Methods
Isolation of BM MNCs, CD34+ cell isolation from the BM MNCs, isolation and culture of BMSCs, colony formation assay, co-culture, and immunofluorescence technique have been described earlier [18]. Briefly, MNCs were isolated from human bone marrow aspirates using density gradient centrifugation (HiSep, HiMedia). The cells were further used to isolate CD34+ HSCs [18,21], or plated in culture-grade Petri dishes of suitable size to obtain BMSCs. BMSCs were used in passage #3.
Interaction between BMSCs and MNCs: This protocol has been described in detail before [18]. Briefly, BMSCs (2X105/well) were seeded in the wells of a 24-well plate and were allowed to attach for 2 hours. They were then treated with TGFβ1 (10ng/ml) or other pharmacological agents, as mentioned in the results section. After incubation, the cells were washed 3X with plain IMDM to remove the treatments, and 150 µl of complete medium (IMDM+20% FBS) was added per well. 1X106 MNCs suspended in 100 µl of complete medium were added per well, and the plates were incubated at 37°C for 1 hour. The MNCs and BMSCs were from different donors, and the samples were not matched for age/gender.
Non-adherent cells were removed by gentle washing with plain IMDM, and the cells adhering to the BMSCs were collected using non-enzymatic cell dissociation solution (Sigma). The cells were then subjected to colony-forming-unit (CFU) assay as described before. Colonies were scored after 14 days using morphological criteria as belonging to CFU-GM (Granulocyte Monocyte), BFU-E (Burst-forming Unit Erythroid), and CFU-GEMM (Granulocyte-Erythroid-macrophage-Megakaryocyte) using a phase contrast microscope (Zeiss).
Co-culture assay and immunofluorescence staining: Co-culture assay and immunostaining of cells were done as described before [18]. Briefly, BMSCs cultured on coverslips placed in 24-well plates were variously treated as described in the results section. After washing to remove the treatments, 1X105CD34+ HSCs were seeded on the BMSCs. After one hour of incubation, non-adherent cells were gently removed, and adherent cells along with the BMSCs were over-layered with 1% methylcellulose supplemented with growth factors (SCF, 50 ng/ml; IL6 and IL3, 20ng/ml). The cells were fixed after 48 hrs. and gently washed to remove the methylcellulose. Non-specific sites were blocked by incubating the cells in 1% BSA prepared in Phosphate buffered saline (PBS; pH 7.4), and the cells were immunostained with antibodies to CD34 and Ki67 and FITC- and PE-tagged secondary antibodies, respectively. The images were acquired on a laser-scanning confocal microscope (Zeiss).
Statistical analysis .
Data are expressed as mean ± SD. All CFU experiments were done in triplicate (n=3) using BM MNCs from different donors (N=3). Statistical significance of the 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). * p≥0.5, **≥ 0.01, ***≥ p0.001. The distribution of data was examined for normality using Shapiro-Wilk normality test. All the graphs were created using Sigma Plot software version 14.0.