Two distinct trophectoderm lineage stem cells from human pluripotent stem cells

Abstract

The trophectoderm layer of the blastocyst-stage embryo is the precursor for all trophoblast cells in the placenta. Human trophoblast stem (TS) cells have emerged as an attractive tool for studies on early trophoblast development. However, the use of TS cell models is constrained by the limited genetic diversity of existing TS cell lines and restrictions on using human fetal tissue or embryos needed to generate additional lines. Here we report the derivation of two distinct stem cell types of the trophectoderm lineage from human pluripotent stem cells. Analogous to villous cytotrophoblasts in vivo, the first is a CDX2- stem cell comparable with placenta-derived TS cells—they both exhibit identical expression of key markers, are maintained in culture and differentiate under similar conditions, and share high transcriptome similarity. The second is a CDX2+ stem cell with distinct cell culture requirements, and differences in gene expression and differentiation, relative to CDX2- stem cells. Derivation of TS cells from pluripotent stem cells will significantly enable construction of in vitro models for normal and pathological placental development. The trophectoderm layer of the blastocyst-stage embryo is the precursor for all trophoblast cells in the placenta. Human trophoblast stem (TS) cells have emerged as an attractive tool for studies on early trophoblast development. However, the use of TS cell models is constrained by the limited genetic diversity of existing TS cell lines and restrictions on using human fetal tissue or embryos needed to generate additional lines. Here we report the derivation of two distinct stem cell types of the trophectoderm lineage from human pluripotent stem cells. Analogous to villous cytotrophoblasts in vivo, the first is a CDX2- stem cell comparable with placenta-derived TS cells—they both exhibit identical expression of key markers, are maintained in culture and differentiate under similar conditions, and share high transcriptome similarity. The second is a CDX2+ stem cell with distinct cell culture requirements, and differences in gene expression and differentiation, relative to CDX2- stem cells. Derivation of TS cells from pluripotent stem cells will significantly enable construction of in vitro models for normal and pathological placental development. Specification of the trophectoderm and the inner cell mass is the first differentiation event during human embryonic development. The trophectoderm mediates blastocyst implantation in the uterus and is the precursor to all trophoblast cells in the placenta. Upon embryo implantation, the trophectoderm forms the cytotrophoblast (CTB), a putative stem cell that can differentiate to form the two major cell types in the placenta, the extravillous trophoblast (EVT) and the syncytiotrophoblast (STB) (1Bischof P. Irminger-Finger I. The human cytotrophoblastic cell, a mononuclear chameleon.Int. J. Biochem. Cel. Biol. 2005; 37: 1-16Crossref PubMed Scopus (125) Google Scholar, 2Benirschke Kurt. Baergen R.N. Burton G. Graham J. Pathology of the Human Placenta [electronic Resource]. Springer, Heidelberg2012Crossref Scopus (48) Google Scholar). The EVTs are involved in remodeling of uterine arteries, which is critical to ensure adequate perfusion of the placenta with maternal blood, whereas the multinucleated STB mediates the nutrient and gas exchange at the maternal–fetal interface (3Yabe S. Alexenko A.P. Amita M. Yang Y. Schust D.J. Sadovsky Y. Ezashi T. Roberts R.M. Comparison of syncytiotrophoblast generated from human embryonic stem cells and from term placentas.Proc. Natl. Acad. Sci. U. S. A. 2016; 113: E2598-E2607Crossref PubMed Scopus (68) Google Scholar, 4Moser G. Orendi K. Gauster M. Siwetz M. Helige C. Huppertz B. The art of identification of extravillous trophoblast.Placenta. 2011; 32: 197-199Crossref PubMed Scopus (29) Google Scholar). Abnormalities in trophoblast development are associated with pregnancy-related pathologies such as miscarriage, preeclampsia, and placenta accreta. Yet, despite its relevance to maternal and fetal health, constraints on research with human embryos and early fetal tissue impede mechanistic insight into early trophoblast development. Trophoblast stem (TS) cells derived from first-trimester human placental samples and blastocyst-stage embryos have emerged as an attractive in vitro model system for early human trophoblast (5Okae H. Toh H. Sato T. Hiura H. Takahashi S. Shirane K. Kabayama Y. Suyama M. Sasaki H. Arima T. Derivation of human trophoblast stem cells.Cell stem PubMed Scopus Google Scholar). However, of embryos and placental samples from early and genetic diversity of existing cell lines the use of human pluripotent stem cells are a for in vitro models of human early samples the is human pluripotent stem cells can models of normal and pathological trophoblast development Yang Y. A. Yang P. A. Y. G. Schust D.J. Ezashi T. Roberts R.M. in a model for human placental Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar). However, trophoblast can from a of R.M. Amita M. K. Alexenko A.P. Schust D.J. Ezashi T. of trophoblast cells from human embryonic stem or to PubMed Scopus Google Scholar). trophoblast derived from and in to studies have R.M. Ezashi T. Yang Y. Specification of trophoblast from embryonic stem cells to PubMed Scopus Google both placental samples and of trophoblast from exhibit and cell and cells derived from C. M. P. B. P. T. Derivation of trophoblast stem cells from human pluripotent stem PubMed Scopus Google Scholar, Y. I. J. H. Arima T. human embryonic stem cells can to cells with a transcriptome and PubMed Scopus Google Scholar, H. of trophoblast from human pluripotent stem cells in cell PubMed Scopus Google Scholar, M. S. T. J. J. J. of and human stem Biol. PubMed Scopus Google Scholar). we report the derivation and of two distinct trophectoderm lineage stem cell types from human embryonic stem cells and in culture The first is a CDX2- stem cell that is comparable with TS cells derived from placental samples and similar to the villous The second is a CDX2+ cell with distinct cell culture requirements, and differences in gene expression and differentiation, relative to CDX2- stem cells. the of stem cell a of placenta-derived TS cells with TS cells from and differentiation high and CDX2- TS cells. The derivation of TS cells from will for mechanistic studies on normal and pathological early trophoblast development. in studies on trophoblast differentiation of such as or that as for have in of B. I. J. H. K. H. of the by PubMed Scopus Google Scholar, K. T. T. PubMed Scopus Google Scholar). the in can a critical in of the trophectoderm in I. A. the trophectoderm lineage at the of PubMed Scopus Google Scholar, S. of the trophoblast lineage in with PubMed Scopus Google Scholar, S. H. Sato H. A. M. K. Sasaki H. is for of trophectoderm in PubMed Scopus Google as as human trophoblast development B. A. P. B. S. A. C. S. S. development by trophoblast in early human Natl. Acad. Sci. Scopus Google Scholar, G. S. J. C. S. A. M. of the in trophoblast of the human Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar). the use of in the of trophoblast differentiation of under culture conditions, by that P. A. the of human embryonic stem Biol. PubMed Scopus Google Scholar, P. A. of in human embryonic stem and in human placental 2016; PubMed Scopus Google Scholar). and in for in with by with and the conditions, we of the trophectoderm and the and of However, in associated with or differentiation that differentiation to and at expression of the and and expression of and The putative cells at for to differentiate to EVTs and using similar to P. A. the of human embryonic stem Biol. PubMed Scopus Google Scholar). of cells from cells a into with and expression of and the and cells in and with and in the of cells the STB and an of from the during differentiation to cells the of EVTs that and under identical differentiation to STB in the of as by of expression of and of the trophectoderm and of of and in cells of differentiation, of that similar to in studies culture P. A. the of human embryonic stem Biol. PubMed Scopus Google can by differentiation of in a of is for differentiation to trophoblast in culture of by in of M. M. Y. the and of human embryonic stem cells.Cell stem PubMed Scopus Google Scholar, of the by PubMed Scopus Google Scholar). associated and a critical in trophectoderm in the K. K. H. M. A. S. H. H. J. and to trophectoderm from inner cell Cel. PubMed Scopus Google Scholar, K. of and of in the Biol. PubMed Scopus Google Scholar). we the of and in trophoblast differentiation of The during differentiation of to cells and differentiation to and STB to the of conditions, expression in cells from expression and in a cells the expression of in cells derived from of STB as by the of expression of and the of in from we an cell that an or a C. M. S. Human pluripotent stem cell culture J. 2016; PubMed Scopus Google Scholar). differentiation to and as by of expression of the is that high cell and expression a in relative to the of the and whereas in relative to the that and are for differentiation of to that can to both EVTs and in culture both and K. T. T. PubMed Scopus Google Scholar, M. S. S. and its in Biol. PubMed Scopus Google Scholar). the of during differentiation to we with in as an for the an A. S. T. S. of the and to and Biol. PubMed Scopus Google Scholar). with similar cells expression of and and Upon differentiation as STB and and and and and that mediates the of during differentiation to trophoblast in K. T. T. PubMed Scopus Google Scholar, M. S. S. and its in Biol. PubMed Scopus Google have to G. B. of placental trophoblast differentiation a 2005; PubMed Scopus Google Scholar). for and in differentiation of and in cells for all and use of in expression of the and and of STB and and and However, we the use of the in expression of and high in at relative to the of multinucleated STB the or as with the the the and the of relative to the that mediates of during trophoblast differentiation of in culture that use of the in expression of markers, and both STB and EVTs the we the for cells by of with and in with the for and maintained under for culture of and placenta-derived TS cells (5Okae H. Toh H. Sato T. Hiura H. Takahashi S. Shirane K. Kabayama Y. Suyama M. Sasaki H. Arima T. Derivation of human trophoblast stem cells.Cell stem PubMed Scopus Google Scholar). Upon in trophoblast stem cell by (5Okae H. Toh H. Sato T. Hiura H. Takahashi S. Shirane K. Kabayama Y. Suyama M. Sasaki H. Arima T. Derivation of human trophoblast stem cells.Cell stem PubMed Scopus Google cells differentiation and a expression is significantly in as as of differentiation by and studies have differentiation of to cells with for M. Y. K. Y. Human pluripotent stem cells as a model of trophoblast differentiation in both normal development and Natl. Acad. Sci. 2016; 113: PubMed Scopus Google Scholar). we the use of a differentiation for cells of differentiation, and and However, by and expression of that all cells are CDX2+ at in to cells at use of a in a significantly of CDX2+ cells in the of in with the on the a of cells CDX2+ at using with significantly expression and with significantly expression in and of such as and in to in at whereas expression of of and gene associated with and gene expression in cells and with differentiation to associated with the for cell and cell differentiation in CDX2+ cells at into a major the the the and and are of for culture of TS is in and placenta-derived TS cells and the early placenta (5Okae H. Toh H. Sato T. Hiura H. Takahashi S. Shirane K. Kabayama Y. Suyama M. Sasaki H. Arima T. Derivation of human trophoblast stem cells.Cell stem PubMed Scopus Google Scholar). in maintained as for the of cells derived from and expression of the trophoblast and and I. A. the trophectoderm lineage at the of PubMed Scopus Google Scholar, S. H. Sato H. A. M. K. Sasaki H. is for of trophectoderm in PubMed Scopus Google Scholar, I. is a of and during early PubMed Scopus Google Scholar, P. B. S. S. B. A. M. M. B. of first cell lineage Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar, A. Sasaki H. P. G. P. J. trophoblast development of and in to PubMed Scopus Google Scholar, H. Y. Takahashi K. J. and trophectoderm 2005; PubMed Scopus Google Scholar). cells the and of expression associated with the trophectoderm and is placental are M. Y. K. Y. Human pluripotent stem cells as a model of trophoblast differentiation in both normal development and Natl. Acad. Sci. 2016; 113: PubMed Scopus Google Scholar, M. P. H. Burton an trophoblast stem cell in the human PubMed Scopus Google Scholar, P. I. K. P. P. the cell of the human blastocyst by PubMed Scopus Google Scholar, M. S. J. J. Human placenta and trophoblast and model Sci. PubMed Scopus Google Scholar). that cells are derived from and to cells from TS cells that cells are as cells. the differentiation of cells using as by for differentiation of TS cells to EVTs and STB (5Okae H. Toh H. Sato T. Hiura H. Takahashi S. Shirane K. Kabayama Y. Suyama M. Sasaki H. Arima T. Derivation of human trophoblast stem cells.Cell stem PubMed Scopus Google Scholar). to form STB that and However, cells form cells a Upon cells and expression cells maintained an with expression cells maintained in for TS cells (5Okae H. Toh H. Sato T. Hiura H. Takahashi S. Shirane K. Kabayama Y. Suyama M. Sasaki H. Arima T. Derivation of human trophoblast stem cells.Cell stem PubMed Scopus Google Scholar). cells in for into cells a in differentiation cell of the cells that of placenta-derived TS cells in that as a (5Okae H. Toh H. Sato T. Hiura H. Takahashi S. Shirane K. Kabayama Y. Suyama M. Sasaki H. Arima T. Derivation of human trophoblast stem cells.Cell stem PubMed Scopus Google Scholar). cells expression of and expression of in cells maintained as of differentiation into into in differentiation, a in similar in the as for cells to and both the cells into and placenta-derived TS cells. and into of differentiation or from high expression of and similar to cells in expression of and the and and The cells in exhibit a similar expression of trophoblast as placenta-derived TS cells and cells are as cells to that are derived from the differentiation of cells using the as by for differentiation of TS cells to EVTs and STB (5Okae H. Toh H. Sato T. Hiura H. Takahashi S. Shirane K. Kabayama Y. Suyama M. Sasaki H. Arima T. Derivation of human trophoblast stem cells.Cell stem PubMed Scopus Google Scholar). to placenta-derived TS cell cells into EVTs and and and and STB and and the expression of to and differentiation of cells similar to in the of placenta-derived TS cell and cells have maintained in for to differentiate into STB and EVTs culture in with differences in culture for differentiation and expression of the trophectoderm that and cells two distinct stem cell transcriptome on and placenta-derived TS cells using and placenta-derived TS cells are under identical conditions, a transcriptome two cell of that and TS cells in gene expression expression gene and placenta-derived TS cells and high transcriptome and placenta-derived TS cells. that exhibit expression in cells relative to placenta-derived TS cells differentiation expression in cells derived from the high transcriptome and placenta-derived TS cells. in with in expression and culture for and differentiation, that are to placenta-derived TS cells. that cells are a distinct cell that from cells and to the trophoblast lineage for of gene expression that significantly and significantly expression and significantly expression in cells and of and associated with and expression in and with differences in and associated with and in cells. with distinct for in cell and differences in differentiation under identical conditions, that and distinct stem cell expression of the and is in cells relative to cells that are to placenta-derived TS cells. the expression of associated with villous is in relative to the the human trophectoderm and we the transcriptome of and placenta-derived TS cells with the transcriptome of trophectoderm cells from human embryos S. B. S. A. S. lineage and in human 2016; PubMed Scopus Google Scholar). The to gene expression trophectoderm cells and or placenta-derived TS cells The similar for all TS cell types and cells and placenta-derived TS cells or and cells. The are to the differences cells in culture and human embryos and for transcriptome trophectoderm cells from human embryos using as to in studies are to cells are to cells of the human we on derivation of and cells from to we to and cells from the cells derived from maintained expression of and with a expression of in cells derived from and in and cells expression of expression of and in The of cells similar to that of placenta-derived TS cells of cells derived from using by (5Okae H. Toh H. Sato T. Hiura H. Takahashi S. Shirane K. Kabayama Y. Suyama M. Sasaki H. Arima T. 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A. and differentiation of human and pluripotent stem cells into embryonic and 2011; PubMed Scopus Google Scholar). studies have the differentiation of to the trophectoderm and report the derivation of TS cells from C. M. P. B. P. T. Derivation of trophoblast stem cells from human pluripotent stem PubMed Scopus Google Scholar, Y. I. J. H. Arima T. human embryonic stem cells can to cells with a transcriptome and PubMed Scopus Google Scholar). to TS cell lines in that is for of and placenta-derived TS cells in that differentiation to the trophectoderm lineage using a with that differentiate to cells of the G. S. Yang J. J. S. J. A. is in human Scopus Google Scholar). However, the differentiation in studies significantly from that in for cells from that by the a critical in differentiation to trophoblast in in blastocyst in S. 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Toh H. Sato T. Hiura H. Takahashi S. Shirane K. Kabayama Y. Suyama M. Sasaki H. Arima T. Derivation of human trophoblast stem cells.Cell stem PubMed Scopus Google trophectoderm cells of into expression of and of in and can differentiate to form EVTs and that TS cells derived from the trophectoderm in the blastocyst-stage embryo expression of (5Okae H. Toh H. Sato T. Hiura H. Takahashi S. Shirane K. Kabayama Y. Suyama M. Sasaki H. Arima T. Derivation of human trophoblast stem cells.Cell stem PubMed Scopus Google Scholar). the to to by in the trophectoderm forms a STB and with the cells form cells under the differentiation for differentiation of and placenta-derived TS cells. the cells a cell to the human trophectoderm or a distinct cell from differences in cell culture are similar to the human we transcriptome for and placenta-derived TS with for trophectoderm cells from human embryos S. B. S. A. S. lineage and in human 2016; PubMed Scopus Google Scholar). However, in expression TS cell types in culture and trophectoderm is to differences cells in culture and human embryos and for transcriptome TS cell lines and TS cell lines derived from human embryonic stem cells in differentiation Y. I. J. H. Arima T. human embryonic stem cells can to cells with a transcriptome and PubMed Scopus Google Scholar). studies are needed to cells are similar to cells of the human trophectoderm a trophoblast cell cells. maintained in are first for with the and the to CDX2+ cells. CDX2+ cells are in to we differentiation of cells into relative to and cells. the in of to differentiation and derivation of cells. 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