Biochemical and Biophysical Research Communications
Roles of CDX2 and EOMES in human induced trophoblast progenitor cells
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.
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