脂肪干细胞的标记

E-Mail karger@karger.com www.karger.com/cto Original Paper Cells Tissues Organs 2013;197:384–398 DOI: 10.1159/000346714 DiI Labeling of Human Adipose-Derived Stem Cells: Evaluation of DNA Damage, Toxicity and Functional Impairment K. Froelich a G. Steussloff a K. Schmidt b M. Ramos Tirado a A. Technau a A. Scherzed a S. Hackenberg a A. Radeloff a R. Hagen a N. Kleinsasser a Departments of a Otorhinolaryngology, Plastic, Aesthetic and Reconstructive Head and Neck Surgery and b Trauma, Hand, Plastic and Reconstructive Surgery, University of Wuerzburg, Wuerzburg , Germany Key Words DiI labeling · Toxicity · Adipose tissue-derived stem cells Abstract Introduction: Adipose tissue-derived stem cells (ASCs) have become the primary focus of tissue engineering research. To understand their functions and behavior in in vitro and in vivo models, it is mandatory to track the implanted cells and distinguish them from the resident or host cells. A common labeling method is the use of fluorescent dyes, e.g. the lipo- philic carbocyanine dye, DiI. This study aimed to analyze po- tential DNA damage, toxicity and impairment of the func- tional properties of human ASCs after labeling with DiI. Methods: Cytotoxicity was measured using the MTT assay and DNA damage was determined by means of the comet assay. Potential apoptotic effects were determined using the annexin V-propidium iodide test. Differentiation potential was evaluated by trilineage differentiation procedures in la- beled and unlabeled ASCs. Proliferation as well as migration capability was analyzed, and the duration and stability of DiI labeling in ASCs during in vitro expansion was observed over a period of 35 days. Results: DiI labeling did not cause geno- toxic effects 15, or 30 min or 24 h after the labeling proce- dure, and there were no cytotoxic effects until 72 h after- Accepted after revision: December 26, 2012 Published online: March 9, 2013 Dr. Katrin Frölich Department of Otorhinolaryngology, Plastic, Aesthetic and Reconstructive Head and Neck Surgery , University of Wuerzburg, Josef Schneider Strasse 11 DE–97080 Wuerzburg (Germany) E-Mail froelich_k   @   klinik.uni-wuerzburg.de © 2013 S. Karger AG, Basel 1422–6405/13/1975–0384$38.00/0 Abbreviations used in this paper ALP alkaline phosphatase APC allophycocyanin ASCs adipose-derived stem cells BrdU 5-bromo-2-deoxyuridine CD cluster of differentiation DiI/CM-DiI chloromethylbenzamido-1,1′-dioctadecyl-3,3,3′,3′ tetramethylindocarbocyanine perchlorate DMEM Dulbecco’s modified Eagle’s medium DMSO dimethylsulfoxide DT % of DNA in the tail FABP4 fatty acid-binding protein 4 FCS fetal calf serum FITC fluorescein isothiocyanate LPL lipoproteinlipase MMS methyl methane sulfonate MTT 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide OTM Olive tail moment PBS phosphate-buffered saline P/S penicillin/streptomycin RUNX-2 Runt-related transcription factor 2 t-BHP tert-butylhydroperoxide TL tail length D ow nl oa de d by : Pe ki ng U ni ve rs ity 21 8. 24 9. 94 .3 - 7/ 23 /2 01 3 4: 11 :1 1 AM Influence of DiI Labeling on ASCs Cells Tissues Organs 2013;197:384–398 DOI: 10.1159/000346714 385 wards. No impairment of proliferation or migration capability or differentiation potential could be determined. However, after 35 days, only 37% of labeled cells could be detected us- ing the fluorescence microscope, which indicates a decrease in staining stability during in vitro expansion. Conclusion: DiI is a convenient method for ASCs labeling which causes no toxic effects and does not impair the proliferation, migration or differentiation potential of ASCs after the labeling proce- dure. Copyright © 2013 S. Karger AG, Basel Introduction Adipose tissue-derived stem cells (ASCs) of humans and other species have become the primary focus of stem cell research and clinical applications over the past decade [Lindroos et al., 2011]. Adipose tissue is abundantly available and ASCs can be easily harvested with low donor-site morbidity, and isolated and expanded extensively in vitro. Similar to bone marrow-derived stem cells, ASCs are able to under- go multilineage differentiation i.e. osteogenic, chondro- genic, adipogenic, myogenic and neurogenic [Zuk et al., 2001; Gimble et al., 2007; Guilak et al., 2004; De Ugarte et al., 2003; Bunnell et al., 2008; Frölich et al., 2011; Lind- roos et al., 2011]. This property predestines ASCs for use in various fields of application [Lindroos et al., 2011]. However, to assign specific behavior and functions to transplanted ASCs within an in vivo model or in cocul- ture systems, it is necessary to distinguish these cells from host, resident or cocultured cells, respectively. Cellular labeling allows the differentiation of resident and im- planted cells as well as observation of cell migration, fate, interaction, differentiation and tissue formation after im- plantation. Labeling methods have to be feasible and the tracking reagents have to be detectable until harvesting and analyzing the cells or tissue. In addition, the viability and migration and differentiation potential of the labeled cells should not be impaired. Various methods and substances for the cellular label- ing of mesenchymal stem cells used for in vitro and in vivo analyses are described. These include luciferase transfection [Bereziat et al., 2005; Wolbank et al., 2007; Bai et al., 2011], green fluorescent protein [Bereziat et al., 2005; Lin et al., 2006; Yan et al., 2007; Wolbank et al., 2007; Bai et al., 2011], fluorescence in situ hybridiza- tion [Li et al., 2008], various fluorochromes [Rieck and Schlaak, 2003; Hemmrich et al., 2006; Yan et al., 2007; Li et al., 2008; Li et al., 2009; Lequeux et al., 2011], iron oxide labeling for MRI [Heymer et al., 2008; Kraitchman and Bulte, 2008; Yan et al., 2007] as well as radionuclide label- ing strategies [Hofmann et al., 2005; Yan et al., 2007]. However, there is no general recommendation for the labeling of ASCs. Fluorochromes such as PKH26, a fluo- rescent surface marker [Rieck and Schlaak, 2003; Hemmrich et al., 2006], BrdU (5-bromo-2-deoxyuri- dine) which replaces thymidine and incorporates into DNA strands during cell proliferation [Lequeux et al., 2011] and CSFE (Vybrant ® CFDA-SE cell tracer kit) which diffuses passively into the cells [Hemmrich et al., 2006] were analyzed for their applicability to ASCs. In addition, gene transfection procedures resulting in the expression of green fluorescence protein or luciferase were also used to label ASCs [Béréziat et al., 2005; Wolbank et al., 2007]. Chloromethylbenzamido-1,1 ′ -dioctadecyl-3,3,3 ′ ,3 ′ - tetramethylindo-carbocyanine perchlorate (CM-DiI; CellTracker TM ) is one of the most commonly used label- ing dyes and was applied in various cell types such as neurons [Honig and Hume, 1989; Kuffler, 1990; Deng et al., 2001; Schmidt and Rathjen, 2011], lymphocytes [An- drade et al., 1996], bone marrow-derived stem cells and ASCs [Dai et al., 2005; Hemmrich et al., 2006; Li et al., 2008] in vivo [Honig and Hume, 1989; Andrade et al., 1996; Deng et al., 2001; Dai et al., 2005] and in vitro [Ho- nig and Hume, 1989; Andrade et al., 1996; Deng et al., 2001; Hemmrich et al., 2006; Li et al., 2008; Schmidt and Rathjen, 2011]. This lipophilic fluorescent carbocyanine dye functions as a phospholipid analog and labels the plasma membrane and the membranes within the cell [Li et al., 2008; Chazotte, 2011]. CM-DiI labeling can be combined with subsequent immunohistochemical or immunfluorescence analyses or electron microscopy, as it is retained in the cells during the fixation processing [Andrade et al., 1996; Hemmrich et al., 2006; Li et al., 2008]. Hemmrich et al. [2006] analyzed CFSE, CM-DiI and PKH26 and applied these fluorochromes for the labeling of human ASCs. They found no impairment in differen- tiation potential and proliferation. However, they report- ed a high toxicity for CFSE and CM-DiI. The cell loss was about 45% using 1 μ M CM-DiI and about 67% using 10 μ M CM-DiI for cell labeling. So the aim of this study was to analyze possible DNA damage, toxicity and impairment of the functional prop- erties of human ASCs and long-term staining stability us- ing CM-DiI for cell tracking. D ow nl oa de d by : Pe ki ng U ni ve rs ity 21 8. 24 9. 94 .3 - 7/ 23 /2 01 3 4: 11 :1 1 AM Administrator 高亮 Administrator 高亮 Administrator 高亮 Administrator 高亮 Administrator 高亮 Froelich et al. Cells Tissues Organs 2013;197:384–398 DOI: 10.1159/000346714 386 Materials and Methods Isolation and Cell Culture of Human ASCs Human ASCs were isolated from subcutaneous adipose tissue of 8 healthy donors undergoing liposuction surgery for aesthetic reasons. Studies were approved by the Ethics Board of the Medical Department of the Julius Maximilian University of Wuerzburg (grant No. 72/06) and informed consent was obtained from all in- dividuals included in the study. The isolation procedure was performed as previously described [Frölich et al., 2011; Technau et al., 2011]. Liposuction material was rinsed with phosphate-buffered saline (PBS; Roche Diagnos- tics GmbH, Mannheim, Germany) plus 1% penicillin/streptomy- cin (P/S; Biochrom AG, Berlin, Germany) and digested with Col- lagenase P (Roche Diagnostics GmbH). The stromal cell fraction was separated from the adipose cell fraction by centrifugation. Af- ter discarding the supernatant and elimination of erythrocytes in the remaining cell pellet by erythrocyte lysis buffer [154 m M am- monium chloride, 10 m M potassium bicarbonate and 0.1 m M eth- ylenediaminetetraacetic acid (EDTA)] further centrifugation and washing steps followed. Subsequently, cells were resuspended in expansion medium consisting of Dulbecco’s modified Eagle’s me- dium (DMEM; Gibco Invitrogen, Karlsruhe, Germany) contain- ing 1% P/S and 10% fetal calf serum (FCS; Linaris, Wertheim-Bet- tingen, Germany). Cells were plated in culture flasks and main- tained at 37   °   C in a humidified atmosphere and 5% CO 2 . Primary cells were defined as passage 0. Medium was replaced every third day during expansion. When the cells reached 80% confluency, they were detached with 0.25% trypsin containing 1 m M EDTA (Gibco Invitrogen), counted using an automated cell counter (Casy ® Technologies, Innovatis AG, Reutlingen, Germany) and resuspended in cryopreservation medium [80% FCS, 10% DMEM and 10% dimethylsulfoxide (DMSO)]. The ASCs (1 × 10 6 ) were resuspended in 1 ml of cryopreservation medium, frozen at –80   °   C in an ethanol-jacketed closed container for 2 days and then stored in liquid nitrogen. For the following experiments, ASCs were rap- idly thawed at 37   °   C in a water bath and centrifuged (1,300 rpm for 7 min) to remove DMSO. They were then resuspended in expan- sion medium, seeded in 175-cm 2 culture flasks at a density of 2,000 cells/cm 2 and maintained at 37   °   C in a humidified atmosphere and 5% CO 2 . Thawed cells were defined as passage 1. When the cells reached 80% confluency, they were detached with 0.25% trypsin and transferred into new flasks for the next passage. ASCs of pas- sage 2 were used for the following experiments. Isolation of Human Lymphocytes Human peripheral blood lymphocytes were isolated from 8 healthy donors after receiving informed consent according to the approval of the Ethics Board of the Medical Department of the Julius Maximilian University of Wuerzburg (grant No. 72/06). Whole blood samples were collected in heparin-containing tubes and lymphocytes were isolated using separation medium and a cell separation tube (Ficoll Paque Plus, GE Healthcare, Freiburg, Ger- many). Flow Cytometry To determine the ASC-specific cell surface marker expression, flow cytometry was performed [Becton Dickinson (BD) FACS- Canto TM ; BD Bioscience, Bedford, Mass., USA]. Cells of passage 2 were incubated with anti-cluster of differentiation (CD)34 [phy- coerythrin (PE), No. 550761], anti-CD44 [fluorescein isothiocya- nate (FITC), No. 555478], anti-CD45 (FITC, No. 555482), anti- CD73 (PE, No. 550257), anti-CD90 [allophycocyanin (APC), No. 559869] and anti-CD105 (FITC, Nos. 555690 and 555988). All an- tibodies were purchased from BD. Flow cytometry was done with and without antibody staining. DiI Labeling CM-DiI was purchased from Molecular Probes, Inc. (Eugene, Oreg., USA). DiI labeling of the ASCs was performed according to the manufacturer’s instructions. Stock solution was prepared in 100% ethanol at a concentration of 2 mg/ml. Immediately before labeling, cells were detached with 0.25% trypsin containing 1 m M EDTA (Gibco Invitrogen), counted using an automated cell coun- ter (Casy ® Technologies, Innovatis AG) and resuspended in DMEM with 10% FCS and 1% P/S with a concentration of 1 × 10 5 cells per milliliter of medium. After this, DiI solution was added to obtain a final concentration of 2 μ M DiI (2 μg DiI/ml medium). Finally, 1 × 10 5 cells were exposed to 2 μg DiI/ml medium. Cells were incubated in the working solution for 5 min at 37   °   C and for an additional 15 min at 4   °   C with occasional mixing, followed by two washing and centrifugation steps. They were then counted again to obtain the correct number of labeled cells after the label- ing procedure for the following experiments. Alkaline Single-Cell Microgel Electrophoresis Assay The alkaline single-cell microgel electrophoresis (comet) assay was performed as previously described [e.g. Tice et al., 2000; Hack- enberg et al., 2011] to detect DNA strand breaks and alkali labile sites as well as incomplete excision repair sites in single cells. For evaluation, a DMLB fluorescence microscope (Leica Microsys- tems, Wetzlar, Germany) with a filter system incorporating a green excitation filter (515–560 nm), a dichromatic beamsplitter (580 nm) and an emission filter (590 nm) at a magnification of 400× was used. To analyze DNA fragmentation, the Komet 5.5 image system (Kinetic Imaging, Liverpool, UK) was used. Tail DNA, tail length (TL) and Olive tail moment (OTM; which represents the product of the median migration distance and the percentage of DNA in the tail [Olive et al., 1993]) were determined to quantify DNA frag- mentation. Statistical evaluation was based on the OTM values. Preliminary analyses of possible DNA strand breaks due to DiI addition were performed using human peripheral lymphocytes. Isolated lymphocytes were treated with a final concentration of 2 μ M DiI according to the manufacturer’s labeling instructions. The comet assay was performed immediately after the labeling procedure, and then after 15 and 30 min and 24 h. Unlabeled cells were used as a negative control. Cells were treated with 200 μ M di- rectly alkylating methyl methane sulfonate (MMS; Sigma-Aldrich, Steinheim, Germany) to serve as a positive control avoiding cyto- toxic effects. ASCs from passage 2 of 8 healthy donors were labeled with DiI according to the manufacturer’s instructions. The comet assay was performed immediately after the labeling procedure, and after 15 and 30 min and 24 h. Unlabeled ASCs were used as a negative con- trol. Positive controls were treated with 200 μ M MMS. Trypan Blue Exclusion Test Immediately after labeling, and after 15 and 30 min and 24 h, the viability of ASCs was determined by the Trypan Blue exclusion test [Philips, 1973] before conducting the comet assay to ensure an D ow nl oa de d by : Pe ki ng U ni ve rs ity 21 8. 24 9. 94 .3 - 7/ 23 /2 01 3 4: 11 :1 1 AM Influence of DiI Labeling on ASCs Cells Tissues Organs 2013;197:384–398 DOI: 10.1159/000346714 387 appropriate viable cell number. Cells were counted in a Neubauer chamber. Viable cells have the ability to exclude the dye and are transparent whereas nonviable cells turn blue due to defects in the cell membrane. The percentage of viable cells was determined in 16 counting fields. MTT Cytotoxicity Assay The effect on the proliferation and viability of ASCs after DiI labeling was studied using the MTT [3-(4,5-dimethylthiazol- 2-yl)-2,5-diphenyl tetrazolium bromide] colorimetric staining method [Mosmann, 1983]. ASCs of passage 2 of 6 patients were used for this experiment. Cells were labeled with DiI as described above. Thus, 10 4 ASCs per well were plated in a 96-well plate for the time points 3 and 24 h. To avoid excessive proliferation and confluence, wells for the analyses of the time points 48 and 72 h after labeling were seeded with 5 × 10 3 ASCs for each group. Un- labeled cells were used as a negative control and cells treated with tert-butylhydroperoxide (t-BHP; Luperox ® TBH70X, Sigma-Al- drich) at a concentration of 1.5 m M for 2 h before performing the MTT assay served as positive control. For each patient, each group and each time point, 8 wells were seeded, i.e. altogether 48 samples for each group and each time point were measured. ASCs were in- cubated with DMEM with 1% P/S and 10% FCS (expansion me- dium) at 37   °   C in a 5% CO 2 atmosphere. The first measurement was performed 3 h after labeling and seeding of the cells and the further analyses were conducted at 24, 48 and 72 h. All wells were incubated with medium containing MTT (Sigma-Adrich) at a con- centration of 1 mg per milliliter of medium for the analyses. There- after, an incubation at 37   °   C in a 5% CO 2 atmosphere took place for 4 h. The medium of each well was replaced by 100 μl isopropa- nol. After 30 min, the color conversion of the blue formazan dye of each well was measured using a multiplate reader [Titertek Mul- tiscan PLUS (MKII), Pforzheim, Germany] at 570 nm. The mean extinction values were calculated from 8 wells per patient, group and time point. The values of the unlabeled cells were equalized to a viability of 100%. The viability of the labeled cells and the positive control was indicated as a percentage of the viability of the unla- beled controls. Annexin V-Propidium Iodide Staining Apoptosis Test An annexin V-propidium iodide kit (BD Bioscience, Heidel- berg, Germany) was used to measure apoptosis and necrosis after DiI labeling by flow cytometry according to the manufacturer’s instructions. DiI-labeled and unlabeled cells were seeded in a 6-well plate at a density of 1 × 10 5 ASCs per well. Analyses were performed in duplicate for each of the 6 patients. The first measurement was performed 3 h after the labeling and seeding of the cells and further analyses followed after 24, 48 and 72 h. Cells treated with t-BHP at a concentration of 2 m M for 2 h before performing the apoptosis test served as a positive control. Adherent cells were trypsinized and added to the preserved medium. Following two washing steps with 1× PBS, the cell pellet was resuspended with ice-cold binding buffer (0.1 M HEPES pH 7.4, 1.4 M NaCl and 25 m M CaCl 2 ). By addition of 5 μl annexin V-APC and 5 μl propidium iodide to each sample, annexin V and propidium iodide staining was performed. Incubation for 15 min at room temperature followed. Fluores- cence was measured using flow cytometry. Multilineage Differentiation Potential Adipogenic, osteogenic and chondrogenic differentiation pro- cedures were performed in order to determine the multilineage differentiation potential and possible impairment due to DiI label- ing. DiI-labeled and nonlabeled ASCs of passage 2 were used for these studies. For