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.