First published online 3 March 2004
doi: 10.1242/dev.01044
Development 131, 1651-1662 (2004)
Published by The Company of Biologists 2004
Development of definitive endoderm from embryonic stem cells in culture
Atsushi Kubo1,*,
Katsunori Shinozaki1,
John M. Shannon2,
Valerie Kouskoff1,
,
Marion Kennedy1,
Savio Woo1,
Hans Joerg Fehling3 and
Gordon Keller1,
1 The Carl C. Icahn Center for Gene Therapy and Molecular Medicine, Mount Sinai
School of Medicine, New York, NY 10029, USA
2 Division of Pulmonary Biology, Children's Hospital Medical Center, Cincinnati,
OH 45229, USA
3 Department of Immunology, Medical Faculty/University Clinics, Ulm,
Germany
* Present address: Department of Public Health, Nara Medical University, Nara
634-8521, Japan
Present address: Paterson Institute for Cancer Research, Christie Hospital NHS
Trust, Wilmslow Road, Manchester M20 4BX, UK
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Fig. 1. Effects of serum on endoderm development and hepatocyte differentiation.
EBs were differentiated either in serum for the entire six-day period (serum)
or initiated in serum for 2.5 days and then passaged to serum-free cultures
for the remaining 3.5 days (SF). (A) RT-PCR expression analysis of different
aged EBs. (B) FACS analysis of GFP-Bry expression in EBs differentiated in
serum-containing (serum) or serum-containing followed by serum-free media
(serum/SF). (C) Hematopoietic progenitor analysis of EBs generated under
different conditions. Numbers represent colonies per 1x105
cells plated. Data represents mean±s.e.m. (n=3). Ep, primitive
erythroid colonies; Mac, macrophage colonies; Mix, multilineage colonies. (D)
RT-PCR analysis of replated cultures from day 10 EBs generated in serum/SF
(S/SF) cultures. EB were replated for 4 days on matrigel with dexamethasone
(10–7 M) in the presence of serum. S, replated cells from day
10 EBs differentiated in the presence of serum for the entire time; FL, day 14
fetal liver; AL, adult liver.
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Fig. 2. Endoderm potential of GFP-Bry+ cells. (A) FACS profile of day
2.5 EBs and reaggregation and culture protocol for differentiation of the
sorted cells. (B) RT-PCR expression analysis of pre-sorted (p),
GFP-Bry– and GFP-Bry+ cells. d2.5, cells analyzed
immediately after isolation by sorting; d6.0, reaggregated EBs; d10, cells
from replated EBs. (C) Immunostaining of GFP-Bry+ and
GFP-Bry– cells. Upper panels shows day 2.5 EB sorted cells
stained with an antibody to Foxa2. Positive cells are red/pink in color.
Nuclei of all cells are stained with DAPI (blue). Bottom panels show cells
from 10-day old cultures stained with an antibody to albumin. Positive cells
are indicated by red color. (D) RT-PCR expression analysis of genes associated
with liver maturation in populations derived from GFP-Bry+ and
GFP-Bry– day 2.5 EB cells. Cells from the pre-sorted
population as well as those from the GFP-Bry+ and
GFP-Bry– fractions were reaggregated and cultured in SF
conditions for 8 days. At this stage, the reaggregated EBs were replated into
serum hepatocyte conditions for 4 days, harvested and analyzed.
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Fig. 3. Effects of activin A on GFP-Bry expression and endoderm induction in EBs.
(A) Kinetics of GFP-Bry expression in EBs differentiated in SF cultures in the
presence (+activin, 100 ng/ml; open circles) or absence (–activin;
closed squares) of activin. (B) Temporal analysis of gene expression in EBs
differentiated in the presence (100 ng/ml) or absence of activin in SF
cultures. C, controls; top six lanes, day 3 serum-stimulated EBs; lower three
lanes, day 6 serum-stimulated EBs. (C) FACS analysis demonstrating the effect
of different activin concentrations on GFP-Bry expression in day 6 EBs. (D)
RT-PCR analysis demonstrating the effect of different activin concentrations
on gene expression profiles in day 7 EBs. (E) Immunostaining demonstrating the
presence of Foxa2 protein in day 6 EBs differentiated in the absence (0 ng/ml)
or presence (3 ng/ml or 100 ng/ml) of activin. Pink color indicates
Foxa2-positive cells.
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Fig. 5. Analysis of kidney capsule grafts of activin-stimulated GFP-Bry+
and GFP-Bry– EB-derived populations. GFP-Bry+ and
GFP-Bry– cells isolated from day 5 EBs differentiated in the
presence of 3 or 100 ng/ml activin were reaggregated and cultured as EBs for
an additional 8 days in SF cultures in the absence of factor. The reaggregated
EBs were replated in hepatocyte conditions for 4 days and then harvested and
transplanted under the kidney capsule of SCID-beige mice. Three weeks
following transplantation, the mice were sacrificed and the kidneys harvested
for analysis. (A) Photograph showing the size of the grafts from the
GFP-Bry+ and GFP-Bry– populations. Size is
indicted by the ruler (mm) at the bottom of the figure. (B) Histological
analysis of grafts from the GFP-Bry+ and GFP-Bry–
populations. SM, skeletal muscle; Gu, gut epithelial-like structure, B, bone;
C, columnar cells; Br, neural tissue; NT, neural tube-like structure.
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Fig. 6. Analysis of endoderm derivatives in kidney grafts of GFP-Bry+
populations. (A,B) Immunohistochemistry showing expression of Foxa2 protein in
tubular structures present in the grafts. (C,D) Immunohistochemistry
demonstrating expression of intestinal fatty acid binding protein (Ifabp) in
the gut-like structures present in grafts. (E,F) In situ hybridization
indicating expression of surfactant protein C (Sftpc) in the grafts: (E)
Bright-field exposure, arrow indicates positive area, (F) dark-field exposure.
(G) Hematoxylin and Eosin, and (H) D-PAS staining of consecutive sections
demonstrating muscin (M) in the gut epithelial-like structure in the grafts.
(I) Skeletal muscle (SM) in a graft from GFP-Bry+ cells induced
with 3 ng/ml of activin. K, kidney of recipient.
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© The Company of Biologists Ltd 2004
-actinin in skeletal
muscle outgrowths generated from EBs differentiated in the presence of 3 ng/ml
activin. EBs were generated as in section (B) above. At day 10, EBs were
plated on gelatin-coated coverslips, cultured for 4 days and then stained with
antibodies to skeletal myosin and