Identification and isolation of embryonic stem cell-derived target cells by adenoviral conditional targeting.
The technical limitations of isolating target cells have restricted the utility of pluripotent embryonic stem (ES) cells. For example, early cardiac (i.e., precontractile) cells have not been isolated from ES cells. Here, we find that direct expression of reporter genes under cell-specific promoters-the currently available strategy for isolating cells lacking cell-specific surface markers-is ineffective for isolating progenitor cells. This was due to the weak activity of cell-specific promoters, particularly in ES cells at early stages. We show that adenoviral conditional targeting efficiently isolates viable ES cell-derived target cells without harmful effects. In this strategy, we employ the alpha-myosin heavy chain and Nkx2.5 promoter to visualize and purify efficiently differentiated and primitive cells of the cardiac lineage, respectively. While the former cells predominantly expressed sarcomeric proteins and maintained contractile function, the latter demonstrated neither of these features, but rather exhibited expression patterns characteristic of a mixture of primitive cells and cardiomyocytes. Interestingly, smooth muscle actin was predominantly expressed in the latter cells, and both functionally known and unknown genes were systematically identified, demonstrating the benefits of this system. Thus, our method facilitates molecular and cellular studies of development and ES cell-derived cell therapy.
Takahashi
Tomoyuki
T
Division of Gene Therapy and Regenerative Medicine, Cognitive and Molecular Research Institute of Brain Diseases, Department of Advanced Therapeutics and Regenerative Medicine, Kurume University, 67 Asahi-machi, Kurume 830-0011, Japan.
Kawai
Takao
T
Ushikoshi
Hiroaki
H
Nagano
Satoshi
S
Oshika
Hatsue
H
Inoue
Makoto
M
Kunisada
Takahiro
T
Takemura
Genzou
G
Fujiwara
Hisayoshi
H
Kosai
Ken-ichiro
K
D000256:Adenoviridae / Q000235:genetics*; D000818:Animals; D002454:Cell Differentiation; D019070:Cell Lineage; D002469:Cell Separation / Q000379:methods*; D002478:Cells, Cultured; D015536:Down-Regulation; D004622:Embryo, Mammalian / Q000166:cytology / Q000378:metabolism; D000072576:Homeobox Protein Nkx-2.5; D018398:Homeodomain Proteins / Q000235:genetics / Q000378:metabolism; D051379:Mice; D046529:Microscopy, Electron, Transmission; D009200:Myocardial Contraction; D009206:Myocardium / Q000378:metabolism; D013234:Stem Cells / Q000166:cytology* / Q000378:metabolism*; D014157:Transcription Factors / Q000235:genetics / Q000378:metabolism; D015854:Up-Regulation
D000072576:Homeobox Protein Nkx-2.5; D018398:Homeodomain Proteins; C484469:Nkx2-5 protein, mouse; D014157:Transcription Factors