title: Illumina Genome Analyzer IIx sequencing; Transcriptional profiling of early differentiation of primary human mesenchymal stem cells into chondrocytes
library_construction_protocol: hMSCs were isolated from a heparinized bone marrow aspirate harvested from the iliac crest of a healthy volunteer, 23 year old male with informed consent. All procedures were approved by the Clinical Research Ethical Committee of Galway University Hospital and by the National University of Ireland Galway Research Ethics Committee. hMSCs were isolated and expanded in vitro by direct plating as described previously by Murphy et al. (2002). Briefly, aspirates were washed 1:1 with Dulbecco's phosphate buffered saline (PBS, Gibco) and centrifuged for 10 minutes at 900g. Following removal of the supernatant a sample of the cell pellet was taken for counting; this was diluted in PBS and added to 4% acetic acid (1:1 ratio) to promote red blood cell lysis. Primary MSCs were plated at a cell density of 40 × 106 to 60 × 106 cells per T-175 flask (~4000-5000 MSCs/cm2) and cultured in alpha-Minimum Essential Medium (α-MEM, Invitrogen) supplemented with 10% (FBS, Lonza) and 1% penicillin/streptomycin. Clonal human MSC (hMSC) populations were established by limiting dilution from the bone marrow of a healthy 23 year old male donor. Briefly, the entire bone marrow aspirate was plated at 5x104 cells per cm2 onto cell culture plastic in alpha MEM (Gibco) supplemented with 10% FBS (Sigma), 1ng/mL FGF2 (Peprotech) and 1% penicillin-streptomycin solution (Gibco). After five days, when colonies were visible, the cells were trypsinized and cloned by limiting dilution into 96 well plates (0.3 cells/well).  These clones were termed P1. Wells with single clones were passaged and expanded to P4. Following characterisation, a single clone with clear trilineage potential was selected for the study of commitment to the chondrogenic lineage. For induction of osteogenesis in hMSCs, 4 x 104 cells were seeded per well in a 24 well plate. Four wells were used for osteogenic induction and four wells were allocated as the control. Control cultures were incubated with hMSC medium and osteogenesis cultures were incubated in low glucose (1mg/ml) DMEM (Gibco) supplemented with 100nM dexamethasone, 100µM ascorbic acid 2-phosphate, 10mM β-glycerophosphate (Sigma-Aldrich), 10% FBS and 1% penicillin/streptomycin. Media was changed twice weekly and the assay was harvested between 10-14 days depending on the condition of the monolayer. For the hMSC clone 1F3   Calcium deposition was quantified in both control and induced cultures using the Calcium (CPC) Liquicolour Kit (Stanbio). Briefly, cells were scraped from each well by adding 0.5M hydrochloric acid (HCL; Sigma-Aldrich) and placed on a shaker overnight at 4°C.The following day samples were centrifuged to pellet any cell debris. Standards ranging between 0-1µg/ml were made up in 0.5M HCL using the supplied calcium standard in the kit. A working solution was also prepared using binding reagent and working dye at a 1:1 ratio. Standards and samples were then added in triplicate to a 96 well microtitre plate and 200µl of working solution was added per well. Absorbance (550-650nm) was read using a Wallace Vicktor plate reader and the amount of calcium in each well was determined based on the standard curve. Calcium deposition in control and induced wells was assessed by fixing cells in ice cold 95% methanol (Sigma-Aldrich) and stained using 2% Alizarin Red S solution (Sigma-Aldrich). For induction of adipogenic differentiation in hMSCs, 4 x 104 cells were seeded per well in a 24 well plate. Control cultures were incubated with hMSC medium. Adipogenic cultures were incubated for 3 days with adipogenic induction medium consisting of 4.5mg/ml DMEM, 1µM dexamethasone, 10µg/ml insulin (Sigma-Aldrich), 200µM indomethacin (Sigma-Aldrich), 500µM 3-Isobutyl-1 Methyl-Xanthine (MIX, Sigma-Aldrich) 1% penicillin/streptomycin and 10% FBS. The medium on the induced cells was then replaced with adipogenic maintenance medium which consisted of 4.5mg/ml DMEM, 10µg/ml insulin, 1% penicillin/streptomycin and 10% FBS for 1 day. This cycle was repeated another two times before finally leaving the cells in maintenance medium for 5-7 days. Oil red O (Sigma-Aldrich) was used to stain for lipid vacuoles. Briefly, Oil Red O stock solution (0.3% of Oil Red O powder in 99% isopropanol) was diluted to a working solution by mixing 6 parts Oil Red O stock solution with 4 parts distilled water (dH2O). The working solution was allowed to stand for 10 mins and then was filtered through a Whatman filter paper. Cells were then fixed in 10% neutral buffered formalin for 20 mins. Following fixation cells were washed with dH2O and then stained with Oil Red O working solution for 5 mins. Excess stain was removed by adding 60% isopropanol and then rinsing in warm tap water. Oil Red) stained lipid droplets were then imaged using the Olympus IX71 inverted microscope. For induction of chondrogenic differentiation in the mouse cell lines, 5 x 105 cells/pellet culture were placed in 1.5ml screw capped micro tubes (Sarstedt) and centrifuged at 100g to pellet the cells. Pellets were cultured at 37°C with 5% CO2 and 95% humidity in 500µl of complete chondrogenic media (CCM).CCM contained 4.5mg/ml DMEM (Gibco), 100nM dexamethasone, 50µg/ml ascorbic acid 2 phosphate, 40µg/ml L-proline (Sigma-Aldrich), 1%  Insulin-Transferrin-Selenium (ITS) + premix supplement (Becton Dickinson, Bedford, MA; 6.25µg/ml bovine insulin, 6.25µg/ml transferring, 6.25µg/ml selenous acid, 5.33ug/ml linoleic acid, 1.25mg/ml BSA), 1mM sodium pyruvate (Gibco),1% penicillin/streptomycin, 10ng/ml transforming growth factor β3 (TGF-β3) and 100ng/ml bone morphogenetic protein 2 (BMP2). Control pellet cultures contained incomplete chondrogenic media (ICM) which did not contain TGF-β3 or BMP2. The medium was replaced on pellets every second day for a period of 21 days. For each cell type 4 CCM and 4 ICM pellets were set up and this procedure was repeated a total of 3 times for n=3. The same protocol was applied for hMSCs with some adjustments to cell number using 2.5 x 105 cells/pellet cultures and excluding BMP2. The glycosaminoglycan (GAG) content of the pellet cultures was assessed by 1, 9 Dimethylmethylene blue (DMMB) analysis. Briefly, chondroitin-6-sulphate (Sigma-Aldrich) was used to prepare standards using a dilution buffer (50mM sodium phosphate (Sigma-Aldrich), 2mM N-acetyl cysteine (Sigma-Aldrich), and 2mM EDTA (Sigma-Aldrich), adjusted to pH 6.5) to achieve concentrations ranging from 0 to 2 µg of GAG.  3 pellets from CCM and ICM groups were each digested in 25µg/ml papain (Sigma-Aldrich) in dilution buffer overnight at 60°C and combined with DMMB stock solution (pH 3).  Samples were measured on a Wallace Victor plate reader at an absorbance of 595nm. The DNA content of each pellet was assessed using the Quant-iT PicoGreen dsDNA assay kit from Invitrogen (Grand Island, USA) according to the manufacturer's instructions. Together, the results from the DMMB and DNA analysis allowed expression of GAG to be normalized to DNA content.  Total RNA was extracted using an miRNeasy Mini kit (QIAGEN). The concentration and purity of the isolated RNA was determined using the Nanodrop ND-1000 (Nanodrop Technologies). RNA integrity was measured using the 2100 Bioanalyzer (Agilent Technologies). Poly(A)+ enrichment to purify mRNA from each sample was performed by hybridization of the RNA with poly(T) oligomers. The mRNA was then converted to cDNA and fragmented creating cDNA library fragments of approximately 250nt in size.  Sequencing adaptors were then added to each cDNA library fragment.