Lineage-reprogramming of pericyte-derived cells of the adult human brain into induced neurons

J Vis Exp. 2014 May 12;(87):51433. doi: 10.3791/51433.

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.

Publication types

  • Research Support, Non-U.S. Gov't
  • Video-Audio Media

MeSH terms

  • Cell Culture Techniques
  • Cell Lineage
  • Cellular Reprogramming / physiology*
  • Cerebral Cortex / cytology*
  • Flow Cytometry
  • Humans
  • Immunohistochemistry
  • Neurons / cytology*
  • Neurons / metabolism
  • Patch-Clamp Techniques
  • Pericytes / cytology*
  • Pericytes / metabolism
  • Retroviridae / genetics
  • SOXB1 Transcription Factors / biosynthesis
  • SOXB1 Transcription Factors / genetics
  • Transduction, Genetic

Substances

  • SOX2 protein, human
  • SOXB1 Transcription Factors