title: Highly efficient generation of induced neurons from human fibroblasts that survive transplantation into the adult rat brain.
abstract: Induced neurons (iNs) offer a novel source of human neurons that can be explored for applications of disease modelling, diagnostics, drug screening and cell replacement therapy. Here we present a protocol for highly efficient generation of functional iNs from fetal human fibroblasts, and also demonstrate the ability of these converted human iNs (hiNs) to survive transplantation and maintain their phenotype in the adult rat brain. The protocol encompasses a delay in transgene activation after viral transduction that resulted in a significant increase in conversion efficiency. Combining this approach with treatment of small molecules that inhibit SMAD signalling and activate WNT signalling provides a further increase in the conversion efficiency and neuronal purity, resulting in a protocol that provides a highly efficient method for the generation of large numbers of functional and transplantable iNs from human fibroblasts without the use of a selection step. When transplanting the converted neurons from different stages of in vitro culture into the brain of adult rats, we observed robust survival and maintenance of neuronal identity four weeks post-transplantation. Interestingly, the positive effect of small molecule treatment observed in vitro did not result in a higher yield of iNs surviving transplantation.
authors.affiliation: Department of Experimental Medical Science, Wallenberg Neuroscience Center and Lund Stem Cell Center, Lund University, 221 84 Lund, Sweden.
authors.forename: Maria
authors.initials: M
authors.lastname: Pereira
authors.affiliation: Department of Experimental Medical Science, Wallenberg Neuroscience Center and Lund Stem Cell Center, Lund University, 221 84 Lund, Sweden.
authors.forename: Ulrich
authors.initials: U
authors.lastname: Pfisterer
authors.affiliation: Department of Experimental Medical Science, Wallenberg Neuroscience Center and Lund Stem Cell Center, Lund University, 221 84 Lund, Sweden.
authors.forename: Daniella
authors.initials: D
authors.lastname: Rylander
authors.affiliation: Department of Experimental Medical Science, Wallenberg Neuroscience Center and Lund Stem Cell Center, Lund University, 221 84 Lund, Sweden.
authors.forename: Olof
authors.initials: O
authors.lastname: Torper
authors.affiliation: Department of Experimental Medical Science, Wallenberg Neuroscience Center and Lund Stem Cell Center, Lund University, 221 84 Lund, Sweden.
authors.forename: Shong
authors.initials: S
authors.lastname: Lau
authors.affiliation: Department of Experimental Medical Science, Wallenberg Neuroscience Center and Lund Stem Cell Center, Lund University, 221 84 Lund, Sweden.
authors.forename: Martin
authors.initials: M
authors.lastname: Lundblad
authors.affiliation: Department of Experimental Medical Science, Wallenberg Neuroscience Center and Lund Stem Cell Center, Lund University, 221 84 Lund, Sweden.
authors.forename: Shane
authors.initials: S
authors.lastname: Grealish
authors.affiliation: Department of Experimental Medical Science, Wallenberg Neuroscience Center and Lund Stem Cell Center, Lund University, 221 84 Lund, Sweden.
authors.forename: Malin
authors.initials: M
authors.lastname: Parmar
mesh_terms: D000818:Animals; D051792:Basic Helix-Loop-Helix Proteins / Q000235:genetics; D001921:Brain / Q000166:cytology*; D002454:Cell Differentiation / Q000502:physiology; D049109:Cell Proliferation; D002470:Cell Survival; D002478:Cells, Cultured; D065150:Cellular Reprogramming / Q000502:physiology*; D004298:Dopamine / Q000096:biosynthesis; D005260:Female; D005347:Fibroblasts / Q000166:cytology; D018398:Homeodomain Proteins / Q000235:genetics; D006801:Humans; D057026:Induced Pluripotent Stem Cells / Q000166:cytology*; D008869:Microtubule-Associated Proteins / Q000096:biosynthesis; D009419:Nerve Tissue Proteins / Q000235:genetics; D009474:Neurons / Q000166:cytology / Q000637:transplantation*; D050809:POU Domain Factors / Q000235:genetics; D051381:Rats; D017207:Rats, Sprague-Dawley; D051785:Smad Proteins / Q000037:antagonists & inhibitors; D014157:Transcription Factors / Q000235:genetics; D019076:Transgenes / Q000235:genetics; D014183:Transplantation, Heterologous / Q000379:methods*; D014404:Tubulin / Q000096:biosynthesis; D060449:Wnt Signaling Pathway / Q000187:drug effects
chemical_list: D051792:Basic Helix-Loop-Helix Proteins; D004298:Dopamine; D018398:Homeodomain Proteins; D008869:Microtubule-Associated Proteins; D009419:Nerve Tissue Proteins; D050809:POU Domain Factors; D051785:Smad Proteins; D014157:Transcription Factors; D014404:Tubulin; C065500:ASCL1 protein, human; C489788:MAP2 protein, human; C561020:MYT1L protein, human; C085076:transcription factor Brn-2