Retroviral Infection and Selection of Culture-Derived Platelets Allows Study of the Effect of Transgenes on Platelet Physiology Ex Vivo and on Thrombus Formation In Vivo

Abstract

Background-We recently reported the development of culture-derived (CD) platelets with the aim to express any protein of interest in these platelets. 1 We now report a specific protocol of retroviral infection into the progenitor cells and subsequent selection, which allows to generate large amounts of highly homogenous transgene-expressing CD platelets and to study transgene function rapidly and reliably at large-scale ex vivo and in vivo settings. Methods and Results-After retroviral infection and selection, the activation-dependent expression profile of surface markers, aggregation, and granule release were investigated. The function of transgene-expressing CD platelets, the precursor cells of which had been retrovirally infected, compared well to noninfected CD platelets or freshly isolated platelets. Hence, the retroviral infection protocol did not alter platelet physiology. In contrast, adenoviral infection of precursors to CD platelets resulted in marked functional alterations that obviated their use in analytic experiments. Additionally, sufficient amounts of selected CD platelets were generated to warrant intravenous injections into living mice. This approach permitted study of their adhesive profile at endothelial lesions and their effect on thrombus formation in vivo by intravital videofluorescence microscopy. Conclusion-The novel selection method allowed us to produce recombinant transgene-expressing platelets in sufficient amounts to study genetically modified platelets in vitro and in vivo. Key Words: platelets Ⅲ gene transfer Ⅲ pharmacology C onsiderable interest has focused on the identification and characterization of pivotal signaling proteins in platelets because they play a central role in a variety of vascular diseases. However, the study of cytosolic platelet proteins has been hampered by the fact that these proteins cannot be accessed easily. One approach consisted in the permeabilization of platelets and the subsequent administration of antibodies to inactivate various proteins, as discussed. 1 Unfortunately, this permeabilization process leads to a severe disturbance of platelet physiology. Alternatively, transgenic mice were created by germline transmission, which constitutively overexpressed recombinant platelets. However, this approach is very time consuming and not feasible for the large set of platelet proteins, the overexpression or knockout of which causes lethal phenotypes.We recently established a method to reliably generate large amounts of culture-derived (CD) platelets in vitro from megakaryocyte progenitor cells. 1 However, so far, recombinant transgene-expressing platelets could not be produced homogenously in sufficient amounts for large-scale in vivo and in vivo analysis.To further improve this system and to express any protein of interest after gene transfer into the precursors to CD platelets, we compared different gene transduction protocols and established a novel system of efficient selection of recombinant CD platelets. Previous studies investigated the possibility ...

Human platelets produced in nonobese diabetic/severe combined immunodeficient (NOD/SCID) mice upon transplantation of human cord blood CD34+ cells are functionally active in an ex vivo flow model of thrombosis

“…5,[11][12][13][14][15] Such produced platelets are functional, as demonstrated in aggregation assays and by expression of P-selectin on the platelet surface or by activation of glycosylphosphatidylinositol (GPI) IbIIIa upon thrombin/ thrombin receptor-activating peptide (TRAP) stimulation. 6,[11][12][13][14] The nonobese diabetic/severe combined immunodeficient (NOD/ SCID) mouse model has long been accepted as the standard tool to reproduce human hematopoiesis after hematopoietic stem cell (HSC) xenoengraftment. 16 These animals present a favorable environment for efficient human progenitor cell engraftment due to various immunologic abnormalities such as T-and B-cell deficiency, defective natural killer cells, macrophage dysfunction, and absence of circulating complement.…”

Three-StageEx VivoExpansion of High-Ploidy Megakaryocytic Cells: Toward Large-Scale Platelet Production

“…Notably, other investigators have transfused culture-derived platelets, with similar in vitro functional activity to that described in this study, into mice and observed culture-derived platelets participating in growing blood clots in response to injury. 30,33,51,75 …”

Adenovirus vector induced innate immune responses: Impact upon efficacy and toxicity in gene therapy and vaccine applications

“…Though technically not cells, blood platelets may figure prominently in these responses. It has been shown that Ads can infect platelets in vitro, and mice intravenously injected with Ad vectors can be found to contain Ads within their platelets as well (Faraday et al, 1999;Gillitzer et al, 2005;Stone et al, 2007). The significance of Ad interactions with platelets (i.e.…”