analysis methods, these observations were likely to be missed, resulting in sub-optimal control of tissue formation. Moreover, the data provide insight into the use of human fetal mesenchymal stromal cells as an alternative to animal models for processes that are not regulated in the same manner in various species. As a model, it is readily accessible for genetic manipulation and might be used for unraveling the molecular mechanisms underlying growth regulation in the human epiphyseal plate during puberty. As such it may find its use in the development of novel treatment strategies for various growth disorders aimed at intervening in growth plate maturation and fusion and bone tissue engineering. Experimental Procedures 4.1 Cell culture The use of human fetal material was approved by the medical ethical committee of the Leiden University Medical Center and written informed consent was obtained from the women under- going elective abortion. Cell suspensions of fetal bone marrow were obtained by flushing the long bones of fetuses with M199 washing medium. For the chondrogenic differentiation and microarray analysis, cells derived from a single 22 week old fetus were used. Red cells were depleted by incubation for 10 minutes in NH4Cl (8.4 g/L)/KHCO3 (1 g/L) buffer at 4uC. Mononuclear cells were plated at a density of 166104 cells/cm2 in M199 culture medium (Gibco) supplemented with 10% fetal bovine serum (FBS), 1% penicillin/streptavidin (P/S), fungizone, endothelial cell growth factor (ECGF) 20 mg/ml (Roche Diagnostics) and heparin 8 U/ml in culture flasks coated with 1% gelatin according to previously established culture conditions for human fetal MSCs [40]. Cultures were kept in a humidified atmosphere at 37uC with 5% CO2. The culture medium was changed twice per week. After reaching near-confluence at passage 4 to 5 (15 population doublings), hfMSCs were harvested by treatment with 0.5% trypsin and 0.5% ethylenediaminetetraacetic acid (EDTA; Gibco) for 5 minutes at 37uC and replated for chondrogenic differenti- ation. 4.2 In vitro chondrogenic differentiation hfMSCs (26105 cells/well) were cultured in cell pellets. Pellets were formed by centrifugation of the cells at 1200 rpm for 4 minutes in U-shaped 96-well suspension culture plates (Greiner). To induce chondrogenesis the pellets were cultured at 37uC with 5% CO2 in 200 ml of serum-free chondrogenic medium consisting of high-glucose (25 mM) Dulbecco’s modified Eagle’s medium (DMEM; Gibco) supplemented with 40 mg/ml proline (Sigma), 100 mg/ml sodium pyruvate (Sigma, USA), 50 mg/ml ITS (insulin-transferrin-selenic acid) with Premix (BD Biosciences), 1% Glutamax (Gibco), 1% penicillin/streptavidin, 50 mg/ml ascorbate-2-phosphate (Sigma), 1027 M dexamethasone (Sigma), 10 ng/ml transforming growth factor-b3 (TGFb3; R&D Systems), 500 ng/ml bone morphogenetic protein 6 (BMP6) and antibiotic and antimycotic mix (0.06% polymixin, 0.2% kanamycin, 0.2% penicillin, 0.2% streptavidin, 0.02% nystatin and 0.5% ampho- tericin) as described by Sekiya et al., 2001 [41]. The medium was changed twice per week for 5 weeks. 4.3 Histological analysis Two pellets per time point (after 1, 2, 3, 4, or 5 weeks of chondrogenesis) were used for histological evaluation. Pellets were fixed in 10% formalin, dehydrated by treatment with graded ethanols and processed for paraffin embedding. 5 mm sections were cut using a Reichert Jung 2055 microtome (Leica). For each pellet, only the midsagittal sections were mounted on glass slides. hfMSCs Differentiate toward Growth Plate Cartilage Before histological (toluidine blue) or immunohistochemical staining, sections were deparaffinized in xylene, treated with graded ethanols followed by three washing steps with phosphate buffered saline (PBS). For immunofluorescence of collagen type II, sections were pre- treated with 10 mM citric acid buffer (pH = 6) for antigen retrieval. Sections were incubated with a collagen type II monoclonal antibody (clone 3HH1-F9, Abnova) at 1:100 dilution in 1% bovine serum albumine (BSA)/PBS buffer overnight at 4uC. After washing, sections were incubated with Alexa Fluor 488-Goat anti-Mouse IgG1 (Invitrogen, Molecular Probes, diluted 1:1000 in PBS/1% BSA) for 1 hour and protected from light. Sections were counterstained with DAPI and mounted with vectashield. For collagen type X immunohistochemistry, sections were preincubated with blocking buffer (1% H2O2 in 40% methanol, 60% tris buffered saline) twice for 15 minutes at room tempera- ture, followed by overnight incubation at 4uC with mouse monoclonal antibody against collagen type X in a 1:100 dilution (Quartett). Next, sections were incubated with the secondary antibody biotinylated rabbit-anti-mouse IgG (DAKO) in a 1:300 dilution, followed by incubation with horseradish-peroxidase- conjugated-streptavidine (Amersham Biosciences). Staining was visualized with 3-amino-9-ethylcarbazole substrate in 0.2 mg/ml acetate buffer (pH 5.2) with 0.04% H2O2. After counterstaining with haematoxylin, the sections were mounted in Histomount (National Diagnostics). Pictures of the stained pellets were taken with a Nikon DXM 1200 digital camera using standardized settings. 4.4 Patients and tissue preparation Human proximal tibia growth plate tissues were collected from patients at Tanner stage B1 undergoing surgery for different medical conditions. The protocol used was approved by the medical ethical committee of the Leiden University Medical Center and written informed consent was obtained from the patients as well as their parents. Epiphyseal samples were embedded in Tissue-Tek (Sakura Finetek Europ BV, Zoeter- woude, The Netherlands) and frozen in liquid nitrogen. RNA was extracted according to the optimized method for RNA extraction from cartilage as described by Heinrichs et al. [42]. 4.5 RNA isolation from hfMSCs Total RNA from 26106 undifferentiated hfMSCs derived from the 22 weeks old fetus was extracted with Trizol (Invitrogen). After 1, 2, 3, 4, or 5 weeks of chondrogenesis, 60 pellets (per time point) were pooled and homogenized in 1 ml 4 M guanidine isothiocy- anate solution (Sigma) and RNA was extracted as described above. The extracted total RNA was purified using the RNeasy kit according to recommendations of the manufacturer (Qiagen). 4.6 Gene expression profiling High RNA quality was confirmed by capillary electrophoresis on an Agilent 2100 bioanalyzer (Agilent). Total RNA (100 ng) was amplified and labeled using the GeneChip Two-Cycle cDNA Synthesis Kit (Affymetrix) and the MEGAscript T7 Kit (Ambion). For gene expression profiling, labeled cRNA was hybridized in duplicate to Affymetrix Human Genome U133 PLUS 2.0 Array Genechips. All procedures were carried out according to the manufacturer’s recommendations. Raw data from Affymetrix CEL files were analyzed using SAS software package Microarray Solution version 1.3 (SAS Institute). Custom CDF version 10 with Entrez based gene definitions [43] was applied to map the probes to genes. Gene annotation was obtained using the Affymetrix NetAffx website (http://www. PLOS ONE | www.plosone.org 9 November 2012 | Volume 7 | Issue 11 | e44561