A New Mechanism of Communication between Stem Cells Involving Vertical Transfer of mRNA by Its Intracellular Delivery within Membrane-Derived Microvesicles.

Abstract

Membrane-derived microvesicles (MV) are spherical membrane fragments that are released from eukaryoctic cells upon their activation. We reported that MV transfer cell membrane-derived receptors between cells and/or directly stimulate target cells by the ligands expressed on their surface (Blood 2001: 98;3143; AIDS 2003: 17;33). Recently we observed that embryonic stem cells (ES) shed MV into culture media and that co-culture of ES with hematopoietic stem cells enhances the latter's expansion. Similarly, co-culture of ES with somatic cells induces their dedifferentiation. Although the mechanisms responsible for these phenomena are not clear, we hypothesized that ES-derived MV (ESMV) could play an important role. To address this we isolated ESMV from murine (ES-D3) and two human (CCTL-12 and CCTL-14) embryonic cell lines and focused on molecules that may be responsible for epigenetic changes of cells co-cultured with ESMV. We found that ESMV as compared to the ES cells from which they originated are highly enriched in mRNA. This increase in mRNA content suggested a segregation mechanism that enriches ESMV in cytoplasmatic mRNA during their shedding from ES. Using real-time RT-PCR we found that ES-MV are highly enriched (x 103−107) in mRNA for early transcription factors that regulate self-renewal of stem cells (e.g., Oct-4, Gata-4, Rex-1 and Nanog). Intrigued by these observations we hypothesized that ESMV could penetrate the cells and deliver ES-derived mRNA and that this could be a novel mechanism for reprogramming target cells. Supporting our hypothesis we found (i) by confocal microscopy that ESMV do indeed penetrate the cells (e.g., BM-derived CD34+ cells or ES themselves), and (ii) by Western blot analysis that mRNA delivered to the target cells by ESMV is not trapped in the endosomal compartment but is delivered to the cytoplasm and actively transcribed into appropriate proteins (e.g., Oct-4). Based on these data we postulate that MV may transfer mRNA between the stem cells and play a role in vertical transfer of genetic information. Our recent similar data on MV derived from other cell types (normal and malignant) lend further support to this novel hypothesis and mechanism of cell to cell signaling/communication.