Efficient definitive endoderm induction from mouse embryonic stem cell adherent cultures: a rapid screening model for differentiation studies.
Definitive endoderm (DE) differentiation from mouse embryonic stem cell (mESC) monolayer cultures has been limited by poor cell survival or low efficiency. Recently, a combination of TGFβ and Wnt activation with BMP inhibition improved DE induction in embryoid bodies cultured in suspension. Based on these observations we developed a protocol to efficiently induce DE cells in monolayer cultures of mESCs. We obtained a good cell yield with 54.92% DE induction as shown by Foxa2, Sox17, Cxcr4 and E-Cadherin expression. These DE-cells could be further differentiated into posterior foregut and pancreatic phenotypes using a culture protocol initially developed for human embryonic stem cell (hESC) differentiation. In addition, this mESC-derived DE gave rise to hepatocyte-like cells after exposure to BMP and FGF ligands. Our data therefore indicate a substantial improvement of monolayer DE induction from mESCs and support the concept that differentiation conditions for mESC-derived DE are similar to those for hESCs. As mESCs are easier to maintain and manipulate in culture compared to hESCs, and considering the shorter duration of embryonic development in the mouse, this method of efficient DE induction on monolayer will promote the development of new differentiation protocols to obtain DE-derivatives, like pancreatic beta-cells, for future use in cell replacement therapies.
Cell Differentiation Unit, Diabetes Research Center, Vrije Universiteit Brussel, Belgium. Electronic address: jmfopouk@vub.ac.be.
Josué Kunjom
JK
Mfopou
Cell Differentiation Unit, Diabetes Research Center, Vrije Universiteit Brussel, Belgium.
Marloes
M
Geeraerts
Cell Differentiation Unit, Diabetes Research Center, Vrije Universiteit Brussel, Belgium.
Roba
R
Dejene
Cell Differentiation Unit, Diabetes Research Center, Vrije Universiteit Brussel, Belgium.
Stijn
S
Van Langenhoven
Cell Differentiation Unit, Diabetes Research Center, Vrije Universiteit Brussel, Belgium.
Asma
A
Aberkane
Liver Cell Biology, Department of Cell Biology, Vrije Universiteit Brussel, Belgium.
Leo A
LA
Van Grunsven
Cell Differentiation Unit, Diabetes Research Center, Vrije Universiteit Brussel, Belgium.
Luc
L
Bouwens
D028341:Activins / Q000494:pharmacology; D000818:Animals; D002352:Carrier Proteins / Q000494:pharmacology; D018929:Cell Culture Techniques; D002454:Cell Differentiation / Q000187:drug effects; D002478:Cells, Cultured; D053595:Embryonic Stem Cells / Q000166:cytology* / Q000187:drug effects / Q000378:metabolism; D004707:Endoderm / Q000166:cytology* / Q000378:metabolism; D006801:Humans; D007211:Indoles / Q000494:pharmacology; D018021:Lithium Chloride / Q000494:pharmacology; D051379:Mice; D016688:Mice, Inbred NOD; D016513:Mice, SCID; D008954:Models, Biological; D010091:Oximes / Q000494:pharmacology; D010179:Pancreas / Q000166:cytology / Q000378:metabolism; D033581:Stem Cell Transplantation; D060449:Wnt Signaling Pathway / Q000187:drug effects; D000099176:Noggin Protein
D028341:Activins; D002352:Carrier Proteins; D007211:Indoles; D018021:Lithium Chloride; D010091:Oximes; D000099176:Noggin Protein; C483321:6-bromoindirubin-3'-oxime; C422405:activin A
1-azakenpaullone; 6-bromo indirubin-3-oxine; AKP; ActA; BIO; CDM; CHIR; CHIR99021; DE; EBs; KOSR; NG; activin A; chemically defined medium; definitive endoderm; embryoid bodies; hESC; human embryonic stem cells; knockout serum replacement; mESC; mouse embryonic stem cells; noggin