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Induction and monitoring of definitive and visceral endoderm differentiation of mouse ES cells

Abstract

Preparation of specific lineages at high purities from embryonic stem (ES) cells requires both selective culture conditions and markers to guide and monitor the differentiation. In this study, we distinguished definitive and visceral endoderm by using a mouse ES cell line that bears the gfp and human IL2Rα (also known as CD25) marker genes in the goosecoid (Gsc) and Sox17 loci, respectively. This cell line allowed us to monitor the generation of Gsc+Sox17+ definitive endoderm and GscSox17+ visceral endoderm and to define culture conditions that differentially induce definitive and visceral endoderm. By comparing the gene expression profiles of definitive and visceral endoderm, we identified seven surface molecules that are expressed differentially in the two populations. One of the seven markers, Cxcr4, to which a monoclonal antibody is available allowed us to monitor and purify the Gsc+ population from genetically unmanipulated ES cells under the condition that selects definitive endoderm.

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Figure 1: Establishment of ES-GscgfpSox17huCD25 cell lines.
Figure 2: Two distinctive Sox17+ endoderm cells were differentially induced by the two distinct serum-free culture conditions.
Figure 3: Identification of cell surface markers for distinguishing definitive and visceral endoderm in ES cell differentiation.
Figure 4: Expression of Cxcr4 in vivo and coexpression of Cxcr4 with goosecoid (Gsc) during differentiation of mesendoderm in vitro.
Figure 5: Purification of definitive endoderm from genetically unmanipulated ES cells.

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Acknowledgements

We are grateful to Hitoshi Niwa for EB5 cells, to Yoshiakira Kanai for the in situ probe of Sox17, to Muneaki Miyata for his help in the cell sorting and to Yasuhiro Tosaka for obtaining qPCR data. This work was supported by grant (to N.S.) from the project for realization of regenerative medicine, grant (No.17045039 to E.T.) from the Ministry of Education, grant (to E.T. and M.Y.) from RIKEN research collaborations with industry program and grant (Kobe city collaboration of regional entities for advancement of technological excellence to M.Y.) from Japan Science and Technology Agency.

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Correspondence to Shin-Ichi Nishikawa.

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Supplementary information

Supplementary Fig. 1

Fate analysis of mesendoderm cells and two derivatives; derivative endoderm progenitors and mesoderm progenitors. (PDF 36 kb)

Supplementary Table 1

List of tenfold upregulated genes in derivative endoderm or visceral endoderm using Affymetrix MGU74v2 Gene Chips. (PDF 40 kb)

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Yasunaga, M., Tada, S., Torikai-Nishikawa, S. et al. Induction and monitoring of definitive and visceral endoderm differentiation of mouse ES cells. Nat Biotechnol 23, 1542–1550 (2005). https://doi.org/10.1038/nbt1167

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