ORIGINAL ARTICLE Embryonic stem cell-derived microvesicles reprogram hematopoietic progenitors: evidence for horizontal transfer of mRNA and protein delivery J Ratajczak 1 , K Miekus 1,2 , M Kucia 1 , J Zhang 1 , R Reca 1 , P Dvorak 3 and MZ Ratajczak 1 1 Stem Cell Biology Program, James Graham Brown Cancer Center, University of Louisville, Louisville, KY, USA; 2 Jagiellominan University, Krakow, Poland and 3 Department of Molecular Embryology, Institute of Experimental Medicine, Academy of Sciences of the Czech Republic, Brno, Czech Republic Membrane-derived vesicles (MV) are released from the surface of activated eucaryotic cells and exert pleiotropic effects on surrounding cells. Since the maintenance of pluripotency and undifferentiated propagation of embryonic stem (ES) cells in vitro requires tight cell to cell contacts and effective inter- cellular signaling, we hypothesize that MV derived from ES cells (ES-MV) express stem cell-specific molecules that may also support self-renewal and expansion of adult stem cells. To address this hypothesis, we employed expansion of hemato- poietic progenitor cells (HPC) as a model. We found that ES-MV (10 lg/ml) isolated from murine ES cells (ES-D3) in serum-free cultures significantly (i) enhanced survival and improved expansion of murine HPC, (ii) upregulated the expression of early pluripotent (Oct-4, Nanog and Rex-1) and early hemato- poietic stem cells (Scl, HoxB4 and GATA 2) markers in these cells, and (iii) induced phosphorylation of MAPK p42/44 and serine-threonine kinase AKT. Furthermore, molecular analysis revealed that ES-MV express Wnt-3 protein and are selectively highly enriched in mRNA for several pluripotent transcription factors as compared to parental ES cells. More important, this mRNA could be delivered by ES-MV to target cells and translated into the corresponding proteins. The biological effects of ES-MV were inhibited after heat inactivation or pretreatment with RNAse, indicating a major involvement of protein and mRNA components of ES-MV in the observed phenomena. We postulate that ES-MV may efficiently expand HPC by stimulating them with ES-MV expressed ligands (e.g., Wnt-3) as well as increase their pluripotency after horizontal transfer of ES-derived mRNA. Leukemia (2006) 20, 847–856. doi:10.1038/sj.leu.2404132; published online 2 February 2006 Keywords: microvesicles; embryonic stem cells; stem cell expansion; mRNA transfer; Oct-4 Introduction Cells communicate by secreted growth factors, cytokines, adhesion molecules and small molecular mediators such as nucleotides or bioactive lipids. 1–4 Recently, we and others have postulated that circular membrane fragments called micro- vesicles (MV) play an important but underappreciated role in cell to cell communication. 5–8 MV are shed from the surface of activated cells or are derived from the endosomal membrane compartment after fusion of secretory granules with the plasma membrane, where they exist as intraluminal membrane-bound vesicles. 9–11 MV released from surface membranes are gener- ated in a calcium flux–calpain-dependent manner and are relatively large (100 nm1 mm), in contrast to smaller mem- brane fragments derived from the endosomal compartment (30– 100 nm). These MV contain numerous proteins and lipids similar to those present in the membranes of the cells from which they originate. We and others reported that MV may affect target cells (i) by stimulating them directly as ‘signaling devices’ by surface- expressed ligands, 5–7 or (ii) by transferring surface receptors between cells. 12,13 This MV-mediated communication between cells may perhaps have developed very early in the course of eucaryocytic evolution, before soluble mediators emerged. Recently, we and others postulated that MV may play a role in the spread of certain infections (e.g., HIV or prions) after they have fused with the target cells and delivered infectious particles inside the MV to the cytoplasm, by a so-called ‘Trojan horse mechanism’ of infection. 12,14–16 Embryonic stem (ES) cells are a rich source of MV. The propagation of ES cells in the undifferentiated state as well as induction of differentiation in developing embryoid bodies is coregulated by intercellular communications within ES cell colonies or embryoid bodies through membrane-bound mole- cules. Hence, we hypothesize that ES-derived MV express various stem cell-specific molecules that may affect the growth of target cells and contribute to the cell-fate decision. Owing to these characteristics, undifferentiated ES-derived MV may represent one of the critical components that support self- renewal and expansion of pluripotent or multipotent stem cells in vitro. Furthermore, it had been demonstrated that mature somatic cells cocultured with intact ES cells or extracts from these cells undergo epigenetic changes. 17,18 We hypothesize that some of these effects could be explained by a biological modification of the target cells via ES-MV. 17–20 If this is true, a reverse mechanism by which pluripotent ES cells influence growth of hematopoietic progenitor cells (HPC) could occur. To test this hypothesis we employed ES-MV obtained either from murine ES cells (D3) or human ES cells (CCTL14) in a model of expansion of murine HPC. We found that both murine D3 cell-derived ES-MV and human CCTL14 cell-derived ES-MV are highly enriched in Wnt-3 protein and mRNA for several early pluripotent transcription factors as compared to the ES cells from which they originated. This selective increase in mRNA content in ES-MV compared to parental ES cells suggests the presence of a mechanism that enriches ES-MV in mRNA molecules before their shedding from ES cells. Intrigued by these observations, we investigated whether ES-MV could enter HPC as a kind of physiological ‘liposome’ and increase their pluripotency after delivering ES-derived mRNA. Received 16 November 2005; revised 21 December 2005; accepted 26 December 2005; published online 2 February 2006 Correspondence: Professor MZ Ratajczak, Director of Stem Cell Biology Program, James Graham Brown Cancer Center, University of Louisville, KY 40202, USA. E-mail: mzrata01@louisville.edu Leukemia (2006) 20, 847–856 & 2006 Nature Publishing Group All rights reserved 0887-6924/06 $30.00 www.nature.com/leu