Ethics statement
This study protocol was approved by the University of Calgary Human Research Ethics Board (REB15–0005 and REB15–0880). All participants provided written consent to participate. All testing was carried out in accordance with the declaration of Helsinki.
Description of patients
MPCs were isolated from the synovial membrane of two groups of patients. The first were patients who had a periacetabular osteotomy (PAO) procedure done to correct acetabular dysplasia (ACD) or femoroacetabular impingement (FAI) (n = 12, 9 female, 3 male, average age = 25.3 years); while the second group was comprised of patients who received a total or partial hip joint replacement due to end-stage OA (n = 22, 9 female, 13 male, average age = 56.7 years).
Experimental design
An overview of the experimental design of the project is presented in Fig. 1.
Synovial membrane tissue digestion
The intimal layer was dissected from the synovial biopsies and was then cut into 5 mm2 pieces. It was subsequently digested in 1 mg/ml type IV collagenase (Sigma) in heat inactivated fetal bovine serum (FBS)(ThermoScientific) for 120 min at 37 °C with shaking, in order to obtain a single cell suspension. The cells were then washed with phosphate buffered saline (PBS) and immediately immunophenotyped with the International Society for Cellular Therapy (ISCT) [15] recommended MPC/MSC cell surface markers. The MSC/MPC markers included were: CD90 (Clone # 5E10, PE), CD271 (Clone # C40–1457, BV421), CD44 (Cone # G44–26, PE-Cy7), CD73 (Clone # AD2, APC), and CD105 (Clone # 266, BV650), a macrophage marker, CD68 (clone # Y1/82A, FITC), and a cell viability marker, fixable viability stain (FVS) 510 (BV510) (all BD Biosciences). UltraComp eBeads (eBioscience) individually stained with each single colour as well as unstained cells were used as compensation controls.
Flow cytometry
The stained cells underwent fluorescent activated cell sorting (FACS) on a BD FACS Aria Fusion (BD Biosciences). Macrophages (CD68+) as well as the dead cells (FVS510+) were excluded. The remaining cells were unbiasedly (e.g. cells were not isolated based on the expression/absence of any marker / combination of markers) indexed-sorted (e.g. single cell into single well) into a 96-well plate containing 100 μL DMEM/F-12 media (Lonza- BioWhittaker) with 10% MSC stimulatory supplement (Stem Cell Technologies) with 1% antibiotic-antimycotic (ThermoFisher). The indexed-sorting recorded the presence/absence of any/all cell surface markers per cell (referred to as in situ marker data). The indexed sorting was undertaken using a “single cell” mask to reduce the chance of having multiple cells per well in addition to using a 100 μM sort nozzle and low flow rate (45% of system maximum) to reduce the pressure on the cells.
Clonal cell expansion
The clonally derived cells within the 96-well plates were incubated at 37 °C and 5% CO2. Cell culture media consisted of DMEM/F-12 media (Lonza- BioWhittaker) with 10% MSC stimulatory supplement (Stem Cell Technologies) with 1% antibiotic-antimycotic (ThermoFisher). Once the cells reached ~ 70% confluency in the 96-well plate, the cells were passaged using trypsin (Corning). Cells were transferred to 12-well plates, then T25 and finally T75 flasks (all Primaria, Corning) with each successive passage.
Differentiation
The clonal cell lines were expanded until ~ 0.75 × 106 cells were obtained (~ 19 population doublings). At this point, they underwent multi-lineage differentiation analysis to determine their osteo/chondro/adipo-genic capacity.
Osteogenesis: For each replicate, 5 × 105 cells were seeded into each well in a 24-well plate and then placed into DMEM/F-12 media that contained Dexamethasone (final concentration (FC): 100 nM) (Sigma), L-Ascorbic Acid (FC: 50 μg/mL) (Sigma), β-Glycerolphosphate (FC: 10 mM) (Sigma).
Adipogenesis: For each replicate, 5 × 105 cells were seeded into each well in a 24-well plate and then placed into DMEM/F-12 media that contained Dexamethasone (FC: 1 μM) (Sigma), Insulin (FC: 10 μM) (Sigma), Indomethacin (FC: 200 μM) (Sigma), and Isobutylmethylxanthine (FC: 500 μM) (Sigma).
Chondrogenesis: For each replicate, 5 × 105 cells were pelleted through centrifugation and placed into DMEM/F-12 media that contained Dexamethasone (FC: 10 nM) (Sigma), L-Ascorbic Acid (FC: 50 μg/mL) (Sigma), MEM Non-Essential Amino Acids (FC: 1%) (MEM-NEAA Gibco), Transforming growth factor (TGF)-β3 (FC: 10 ng/mL) (Peprotech), Bone morphogenetic protein (BMP)-2 (FC: 500 ng/mL) (Peprotech), insulin transferrin selenium (FC: 1%) (Lonza- BioWhittaker), and sodium pyruvate (FC: 1%) (ThermoFisher). Media was adjusted to neutral pH (7.0–7.6).
After 21 days of osteogenic, adipogenic or chondrogenic differentiation, with media changes performed twice a week, differentiation was assayed using reverse transcriptase quantitative polymerase chain reaction (RT-qPCR) and histological staining.
RT-qPCR
mRNA was isolated using the TRIzol reagent protocol (ThermoFisher) following the manufactures instructions with the addition of glycogen solution (Amresco) to increase the yield of mRNA. Chondrogenic cultures alone went through an additional spin column step (OMEGA bio-tek E.Z.N.A. Total RNA Kit I) to remove additional ECM proteins which could potentially interfere with downstream applications. For first strand synthesis, mRNA was then added cDNA Master Mix (High Capacity cDNA kit, Applied Biosystems) following the manufactures instructions. The cDNA was stored at − 20 °C until use.
RT-qPCR analysis was employed to quantify the gene expression levels of each of the markers expressed by different lineages (osteoblasts, chondrocytes, adipocytes) as a surrogate outcome to measure the multipotent differentiation capacity of the sMPCs. For osteogenesis, gene expression of Osterix (Sp7) (Probe set # Mm00504574_m1) and Runx2 (Probe set # Mm00501584_m1) were quantified. For adipogenesis, ADIPOQ (Probe set # Mm00456425_m1) was quantified. For chondrogenesis, Sox9 (Probe set # Mm00448840_m1) and Col2a (Probe set # Mm01309565_m1) were quantified. Ribosomal 18S (Probe set # Mm03928990_g1) was employed as a housekeeping gene. All TaqMan Gene Expression Assays were obtained from Applied Biosystems. TaqMan Universal PCR Master Mix No AmpErase (Applied Biosystems) was used following the manufacturers instructions. Three replicates were run per sample and all samples were run on an ABI 7900 (Applied Biosystems) using the following program: UNG incubation - 50 °C 2 min; Enzyme activation − 95 °C 20 s; Denaturation - 95 °C 3 s; Annealing / Extending - 60 °C 30 s (40 cycles). Resulting threshold (Ct) values were analyzed using the ΔΔCt method against 18S endogenous control and undifferentiated cells as the reference sample.
Histological staining
For further analysis of differentiation, histological staining were performed post differentiation. For osteogenic and adipogenic differentiations, the wells were fixed with 10% neutral buffered formalin (NBF) for one hour. The osteogenic wells were stained with a 0.2% Alizarin Red S (Sigma) solution in the dark for 10–15 min. The adipogenic wells were stained with a 0.5% Oil Red O solution (Sigma) for 15 min. For chondrogenic pellets, whole-mount staining was performed as follows. Pellets were fixed with 10% NBF for three hours, then washed with distilled water. The pellets were then stained with 0.1% Safranin O solution (Fisher Chemical) for 45 min in the dark. The pellets were then de-stained and transferred to PBS.
Controls for enzymatic digestion, cell sorting, and antibody staining
To control for artefacts in the clonal MPCs induced by enzymatic digestion of the synovium, cells were plated on a 12-well plate before tissue digestion (e.g. cell outgrowth from the intact synovial tissue) to demonstrate that the tissue contained viable cells. Cells were also plated after tissue digestion in order to demonstrate that the digestion procedure did not negatively affect cell viability. And lastly, cells were plated after the immunophenotyping staining procedure (but without cell sorting) to demonstrate that the staining procedure did not reduce cell viability. The cells under all of these conditions were then allowed to proliferate under the same conditions and the same outcome procedures (e.g. differentiation analysis) were performed as the index sorted sMPCs.
In vitro analysis of cell surface markers by flow cytometry
At the point the individual sMPC clones were ready to be placed under differentiation conditions (e.g. ~ 0.75 × 106 cells) the cells were re-immunophenotyped with the same MPC markers (CD90, CD73, CD44, CD271, and CD105) and analyzed on the BD Fusion using the same settings as the indexed sorting described previously.
Non-clonal FACS of sMPC populations
Once information regarding the cell surface markers present on clonal MPCs with chondrogenic potential was determined, this was used to isolate and expand MPCs using non-clonal FACS. Cell suspensions from 4 new patients (n = 2 POA, 1 female, 1 male, average age = 34.2 years) (n = 2 OA, 1 female, 1 male, average age = 63.1 years) were derived using methods described previously. The cell suspension was stained with CD90, CD73, CD44, CD68 and the cell viability marker FVS510. CD68+ and FVS510+ cells were excluded and then the remaining cells were sorted to obtain a purified populations of cells that were positive for all three MPC markers (CD90, CD73, and CD44). The cells were then expanded until ~ 0.75 × 106 cells were obtained (~ 8 population doublings). They then underwent immuno-phenotyping by flow cytometry and differentiation analysis followed by RT-qPCR and histology as described above.
Data analysis
For a cell surface marker to be positive for flow cytometry/FACS, the given cell (or population) had to demonstrate a fluorescence signal above the 95th percentile of the unstained/isotype control. For an mRNA lineage marker (e.g. Sox9, Sp7) to be positive in RT-qPCR analyses, there had to be a statistical increase of a significance value set at p < 0.05 versus the undifferentiated control (derived from the same clonal population). For a histological marker to be positive, the cells had to demonstrate a dark robust staining compared to the undifferentiated/negative control. In order for a given clonal MPC line to be considered positive for any of the three lineages tested (e.g. osteoblast, chondrocyte, adipocyte), a given MPC line had to demonstrate a positive result for both the RT-qPCR data (at least one expressed gene per lineage) in addition to the histological stain. If a cell line was only positive for RT-qPCR or histology, the cell line was considered negative for that lineage.
Statistics
The RT-qPCR data were analyzed using GraphPad Prism 7 (GraphPad Software). The data had been reported as ± standard deviation (SD). Statistical analysis was performed with a paired t-test since the undifferentiated controls for each experiment performed are derived from the same clone as the differentiated cells. An alpha value of p < 0.05 was regarded as statistically significant.