Pharmacological activation of PKC and increase in intracellular
Calcium levels boost the hematopoiesis-supportive ability of BMSCs.
Since inhibition of PKC and buffering of [Ca2+]i
significantly affected the potency of TGF-primed BMSCs, I examined
whether pharmacological activation of PKC and increase in
[Ca2+]i can boost the hematopoiesis-supportive
ability of BMSCs. In this set of experiments, I used Indo (-) (Sigma) to
activate PKC in the BMSCs [25]. BMSCs were treated with Indo
(-)/Indo (+) for 1 hr. (both 5 µM for 30 minutes). Indo (+) (Sigma) was
used as a negative control for Indo (-). As seen in Fig. 2a, the BMSCs
primed with Indo (-) but not with Indo (+) robustly enhanced the colony
formation from BM MNCs that interacted with them.
In another set of experiments, I used CPA (Sigma; 5µM for 30 minutes; )
[26]and Thapsigargin (Tsg; Molecular Probes; 0.5µM for 30 minutes)
[27]to increase the
[Ca2+]i in
BMSCs. I found that treating BMSCs with CPA and Tsg also resulted in a
significantly higher colony formation from the BM MNCs that interacted
with them (Fig. 2b). Then I co-cultured CD34+ HSCs
with the BMSCs grown on coverslips and treated or not with CPA
(BMSC*CPA) and Tsg (BMSC*Tsg), and after 48 hrs., subjected them to
immunostaining with an antibody to CD34. I observed that the
CD34+ HSCs co-cultured with BMSC*CPA or BMSC*Tsg
proliferated extensively compared to those co-cultured with control
BMSCs (Fig. 2c).
These data show that pharmacological activation of PKC and an increase
in [Ca2+]i in BMSCs boost their
hematopoiesis-supportive ability. Notably, the effect of these
modulators was rapid – less than 1 hr., making the processtime-efficient . The use of such synthetic compounds could also
make the process cost-effective .
In my earlier work, I have shown that treating BMSCs with bFGF also
boosts their hematopoiesis-supportive ability [19]; however, a
combination of TGFβ1 and bFGF was counterproductive. I plan to perform
similar studies using downstream effectors of the bFGF pathway. These
studies would increase the gamut of agents for priming the BMSCs and
help in understanding the cause of inhibitory effects of the combined
treatment of TGFβ1 and bFGF on BMSCs [19]. Such understanding is
necessary to avoid the combined application of priming agents having
antagonistic effects on the desired therapeutic activity of BMSCs.