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First published online 1 September 2005
doi: 10.1242/dev.02005

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Characterization of mesendoderm: a diverging point of the definitive endoderm and mesoderm in embryonic stem cell differentiation culture

¹ Laboratory for Stem Cell Biology, RIKEN Center for Developmental Biology, 2-2-3 Minatojima-minamimachi, Chuo-ku, Kobe, 650-0047, Japan
² Department of Gastroenterology and Hepatology, Graduate school of Medicine, Kyoto University, 54 Shogoinkawara-cho, Sakyo-ku, Kyoto, 606-8507, Japan
³ Department of Bioinformatic Engineering, Graduate School of Information Science and Technology, Osaka University, 2-1 Yamada-oka, Suita City, Osaka, 565-0871, Japan
⁴ Laboratory for Animal Resources and Genetic Engineering, RIKEN Center for Developmental Biology, 2-2-3 Minatojima-minamimachi, Chuo-ku, Kobe, 650-0047, Japan

View larger version (52K): [in a new window]   Fig. 1. Induction and monitoring of Gsc expression. (A) Our strategy for inserting GFP gene into Gsc allele. Southern blots demonstrate correct insertion of Gsc-gfp transgene and removal of Neo cassette. (B-F) Gsc induction under various culture conditions. Cultures were basically performed on collagen IV-coated dishes, except embryoid body (EB) formation experiments (right panels in B, part i and F). Gscgfp/+ES cells were cultured for 4 days (D4) or 6 days (D6). (B, part i) Day 4 FACS analysis of cells cultured in serum containing medium (SCM). (B, part ii) RT-PCR expression analysis of GFP+ and GFP- population induced in SCM for 4 days. The specific molecules for the organizer were only expressed in GFP+ population. (B, part iii) GFP expression in tetraploid embryo. GFP expression was strongly detected in the organizer region of mid-streak stage embryo. (C-F) SF-O3 serum-free media (SFM) were used in Gsc induction. (C, part i) Selective induction of Gsc-GFP. Ninety-three percent of cells expressed Gsc-GFP on day 6 in the monolayer culture with activin. (C, part ii) RT-PCR analysis of GFP+ and GFP- population on day 4. (C, part iii) Expression pattern of visceral endoderm markers. Upper panel shows RT-PCR analysis of total cells differentiated in SFM with activin for 4 days. Lower panel shows that of EBs differentiated in SCM for 4 days. (C, part iv) Immunostaining showing that almost all of cells on day 6 in SFM plus activin expressed brachyury protein. (D) Effect of Nodal on the generation of Gsc+ cells. Nodal showed a similar activity to induce Gsc+ cells. (E,F) Bmp4 and serum inhibited the generation of Gsc+ cells even in the presence of a large amount of activin. The culture with EB formation is less efficient for the generation of Gsc+ cells even in SFM (F), than the monolayer culture (C). The units displayed for activin (Act), nodal and Bmp4 are ng/ml.

View larger version (36K): [in a new window]   Fig. 2. Differentiation course of Gsc+ mesendoderm cells analyzed by FACS. (A) Expression of ECD and GFP from day 4 to day 6. The earliest GFP+ cells were detected on day 3.5 and co-expressed ECD (data not shown). On day 4, more than 90% of GFP+ cells are ECD+. Two major populations of GFP+ cells, ECD+ and ECD-, were observed from day 5. (B) Expression pattern of ECD and R of GFP+ cells. The two molecules are co-expressed in the day 4 GFP+ cells but segregate into ECD+R- and ECD-R+ cells on day 6. (C) The FACS pattern of ECD, Vegfr2 and ßR of GFP+ cells on day 5. Vegfr2 and ßR are expressed in the ECD- population exclusively. (D) Cell sorting and re-culture experiments of Gsc+ cells. ECD+ and ECDlow populations in day 4 GFP+ cells were purified and re-cultured for 1 day in SFM containing activin. ECDhigh population could give rise to both ECD+ and ECD- cells.

View larger version (59K): [in a new window]   Fig. 3. Differentiation of mesendoderm cells into definitive endoderm and mesoderm lineages. (A) RT-PCR analysis of gene expression in GFP+ECD+ and GFP+ECD- cells on day 5. (B) Multicolour immunofluorescent staining of cells in day 5 cultures. Gsc+ECD+Foxa2+ cells are within the epithelial sheet, whereas Gsc+ECD-Foxa2- cells disperse around the sheet. (C) RT-PCR analysis of gene expression in differentiated Gsc+ECD+ cells. Gsc+ECD+ cells were purified from cultures with activin on day 6 and re-cultured in SCM. Expression level of Gsc, Mixl1 and Afp changes during culture. Gsc is positive only in the day 5 culture (A), Mixl1 is in the day 5 (A) and 12 cultures, and AFP is only in the day 12 culture. (D) Albumin-producing cells are generated from Gsc+ECD+ population. (i) Gsc+ cells were induced in SFM containing activin. On day 6, Gsc+ECD+ cells were purified by cell-sorter and re-cultured for 3 days on collagen I-coated dish in the presence of Egf, Bmp4, acid Fgf and basic Fgf. (ii) The expression of albumin in the Gsc+ECD+-derived cells detected by immunostaining. (iii) The expression of albumin in the Gsc+ECD+-derived cells detected by RT-PCR. Hnf6, a marker of precursor of liver and pancreatic cells, was stimulated to be expressed.

View larger version (88K): [in a new window]   Fig. 4. Differentiation potential of Gsc+ mesendoderm cells. (A) Fate of Gsc+ECD- cells. Potential of Gsc+ECD- cells to give rise to osteocytes (left, Alizarin Red staining) and endothelial cells (right). Osteoblastic markers such as Bglap1, Bglap2 and osteopontin (Opn) are expressed in the case of osteogenic induction. (Right) Endothelial colonies were visualized by immunostaining of Vecad and Pecam1, which are specific markers for endothelial cells. Arrowheads indicate colonies of endothelial cells. (B) Multicolour immunofluorescent images of day 6.5 culture. There are three different types of cells in this picture. Gsc-ECD+Foxa2+ mature endoderm cells are larger than Gsc+ECD+Foxa2+ cells, but both form a continuous epithelial sheet. Gsc+ECD-Foxa2- cells outside the epithelial sheet correspond to mesoderm cells. (C) Fate analysis of day 4 Gsc+ECD+ cells at single cell level. Two-colour immunostaining of wells containing both Foxa2+ (dark purple, arrowheads) epithelial cells and ßR+/Vegfr2+ (red, arrows) mesoderm cells. All Foxa2+ cells form a single cluster, whereas all ßR+/Vegfr2+ cells are dispersed.

View larger version (97K): [in a new window]   Fig. 5. Characterization of an ES cell-derived endoderm cell line. (A) Sustained growth of endoderm cell line under serum-containing condition. The Gsc+ECD+ population was isolated by FACS and re-cultured in SCM. Cells were passaged every 3 days and cell number was measured at each passage day. (B) Morphology of a endoderm cell line after ten passages. (C) Multicolour immunofluorescent staining of the endoderm cell line after ten passages. Homogenous epithelial cells co-expressing ECD, Gata4, Foxa2 and cytokeratin 18 are present. (D) Stable expression of endoderm markers in the cell line. The expression pattern of this cell line is almost identical to that of endoderm cells on day 26 in Fig. 3D.

View larger version (54K): [in a new window]   Fig. 6. Isolation of mesendoderm cells and endoderm cells from genetically unmanipulated ES cells. (A) Purification of ECD+R+ mesendoderm cells. EB5 unmodified ES cells were cultured in SFM containing activin. On day 4, ECD+R+ cells were purified by FACS and re-cultured for 2 days. Both ECD- and ECD+ populations (labelled A and B, respectively) derived from day 4 ECD+R+ cells were sorted again by FACS for further analyses. (B) The expression of mesoderm and endoderm markers in ECD- and ECD+ populations (labelled A and B, respectively). A set of endoderm markers such as Foxa2 and Sox17 was exclusively expressed in B, whereas the expression of mesoderm markers such as ßR and Vegfr2 was detected only in A. (C) The morphology of endoderm cell line derived from wild-type ES cells. Epithelial-like morphology with clear cell-cell boundary is present. This morphology is similar to endoderm cell lines generated from Gsc+ECD+ cells (Fig. 4B).

View larger version (24K): [in a new window]   Fig. 7. The differentiation processes from ES cells to mesendoderm cells. ES cells differentiate selectively into the Gsc+ECD+R+ intermediates in serum-free media containing activin. In vitro clonal analysis indicates that this population contains mesendoderm. Gsc+ECD+R+ intermediates diverge to Gsc+ECD+R- and Gsc+ECD-R+ cells, which further differentiate to endoderm and mesoderm cells, respectively. With this highly selective culture condition and surface markers, it is possible to obtain mesendoderm and their progenies without using Gsc as a marker.