BAC transgenesis in human embryonic stem cells as a novel tool to define the human neural lineage.
Human embryonic stem cells (hESCs) have enormous potential for applications in basic biology and regenerative medicine. However, harnessing the potential of hESCs toward generating homogeneous populations of specialized cells remains challenging. Here we describe a novel technology for the genetic identification of defined hESC-derived neural cell types using bacterial artificial chromosome (BAC) transgenesis. We generated hESC lines stably expressing Hes5::GFP, Dll1::GFP, and HB9::GFP BACs that yield green fluorescent protein (GFP)(+) neural stem cells, neuroblasts, and motor neurons, respectively. Faithful reporter expression was confirmed by cell fate analysis and appropriate transgene regulation. Prospective isolation of HB9::GFP(+) cells yielded purified human motor neurons with proper marker expression and electrophysiological activity. Global mRNA and microRNA analyses of Hes5::GFP(+) and HB9::GFP(+) populations revealed highly specific expression signatures, suggesting that BAC transgenesis will be a powerful tool for establishing expression libraries that define the human neural lineage and for accessing defined cell types in applications of human disease.
Placantonakis
Dimitris G
DG
Department of Neurosurgery, Sloan-Kettering Institute for Cancer Research, New York, New York, USA. dip9011@nyp.org
Tomishima
Mark J
MJ
Lafaille
Fabien
F
Desbordes
Sabrina C
SC
Jia
Fan
F
Socci
Nicholas D
ND
Viale
Agnes
A
Lee
Hyojin
H
Harrison
Neil
N
Tabar
Viviane
V
Studer
Lorenz
L
D051792:Basic Helix-Loop-Helix Proteins / Q000235:genetics / Q000378:metabolism; D002454:Cell Differentiation / Q000235:genetics / Q000502:physiology; D002460:Cell Line; D002478:Cells, Cultured; D022202:Chromosomes, Artificial, Bacterial / Q000235:genetics*; D004594:Electrophysiology; D053595:Embryonic Stem Cells / Q000166:cytology* / Q000378:metabolism*; D005434:Flow Cytometry; D005455:Fluorescent Antibody Technique; D005786:Gene Expression Regulation / Q000187:drug effects; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D006801:Humans; D036341:Intercellular Signaling Peptides and Proteins / Q000235:genetics / Q000378:metabolism; D009046:Motor Neurons / Q000166:cytology / Q000378:metabolism; D009474:Neurons / Q000166:cytology* / Q000378:metabolism*; D051880:Receptors, Notch / Q000378:metabolism; D012097:Repressor Proteins / Q000235:genetics / Q000378:metabolism
D051792:Basic Helix-Loop-Helix Proteins; D049452:Green Fluorescent Proteins; D036341:Intercellular Signaling Peptides and Proteins; D051880:Receptors, Notch; D012097:Repressor Proteins; C077478:HES5 protein, human