Reagents
Iscove's Modified Dulbecco's Medium(IMDM) was from HiMedia (Mumbai, India), Fetal Bovine Serum (FBS) was from Invitrogen, MA, USA; anti-Glycophorin A and B (raised in mouse), FITC-tagged anti-rabbit antibody (raised in goat), Collagenase Type 1A, EPO, TGFβ1, protease and phosphatase inhibitor cocktails and Histopaque were from Sigma-Aldrich (MO, USA); recombinant human soluble EPO-R and anti-EPO-R antibodies (raised in mouse and goat) were from R&D Systems (MN, USA); anti-EPO-R antibody raised in rabbit and its corresponding neutralizing peptide, and anti-TGFβ1 antibodies (raised in mouse and rabbit) were from Santacruz Biotech (TX, USA); anti-biotin-HRP and anti-rabbit IgG-HRP antibodies were from Cell Signalling Technology (MA, USA), antibodies to CD41a and CD41b were from Pharmingen ( CA, USA); all tissue culture grade plastic ware was from Nunc/Thermo Fisher Scientific ( MA, USA); and microfuge tubes were from Eppendorf (Hamburg, Germany). TGFβ1 ELISA kit was from Genzyme (MA, USA). Dynabeads® immunomagnetic beads conjugated to antibodies to CD2, CD4, CD8, CD14, CD15, CD19, and anti-mouse IgG were from Invitrogen/Thermo Fisher Scientific (MA, USA). All flow-qualified antibodies (Table S1), their respective isotypes, and Human BD FC block were from Beckton Dickinson (Franklin Lakes, NJ, USA). Protein A/G agarose was from Thermo Fisher, Scientific (MA, USA). PVDF membrane (Sequiblot) was from Biorad (CA, USA). LumiGLO™ substrate was from (Cell Signaling Technology).
Isolation of Bone Marrow and peripheral blood mononuclear cells:
Left-over iliac crest pieces from grafts used in spine surgeries were collected in IMDM supplemented with 2% FBS and cut into small pieces. The pieces were subjected to collagenase (Type 1A; 1 mg/ml prepared in IMDM) digestion for 15 minutes at room temperature with constant gentle stirring. The dissociated cells were collected by centrifugation, washed twice with plain IMDM, and MNCs were isolated using density-gradient centrifugation using Histopaque. The MNCs were suspended in 1 ml of complete growth medium (IMDM supplemented with 20%FBS) or cell isolation buffer (1X PBS supplemented with 1%BSA and 0.6% Sodium citrate, pH 7.4). Viable cell count was taken using Trypan Blue, and the cell suspension was adjusted to 1 X 107 MNCs/ml using the growth medium or cell isolation buffer as required for the experiment. Cell viability was typically ≥ 95%.
10 ml of peripheral blood samples were collected from healthy volunteers, and mononuclear cells were isolated using a density gradient centrifugation using Histopaque. The cells were washed and processed for flow cytometry analysis.
Detection of EPO-R on BM and PBL MNCs by flow cytometry
MNCs (BM or PBL) were suspended (5x106 cells/ml) in 1%BSA-PBS supplemented with 0.01% sodium azide (staining buffer), and aliquoted in Eppendorf tubes (200µl/tube). Fc block (1 µg) was added to each tube, and the tubes were incubated for 20 minutes at RT. The tubes were cooled on ice, and 1µg/ml anti-EPO-R antibody (raised in rabbit) was added. After incubation on ice for 45 minutes with intermittent mixing, the cells were washed and suspended in the staining buffer. PE-tagged antibodies to specific cell surface markers (or their isotypes) (TableS1) and the FITC-tagged anti-rabbit antibody were added (all 1µg/ml) in all tubes. The cells were incubated for 45 minutes on ice with intermittent mixing. The cells were washed, suspended in sheath fluid, and immediately acquired on a flow cytometer (CantoII, BD). % EPO-R+ cells present in a particular lineage was determined.
Depletion of specific cell types:
One ml of BM MNCs suspended in pre-cooled isolation buffer (1 X 107 MNCs/ml) were kept on ice, and 100 µl of pre-washed paramagnetic beads tagged to specific antibodies were added to them. This ratio of cells to beads was standardized by flow analysis of the depleted population using specific antibodies. The cells and beads were mixed by gentle tapping and gently inverting the tubes a few times.
For depletion of glycophorin+ erythroblasts and CD41+ megakaryocytes, a two-step procedure was followed. First, the cells were incubated with antibodies to glycophorin a and b, or CD41 a and b (raised in mouse, 2 µg each) for 45 minutes on ice, followed by washing. Then 100 µl of paramagnetic beads tagged to anti-mouse IgG were added to each tube to deplete the positive cells.
The tubes were incubated on ice for 45 minutes with intermittent tapping. The tubes were then held on a magnet for 2 minutes to allow the cells attached to the beads to settle. Supernatant unbound cells were collected, washed, and suspended in one ml of complete growth medium. These cells were labeled as CD**-dep MNCs, depending on the antibody used (e.g. CD2-dep MNCs). Equivalent cell suspension (1 ml) of un-manipulated MNCs was used as controls (undep MNCs).
Preparation of Conditioned media (CM):
BM MNCs (1X107/ ml), depleted of specific cell type or not, were incubated in the growth medium with or without the addition of EPO (2U/ml). After 48 hours, the cells were harvested and centrifuged at 3000 rpm in a cold centrifuge. The cells were suspended in the growth medium and incubated for a further 24 hrs. without the addition of EPO. In the experiments involving immune-precipitation of TGFβ1, this second step was performed in a serum-free medium. The cell-free supernatants (conditioned medium, CM) were collected and aliquoted in polypropylene microfuge tubes. Aliquots were stored at -80°C till further use. An equal amount of growth medium was also incubated in parallel and aliquoted.
Quantification of TGFβ1 in the CM
CM preparations were subjected to ELISA assay using Genzyme TGFβ1 ELISA kit as per the manufacturer’s instructions. A standard curve was generated using purified TGFβ1 supplied with the kit. Readings for only growth medium (IMDM supplemented with 20% FBS, incubated for 48 hrs.) were subtracted from the readings obtained for CM preparations.
Immuno-precipitation of CM
Aliquots of CM were precleared using protein A/G agarose and then subjected to immune-precipitation using anti-EPO-R or anti-TGFβ1 antibodies (5 µg/500 µl each, raised in mouse and rabbit, respectively). The immune-precipitate was dissolved in 30µl of lysis buffer, boiled in sample buffer at 95°C for 5 minutes, and subjected to western blot analysis using anti-EPO-R or anti-TGFβ1 antibodies (raised in goat and mouse, respectively). The IP of TGFβ1 and rTGFβ1 were boiled in the sample buffer prepared without the addition of 2-ME.
Western blot
The samples were electrophoretically separated on polyacrylamide gel containing SDS (SDS PAGE). The separated proteins were then electrically transferred onto PVDF membrane (Sequiblot, Biorad). The membranes were blocked with 5%BSA prepared in TBST (Tris-buffered saline supplemented with 0.1% Tween 20), for 3 hours, washed thrice with TBST, and probed with antibodies to EPO-R or TGFβ1 followed by incubation with appropriate HRP-conjugated secondary antibodies (Cell Signaling Technology). Immuno-reactive conjugates were detected using LumiGLO™ substrate (Cell Signaling Technology) according to the manufacturer’s instructions.
Statistical Analysis
Data are expressed as mean ± SD. Statistical significance of all data was analyzed by One-Way Repeated Measure Analysis of Variance (One-Way RM ANOVA) using Sigma Stat software version 3.5 (Jandel Scientific, CA, USA) and is denoted as * p≥0.5, **≥ 0.01, ***≥ p0.001. The distribution of data was examined for normality using the Shapiro-Wilk normality test.