Reprogrammation of eukaryotic cells with engineered microvesicles
The present invention relates to a non-genetic, detergent-free, bacteria-free method for reprogramming a eukaryotic cell, in particular for obtaining induced pluripotent stem cells (iPS), by using engineered microvesicles carrying at least one reprogramming transcription factor, wherein said engineered microvesicles are virus-free.
1. An in vitro method for delivering a reprogramming factor into a target cell comprising i) providing an eukaryotic cell overexpressing a viral membrane fusion protein and at least one reprogramming transcription factor, wherein said eukaryotic cell secretes a microvesicle comprising said viral membrane fusion protein and said at least one reprogramming transcription factor and wherein said eukaryotic cell does not express any viral structural proteins; and ii) contacting said microvesicle to a target cell, wherein said contacting delivers said at least one reprogramming transcription factor into said target cell.
2. The method according to claim 1 , wherein said viral membrane fusion protein is VSV-G.
3. The method according to claim 2 , wherein said at least one transcription factor is selected from the group consisting of Oct-4, Klf4, Sox2, Lin28, and c-Myc.
4. The method according to claim 1 , wherein said contacting comprises co-culturing said eukaryotic cell with said target cell.
5. A method of obtaining induced pluripotent stem cells from a target cell comprising: i) providing an eukaryotic cell overexpressing a viral membrane fusion protein and reprogramming transcription factors, where said eukaryotic cell secretes a microvesicle comprising said viral membrane fusion protein and said reprogramming transcription factors and wherein said eukaryotic cell does not express any viral structural proteins; and ii) contacting said microvesicle to a target cell, wherein said contacting delivers said reprogramming transcription factors into said target cell and reprograms said target cell into an induced pluripotent cell.
6. A method of obtaining an induced pluripotent cell comprising the steps of:
(i) contacting a target cell with a microvesicle comprising a viral fusion protein and an ecotropic viral receptor (EcoR), wherein the microvesicle delivers the EcoR into said target cell and renders said target cell permissive to an ecotropic virus; and
(ii) transducing said target cell with an ecotropic virus comprising a nucleic acid encoding reprogramming transcription factors,
wherein said target cell expresses said nucleic acid encoding reprogramming transcription factors and said reprogramming transcription factors reprogram said target cell into an induced pluripotent cell.
7. The method according to claim 6 , wherein said ecotropic virus is a rodent ecotropic virus that can only replicate in a cell expressing EcoR.