Direct interaction of integrin-specific peptides with the MNCs is not very effective.
Akin to BMSCs, HSC and HSPCs also express several types of integrins [36,37]. Integrins are known to have homophilic interactions [39]. Hence, I examined whether such homophilic interaction of bioactive cyclic peptides with the integrins expressed on the HSPCs would also simulate them to form more colonies. The MNCs were subjected to CFU assay in methylcellulose-based media supplemented with α5β1, αIIbβ3 and α4β1-ada peptides (10µg/ml). I found that the MNCs incubated with α5β1peptide did not yield a higher number of CFU, while those incubated with αIIbβ3 and α4β1-ada peptides yielded only a marginally higher number of CFU, as compared to the untreated MNCs (Fig. S1b).
Direct actions of pharmacological compounds on HSCs could differ from those mediated via stromal cells primed with the same compounds. We have shown earlier that the nitric oxide (NO) donors exert an age-specific effect on HSCs by differential induction of various transcription factors involved in commitment vis-à-vis self-renewal in adult and juvenile HSCs, respectively [40]. On the other hand, NO-primed BMSCs stimulate the expansion of HSCs without showing any age-specific differences [41]. The data obtained in the experiments involving the direct action of integrin-specific compounds also show such a difference. The inhibitory effect of α4β1-ada peptide seen in earlier experiments was not seen when it directly interacted with the HSCs/HSPCs. In fact, an increase in CFU, albeit marginal, was seen. Nonetheless, these data confirm that the inhibitory effect of α4β1-ada peptide seen in the earlier experiments was primarily due to its interference in FN-VCAM-mediated stromal cell-HSC interaction, and the peptide itself did not have any inhibitory activity on HSPCs.
The milder effect shown by the direct addition of αIIbβ3 peptide seen in this set of experiments also indicates that its action on BMSCs evoked more robust signaling, leading to the extensive proliferative response in the HSCs, perhaps due to its interaction with its ligand rather than homophilic interaction with its counterpart on the BMSCs. In the future, I plan to examine the direct effect of various ECM-specific bioactive peptides on the expansion of HSC/HSPCs. Such ligand-integrin interactions could be perhaps more effective in expanding HSCs.
In this study, I have not examined the in vivo functionality of CD34+ cells co-cultured with the primed BMSCs. Although at 48-hr. co-culture, extensive proliferation of these HSCs was seen, I need to examine their engraftment potential using an immuno-compromised mouse model formally.
Our previous studies [41-43] showed that the extracellular vesicles (EVs) secreted by variously primed BMSCs are very effective in improving the functionality of the HSCs. It will be worthwhile to study the composition of the EVs secreted by the BMSCs primed with these pharmacological modulators and examine their effect on the HSCs’ functionality. Since the EVs can be effectively cryopreserved without losing their functionality [44], they could serve as “ready-to-use” biologics for priming HSCs.
Conclusion: My data show that priming the BMSCs with various pharmacological reagents involved in the TGFβ1 pathway boosts their hematopoietic supportive ability. This cost-effective and time-efficient approach can be used in clinical settings to improve the outcome of HSC transplantation.
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