Genetic engineering of mouse embryonic stem cells by Nurr1 enhances differentiation and maturation into dopaminergic neurons.
Nurr1 is a transcription factor critical for the development of midbrain dopaminergic (DA) neurons. This study modified mouse embryonic stem (ES) cells to constitutively express Nurr1 under the elongation factor-1alpha promoter. The Nurr1-expression in ES cells lead to up-regulation of all DA neuronal markers tested, resulting in about a 4- to 5-fold increase in the proportion of DA neurons. In contrast, other neuronal and glial markers were not significantly changed by Nurr1 expression. It was also observed that there was an additional 4-fold increase in the number of DA neurons in Nurr1-expressing clones following treatment with Shh, FGF8 and ascorbic acid. Several lines of evidence suggest that these neurons may represent midbrain DA neuronal phenotypes; firstly, they coexpress midbrain DA markers such as aromatic L-amino acid decarboxylase, calretinin, and dopamine transporter, in addition to tyrosine hydroxylase and secondly, they do not coexpress other neurotransmitters such as GABA or serotonin. Finally, consistent with an increased number of DA neurons, the Nurr1 transduction enhanced the ability of these neurons to produce and release DA in response to membrane depolarization. This study demonstrates an efficient genetic manipulation of ES cells that facilitates differentiation to midbrain DA neurons, and it will serve as a framework of genetic engineering of ES cells by key transcription factor to regulate their cell fate.
Chung
Sangmi
S
Udall Parkinson's Disease Research Center of Excellence, Neuroregeneration Laboratory, McLean Hospital/Harvard Medical School, Belmont, MA 02478, USA.
Sonntag
Kai-C
KC
Andersson
Therese
T
Bjorklund
Lars M
LM
Park
Jae-Joon
JJ
Kim
Dong-Wook
DW
Kang
Un Jung
UJ
Isacson
Ole
O
Kim
Kwang-Soo
KS
D000818:Animals; D001933:Brain Stem / Q000378:metabolism; D018929:Cell Culture Techniques; D002454:Cell Differentiation / Q000235:genetics; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D004298:Dopamine / Q000378:metabolism*; D005818:Genetic Engineering / Q000379:methods; D007150:Immunohistochemistry; D051379:Mice; D009474:Neurons / Q000378:metabolism*; D057126:Nuclear Receptor Subfamily 4, Group A, Member 2; D010957:Plasmids; D039904:Pluripotent Stem Cells / Q000378:metabolism*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012701:Serotonin / Q000378:metabolism; D014157:Transcription Factors / Q000235:genetics / Q000378:metabolism*; D014162:Transfection; D014446:Tyrosine 3-Monooxygenase / Q000378:metabolism
D004268:DNA-Binding Proteins; C539883:Nr4a2 protein, mouse; D057126:Nuclear Receptor Subfamily 4, Group A, Member 2; D014157:Transcription Factors; D012701:Serotonin; D014446:Tyrosine 3-Monooxygenase; D004298:Dopamine