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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