Korean J. Microbiol. Biotechnol. Vol. 40, No. 2, 117–127 (2012) http://dx.doi.org/10.4014/kjmb.1204.04001 pISSN 1598-642X eISSN 2234-7305 Cell Biological Function of Secretome of Adipose-Derived Stem Cells on Human Dermal Fibroblasts and Keratinocytes Lee, Jae Seol1 and Jong-Hwan Lee1,2,3* 1Department of Biomaterial Control, Dong-Eui University, Busan 614-714, Korea 2Blue-Bio Regional Innovation Center, Dong-Eui University, Busan 614-714, Korea 3Department of Biotechnology and Bioengineering, Dong-Eui University, Busan 614-714, Korea Received : April 3, 2012 / Revised : June 7, 2012 / Accepted : June 9, 2012 The beneficial effects of adipose-derived stem cell conditioned media (ADSC-CM) for skin regeneration have previously been reported, despite the precise mechanism of how ADSC-CM promotes skin regeneration remaining unclear. ADSC-CM contains various secretomes and this may be a factor in it being a good resource for the treatment of skin conditions. It is also known that ADSC-CM produced in hypoxia conditions, in other words Advanced Adipose-Derived Stem cell Protein Extract (AAPE), has excellent skin regenerative properties. In this study, a human primary skin cell was devised to examine how AAPE affects human dermal fibroblast (HDF) and human keratinocyte (HK), which both play fundamental roles in skin regeneration. The promotion of collagen formation by HDFs was observed at 0.32 mg/ml of AAPE. AAPE treatment signifi- cantly stimulated stress fiber formation. DNA gene chips demonstrated that AAPE in HKs (p<0.05) affected the expression of 133 identifiable transcripts, which were associated with cell proliferation, migration, cell adhesion, and response to wounding. Twenty five identified proteins, including MMP, growth factor and cytokines such as CD54, FGF-2, GM-CSF, IL-4, IL-6, VEGF, TGF-β2, TGF-β3, MMP-1, MMP-10, and MMP-19, were contained in AAPE via antibody arrays. Thus, AAPE might activate the HK biological func- tion and induce the collagen synthesis of HDF. These results demonstrate that AAPE has the potential to be used for clinic applications aimed at skin regeneration. Keywords: ADSC-CM, skin regeneration, stress fiber, collagen synthesis Introduction for biotechnology such as skin care product like cosmetic, protein drug industries. Adipose-derived stem cells (ADSC) derived from human Oxygen deficiency, i.e., hypoxia, may have negatively subcutaneous adipose tissue are a population of pluripotent affected cell biological action. However, cellular functions mesenchymal cells and possess the ability to differentiate to hypoxic stress are highly dependent in cell type, position into various lineages. The secretome of ADSCs repair and and micro-environment. ADSCs are thought to reside in replace the defective surrounding cells. Thus, secretome hypoxic position covered with various tissues in complicated itself is a good resource for skin regeneration, re-epitheli- 3-dimensional space of the human body. Therefore, when alization, wound healing, and wrinkling care. Conditioned ADSCs are cultured under hypoxic conditions in vitro their medium from ADSCs (ADSC-CM) contained various growth proliferative and self-renewal capacities are significantly factors secreted from ADSC [12] and has excellent advan- improved [18] and hypoxia enhanced the expression of tages for the treatment of skin problems such as skin scar, certain secretome [7]. replacement and regeneration. ADSC-CM can be applied Therefore, we focused on Advanced Adipose-Derived Stem cell Protein Extract (AAPE), which is conditioned medium produced under a hypoxia of ADSCs. Human *Corresponding author dermal fibroblasts (HDF) and human HKs (HK) play an Tel: +82-51-890-2280; Fax: +82-51-890-2632 E-mail: [email protected] important role in skin biology such as wound re-epitheliali- 118 LEE AND LEE zation, the re-establishment and wound healing of the skin human fetal skin was grown in DMEM medium supplemented [15, 17, 26]. HKs with normal dermal fibroblasts must be with 10% fetal bovine serum, 2 mM glutamine and 100 µg/ o lead to upregulation of mRNA for collagen type I and III, ml penicillin-streptomycin at 5% CO2 and 37 C humidified increased fibroblast proliferation, and extracellular matrix atmosphere. accumulation [27]. Thus, cell biological function of both cells is essential for performing regenerative processes on Cell proliferation the skin surface. In this study, we examined the cell bio- Cell viability and proliferation was determined using logical function of AAPE on HDF and HK in vitro, and the CellTiter 96 Aqueous One Solution Reagent (Promega, components of AAPE through antibody array analysis. Madison, WI, US). Briefly, cells (2×105) were placed in 96-well plastic culture plates and incubated at 37oC in 5% Materials and Methods CO2 for 24 h, at which point 100 µL of 0.5 mg/mL MTS solution was added to each well and incubated for 4h at Stem cell culture and AAPE production 37oC. Formazan absorbance was read at 490 nm using a Human subcutaneous adipose tissue derived stem cells plate reader. (ADSC) were prepared from Prostemics Research Institute (Sungnam, Korea) followed by characteristic expressions Collagen synthesis assay of stem cell-related surface markers were confirmed by HDF was inoculated into 48-well plate (1×105 cells/well) flow cytometry [15, 16] and adipogenic, osteogenic, and and cultivated for 24 h. After culture, the medium was chondrogenic differentiation were also checked by the changed to serum-free medium containing vitamin C as a conventional method [11, 13]. ADSCs were cultured and positive control and AAPE at several concentrations, and expanded in normal control medium, and used for the ex- then HDF were cultivated for 48 h. Control cells were periments at passages 4. Cells were finally frozen in aliquots cultivated without samples. After culture, supernatants of using CellFreezerTM (Genenmed, Seoul, Korea) for the each well were collected, and the amount of procollagen future. To produce a ADSC-CM (AAPETM), a frozen vial type I C-peptide was measured by using a type I C-peptide containing 1×106 cells were launched onto culture medium EIA kit (TakaRa, Kyoto, Japan). Finally, samples were containing 10% FBS. After repeating subcultures to reach measured by ELISA reader at 450 nm wave length. 5×108 cells, the expanded ADSCs were introduced into CellFactoryTM CF10 (Nunc, Rochester, NY, US) in DMEM/ Fluorescence microscopy of stress fibers F12 serum-free medium (Welgene, Taegu, Korea). Cultures HDF and HK on collagen coated chamber slides (Lab- were conducted under a hypoxia by providing 2% O2 using Tek, Nalge Nunc Int. Naperville, IL, US) were cultured in N2 gas supply in a humidified multichannel incubator serum-free DMEM and growth factor free HK media for during 2 weeks. The conditioned media were collected and 12 h, respectively. The cells were treated with the AAPE micro-filtered, followed by quantitated total protein produc- for 24 h, then fixed in formalin and treated with ice-cold tion. Finally, for fresh use, 4 ml vials containing equal methanol for 10 min. The cells were then stained with rho- protein concentration were freeze-dried as a single lot sample damine phalloidin and observed by fluorescence microscopy. preparation of AAPE (Prostemics Research Institute, Sungnam, Korea) for this study. DNA chip analysis For this analysis with GeneChip Human Gene 1.0 ST Cell culture array (Affymetrix, Santa Clara, CA, US) containing 28,869 Normal HK was purchased from ATCC cell bank (ATCC# gene-level probe set, total cellular RNA was isolated from PCS-200-011). HKs were cultured in serum-free HK media HK incubated with AAPE (1.25 µg/ml) for 24 h using with epidermal growth factor at concentrations of 0.2% (v/ Trizol reagent (Invitrogen, Foster, CA, US) according to v) of bovine pituitary extract, 5 g/mL bovine insulin, 0.18 manufacturer's directions. The RNA samples were submitted g/mL hydrocortisone, 5 g/mL bovine transferrin, 0.2 ng/mL to the Advanced Medical Technology Center for Diagnosis human epidermal growth factor (EGF) at 37oC in a 5% & Prediction (School of Medicine, Kyungpook National CO2 humidified atmosphere. HDF cultured primarily from University, Taegu, Korea) for array preparation and analysis. CELL BIOLOGICAL FUNCTION OF SECRETOME OF ADIPOSE-DERIVED STEM CELLS 119 The signal intensity of the gene expression level was calculated by Expression Console software, Version 1.1 (Affymetrix). The list was filtered first for the absent genes, secondly for a fold change cutoff of 2, and thirdly for p value of ≤0.05 using Welch's t-test. Antibody array The freeze dried protein sample was submitted to the Ebiogen (Kyung Hee business center, Kyung Hee University, Seoul, Korea) for array preparation and analysis. The explorer antibody microarray slide (Fullmoon biosystems, Fig. 1. Human dermal fibroblast (HDF) proliferation. The Sunnyvale, CA, US) with 656 antibodies and the cytokine- amount of HDF proliferation is represented by the cell prolifera- focused microarray slide with 77 antibodies were treated 30 tion (%) in the MTS assay (n=3). The amount HDF proliferation was significantly less in AAPE (more than 0.625 µg/ml) treated ml of blocking solution and incubated on shaker for 45 group compared to the group treated with AAPE (from 0 to 0.313 minutes at room temperature. After blocking and coupling, µg/ml) by Student’s t-test. the slide was washed 2 times with 30 ml of washing solution. The 30 µL of 0.5 mg/ml Cy3-streptavidin (GE Healthcare, Chalfont St. Giles, UK) was mixed in 30 ml of detection buffer. After detecting, the slide was washed 2 times with 30 ml of washing solution. The slide scanning was performed using RevolutionTM 4200 microarray scanner (Vidar Systems, Herndon, VA, US) and ArraySifter Express 1.3 (Vidar Systems). The slide was scanned at 10 µm resolution, optimal laser power and PMT. After got the scan image, it was grided and quantified with ArraySifter Express 1.3.