Megakaryocytic microparticles-mediated nucleic acid delivery for gene therapy

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 (1). We have demonstrated that, in vitro, MkMPs specifically targeted and were taken up by human hematopoietic stem & progenitor cells (HSPCs) via fusion or endocytosis following specific receptor recognition (2). MkMPs transfer cargo to HSPCs and induce potent Mk differentiation of HSPCs in the absence of thrombopoietin (3). Here, we explored the capability of human MkMPs to transfer DNA and siRNA to HSPCs, and developed MkMP-based strategies for gene therapy. From our current protocol, we were able to achieve loading of plasmid DNA (pGFPns: encoding eGFP) into over 80% of MPs and functional delivery to HSPCs though co-culture to perform eGFP expression. DNA delivery efficiency were increased by optimized co-culture methods, chemically via fusogens to enhance membrane fusion, and physically to enhance contact between MPs and HSPCs. As a result, we were able to make possible that more than 20% of HSPCs express GFP. Functional RNA delivery was also studied by examining the impact of siR-MYB mediated c-myb silencing in enhancing Mk differentiation of CD34+ cells. Our data demonstrate that MkMP-based delivery of siR-MYB to HSPCs enabled c-myb silencing that resulted in enhanced Mk differentiation beyond that of unloaded MkMPs, as assessed by a 29% increase of CD41 expression (an Mk marker), indicating functional siRNA delivery. To sum, functional pDNA and siRNA delivery to HSPCs via MkMPs demonstrate the potential of this delivery system for targeting the stem-cell compartment, suggesting that MkMPs constitute a potentially useful therapeutic delivery system for gene therapy, with applications in regenerative and transfusion medicine. References: 1. Flaumenhaft, R., Dilks, J. R., Richardson, J., Alden, E., Patel-Hett, S. R., Battinelli, E., Klement, G. L., Sola-Visner, M., and Italiano, J. E., Jr. (2009) Megakaryocyte-derived microparticles: direct visualization and distinction from platelet-derived microparticles. Blood 113, 1112-1121 2. Jiang, J., Kao, C., and Papoutsakis, E. T. (2017) How do megakaryocytic microparticles target and deliver cargo to alter the fate of hematopoietic stem cells? Journal of Controlled Release 247, 1-18. doi:10.1016/j.jconrel.2016.1012.1021 3. Jiang, J., Woulfe, D. S., and Papoutsakis, E. T. (2014) Shear enhances thrombopoiesis and formation of microparticles that induce megakaryocytic differentiation of stem cells. Blood 124, 2094-210

Supersymmetric Electroweak Parity Nonconservation in Top Quark Pair Production at the Fermilab Tevatron

We evaluate the supersymmetric (SUSY) electroweak corrections to the effect of parity nonconservation in $q {\bar q} \to t {\bar t}$ production at the Fermilab Tevatron predicted by the Minimal Supersymmetric Model (MSSM). We find that the parity nonconserving asymmetry from the SUSY electroweak and SUSY Yukawa loop corrections predicted by the minimal supergravity (mSUGRA) model and the MSSM models with scenarios motivated by current data is about one percent. It will be challenging to observe such a small asymmetry at the Tevatron with 10 fb^{-1} of luminosity. It could however be observable if both the top- and bottom-squarks are light and $\tan \beta$ is smaller than 1, though theses parameters are not favored by mSUGRA.Comment: revised version, some new numerical results adde

Human-cell microparticles for cell-therapy and cargo delivery to stem cells

Cell-derived microparticles (MPs) are vesicles budding from cellular plasma membrane under stress or activation, range from 0.1 to 1 micron in size. MPs, which are distinct from the smaller exosomes, are found to be important in cell-cell communication through transferring cargo such as RNAs, proteins, and lipids from parent cells to target cells. We work with megakaryocytic MPs (MkMPs), which are the most abundant MPs in circulating blood. MkMPs are derived from megakaryocytes (Mks) which are derived from hematopoietic stem and progenitor cells (HSPCs). We have previously shown that MkMPs target HSPCs to program them into generating megakaryocytes (Mks) leading to platelet generation without addition of thrombopoietin. The recognition is specific for HSPCs and the outcomes specific for Mk and platelet generation. We have also shown that uptake of MkMPs by HSPCs is though both some endocytotic process had membrane fusion. We carried out studies to enhance our understanding of the process of target recognition and specificity with the ultimate goal of using MkMPs for cell therapy and cargo-delivery applications. Using various inhibitors, we show that macropinocytosis and lipid rafts play an important role in the uptake of MkMPs by HSPCs. We also describe that MkMPs target and enter HSPCs in the HSPC uropod (“tail”), which helped us identify the specific surface antigens, mostly on HSPCs, that are responsible of mediating the recognition and uptake process of MkMPs. To pursue therapeutic applications, we present data involving transfusion of MkMPs in a murine model with and without induced thrombocytopenia. We also discuss methods for loading MkMPs with exogenous cargo for delivery to HSPCs, and the development of tools to visualize the cargo delivery

Cell-derived microparticles for cell therapy, cargo delivery, and applications in CHO-cell biotechnology

Mammalian cells release into the extracellular environment microparticles (MPs; less than 1 micron) under some stress or activation process. MPs result from direct budding off the plasma membrane, and are important in intercellular communication by transferring RNA, proteins, and lipids between cells. Cells endow their MPs with signaling or functional molecules, to target specific cell types. They enrich their MPs in specific miRNAs, piRNAs, and long ncRNAs to program or reprogram target cells towards functional differentiation or specific cellular actions. Cells also use MPs to get rid of “death molecules”, and/or promote cell survival and “renewal” of target cells. We will discuss the characterization and potential applications of MPs from two biological systems: megakaryocytic MPs (MkMPs) derived from human hematopoietic stem and progenitor cells (HSPCs) and CHO-cell MPs (ChocMPs). Megakaryocytes (Mks) derive from the differentiation of HSPCs in the bone marrow, and as they mature, they achieve high-ploidy status. Platelets are produced from polyploid Mks under the action of biomechanical forces. We showed that mature Mks also shed MkMPs, whose generation is dramatically enhanced by shear flow. Co-culture of MkMPs with HSPCs promotes HSPC differentiation to Mks without exogenous thrombopoietin (the growth factor that stimulates Mk production from HSPCs), thus identifying a novel and previously unexplored physiological role for MkMPs. We show that MkMPs target HSPCs with exquisite specificity, and discuss mechanisms by which MkMPs target and act upon HSPCs. We argue for using MkMPs for regenerative-medicine applications, notably for the treatment of thrombocytopenia, as well as in as vectors for delivering nucleic-acid and protein “cargo” to HSPCs. The mechanism and kinetics of ChocMP generation and their biological role remain unexplored. We will discuss the first characterization of ChocMPs by examining the kinetics and mechanism of formation, their RNA content and efforts to identify their functional role. CHO cells produce ChocMPs from the very beginning of the culture, with different kinetics for attached versus suspension CHO cells. E.g., in suspension culture, ChocMPs concentration decreases with culture progression, thus suggesting that ChocMPs are fused into or endocytosed by CHO cells. In attached CHO cells, ChocMP generation is associated with “star- or bubble-studded” cellular images, characteristic of tumor-cell MP generation, thus providing first clues as to of the role of ChocMPs, since tumor-cell MPs have rather unique make up and role. ChocMP generation is promoted by stationary-phase conditions (notably, serum starvation), and biomechanical forces. Based on paradigms from other cells, we envision using ChocMPs as a means to predict culture fate, identify the role of small RNAs they are enriched in, and enhance culture performance