人胚胎干细胞在肠道中的分化

Soichiro  Ogaki; Shoen  Kume

doi:10.21769/BioProtoc.1010

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Peer-reviewed

Intestinal Differentiation of Human ESCs

人胚胎干细胞在肠道中的分化

SO Soichiro Ogaki

SK Shoen Kume email

发布: 2013年12月20日第3卷第24期 DOI: 10.21769/BioProtoc.1010 浏览次数: 7937

评审: Anonymous reviewer(s)

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The authors used this protocol in:

Cover of Stem Cells, featuring study using the protocol.

Jun 2013

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Abstract

ES cells (ESCs) are pluripotent and offer a good tool to study early embryonic development. Intestinal cells are derived from the definitive endoderm. In contrast to adult tissue stem cells, embryonic development and differentiation from ES cells have not been as well investigated in the intestine. There are four differentiated cell types of non-proliferative epithelial cells: enterocytes, goblet cells, enteroendocrine cells, and Paneth cells. Intestinal stem cells (ISCs) and progenitor cells reside in crypts, proliferate vigorously, and function as the source of differentiated epithelial cells. Here, we describe a protocol, in which differentiated cell types of the intestine are derived from human ESCs. In this protocol, we describe a protocol to differentiate mouse ES cells into Cdx2-expressing intestinal endoderm efficiently by co-culturing with M15, a mouse mesonephric cell line, and treatment with two chemical compounds. The chemical compounds used are BIO and DAPT. BIO is a Gsk3 inhibitor, that activate Wnt signaling pathway, and DAPT is a-secretase inhibitor that inhibit Notch signaling pathway. BIO and DAPT treatment yielded all representative cell lineages, enterocytes, goblet cells, enteroendocrine cells and paneth cells, to be derived from human ESCs. The protocol for human ESCs is principally very similar with that for the mouse ESCs, with some modifications.

Keywords: Intestine (肠)

Embryonic stem cells (胚胎干细胞)

differentiation (分化)

endoderm (内胚层)

human (人类)

Materials and Reagents

Human embryonic stem cells (ESCs)
M15 cells (ECACC, catalog number: 95102517 )
Mouse embryonic fibroblast (MEF)
0.1% gelatin
PBS
Dulbecco’s Modified Eagle Medium (DMEM) high glucose (Gibco^®, catalog number: 11995-075 )
DMEM low glucose (Gibco^®, catalog number: 11885-084 )
KnockOut DMEM/F12 (Gibco^®, catalog number: 12660-012 )
RPMI1640 (Gibco^®, catalog number: 11875-093 )
Fetal Bovine Serum (FBS) (Hyclone, catalog number: SH30310.03 )
200 mM L-glutamine (L-Gln) (Nacalai Tesque, catalog number: 16948-04 )
5,000 units/ml mixture of penicillin and streptomycin (PS) (Nacalai Tesque, catalog number: 26252-94 )
10 mM MEM Non-Essential Amino Acids Solution (NEAA) (Gibco^®, catalog number: 11140-050 )
2-mercaptoethanol (2-ME) (Sigma-Aldrich, catalog number: M7522 )
D-(+)-Glucose (Sigma-Aldrich, catalog number: G5146 )
KnockOut Serum Replacement (KSR) (Gibco^®, catalog number: 10828 )
B27 supplements (Gibco^®, catalog number: 17504044 )
Recombinant human Activin A (R&D Systems, catalog number: 338-AC )
Recombinant human bFGF (Pepro Tech, catalog number: 100-18B )
BIO (Calbiochem^®, catalog number: 361550 )
DAPT (Wako Pure Chemical Industries, catalog number: 041-30983 )
Y-27432 (Wako Pure Chemical Industries, catalog number: 688000 )
Mitomycin C (MMC) (Sigma-Aldrich, catalog number: M4287 )
0.05% Trypsin-EDTA (Life Technologies, catalog number: 2014-11 )
0.25% Trypsin-EDTA (Life Technologies, catalog number: 25200-072 )
EF medium (see Recipes)
hESC Medium (see Recipes)
EB medium (see Recipes)
hESCs Endoderm Medium (see Recipes)
Intestinal Medium (see Recipes)