Figure legends
Fig 1 TGFβ1 boosts the hematopoiesis-supportive ability of BMSCs
via activation of PKC and rise in intracellular calcium levels (a, b) 1
X 106 BM MNCs were interacted with BMSCs treated or
not with TGFβ1 and PKC inhibitor, Myristoylated RKRTLRRL, or a calcium
chelator, BAPTA-AM. After 1 hr., the non-adherent cells were gently
washed, and the cells adhering to the BMSCs were subjected to CFU assay.
The graphs depict that the increase in the number of CFU formed from the
BM MNCs interacting with TGF-primed BMSCs gets significantly reduced in
the presence of the PKC inhibitor (a) and
BAPTA-AM (b). (c) Differential
scoring of the colonies formed in various sets indicates that inhibition
of PKC and buffering of intracellular calcium affected the formation of
all types of colonies. Colonies
belonging to BFU-E, GM, and GEMM were manually scored using a phase
contrast microscope (Zeiss). All experiments were done on at least 3
independent BM samples (N=3); each set had triplicate plates (n=3). The
data are represented as mean ± S.D. *** and ### p ≤ 0.001. (d)Inhibition of PKC and buffering of intracellular calcium in
TGF-primed BMSCs abrogates their HSC-supportive ability 1 x
105 CD34+ HSCs isolated from human
bone marrow were co-cultured with variously treated BMSCs grown on
coverslips. After 3 days of incubation, the cells were fixed and
immuno-stained with an antibody to human CD34 (raised in mouse; green)
followed by anti-mouse-FITC antibody. DAPI was used to demarcate the
nuclei (Blue.) Images show that CD34+ cells grew
luxuriantly on TGF-primed BMSCs (BMSC*TGF). This effect was abolished by
applying a PKC inhibitor and a calcium chelator to them before the
addition of TGFβ1. The images were acquired on a confocal laser-scanning
microscope (Zeiss), and represent experiments done on 3 independent BM
samples (N=3). Bar represents 10 µM.
Fig 2 Pharmacological activation of PKC and an increase in
intracellular calcium boost the hematopoiesis-supportive ability of
BMSCs (a, b, c) BMSCs were treated or not with Indo (-), its inactive
counterpart, Indo (+) (both 5 µM for 30 minutes), CPA, or Thapsigargin
(both 5µM for 30 minutes). After removing the treatments,
1X106 BM MNCs were interacted with them for 1 hr.
After incubation, the non-adherent cells were gently removed, and the
cells closely interacting with the stromal cells were subjected to CFU
assay. The graphs show that the MNCs interacting with the BMSCs treated
with a PKC activator Indo (-), but not with its inactive counterpart
Indo (+), (a), and agonists of intracellular calcium, cyclopiazonic acid
(CPA) and Thapsigargin (Tsg) form a significantly higher number of CFU
(b). All experiments were done on at least 3 independent BM samples
(N=3); each set had triplicate plates (n=3). The data are represented as
mean ± S.D. *** p ≤ 0.001. (c) A pharmacological activation of
intracellular calcium in BMSCs boosts their HSC-supportive abilityBMSCs grown on coverslips were treated with CPA or Tsg (both 5µM for 30
minutes). After removing the treatments, bone marrow-derived
CD34+ HSCs (1x105) were co-cultured
with them. After 3 days of co-culture, the cells were fixed and
immuno-stained with an antibody to CD34 (raised in mouse, green)
followed by anti-mouse-FITC antibody. DAPI was used to demarcate the
nuclei (blue). The images show that BMSCs having increased intracellular
calcium support an extensive proliferation of CD34+HSCs. The images were acquired on a confocal laser-scanning microscope
(Zeiss) and represent 3 experiments done on independent BM samples
(N=3). Bar represents 10 µM.
Fig 3 Peptide-mediated enhanced adhesion to fibronectin
boosts the functionality of BMSCs (a, b) BMSCs were primed with an
FN-adhesion-promoting peptide or cyclic RGD (both 10µM, overnight).
1x106 BM MNCs were seeded on them and incubated for 1
hr. Non-adherent cells were removed by gentle washing, and the cells
closely associated with the stromal cells were subjected to CFU assay.
The graphs show that the BMSCs treated with FN-adhesion-promoting
peptide (a) or cyclic RGD (b) support a significantly higher colony
formation from the BM MNCs interacting with them.Integrin-specific bioactive peptides increase the potency of
BMSCs (c-f) BMSCs grown in a 24-well plate were primed with peptides
specific to α5β1, αIIbβ3, and
α4β1 integrins (all 10µM, overnight). After removing the treatment,
1X106 BM MNCs were seeded on them and incubated for 1
hr. Non-adherent cells were gently washed, and the cells closely
interacting with the stromal cells were subjected to CFU assay. The
graphs show that both α5β1- and
αIIbβ3-primed BMSCs gave a significantly higher output of CFU from the
BM MNCs interacting with them, but those primed with α4β1-ada peptide
suppressed the colony formation (c). (d, e) The graphs show that the
α4β1-ada peptide exerts a
dose-dependent inhibitory effect on the hematopoiesis-supportive ability
of BMSCs (d) and exerts a dominant effect over that of α5β1-specific
peptide (e). (f) A differential scoring of colonies into BFU-E, GM, and
GEMM shows that the effect of both α5β1-and αIIbβ3-primed BMSCs is
across all types of colonies formed. All experiments were done on 3
independent BM samples (N=3); each set had triplicate plates (n=3). The
data are represented as mean ± S.D. * p≤ 0.05, ** p≤0.01; *** p ≤ 0.001.(g) BMSCs primed with α5β1 and αIIbβ3 expand
CD34+ HSCs CD34+ HSCs isolated from
human bone marrow were co-cultured with α5β1-and αIIbβ3-primed BMSCs for
3 days. The cells were fixed and immuno-stained with antibodies to CD34
(raised mouse; green) and Ki67 (raised in rabbit, red) followed by
anti-mouse-FITC and anti-rabbit-PE antibodies, respectively. DAPI was
used to demarcate the nuclei (Blue). The images were acquired on a
confocal laser-scanning microscope (Zeiss), and represent experiments
done on 3 independent BM samples (N=3). The images show that both
α5β1-and αIIbβ3-primed BMSCs support an extensive expansion of
CD34+ HSCs co-cultured with them. The presence of Ki67
in several CD34+cells showed that they are still in a
proliferative state. The images were acquired on a confocal
laser-scanning microscope (Zeiss). Bar represents 10µM.
Fig S1 (a) BMSCs secrete copious amounts of fibronectin in
response to TGFβ1 BMSCs were treated with TGFβ1 (10 ng/ml) overnight,
and then the cells were fixed using freshly prepared buffered
paraformaldehyde (pH 7,4). The cells were immuno-stained with an
antibody to cellular fibronectin (raised in mouse, green) followed by
anti-mouse-FITC antibody. The images were acquired on a confocal
laser-scanning microscope. Bar represents 10 µM. The images depict that
the TGF-treated BMSCs showed a dense network of cellular fibronectin
compared to the control BMSCs (upper panels). Use of monensin (2 µM,
added 1 hr. before TGF) to block trans-Golgi transport to visualize
intracellular proteins before the addition of TGFβ1 facilitated the
detection of intracellular FN (lower panels). (b)Direct interaction of integrin-specific peptides with the MNCs
is ineffective The BM MNCs were subjected to CFU assay in
methylcellulose-based media supplemented with α5β1, αIIbβ3 and α4β1-ada
peptides (10µg/ml). The graph shows that MNCs treated with the
α5β1-specific peptide did not yield a higher number of CFU, while those
treated with αIIbβ3- and α4β1-ada-specific peptides yielded only a
marginally higher number of CFU, as compared to the untreated MNCs. The
data are represented as mean ± S.D. (N=3) * p ≤ 0.05.
Graphical abstract Hematopoiesis-supportive ability of BMSCs can
be boosted by pharmacological means (a, b) Priming BMSCs with TGFβ1
boosts their hematopoiesis-supportive ability resulting in an extensive
proliferation of CD34+ HSCs interacting with them. (c)
Inhibition of PKC and buffering intracellular calcium abrogates the
hematopoiesis-supportive ability of TGFβ1-primed BMSCs. (e, f) Priming
of naïve BMSCs with various pharmacological compounds such as PKC
activators, boosters of intracellular calcium levels,
Fn-adhesion-promoting peptides, Cyclic RGD peptide, and
integrin-specific bioactive peptides also boost the
hematopoiesis-supportive ability of BMSCs leading to expansion of
CD34+ HSCs interacting with them.