Lineage Conversion of Murine Extraembryonic Trophoblast Stem Cells to Pluripotent Stem Cells

Abstract

In mammals, the first cell fate decision is initialized by cell polarization at the 8-to 16-cell stage of the preimplantation embryo. At this stage, outside cells adopt a trophectoderm (TE) fate, whereas the inside cell population gives rise to the inner cell mass (ICM). Prior to implantation, transcriptional interaction networks and epigenetic modifications divide the extraembryonic and embryonic fate irrevocably. Here, we report that extraembryonic trophoblast stem cell (TSC) lines are converted to induced pluripotent stem cells (TSC-iPSCs) by overexpressing Oct4, Sox2, Klf4, and cMyc. Methylation studies and gene array analyses indicated that TSC-iPSCs had adopted a pluripotent potential. The rate of conversion was lower than those of somatic reprogramming experiments, probably due to the unique genetic network controlling extraembryonic lineage fixation. Both in vitro and in vivo, TSC-iPSCs differentiated into tissues representing all three embryonic germ layers, indicating that somatic cell fate could be induced. Finally, TSC-iPSCs chimerized the embryo proper and contributed to the germ line of mice, indicating that these cells had acquired full somatic differentiation potential. These results lead to a better understanding of the molecular processes that govern the first lineage decision in mammals.Life starts with a single totipotent cell, followed by several cleavages and the specification of the earliest cell lineages at the blastocyst stage. The trophectoderm (TE) represents the extraembryonic trophoblast lineage and envelops the inner cell mass (ICM), which gives rise to the embryonic tissues and the primitive endoderm. Embryonic stem cells (ESCs) derived from the ICM are pluripotent because they differentiate into all embryonic tissues, including the germ line, but have lost the ability to form trophoblast derivatives (10). Likewise, trophoblast stem cells (TSCs) derived from the TE layer exclusively recapitulate the developmental potency of their parental lineage and contribute to all trophoblast cell types of the conceptus (40, 50).The first cell fate decision is initiated by cell polarization at the 8-to 16-cell stage, where inner and outer cells are differentiated from each other. Outer cells adopt the trophoblast fate, whereas the inner cell population gives rise to the ICM. Differentiation is established by the expression of lineage-specific transcription networks and fixed by epigenetic asymmetries at the late blastocyst stage (4,42,43,54).The caudal-related homeobox 2 factor, Cdx2, is regarded as one key transcription factor for TE specification and maintenance. Cdx2 is expressed in the outer blastomeres and later in TE (7, 32). Cdx2-null embryos lose trophectoderm integrity, and TSCs cannot be derived (48). The ectopic expression of Cdx2 in ESCs leads to lineage conversion to the extraembryonic trophoblast fate by the direct downregulation of pluripotency-associated transcription factors, such as Oct4 and Nanog, and the activation of the trophectodermal transcription network (5, 35). Oct4 (...

Trophoblast development

“…Furthermore, overexpression of Pou5f1 in newly derived TS cells (ten passages) can drive these into the pluripotent ES (ICM-like) state (Wu et al 2011). Even well-established TS cells can be reverted to ES cells, though requiring a cocktail of pluripotency genes including Pou5f1 (Kuckenberg et al 2011). It follows that shutdown of Pou5f1 (and Nanog) activity and thereby of the pluripotency network, while not necessary for the initial specification of the TE network , is required for the TE network to achieve stability.…”

Transcriptional Reprogramming and Chromatin Remodeling Accompanies Oct4 and Nanog Silencing in Mouse Trophoblast Lineage

“…Studies in embryonic stem (ES) cells showed that forced expression of Cdx2 or ablation of Oct4 induces differentiation toward a TE cell fate via CDX2-OCT4 and CDX2-Oct4 enhancer interactions [6,8]. Alternatively, in trophoblast stem (TS) cells, forced expression of Oct4 alone or in combination with other reprogramming factors promotes an ES cell fate through suppression of Cdx2 and other TS cell regulators [10,11].…”

Cdx2Efficiently Induces Trophoblast Stem-Like Cells in Naïve, but Not Primed, Pluripotent Stem Cells

“…Importantly, overexpression of Cdx2 in ESCs leads to TSC-like cells with highly similar morphology, developmental potential, and gene expression as embryoderived TSCs [16,22,23]. Similarly, TSCs can be converted to ESC-like iPSC by overexpressing Oct4 [24,25]. Likewise, ESCs can be converted to XEN cells using growth factors or PE transcription factors [12,[26][27][28][29].…”