKP
• Passaging of sphere-forming SKP
• Adipogenic and neural differentiation for characteri-zation of SKPs
• Treatment of LIF
• Total RNA extraction
• First-strand cDNA synthesis
• Real time RT-PCR
• Statistical analysis
• Results
• Characterization of SKPs by in vitro differentiation
• Expression of pluripotency and neural crest marker genes
• Discussion
• Acknowledgments

Introduction

 Stem cells have properties of self-renewal and various lineage differentiations in the body [1,2]. Recently, variety of multipotent stem or precursor cells was found in the region of skin such as bulge, hair follicle and dermis [3,4]. Among them, skin-derived precursor cells (SKPs) are origi-nated from juvenile and adult dermis in mammals. In SKPs, Sox2,  Klf4 and  c-Myc, which are key factors for induced pluripotent stem cells [5], and  Snai2 and  Ngfr, neural crest-related genes, are highly expressed and they have both properties of stem cells and neural crest cells [6-8]. SKPs are capable of differentiating neural and mesodermal lineage cells in human, mouse and pig, corresponding to the multipo-tency of embryonic neural crest stem cells [4,7-10]. The cells are isolated from the dermis and cultured in vitro as suspen-ding condition in the existence of bFGF and EGF. SKPs can be differentiated into neurons, glia, smooth muscle cells and adipocytes  in vitro [7]. The cells are also able to form sch-wann cells which can be potentially used for injured spinal cord regeneration [7,11]. In the mouse, the pluripotency of embryonic stem (ES) cells is maintained by leukemia inhi-bitory factor (LIF), a member of IL-6 cytokine family, by preventing cell differentiation [12]. However, unlike murine ES cells, LIF is not necessary for maintaining pluripotency in human ES cells [13]. In addition, LIF plays a crucial role for the development of preimplantation stage embryos in vivo [14]. In contrast to embryonic cells, myeloid cells and neuro-blastoma cells were induced differentiation by LIF treatment [15,16]. Although LIF have been used to culture SKPs in human, pig and mouse [4,8,17], little is understood about the effect of LIF in SKPs. In this study, to evaluate the effect of LIF on the culture of SKPs in vitro, changes of the expres-sion level of pluripotent stem cell- and neural crest-related genes in murine SKPs by LIF, a stemness maintaining factor, was investigated.

Materials and Methods

Chemicals

 All inorganic and organic compounds were obtained from Sigma-Aldrich Korea (Yong-in, Korea) and all liquid solu-tions were purchased from Invitrogen Korea (Seoul, Korea) unless otherwise stated.

Isolation and propagation of SKP

 The murine SKPs were isolated by a previously described method with a few modifications [4,18]. To obtain skin from 5-6 weeks C57BL6 X DBA2 F1 mice, hairs of back were re-moved. After back skin was dissected and washed 4 times in phosphate-buffered saline with penicillin, skin pieces were minced into small pieces with blade. Small pieces of skin were transferred to a 100 mm petri dish containing 5 ml of 0.1% (w/v) trypsin solution and 5 ml of Hank’s buffered salt solution with 2.4 mg/ml of dispase and 1 mg/ml collagenase type IV, and then, incubated for 60 min in 37℃, 5% CO₂ cell culture incubator. The incubated skin pieces were mixed by 30 times of pipetting using 10 ml glass pipette and the me-dium consisting of Dulbecco’s modified Eagle medium and nutrient mixture F-12 (1:1, v/v; DMEM/F-12) was added. Skin cell suspension poured through a 100 and 40 μm Nylon cell strainer over 50 ml conical centrifuge tube. Dissociated cells were centrifuged at 1000 rpm for 5 min. After super-natant was removed, dissociated cells were re-suspended in 10 ml of DMEM/F-12 containing 2% B-27, 20 ng/ml bFGF and EGF (SKP medium). These  cells were cultured in 25 cm² uncoated cell culture flasks in a 37℃, 5% CO₂ atmosp-here (Fig. 1A). Fresh SKP medium was replaced every 2-3 days, and the cells started sphere forming at passage 1 (Fig. 1B).

Fig. 1. Culture of murine skin precursor cells (SKPs). (A) Iso-lated single cells from murine back skin (passage 0). (B) For-mation of primary spheres after 1 week of culture (passage 1). (C) Large spheres after 3 week of culture (passage 3). Bar, 40 μm.

Passaging of sphere-forming SKP

 The medium containing suspending spheres was gently mixed by 10 ml glass pipette and moved to 15 ml conical tube. The spheres were centrifuged at 1000 rpm 5 min. After supernatant was removed, 1 ml of accutase was supplemen-ted and the pellet was mechanically dissociated with a pipet-te and the clusters of cells with accutase were incubated for 3-5 min in a 37℃, 5% CO₂. The cells were t hen centrifu-ged at 1500 rpm for 3 min, and the pellet was mechanically dissected with a pipette. The single cells were cultured in 10 ml of SKP medium. The cells were passaged every 7 days. After the 2-3 weeks, proliferating cells formed spheres in suspension culture and size of the sphere increased with sub- cultural steps (Fig. 1C).

Adipogenic and neural differentiation for characteri-zation of SKPs

 To confirm the character of isolated cells from dermis as SKPs, adipogenic and neural differentiation were induced. For adipogenic differentiation, SKPs were attached on the bottom of 6-well plate containing SKP medium supplemen-ted with 10% FBS and 10 ng/ml bFGF. When the cells grew to confluence, they were cultured in the same medium exclu-ding bFGF for additional 5 days. For staining, differentiated cells were fixed with 10% formaldehyde in a 4℃ for 1 hr and then washed twice with PBS. The cells were then stai-ned with Oil red O. For neural differentiation, SKPs were atta-ched on laminin- and PDL-coated 6-well plate with neuro-basal medium^TM including B27 supplement and 0.5 mM dibu-tyryl cAMP for 14 day.

Treatment of LIF

 After 21 day of culture (Passage 3), 1,000 IU murine LIF (Millipore, Billerica, MA, USA) was treated to SKPs in culture for 72 hr.

Total RNA extraction

 Total RNA was isolated from sphere forming SKP using RNeasy^TM  Mini Kit (QIAGEN, Hilden, Germany). Briefly, the appropriate number of cells was pelleted by centrifuga-tion and all supernatant was removed. The cells were dis-rupted by adding Buffer RLT Plus. The lysate was moved directly into a QIA shredder column, centrifuged for 2 min at 15,000 rpm to fully homogenize the lysate. Then the homo-genized lysate was transferred to a gDNA eliminator spin column and centrifuged for 30 sec at ≥10,000 rpm. Ethanol (70%) was added to the flow-through and the sample inclu-ding the precipitate was transferred to an RNeasy spin co-lumn placed in a 2 ml collection tube and centrifuged for 15 sec at  ≥10,000 rpm and the flow-through was discarded. Buffer RW1 and buffer RPE were treated the same as etha-nol. Then, buffer RPE was added again to the column and centri-fuged for 2 min at ≥10,000 rpm and the flow-through was discarded. The RNeasy spin column was placed in a new 1.5 ml collection tube and RNase-free water added directly to the spin column membrane and centrifuged for 1 min at ≥10,000 rpm to elute the RNA.

First-strand cDNA synthesis

 For the synthesis of first-strand cDNA by reverse transcrip-tase, reverse transcription was performed for 1 hr at 42℃ in a final reaction volume of 25 μl containing purified total RNA, 5 μl of 5X reaction buffer (Promega, Madison, Wi, USA), 5 μl of dNTPs (each 2.5 mM), 2.5  μl of 10 mM synthesis primer, 0.5  μl of RNasin plus RNase inhibitor (40 U/ml; Promega), and 1 μl of M-MuLV reverse transcriptase (20 U/μl, Invitrogen).

Real time RT-PCR

 For optimal quantification,  primers were designed using Primer Express software (Applied Biosystems, Foster City, CA, USA). The real time RT-PCR reaction was performed using the ABI PRISM 7500 system and SYBR Green PCR Master Mix (Applied Biosystems). All points of the standard curve and all samples were run in triplets as technical replicates. The standard curves were calculated using the verified DNA as template for murine GAPDH. In each run 1 μl of cDNA was used as template and the sample was added to 5 μl dou-ble-distilled water, 2 μl of forward and reverse primers (20 pmol/ml) and 10  μl SYBR Green PCR Master Mix. The following amplification procedure was employed: denatura-tion stage (95℃ for 10 min), amplification and quanti-fication stage repeated 40 times (94℃ for 15 sec, 60℃ for 1 min with single fluorescence measurement), dissociation curve stage (temperature increments of 0.1℃ per 30 sec starting from 60 to 95℃ with fluorescence measurement). Data was analyzed with 7500 System Sequence Detection software (Applied Biosystems), which for all samples calculated that starting quantities of all candidate reference genes, based on the standard curves for these genes.

Statistical analysis

 Each experiment was replicated three to five times. Mean gene expression values were analyzed by  t-test to compare parameters between the different study groups. The interac-tion between replicate and treatment was also tested using two-way ANOVA. Difference at  P < 0.05 was considered significant.

Results

Characterization of SKPs by in vitro differentiation

 The character SKPs were confirmed by neural and adipo-genic differentiation. The murine SKPs which attached on the laminin and PDL coated plate in neural differentiation medium were differentiated into neural cells after 14 day of culture. Differentiated cells from SKPs showed heterogene-ous population of various types of neural cells and schwann cells. The cells of adipogenic differentiation induction showed morphology of adipocytes and this was confirmed by Oil red O staining (Fig. 2).

Fig. 2. In vitro differentiation of SKPs. (A) Heterogeneous neural‐like progeny derived from SKPs. (B) Adipogenic cells stained with Oil Red O.

Expression of pluripotency and neural crest marker genes

 The expression of Oct4 significantly increased by LIF trea-tment whereas the expression of Nanog and Ngfr decreased by the treatment (P < 0.05). There was no significant change on the expression level of c-Myc, Klf4 and Snai2; Fig. 3).

Fig. 3. Gene expression analyses in murine SKPs after LIF (1,000 IU) treatment. Quantitative real‐time PCR for genes of pluripotent stem cells (Oct4, Nanog, Klf4 and c-Myc) and neural crest cells (Snai2 and Ngfr). All values were depicted by the ratio to the expression in the control group (values in SKPs without LIF treatment = 1). Data are expressed as mean ± SD (n = 4). *P < 0.05.

Discussion

 SKPs which are able to form sphere in suspension culture can differentiate to various lineage progeny [4]. In the previ-ous study, we showed that valproic acid, histone deacety-lation inhibitor, enhances the expression level of neural crest related genes, whereas reduces the expression level of plu-ripotency-related genes in murine SKPs [18]. In this study, the change of gene expression was observed after LIF treat-ment. LIF is a critical factor for maintaining the pluripo-tency of murine ES cells by inhibiting cell differentiation th-rough JAK/STAT pathway [13,19,20]. The JAK/STAT signa-ling pathway is essential for self-renewal of stem cells [13]. When the ES cells are maintained undifferentiated state by LIF, they are expressed pluripotency-related genes such as Oct4 and Nanog [19,21-23]. LIF signaling also significantly en-hance the STAT3 expression in porcine SKPs [24]. As sp-heres of SKPs include progeny of neural crest cells, the cells expressed neural crest-related genes such as Snai2 and Ngfr [4,7,8]. In this study, LIF increased the expression level of Oct4 which is POU domain transcription factor and sustain self-renewal and pluripotency. This shows that LIF enhan-ces stemness of SKPs. However, expression level of Nanog was decreased by LIF treatment. Although Nanog is impor-tant for maintaining pluripotency in ES cells, it also acce-lerates oesteogenic lineage differentiation in the human me-senchymal cells [25,26]. In the present experiment,  Ngfr, neural crest cell marker, was decreased by LIF and the result represents that the potential of neural crest lineage differen-tiation was suppressed following LIF treatment. However, Snai2 expression was unchanged by LIF treatment. There is a report claiming that the STAT3 expression was increased by LIF treatment in porcine SKPs, whereas the same treatment did not change the level of Snai2 expression [24], and the data of the present study also imply that LIF signaling does not affect  Snai2 expression in the murine SKPs. Enhanced Oct4 expression may keep pluri- or multi-potency by alle-viating differentiation potential in murine SKPs. The previ-ous study showed that cell proliferation of human SKPs was decreased by LIF [17]. In the present study, decreased exp-ression of Nanog following LIF treatment may also reduce proliferative capacity of murine SKPs because Nanog is a cru-cial factor for cell proliferation in adult stem cells [21,23].

 In conclusion, the finding implicates that LIF may sup-port maintenance of stemness in SKPs by suppressing linea-ge differentiation and partly by controlling cell proliferation and this may contribute to controlling stemness of SKPs and other stem cells. In addition, The LIF treatment resulting stem-ness elevation can be a tool for efficient production of pluri-potent stem cells using SKPs which can be collected non- invasively from the patient.

Acknowledgments

 This work was supported by the National Research Foun-dation of Korea (NRF) grant funded by the Korea govern-ment (MEST; Grant number 2011-0027807) and Techno-logy Development Program for Agriculture and Forestry, Mi-nistry for Agriculture, Forestry and Fisheries (MAFF; Grant number 111160-4), Korea.