title: Targeted release of transcription factors for cell reprogramming by a natural micro-syringe.
abstract: Ectopic expression of defined transcription factors (TFs) for cell fate handling has proven high potential interest in reprogramming differentiated cells, in particular for regenerative medicine, ontogenesis study and cell based modelling. Pluripotency or transdifferentiation induction as TF mediated differentiation is commonly produced by transfer of genetic information with safety concerns. The direct delivery of proteins could represent a safer alternative but still needs significant advances to be efficient. We have successfully developed the direct delivery of proteins by an attenuated bacterium with a type 3 secretion system that does not require challenging and laborious steps for production and purification of recombinant molecules. Here we show that this natural micro-syringe is able to inject TFs to primary human fibroblasts and cord blood CD34+hematopoietic stem cells. The signal sequence for vectorization of the TF Oct4 has no effect on DNA binding to its nucleic target. As soon as one hour after injection, vectorized TFs are detectable in the nucleus. The injection process is not associated with toxicity and the bacteria can be completely removed from cell cultures. A three days targeted release of Oct4 or Sox2 embryonic TFs results in the induction of the core pluripotency genes expression in fibroblasts and CD34+hematopoietic stem cells. This micro-syringe vectorization represents a new strategy for TF delivery and has potential applications for cell fate reprogramming.
authors.affiliation: TIMC-TheREx Laboratory UMR 5525, CNRS, Université Grenoble Alpes, Grenoble F-38041, France. Electronic address: lionel.berthoin@ucsf.edu.
authors.forename: Lionel
authors.initials: L
authors.lastname: Berthoin
authors.affiliation: TIMC-TheREx Laboratory UMR 5525, CNRS, Université Grenoble Alpes, Grenoble F-38041, France. Electronic address: BToussaint@chu-grenoble.fr.
authors.forename: Bertrand
authors.initials: B
authors.lastname: Toussaint
authors.affiliation: TIMC-TheREx Laboratory UMR 5525, CNRS, Université Grenoble Alpes, Grenoble F-38041, France; Etablissement Français du Sang, 29 av du Maquis du Grésivaudan, BP35, 38701 La Tronche, France. Electronic address: FGarban@chu-grenoble.fr.
authors.forename: Frédéric
authors.initials: F
authors.lastname: Garban
authors.affiliation: TIMC-TheREx Laboratory UMR 5525, CNRS, Université Grenoble Alpes, Grenoble F-38041, France. Electronic address: ALegouellec@chu-grenoble.fr.
authors.forename: Audrey
authors.initials: A
authors.lastname: Le Gouellec
authors.affiliation: TIMC-TheREx Laboratory UMR 5525, CNRS, Université Grenoble Alpes, Grenoble F-38041, France; Etablissement Français du Sang, 29 av du Maquis du Grésivaudan, BP35, 38701 La Tronche, France. Electronic address: benjamin.caulier@gmail.com.
authors.forename: Benjamin
authors.initials: B
authors.lastname: Caulier
authors.affiliation: TIMC-TheREx Laboratory UMR 5525, CNRS, Université Grenoble Alpes, Grenoble F-38041, France. Electronic address: BPolack@chu-grenoble.fr.
authors.forename: Benoît
authors.initials: B
authors.lastname: Polack
authors.affiliation: TIMC-TheREx Laboratory UMR 5525, CNRS, Université Grenoble Alpes, Grenoble F-38041, France; Etablissement Français du Sang, 29 av du Maquis du Grésivaudan, BP35, 38701 La Tronche, France. Electronic address: david.laurin@efs.sante.fr.
authors.forename: David
authors.initials: D
authors.lastname: Laurin
mesh_terms: D065150:Cellular Reprogramming; D004247:DNA / Q000235:genetics; D005347:Fibroblasts / Q000378:metabolism; D015870:Gene Expression; D018014:Gene Transfer Techniques; D006412:Hematopoietic Stem Cells / Q000378:metabolism; D006801:Humans; D010957:Plasmids; D011550:Pseudomonas aeruginosa; D014157:Transcription Factors / Q000235:genetics*; D000069296:Type III Secretion Systems / Q000008:administration & dosage*; D001618:beta-Lactamases / Q000235:genetics / Q000378:metabolism
chemical_list: D014157:Transcription Factors; D000069296:Type III Secretion Systems; D004247:DNA; D001618:beta-Lactamases
keywords: Carbenicillin disodium (PubChem CID 20933); Ciprofloxacin (PubChem CID: 2764); EGTA: Ethylenebis(oxyethylenenitrilo)tetraacetic acid (PubChem CID: 6207); Embryonic transcription factors; Gentamicin (PubChem CID: 3467); IPTG: Isopropyl-beta-d-thiogalactopyranoside (PubChem CID: 656894); Induced pluripotent stem cells; Magnesium chloride hexahydrate (PubChem CID: 24644); Protein delivery systems; Pseudomonas aeruginosa; Reprogramming cell fate; Type 3 secretion system; Valproic acid (PubChem CID: 3121)