Generation of functional culture-derived platelets from CD34+ progenitor cells to study transgenes in the platelet environment.
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(+) progenitor cells that allow expression of any foreign protein of interest. We have evolved an effective cytokine cocktail (thrombopoietin, stem cell factor, interleukin [IL]-1beta, IL-6) that induces a high yield of CD platelets and optimal shedding from cultivated megakaryocytes generated from CD34(+) 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 alpha- 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(+) 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.
ProCorde GmbH, Fraunhoferstrasse 9, 82152 Martinsried, Germany. ungerer@procorde.com
Martin
M
Ungerer
Mario
M
Peluso
Angelika
A
Gillitzer
Steffen
S
Massberg
Ulrich
U
Heinzmann
Christian
C
Schulz
Götz
G
Münch
Meinrad
M
Gawaz
D000256:Adenoviridae / Q000235:genetics; D000818:Animals; D018952:Antigens, CD34 / Q000032:analysis*; D001792:Blood Platelets / Q000166:cytology* / Q000378:metabolism* / Q000502:physiology; D018929:Cell Culture Techniques; D015550:Cell Degranulation; D002478:Cells, Cultured; D005822:Genetic Vectors; D006412:Hematopoietic Stem Cells / Q000737:chemistry / Q000166:cytology / Q000502:physiology*; D006801:Humans; D051379:Mice; D019039:Platelet Glycoprotein GPIIb-IIIa Complex / Q000378:metabolism; D034061:Thrombopoiesis; D019076:Transgenes
D018952:Antigens, CD34; D019039:Platelet Glycoprotein GPIIb-IIIa Complex