Generation of enhanced definitive endoderm from human embryonic stem cells under an albumin/insulin-free and chemically defined condition.
OBJECTIVE
To enhance survival and generation of definitive endoderm cells from human embryonic stem cells in a simple and reproducible system.


METHODS
Definitive endoderm (DE) differentiation from human embryonic stem cells (hESCs) was induced under a chemical-defined condition withdrawn insulin supplement and serum albumin. We dissected influence of "alternative growth factors", WNT3A, BMP4 and bFGF in activin A-driven differentiation by detection of DE-associated genes expression and cell viability. Expression of DE-associated SOX17 and FOXA2 genes was analyzed by real time reverse transcription polymerase chain reaction (RT-PCR) and Western blot assays. Quantitative evaluation of DE efficiency was performed by flow cytometry analysis of CXCR4-expressed cell population. Cell viability during DE differentiation was analyzed by an Annexin V/PI double staining test.


RESULTS
Supplementation with WNT3A, BMP4 or bFGF promoted DE generation in a dose- and time-dependent manner. Cell apoptosis elicited by activin A was significantly ameliorated by a cocktail with WNT3A, BMP4 and bFGF. This allowed for sustained cell viability without insulin-containing supplements, thereby indirectly improving the efficiency of DE generation. Therefore, the cocktail containing is optimal for efficient DE generation in the presence of activin A and an insulin/albumin-free condition.


CONCLUSIONS
This optimal condition facilitates the balance between the productivity and the viability maintenance, and could be valuable for mass production of DE with minimal variation.
The Key Laboratory of Molecular Medicine of Ministry of Education, School of Basic Medical Sciences, Fudan University, Shanghai, China.
Su
S
Qu
Key Laboratory of Medical Molecular Virology of Ministry of Education and Ministry of Health, School of Basic Medical Sciences, Fudan University, Shanghai, China.
Liang
L
Yan
The Key Laboratory of Molecular Medicine of Ministry of Education, School of Basic Medical Sciences, Fudan University, Shanghai, China.
Bo
B
Fang
The Key Laboratory of Molecular Medicine of Ministry of Education, School of Basic Medical Sciences, Fudan University, Shanghai, China.
Shoudong
S
Ye
The Key Laboratory of Molecular Medicine of Ministry of Education, School of Basic Medical Sciences, Fudan University, Shanghai, China.
Ping
P
Li
Key Laboratory of Medical Molecular Virology of Ministry of Education and Ministry of Health, School of Basic Medical Sciences, Fudan University, Shanghai, China.
Shengyang
S
Ge
Key Laboratory of Medical Molecular Virology of Ministry of Education and Ministry of Health, School of Basic Medical Sciences, Fudan University, Shanghai, China. Electronic address: jian.wu@fudan.edu.cn.
Jian
J
Wu
Key Laboratory of Medical Molecular Virology of Ministry of Education and Ministry of Health, School of Basic Medical Sciences, Fudan University, Shanghai, China. Electronic address: dqu@shmu.edu.cn.
Di
D
Qu
The Key Laboratory of Molecular Medicine of Ministry of Education, School of Basic Medical Sciences, Fudan University, Shanghai, China. Electronic address: hysong@shmu.edu.cn.
Houyan
H
Song
D055415:Bone Morphogenetic Protein 4 / Q000494:pharmacology; D002454:Cell Differentiation / Q000187:drug effects*; D002460:Cell Line; D003470:Culture Media / Q000494:pharmacology*; D004707:Endoderm / Q000166:cytology / Q000378:metabolism*; D016222:Fibroblast Growth Factor 2 / Q000494:pharmacology; D018507:Gene Expression Regulation, Developmental / Q000187:drug effects*; D051542:Hepatocyte Nuclear Factor 3-beta / Q000096:biosynthesis*; D000066449:Human Embryonic Stem Cells / Q000166:cytology / Q000378:metabolism*; D006801:Humans; D007328:Insulin; D055760:SOXF Transcription Factors / Q000096:biosynthesis*; D012709:Serum Albumin; D060509:Wnt3A Protein / Q000494:pharmacology
C525809:BMP4 protein, human; D055415:Bone Morphogenetic Protein 4; D003470:Culture Media; C496502:FOXA2 protein, human; D007328:Insulin; C109188:SOX17 protein, human; D055760:SOXF Transcription Factors; D012709:Serum Albumin; C555510:WNT3A protein, human; D060509:Wnt3A Protein; D016222:Fibroblast Growth Factor 2; D051542:Hepatocyte Nuclear Factor 3-beta
Cell survival; Chemically defined condition; Definitive endoderm; Human embryonic stem cell