Fischer Stefanie, Cornils Kerstin, Speiseder Thomas, Badbaran Anita, Reimer Rudolph, Indenbirken Daniela, Grundhoff Adam, Brunswig-Spickenheier Bärbel, Alawi Malik, Lange Claudia
Research Department Cell and Gene Therapy, Clinic for Stem Cell Transplantation, University Medical Center Hamburg-Eppendorf, Hamburg, Germany.
Research Unit Viral Transformation, Heinrich Pette Institute, Leibniz Institute for Experimental Virology, Hamburg, Germany.
PLoS One. 2016 Sep 29;11(9):e0163665. doi: 10.1371/journal.pone.0163665. eCollection 2016.
The biological relevance of extracellular vesicles (EV) in intercellular communication has been well established. Thus far, proteins and RNA were described as main cargo. Here, we show that EV released from human bone marrow derived mesenchymal stromal cells (BM-hMSC) also carry high-molecular DNA in addition. Extensive EV characterization revealed this DNA mainly associated with the outer EV membrane and to a smaller degree also inside the EV. Our EV purification protocol secured that DNA is not derived from apoptotic or necrotic cells. To analyze the relevance of EV-associated DNA we lentivirally transduced Arabidopsis thaliana-DNA (A.t.-DNA) as indicator into BM-hMSC and generated EV. Using quantitative polymerase chain reaction (qPCR) techniques we detected high copy numbers of A.t.-DNA in EV. In recipient hMSC incubated with tagged EV for two weeks we identified A.t.-DNA transferred to recipient cells. Investigation of recipient cell DNA using quantitative PCR and verification of PCR-products by sequencing suggested stable integration of A.t.-DNA. In conclusion, for the first time our proof-of-principle experiments point to horizontal DNA transfer into recipient cells via EV. Based on our results we assume that eukaryotic cells are able to exchange genetic information in form of DNA extending the known cargo of EV by genomic DNA. This mechanism might be of relevance in cancer but also during cell evolution and development.
细胞外囊泡（EV）在细胞间通讯中的生物学相关性已得到充分证实。到目前为止，蛋白质和RNA被描述为主要货物。在这里，我们表明，人骨髓间充质基质细胞（BM-hMSC）释放的EV还携带高分子量DNA。广泛的EV表征显示，这种DNA主要与EV外膜相关，在较小程度上也存在于EV内部。我们的EV纯化方案确保DNA不是来自凋亡或坏死细胞。为了分析EV相关DNA的相关性，我们将拟南芥DNA（A.t.-DNA）作为指示剂通过慢病毒转导到BM-hMSC中并产生EV。使用定量聚合酶链反应（qPCR）技术，我们在EV中检测到高拷贝数的A.t.-DNA。在用标记的EV孵育两周的受体hMSC中，我们鉴定出转移到受体细胞中的A.t.-DNA。使用定量PCR对受体细胞DNA进行研究并通过测序验证PCR产物，表明A.t.-DNA稳定整合。总之，我们的原理验证实验首次表明通过EV将水平DNA转移到受体细胞中。基于我们的结果，我们假设真核细胞能够以DNA的形式交换遗传信息，从而通过基因组DNA扩展已知的EV货物。这种机制可能与癌症有关，但也与细胞进化和发育有关。
