Generation of Functional Culture–Derived Platelets From CD34 ⁺ Progenitor Cells to Study Transgenes in the Platelet Environment

Abstract

Abstract:-The possibility of evaluating the function of transgenes in platelets requires the generation of platelets from nucleated progenitor cells in vitro. In this article, we provide effective culture conditions for generating functional culture-derived (CD) human and mouse platelets from CD34 ϩ progenitor cells that allow expression of any foreign protein of interest. We have evolved an effective cytokine cocktail (thrombopoietin, stem cell factor, interleukin [IL]-1␤, IL-6) that induces a high yield of CD platelets and optimal shedding from cultivated megakaryocytes generated from CD34 ϩ progenitor cells. CD platelets showed similar functional and morphological characteristics compared with isolated blood platelets, including surface expression of platelet antigens (CD41, CD42, CD62P), aggregation, release of granule constituents (P-selectin, platelet factor 4, serotonin). Moreover, transmission electron microscopy revealed the presence of typical ␣-and dense granules and dense tubular system in CD platelets. Additionally, we showed that stable transgene expression in CD platelets can be performed through infection of CD34 ϩ progenitor cells using adenoviral vectors. Thus, we describe a methodology that enables studying functional consequences of transgenes of interest in the natural environment of platelets that may impose substantial impact on potential future platelet research and therapeutic target evaluation. The full text of this article is available online at http://circres.ahajournals.org.

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

“…The results compared well to those obtained with noninfected CD platelets or freshly isolated platelets. 1 Also, secretion of vasoactive agents from CD platelets, the precursors of which had been retrovirally infected, compared well to that from noninfected CD platelets or freshly isolated platelets. Figure 3C shows that stimulation with thrombin clearly induced the release of serotonin from CD platelets, the precursors of which had been retrovirally infected, well comparable to that from noninfected platelets.…”

“…6,7 Also, human peripheral blood mononuclear cells and murine bone marrow cells were obtained, cultured, and differentiated to megakaryocytes as described. 1 Recombinant (E1/E3-deficient) adenoviruses were generated as described. 8 Retroviruses were cloned using the plasmid pLEGFP-C1 (obtained from Clontech).…”

“…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.…”

“…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.…”

“…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.…”