Introduction: Cell-derived microparticles (MPs) are 0.1 to 1 micron extracellular vesicles (EVs), budding off cellular plasma membranes under stress or activation. MPs play an important role in cell-to-cell communication by transferring cargo from parent to target cells. Among circulating MPs, megakaryocyte-derived MPs (MkMPs) are the most abundant MPs in circulation (Flaumenhaft et al., 2009). We have shown that, in vitro, MkMPs specifically target and are taken up by human hematopoietic stem & progenitor cells (HSPCs) via fusion or endocytosis following specific receptor recognition (Fig.1A) (Jiang et al., 2017). MkMPs transfer cargo to HSPCs and induce potent Mk differentiation of HSPCs in the absence of thrombopoietin (Jiang et al ., 2014). Here, we explore the capability of human MkMPs to transfer DNA and siRNA to HSPCs, and their ability to interact with HSPCs in vivo using a simple murine model. We aim to develop MkMP-based strategies for gene- and cell- therapies.
Methods: Human MkMPs (huMkMPs) were isolated and purified from CD34+-derived Mk cells (Jiang et al., 2014). Electroporation was used for cargo loading to MkMPs. Cy5-labeled plasmid DNA (pGFPns; expressing eGFP) was loaded into MPs at two pDNA-to-MkMP ratios. siRNA (siR-MYB) targeting c-myb or Alexa 647 non-targeting siRNA were separately loaded into MkMPs. Silencing of c-myb enhances Mk differentiation of HSPCs (Bianchi et al ., 2010). Loading efficiencies were calculated after pDNA purification and quantification, or analyzed by flow cytometry (FC). Cargo delivery to HSPCs was enabled by co-culturing loaded MkMPs with HSPCs using an optimized protocol. Delivery of pGFPns was assessed by pDNA quantification, eGFP RNA levels by qRT-PCR, and eGFP expression by FC. siR-MYB-loaded MkMPs were co-cultured with HSPCs, and cells were analyzed by FC for CD41 (an Mk marker) expression at days 3, 5 and 8. To test the hypothesis that huMkMPs interact with murine HSPCs (muHSPCs) in vivo, 6×106 of MkMPs were administered into BALB/c mice intravenously via tail vein injection. At day 3, blood was collected and murine platelet (muPLT) levels measured by FC.
Results: We developed a protocol to enable us to load ca. 1700 copies or 30 ng of pGFPns into MkMPs, with over 77% of MkMPs becoming Cy5+ after electroporation (Fig. 1B). In MkMP-huHSPC co-culture experiments, delivery of and expression from pGFPns was first confirmed by isolating pGFPns from and measuring eGFP mRNA in HSPCs at days 1 and 3, respectively. At day 3, over 45% of HSPCs were Cy5+ while 20% expressed eGFP (Fig. 1C), indicating effective, MkMP-mediated pGFPns delivery to HSPCs with functional biological performance. We assessed functional RNA delivery by examining the impact of siR-MYB mediated c-myb silencing in enhancing Mk differentiation of CD34+ cells beyond the enhancing impact of unloaded MkMPs. MkMP delivery of siR-MYB enabled c-myb silencing that resulted in enhanced Mk differentiation beyond that of unloaded MkMPs: the % of CD41+ and CD34-CD41+ cells increased by 29 % (Fig. 1D) and 25 %, respectively, at day 8 of co-culture versus control (unloaded MkMPs), indicating functional delivery of siR-MYB via MkMPs. In vivo murine experiments in wild-type mice were to test if huMkMPs would interact with muHSPCs, and if so, if they would induce muHSPCs into murine Mk differentiation that would result in increased muPLT numbers. Injection of huMkMPs enhanced muPLT levels by 28 % at day 3 (Fig. 1E), indicating huMkMPs target and induce muHSPCs into Mk differentiation and de novo muPLT generation. In another set of experiments, anti-CD41-antibody induced thrombocytopenia in mice (Apostolidis et al ., 2012) was rescued by huMkMP injection, further supporting the hypothesis that huMkMPs target and deliver cargo to muHSPCs.
Conclusions: This is the first report showing functional MP-mediated pDNA delivery to any cell type. This is also the first report showing that HSPCs can be specifically targeted and cargo delivered using MPs. Moreover, functional siRNA delivery via these MkMPs demonstrate the potential of this delivery system for targeting the stem-cell compartment. Surprisingly, huMkMPs were able to target muHSPCs resulting in higher muPLT levels. Overall, based on our data and the unique characteristics of huMkMP, MkMPs constitute a potentially useful therapeutic delivery system for gene- and cell-therapy, with applications in regenerative and transfusion medicine.
No relevant conflicts of interest to declare.
Asterisk with author names denotes non-ASH members.