Slice Culture Method for Studying Migration of Neuronal Progenitor Cells Derived from Human Embryonic Stem Cells (hESC).
In this unit we describe an overlay brain slice culture assay for studying migration of transgenic neurospheres derived from human embryonic stem cells (hESC). Neuronal progenitor cells were generated from hESC by derivation of embryoid bodies and rosettes. Rosettes were transfected using the PiggyBac transposon system with either control plasmids (GFP) or plasmid encoding a gene important for migration of neuronal progenitor cells, Doublecortin (DCX). Transfected cells were subsequently grown in low-adhesion plates to generate transgenic human neurospheres (t-hNS). Organotypic slice cultures were prepared from postnatal rat forebrain and maintained using the interface method, before transfected t-hNS were overlaid below the cortex of each hemisphere. After 1 to 5 days, forebrain slices were fixed and processed for immunofluorescence. The distance at which cells migrated from the center of neurospheres to the host forebrain tissue was measured using Image J software. This protocol provides details for using the slice culture method for studying migration and integration of human neuronal cells into the host brain tissue.
Department of Physiology and Neurobiology, University of Connecticut, Storrs, Connecticut.
Radmila
R
Filipovic
Saranya Santosh
SS
Kumar
Ben A
BA
Bahr
Joseph
J
Loturco
D000818:Animals; D002465:Cell Movement; D000089662:Doublecortin Protein; D064593:Heterografts; D000066449:Human Embryonic Stem Cells / Q000166:cytology / Q000378:metabolism / Q000637:transplantation; D006801:Humans; D007091:Image Processing, Computer-Assisted / Q000379:methods; D058953:Neural Stem Cells / Q000166:cytology / Q000378:metabolism; D009924:Organ Culture Techniques / Q000379:methods*; D016548:Prosencephalon / Q000166:cytology / Q000196:embryology; D051381:Rats; D033581:Stem Cell Transplantation
human embryonic stem cell; migration; neuronal progenitors; organotypic slice cultures; overlay essay; piggyBac