title: Lineage-reprogramming of pericyte-derived cells of the adult human brain into induced neurons.
abstract: Direct lineage-reprogramming of non-neuronal cells into induced neurons (iNs) may provide insights into the molecular mechanisms underlying neurogenesis and enable new strategies for in vitro modeling or repairing the diseased brain. Identifying brain-resident non-neuronal cell types amenable to direct conversion into iNs might allow for launching such an approach in situ, i.e. within the damaged brain tissue. Here we describe a protocol developed in the attempt of identifying cells derived from the adult human brain that fulfill this premise. This protocol involves: (1) the culturing of human cells from the cerebral cortex obtained from adult human brain biopsies; (2) the in vitro expansion (approximately requiring 2-4 weeks) and characterization of the culture by immunocytochemistry and flow cytometry; (3) the enrichment by fluorescence-activated cell sorting (FACS) using anti-PDGF receptor-β and anti-CD146 antibodies; (4) the retrovirus-mediated transduction with the neurogenic transcription factors sox2 and ascl1; (5) and finally the characterization of the resultant pericyte-derived induced neurons (PdiNs) by immunocytochemistry (14 days to 8 weeks following retroviral transduction). At this stage, iNs can be probed for their electrical properties by patch-clamp recording. This protocol provides a highly reproducible procedure for the in vitro lineage conversion of brain-resident pericytes into functional human iNs.
authors.affiliation: Department of Physiological Genomics, Institute of Physiology, Ludwig Maximilians University Munich; marisa.karow@med.uni-muenchen.de.
authors.forename: Marisa
authors.initials: M
authors.lastname: Karow
authors.affiliation: Tumor Biology Lab, Neurosurgical Clinic, Ludwig-Maximilians University Munich.
authors.forename: Christian
authors.initials: C
authors.lastname: Schichor
authors.affiliation: Institut für Biochemie, Emil-Fischer-Zentrum, Friedrich-Alexander-Universität Erlangen-Nürnberg.
authors.forename: Ruth
authors.initials: R
authors.lastname: Beckervordersandforth
authors.affiliation: Department of Physiological Genomics, Institute of Physiology, Ludwig Maximilians University Munich; Institute of Physiological Chemistry and Focus Program Translational Neuroscience, Johannes Gutenberg University Mainz; berningb@uni-mainz.de.
authors.forename: Benedikt
authors.initials: B
authors.lastname: Berninger
mesh_terms: D018929:Cell Culture Techniques; D019070:Cell Lineage; D065150:Cellular Reprogramming / Q000502:physiology*; D002540:Cerebral Cortex / Q000166:cytology*; D005434:Flow Cytometry; D006801:Humans; D007150:Immunohistochemistry; D009474:Neurons / Q000166:cytology* / Q000378:metabolism; D018408:Patch-Clamp Techniques; D020286:Pericytes / Q000166:cytology* / Q000378:metabolism; D012190:Retroviridae / Q000235:genetics; D055748:SOXB1 Transcription Factors / Q000096:biosynthesis / Q000235:genetics; D014161:Transduction, Genetic
chemical_list: C527782:SOX2 protein, human; D055748:SOXB1 Transcription Factors