Isolation and expansion of bone marrow hMSC clones
hMSCs were isolated from a heparinized bone marrow aspirate harvested from the iliac crest of a healthy 23 year old male volunteer. hMSCs were isolated in vitro by direct plating as described previously19. Cells were plated at a density of 5 × 104 mononuclear cells per cm2 in alpha MEM (Gibco) supplemented with 10% fetal bovine serum (FBS; Hyclone) and 1 ng/mL FGF2 (Peprotech). After five days, when colonies were visible, 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. The trilineage differentiation potential of the clonal populations were examined, which showed clones with differing propensity for differentiation to the chondrocytic, osteoblastic and adipocytic lineages. A single clone, termed 1F3, with trilineage potential was selected for the study of commitment to chondrogenesis. This clone, as well as having the ability to differentiate to osteoblasts, adipocytes and chondrocytes, also displayed the typical fibroblastic-like morphology and surface marker profile of an MSC.
Characterisation of MSC clones using trilineage differentiation and surface marker expression
MSC clones were characterised by multi-lineage differentiation and flow cytometry for cell surface marker expression. The multi-lineage differentiation capacity of the MSC clones was assessed by their differentiation to the classical MSC lineages i.e., chondrogenic, osteogenic and adipogenic using established protocols19.
Chondrogenic potential was examined using a 3-D pellet culture system, where, 2.5 × 105 cells were pelleted at 100 xg for 5 minutes in complete chondrogenic medium (CCM) consisting of Dulbecco’s Modified Eagle Serum (DMEM, 4.5 g/L glucose), 2 mM glutamine, 100 mM dexamethasone, 50 μg/mL ascorbic acid, 40 μg/mL L-Proline, 1% ITS + (Insulin, Transferrin, Selenium) (Corning), 1 mM sodium pyruvate and penicillin-streptomycin (100 U/mL), supplemented with 10 ng/mL TGF-β3 (Peprotech). Control pellets were incubated without TGF-β3 incomplete chondrogenic medium (ICM). After 21 days, pellets were fixed in 10% neutral buffered formalin, and processed for histology in a Leica ASP300S tissue processor and embedded in paraffin. Sections were stained with Safranin O and Fast Green FCF and imaged with an Olympus BX43 microscope.
For osteogenesis, cells were seeded in culture medium and when the monolayer reached 90% confluence, the medium was replaced with osteogenic medium containing DMEM (1 g/L glucose; Sigma Aldrich), 2 mM l-glutamine, 100 nM dexamethasone, 100 μM ascorbic acid, 10 mM β-glycerophosphate, 10% FBS (Hyclone) and penicillin-streptomycin (100 U/ml). Medium was replaced every 3–4 days for up to 14 days, when monolayers were fixed with 10% ice cold methanol, then stained with 2% Alizarin Red and imaged using an Olympus IX71 microscope.
Adipogenesis was assessed by incubating confluent cultures in adipogenic induction medium comprising DMEM (4.5 g/L glucose), 2 mM l-glutamine, 10% FBS (Hyclone), 1 μM dexamethasone, 10 μg/mL insulin (Roche), 200 μM indomethacin, 500 μM 3-isolbutyl-1-methylxanthine and penicillin-streptomycin (100 U/mL). After 3 days, the culture was transferred to adipogenic maintenance medium comprising DMEM (4.5 g/L glucose), 10% FBS (Hyclone), 10 μg/mL insulin (Roche) and penicillin-streptomycin (100 U/mL) for 1 day. This cycle was repeated 3 times after which the cells were maintained in adipogenic maintenance medium for a further 5 days. Cultures were fixed in 10% neutral buffer formalin and stained with Oil Red O before imaging using an inverted Olympus IX71 microscope.
Surface marker expression of MSC clones was carried out by flow cytometry using the BD FACS Canto II flow cytometer (BD Biosciences) using antibodies against CD3, CD14, CD19, CD34, CD45, HLA-DR and the MSC positive markers CD73, CD90 and CD105 (BD Biosciences) as described previously20. Post-acquisition analysis was carried out using the FlowJo software (Treestar Inc.).
Study of the commitment towards chondrogenesis
To examine the commitment towards chondrogenesis, clonal cell cultures were switched to a culture medium containing 1% FBS twelve hours prior to chondrogenic induction. Chondrogenic differentiation was carried out in a pellet culture format as described above. Three pellets were set up for each time point. Pellets from the 0 h time point remained in ICM, while pellets at 15 min, 30 min, 1, 2, 4, 8 and 16 h time points were cultured in CCM. At each time point the 3 pellets were combined into 1 tube, the pooled pellets were washed in PBS and snapped frozen in liquid nitrogen. Clonal cells in monolayer and 21d chondrogenic pellets were included as controls. The time course experiment was carried out three times and each time course served as a technical replica for the experiment.
RNA extraction and sequencing
Total RNA was extracted using an miRNeasy Mini kit (QIAGEN). For day 21 samples, due to the amount of matrix deposition around the chondrocytes in this late time point pellet, mechanical homogenisation by pulverizing in a chilled steel mortar and pestle was required prior to RNA extraction21. 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). Bioanalyzer and Nanodrop results were deposited on Zenodo as lab_qc.zip22. 400 ng of total RNA was included in each sequencing reaction.
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. The libraries were sequenced on an Illumina GAIIx using TruSeq Single read Cluster Generation Kit v5 and TruSeq Single read 36 cycle Sequencing kit v5. The sequencing mode was 35 nt single-read plus 7 nt for indices. The raw reads were aligned to Human Gencode23 (v23/h38.p3) reference genome with STAR aligner24 (v2.5.0a) and summarized with featureCounts25 (v.1.6.2).
Ethics approval and consent to participate
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, including written consent to participate and publish sequenced data.