Roles of CDX2 and EOMES in human induced trophoblast progenitor cells

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Abstract

Abnormal trophoblast lineage proliferation and differentiation in early pregnancy have been associated with the pathogenesis of placenta diseases of pregnancy. However, there is still a gap in understanding the molecular mechanisms of early placental development due to the limited primary trophoblast cultures and fidelity of immortalized trophoblast lines. Trophoblasts stem (TS) cells, an in vitro model of trophectoderm that can differentiate into syncytiotrophoblasts and extravillous trophoblasts, can be an attractive tool for early pregnancy research. TS cells are well established in mouse but not in humans due to insufficient knowledge of which trophoblast lineage-specific transcription factors are involved in human trophectoderm (TE) proliferation and differentiation. Here, we applied induced pluripotent stem cell technique to investigate the human trophoblast lineage-specific transcription factors. We established human induced trophoblast progenitor (iTP) cells by direct reprogramming the fibroblasts with a pool of mouse trophoblast lineage-specific transcription factors consisting of CDX2, EOMES, and ELF5. The human iTP cells exhibit epithelial morphology and can be maintained in vitro for more than 2 months. Gene expression profile of these cells was tightly clustered with human trophectoderm but not with human neuron progenitor cells, mesenchymal stem cells, or endoderm cells. These cells are capable of differentiating into cells with an invasive capacity, suggesting extravillous trophoblasts. They also form multi-nucleated cells which secrete human chorionic gonadotropin and estradiol, consistent with a syncytiotrophoblast phenotype. Our results provide the evidence that transcription factors CDX2 and EOMES may play critical roles in human iTP cell generation.

Highlights

CDX2 and EOMES play critical roles in human induced trophoblast progenitors (iTP). ► iTP cells directly transformed from fibroblasts. ► Differentiation of iTP cells into extravillous trophoblasts and syncytiotrophoblasts.

Introduction

It is well accepted that aberrant trophoblast proliferation and differentiation are two of the major causes of placenta-associated diseases, but the pathogenesis of these diseases are still largely unknown. The molecular mechanisms of human trophoblast lineage proliferation and differentiation are difficult to study due to the existing ethical (use of human embryos) and practical (use of <6-week placenta) issues. Trophoblast stem (TS) cells, which represent trophectoderm (TE) in vivo, can be a useful tool for the study of trophoblast lineage proliferation and differentiation in vitro [1]. However, unlike mouse TS cells which are well established and extensively studied, established human TS cell line does not exist. Numerous studies have been attempted to use human embryonic stem (ES) cells or 1st trimester placenta (8–12 week) to generate human TS cells [2], [3], [4], [5], [6], [7], [8], [9], [10]. Other studies have focused on analyzing transcriptomes between human inner cell mass (ICM) and TE or differentiation of human ES cells into trophoblasts over time in order to identify the transcription factors involved in human trophoblast lineage commitment and differentiation [11], [12], [13], [14], [15], [16]. It has been shown that mouse TS cells and human TE share similar lineage transcription factors. However, applying similar culture conditions which are effective in mouse ES cells/blastocysts differentiation into TS cells are ineffective for human ES cells, indicating the existence of different transcription factor loops/pathways between humans and mice. Thus, there is an urgent need to identify human trophoblast lineage-specific transcription factors and generate viable human TS cell lines to advance reproductive research.

Induced pluripotent stem (iPS) cell technique is the direct reprogramming of fibroblasts into various cell types via transduction with different groups of lineage-specific transcription factors [17]. iPS technique shows promise in clinical applications; for example, dopaminergic neurons, cardiac cells, and hematopoietic cells have been successfully generated directly from fibroblasts using this technique [18], [19], [20]. iPS technique has also been proven to be a useful tool to investigate the biofunction of transcription factors; over-expression of POU5F1 in mouse TS cells can lead to generation of mouse ES cells, suggesting POU5F1 as a critical transcription factor in ES cells [21]. A similar study identified three transcription factors as a group of critical loop for induction of human cardiomycytes [19]. Therefore, it is rational to use this strategy to examine the transcription factors required for establishing human trophoblast cells directly from the fibroblasts. In this study, we transduced the well-documented mouse trophoblast lineage-specific transcription factors: caudal-type homeobox transcription factor 2 (CDX2) [22], [23], [24], eomesodermin (EOMES) [25], [26], and E74-like factor 5 (ELF5) [27], [28], [29], which are known to maintain mouse TS cells pluripotency and lineage specificity as a loop. CDX2, EOMES, and ELF5 are also found expressed in human 1st trimester placental trophoblast [2], [10]. Additionally, we included two oncogenes: MYC avian myelocytomatosis viral oncogene homolog c (C-MYC) and Kruppel-like factor 4 (KLF4), which are essential for cell proliferation and transformation [17]. The iTP cells generated in this study represent a useful tool for the study human trophoblast lineage-specific transcription factor biofunction.

Section snippets

Generation of human induced trophoblast progenitor (iTP) cells

Human lenti-virus constructs for CDX2, ELF5, C-MYC, KLF4 (Open Biosystems) and EOMES (home-made) were used to generate iTP cells from human fetal fibroblasts (IMR90, ATCC). 1.25 × 105 fibroblasts were transducted for 24 h in a mixture of 5 viral genes with fibroblast medium: DMEM supplemented with 10% fetal bovine serum(FBS), 1% l-glutamine, 1% non-essential amino acids (NEAA), 1% penicillin, and 1% streptomycin. After 96 h, cells were passaged onto in-activated CF-1 mouse embryonic fibroblast

Characterization of human iTP cell lines

Human iTP cell lines were successfully established from human fetal fibroblasts (IMR90) by lenti-viral transduction with a pool of transcription factors (CDX2, EOMES, ELF5, KLF4, and c-MYC) (Fig. 1A and B). Four iTP cell lines showed similar growth and morphological characteristics and two (iTP1 and iTP2) were further characterized in detail. 25–30 days after initial transduction, the donor fibroblasts were reprogrammed into iTP cells which formed distinct colonies composed of small round cells

Discussion

In this study, we generated human iTP cells to elucidate the trophoblast lineage-specific transcription factors required for human trophoblast lineage proliferation and differentiation. Here, we used 3 transcription factors (CDX2, EOMES, and ELF5) which were previously shown to form a transcription loop in mouse trophoblast lineage [32], [33]. CDX2 is critical for generating mouse TS cells; mouse ES cells could be transformed into TS cells solely by over-expressing CDX2 [23], [34] whereas CDX2

Acknowledgment

The authors would like to thank Dr. Trixie Smith and Susan Ferguson for critically reading the manuscript and providing constructive criticism.

References (44)

  • M. Hemberger, R. Udayashankar, P. Tesar, H. Moore, G.J. Burton, ELF5-enforced transcriptional networks define an...
  • R. Harun et al.

    Cytotrophoblast stem cell lines derived from human embryonic stem cells and their capacity to mimic invasive implantation events

    Hum. Reprod.

    (2006)
  • R.H. Xu et al.

    BMP4 initiates human embryonic stem cell differentiation to trophoblast

    Nat. Biotechnol.

    (2002)
  • M.M. Matin et al.

    Specific knockdown of Oct4 and beta2-microglobulin expression by RNA interference in human embryonic stem cells and embryonic carcinoma cells

    Stem Cells

    (2004)
  • B. Gerami-Naini et al.

    Trophoblast differentiation in embryoid bodies derived from human embryonic stem cells

    Endocrinology

    (2004)
  • T. Ezashi et al.

    Low O2 tensions and the prevention of differentiation of hES cells

    Proc. Natl. Acad. Sci. USA

    (2005)
  • T. Ezashi, B.P. Telugu, R.M. Roberts, Model systems for studying trophoblast differentiation from human pluripotent...
  • O. Genbacev, M. Donne, M. Kapidzic, M. Gormley, J. Lamb, J. Gilmore, N. Larocque, G. Goldfien, T. Zdravkovic, M.T....
  • Q. Bai, S. Assou, D. Haouzi, J.M. Ramirez, C. Monzo, F. Becker, S. Gerbal-Chaloin, S. Hamamah, J. De Vos, Dissecting...
  • V. Chickarmane et al.

    A computational model for understanding stem cell, trophectoderm and endoderm lineage determination

    PLoS One

    (2008)
  • M. Marchand, J.A. Horcajadas, F.J. Esteban, S.L. McElroy, S.J. Fisher, L.C. Giudice, Transcriptomic signature of...
  • J. Adjaye et al.

    Primary differentiation in the human blastocyst: comparative molecular portraits of inner cell mass and trophectoderm cells

    Stem Cells

    (2005)
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