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.