In vitro differentiation of mouse embryonic stem cells: enrichment of endodermal cells in the embryoid body.
Embryonic stem (ES) cells have the potential to differentiate into all three germ layers, providing new perspectives not only for embryonic development but also for the application in cell replacement therapies. Even though the formation of an embryoid body (EB) in a suspension culture has been the most popular method to differentiate ES cells into a wide range of cells, not much is known about the characteristics of EB cells. To this end, we investigated the process of EB formation in the suspension culture of ES cells at weekly intervals for up to 6 weeks. We observed that the central apoptotic area is most active in the first week of EB formation and that the cell adhesion molecules, except beta-catenin, are highly expressed throughout the examination period. The sequential expression of endodermal genes in EBs during the 6-week culture correlated closely with that of normal embryo development. The outer surface of EBs stained positive for alpha-fetoprotein and GATA-4. When isolated from the 2-week-old EB by trypsin treatment, these endodermal lineage cells matured in vitro into hepatocytes upon stimulation with various hepatotrophic factors. In conclusion, our results demonstrate that endodermal cells can be retrieved from EBs and matured into specific cell types, opening new therapeutic usage of these in vitro differentiated cells in the cell replacement therapy of various diseases.
Choi
Dongho
D
Department of Surgery, Stem Cell Therapy Center, Soonchunhyang University Hospital, Seoul, Korea.
Lee
Hye-Ja
HJ
Jee
Seunghyun
S
Jin
Soojung
S
Koo
Soo Kyung
SK
Paik
Seung Sam
SS
Jung
Sung Chul
SC
Hwang
Sue-Yun
SY
Lee
Kwang Soo
KS
Oh
Bermseok
B
D000818:Animals; D017209:Apoptosis; D002448:Cell Adhesion; D002454:Cell Differentiation; D002460:Cell Line; D019070:Cell Lineage; D017931:DNA Primers / Q000737:chemistry; D004268:DNA-Binding Proteins / Q000378:metabolism; D004475:Ectoderm / Q000378:metabolism; D004622:Embryo, Mammalian / Q000166:cytology*; D047108:Embryonic Development; D004707:Endoderm / Q000166:cytology* / Q000378:metabolism; D005347:Fibroblasts / Q000378:metabolism; D050991:GATA4 Transcription Factor; D005786:Gene Expression Regulation; D022781:Hepatocytes / Q000166:cytology / Q000378:metabolism; D007150:Immunohistochemistry; D066298:In Vitro Techniques; D007208:Indocyanine Green / Q000493:pharmacokinetics; D008648:Mesoderm / Q000378:metabolism; D051379:Mice; D010504:Periodic Acid / Q000494:pharmacology; D010503:Periodic Acid-Schiff Reaction; D016133:Polymerase Chain Reaction; D012333:RNA, Messenger / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012394:Rosaniline Dyes / Q000494:pharmacology; D013234:Stem Cells / Q000166:cytology*; D014157:Transcription Factors / Q000378:metabolism; D014357:Trypsin / Q000378:metabolism / Q000494:pharmacology; D000509:alpha-Fetoproteins / Q000378:metabolism
D017931:DNA Primers; D004268:DNA-Binding Proteins; D050991:GATA4 Transcription Factor; D012333:RNA, Messenger; D012394:Rosaniline Dyes; C476677:Schiff's reagent; D014157:Transcription Factors; D000509:alpha-Fetoproteins; D010504:Periodic Acid; D014357:Trypsin; D007208:Indocyanine Green