Comparative analysis of endoderm formation efficiency between mouse ES cells and iPS cells.
Definitive endoderm (DE) derived from stem cells holds potential to differentiate into hepatocytes. Stem cell therapy using those cells has potential for a treatment of liver disease. To date, various ways of inducing hepatocytes from embryonic stem (ES) cells have been reported by researchers. However, it has not been proved enough that induced pluripotent stem (iPS) cells behave in the same manner as ES cells in endoderm differentiation. The purpose of this study was to establish an efficient method to induce DE from iPS cells, through comparatively analyzing the efficacy of endoderm formation from mouse ES cells. Furthermore, the efficiency of a serum-free medium in the differentiation into DE was investigated. Mouse ES cells and iPS cells were floated in culture medium for 2 or 5 days and embryoid bodies (EB) were formed. Subsequently, DE was induced with 100 ng/ml activin A and 100 ng/ml basic fibroblast growth factor (bFGF). RT-PCR and real-time PCR analyses were carried out at each step to determine the gene expression of EB markers. The difference in cellular proliferation between serum-containing and serum-free media was examined by an MTS assay in EB and DE induction. iPS cells showed the paralleled mRNA expression to ES cells in each step of differentiation into EB, but the levels of expression of Sox17 and Foxa2 were relatively higher in ES cell-derived DE, whereas Cxcr4 expression was higher in iPS cell-derived DE. The utilization of serum-free medium for iPS cells showed significantly favorable cellular proliferation during EB formation and subsequent DE induction. Forming EB for 5 days and subsequently DE induction with activin A and bFGF with serum-free medium was an appropriate protocol in iPS cells. This may represent an important step for generating hepatocytes from iPS cells for the development of cell therapy.
Iwamuro
Masaya
M
Department of Gastroenterology and Hepatology, Okayama University Graduate School of Medicine, Dentistry, and Pharmaceutical Sciences, Okayama, Japan. iwamuromasaya@yahoo.co.jp
Komaki
Toshiyuki
T
Kubota
Yasuhiro
Y
Seita
Masayuki
M
Kawamoto
Hironobu
H
Yuasa
Takeshi
T
Shahid
Javed M
JM
Hassan
Reham A R A
RA
Hassan
Wael A R A
WA
Nakaji
Shuhei
S
Nishikawa
Yuriko
Y
Kondo
Eisaku
E
Yamamoto
Kazuhide
K
Kobayashi
Naoya
N
D000818:Animals; D001681:Biological Assay; D049109:Cell Proliferation / Q000187:drug effects; D048430:Cell Shape / Q000187:drug effects; D003470:Culture Media / Q000494:pharmacology; D058732:Embryoid Bodies / Q000166:cytology / Q000187:drug effects / Q000378:metabolism; D053595:Embryonic Stem Cells / Q000166:cytology* / Q000187:drug effects / Q000378:metabolism; D004707:Endoderm / Q000166:cytology / Q000187:drug effects / Q000196:embryology* / Q000378:metabolism; D018507:Gene Expression Regulation, Developmental / Q000187:drug effects; D057026:Induced Pluripotent Stem Cells / Q000166:cytology* / Q000187:drug effects / Q000378:metabolism; D051379:Mice; D020133:Reverse Transcriptase Polymerase Chain Reaction
D003470:Culture Media