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.