How do megakaryocytic microparticles target and deliver cargo to alter the fate of hematopoietic stem cells?

Abstract

Megakaryocytic microparticles (MkMPs), the most abundant MPs in circulation, can induce the differentiation of hematopoietic stem and progenitor cells (HSPCs) into functional megakaryocytes. This MkMP capability could be explored for applications in transfusion medicine but also for delivery of nucleic acids and other molecules to HSPCs for targeted molecular therapy. Understanding how MkMPs target, deliver cargo and alter the fate of HSPCs is important for exploring such applications. We show that MkMPs, which are distinct from Mk exosomes (MkExos), target HSPCs with high specificity since they have no effect on other ontologically or physiologically related cells, namely mesenchymal stem cells, endothelial cells or granulocytes. The outcome is also specific: only cells of the megakaryocytic lineage are generated. Observation of intact fluorescently-tagged MkMPs inside HSPCs demonstrates endocytosis as one mechanism of cargo delivery. Fluorescent labeling and scanning electron microscopy (SEM) imaging show that direct fusion of MkMPs into HSPCs is also engaged in cargo delivery. SEM imaging detailed the membrane-fusion process in four stages leading to full adsorption of MkMPs into HSPCs. Specifically, macropinocytosis and lipid raft-mediated were shown here as mechanisms of MkMP uptake by HSPC. In contrast, the ontologically related platelet-derived MPs (PMPs) cannot be taken up by HSPCs although they bind to and induce HSPC aggregation. We show that platelet-like thrombin activation is apparently responsible for the different biological effects of MkMPs versus PMPs on HSPCs. We show that HSPC uropods are the preferential site for MkMP binding, and that CD54 (ICAM-1), CD11b, CD18 and CD43, localized on HSPC uropods, are involved in MkMP binding to HSPCs. Finally, we show that MkMP RNA is largely responsible for HSPC programming into Mk differentiation.

Combinatorial effect of miR-486-5p & miR-22-3p mimics thrombopoietin’s impact on hematopoietic stem & progenitor cells

“…We have previously shown that MkMPs were enriched in small RNAs, 16 which play an important role in triggering Mk differentiation of HSPCs. 16,17 We hypothesized that miRs are the dominant MkMP components in inducing and promoting Mk differentiation.…”

“…We have previously shown that MkMPs were enriched in small RNAs, 16 which play an important role in triggering Mk differentiation of HSPCs. 16,17 We hypothesized that miRs are the dominant MkMP components in inducing and promoting Mk differentiation. To characterize and carry out comparative analysis of the MkMP miR profile, total RNA was extracted from day-12 cultured Mks (starting from CD34 + cells of 3 donors), MkMPs and platelet-like particles (PLPs) generated from the corresponding day-12 cultured Mks.…”

“…Total cell and Mk cell numbers were measured at day 10 of coculture. 60,000 CD34 + HSPCs were pretreated with signaling inhibitors for 30 min, followed by coculture 16,17…”

“…MkMPs are highly enriched in small RNAs. RNase treatment, differentially depleting the small RNA pool, attenuated the ability of MkMPs to trigger Mk differentiation of HSPCs, 16 thus suggesting that small RNAs, possibly miRs, can mimic TPO signaling in HSPCs, a hitherto unknown possibility and mechanism. TPOinduced signaling starts with the binding of TPO to its receptor c-Mpl, which activates Janusfamily kinases (Jaks).…”

“…We have previous shown that megakaryocytic microparticles (MkMPs) can induce Mk differentiation of mobilized peripheral blood CD34 + Hematopoietic Stem and Progenitor cells (HSPCs; the term used henceforth to refer to these cells) 16,17 in the absence of exogenous Thrombopoietin (TPO), yet similar in potency and effect as TPO. MkMPs are highly enriched in small RNAs.…”