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Review
. 2012 Sep;349(3):809-24.
doi: 10.1007/s00441-012-1371-2. Epub 2012 Mar 17.

Model systems for studying trophoblast differentiation from human pluripotent stem cells

Toshihiko Ezashi  1 , Bhanu Prakash V L Telugu, R Michael Roberts
Affiliations
Review

Model systems for studying trophoblast differentiation from human pluripotent stem cells

Toshihiko Ezashi et al. Cell Tissue Res. 2012 Sep.

Abstract

This review focuses on a now well-established model for generating cells of the trophoblast (TB) lineage by treating human embryonic stem cells (ESC) and induced pluripotent stem cells (iPSC) with the growth factor BMP4. We first discuss the opposing roles of FGF2 and BMP4 in directing TB formation and the need to exclude the former from the growth medium to minimize the co-induction of mesoderm and endoderm. Under these conditions, there is up-regulation of several transcription factors implicated in TB lineage emergence within 3 h of BMP4 exposure and, over a period of days and especially under a high O(2) gas atmosphere, gradual appearance of cell types carrying markers for more differentiated TB cell types, including extravillous TB and syncytioTB. We describe the potential value of including low molecular weight pharmaceutical agents that block activin A (INHBA) and FGF2 signaling to support BMP4-directed differentiation. We contend that the weight of available evidence supports the contention that BMP4 converts human ESC and iPSC of the so-called epiblast type unidirectionally to TB. We also consider the argument that BMP4 treatment of human ESC in the absence of exogenous FGF2 leads only to the emergence of mesoderm derivatives to be seriously flawed. Instead, we propose that, when signaling networks supporting pluripotency ESC or iPSC become unsustainable and when specification towards extra-embryonic mesoderm and endoderm are rendered inoperative, TB emerges as a major default state to pluripotency.

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Figures

Fig. 1
Fig. 1
Representation of the human placenta during the process of uterine invasion. After initial penetration into maternal tissues by cells derived from blastocyst trophectoderm, which are believed to form a short-lived syncytium, underlying cytoTBs push through the syncytial mass and form chorionic villi (CV). Early CV form multilayered structures that ultimately give rise to anchoring and “floating” (ones not immediately connected to maternal endometrial tissues) CV. Here, for simplification, only anchoring villi are shown. a The early stages of placental development take place in a relatively hypoxic environment that favors cytoTB proliferation rather than differentiation along the invasive pathway. Accordingly, this cell population (light green cells) rapidly increases in number as compared with the embryonic lineages. CytoTB also differentiate into syncytioTB, which lines the interface between the placental villous and the intervillous space. This space ultimately becomes filled with maternal blood. b As development continues, cytoTBs (dark green cells) invade the uterine wall and plug the maternal vessels, a process that helps maintain a state of physiological hypoxia. As indicated by the short black arrows, cytoTBs migrate farther up arteries than veins. c By 10–12 weeks of human pregnancy, blood flow to the intervillous space begins. As the endovascular component of cytoTB invasion progresses, the cells migrate along the lumina of spiral arterioles, replacing the maternal endothelial lining. CytoTBs are also found in the smooth muscle walls of these vessels. In normal pregnancy, the process whereby placental cells remodel uterine arterioles involves the decidual and inner third of the myometrial portions of these vessels. As a result, the diameter of the arterioles expands to accommodate the dramatic increase in blood flow that is needed to support rapid fetal growth later in pregnancy. It is likely that failed endovascular invasion leads, in some cases, to abortion, whereas an inability to invade to the appropriate depth is associated with preeclampsia and a subset of pregnancies in which the growth of the fetus is restricted. The figure is reproduced from Red-Horse et al. (2004)
Fig. 2
Fig. 2
Relative transcript concentrations of several genes for growth factors and their cognate receptors whose expression could influence differentiation events in BMP4-treated H1 cells. Data (GEO GSE10469) were obtained by microarray over time and in response to O2. Values were normalized to the median intensity of the array. H1 hESC were supplemented with BMP4 (10 ng/ml) under either 4% (blue) or 20% (red) O2 conditions. RNA isolated at 0, 3, 12, 24, 72 and 120 h (Schulz et al. 2008). These data were obtained on Agilent Whole Human Genome Oligo microarrays. The value represents a normalized expression (1.0 being the signal at the 50th percentile). For details of methods, see Schulz et al. (2008). Values shown are for a (FGF2), b (FGF4), c (FGFR2), d (BMP4), e (BMP7), f [BMPR1A (ALK3)], g (INHBA) (gene encoding activin A subunits), h [ACVR1 (ALK2)] and i [TGFBR1 (ALK5)]. Note that the cells may continue to have the potential to respond to endogenously produced FGF2 and activin A even when exogenous sources are absent
Fig. 3
Fig. 3
Relative transcript concentrations of genes encoding transcription factors that have been implicated in TB lineage emergence (Roberts and Fisher 2011). Values are shown for a (CDX2), b (EOMES), c (ETS2), d (GATA3), e (TFAP2C) and f (ELF5). The data were obtained as described in the legend to Fig. 2 from cells maintained under either 4% (blue) or 20% (red) O2 conditions. RNA was isolated at 0, 3, 12, 24, 72 and 120 h after BMP4 exposure (GEO GSE10469)
Fig. 4
Fig. 4
Relative transcript concentrations of genes encoding mesoderm-associated markers: a (T), b (KDR), c (VCAM1) and d (TBX4) (Bernardo et al. 2011). The data were obtained as described in the legend to Fig. 2 from cells maintained under either 4% (blue) or 20% (red) O2 conditions. RNA was isolated at 0, 3, 12, 24, 72 and 120 h after BMP4 exposure (GEO GSE10469)
Fig. 5
Fig. 5
The opposing roles of FGF2 and BMP4 in directing TB formation from epiblast type ES cells and iPS cells and the need to exclude FGF2 from the growth medium to minimize the co-induction of mesoderm and endoderm. Also indicated are the potential values of including low molecular weight pharmaceutical agents that block activin A (INHBA) and FGF2 signaling to support BMP4-directed differentiation to TB. Whereas FGF2 and conditioned medium (CM) from mouse embryonic fibroblasts, which contains activin A and other growth factors, enable maintenance of the pluripotent state of epiESC (top left), adding high concentrations of BMP4 leads to TB differentiation and possibly also some endoderm and mesoderm differentiation (bottom left). We argue that when BMP4 is present with FGF2 the extent of emergence of TB will depend on the relative concentrations of the two growth factors. By contrast, a defined medium supplemented with BMP4, FGF2 and activin A appears mainly to generate endoderm (upper right). However, the same defined medium formulated for minimal activin A signaling gives rise predominantly to mesoderm (lower right). Addition of BMP4 but minimizing FGF2 contribution to signaling either by excluding it from the culture medium or blocking MEK1/2 signaling, strongly favors TB formation, as does the inhibition of the activin A-mediated SMAD2/3 pathway (triad cluster in center). Blocking signaling through both the SMAD2/3 and MEK1/2 pathways in the presence of BMP4 will most likely provide optimal, unidirectional differentiation towards TB and the HLAG+ /cytoTB and CG+/syncytioTB sublineages (dashed circle in center)
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