Trophoblast Stem Cells: Scientific review
Stem Cell Biology, Fall 2006

Prepared by: Mary Gergis and Sharon Soh
TA: Helen Liou

Introduction
The area of stem cell research is expanding rapidly with the discovery of stem cells in all organs, including the developing embryo.  A basic stem cell can be defined as a cell that is clonogenic, can self-renew unlimitedly, can be derived from adult or embryonic tissue, can divide symmetrically or asymmetrically, is able to re-populate an organism, and has no differentiation markers.  Trophoblast stem cells (TS cells) are gaining interest in the scientific community because TS cells can proliferate in culture for many generations and can differentiate into a range of trophoblast subtypes in vivo and in vitro , suggesting they are multi-potent (1).

In mammalian development, the embryo divides to form a blastocyst with an inner cell mass (ICM) and the trophoectoderm, which is surrounding the ICM (2, Fig. 1).  Embryonic stem cells (ESC) are derived from the ICM and later form all three germ layers of the embryo, while the trophoectoderm forms the TS cells (2).  The cells derived from TS cells, such as the trophoblast giant cells and spongiotrophoblast cells, function to mediate implantation and physiological adaptations during pregnancy, such as regulation of maternal blood flow, and form layers of the placenta (1).  Studying TS cells is important to understanding embryonic development and the factors that maintain its growth and survival (3). 

Development
In development, the zygote divides several times to give rise to the blastocyst (4).  The blastocyst is made up of the inner cell mass (ICM) and the trophoectoderm.  Trophoblast stem cells come from the polar trophoectoderm that surrounds the inner cell mass in the blastocyst and can be observed after four or five days after fertilization (1, Fig. 1).  Maintaining trophoblast proliferation and self-renewal depends on signals from the ICM (5).  Trophoblast stem cell maintenance is also dependent on cross-talk between the epiblast and extraembryonic endoderm (5). 

Trophoblast stem cells differentiate into three main subtypes; trophoblast giant cells (TGC), spongiotrophoblast cells, and labyrinth trophoblast cells (5).  TGC form the periphery of the conceptus and are in contact with maternal tissue and blood (5).  They assist in adapting maternal physiology during pregnancy, synthesizing angiogenic factors, vasodilators, and anticoagulants which help to promote local blood flow to the implantation site (5).   The formation of TGC is considered a “default” differentiation pathway in the absence of TS cell maintenance because when factors important in TS cell maintenance such as nodal, activin, and TGFβ are secreted, TGC differentiation is suppressed (5).  A second cell type is the spongiotrophoblast cell (SpT), which constitutes the middle layer of the placenta.  SpT cells lie between the outer TGC and inner labyrinth layer (5, Fig. 1).  Their function is not well understood except that they may act as structural support for the developing villous structures of the labyrinth and they may act to suppress several genes (5).  The innermost layer in the murine placenta is the labyrinth layer, which consists of branched villi that provide a large surface area for nutrient, waste and gas exchange (5).  Within the labyrinth are several types of trophoblast cells with specific positions and morphology such as the syncytiotrophoblast (5).  TS cells possess the ability to differentiate into trophoblast subtypes in vitro and contribute to the trophoblast lineage in vivo (3).

Basic Biology
Embryonic stem cells (ESC) are derived from the ICM. The transcription factor, Oct3/4, is a marker of pluripotency in both mouse and human ESC (6).  It has been shown that ESC can be induced to differentiate into the trophoectoderm and therefore, trophoblast stem cells by repression of Oct3/4 (7).  Studies show that Oct3/4- repressed cells transform into trophoblast giant cells rather than cells of embryonic lineage (4).  On the contrary, the HMG box transcription factor, Sox2, has been shown to prevent trophoblast specification (4).  Also, Cdx2 is important in trophoectoderm differentiation and its over expression in ESC results in trophoblast giant cell formation (4).  This was reinforced in other studies, which showed that depletion of Oct3/4 in murine ESC induced expression of the trophoblast stem cell marker, Cdx2, leading to the appearance of flattened epithelial-like cells, which are typical of differentiated trophoblast cells (6).  A gain of Cdx2 expression is therefore associated with loss of pluripotency-associated gene expression (8).  Like Cdx2, eomesodermin (eomeso), is another gene that has been found to induce differentiation (7).  Another important differentiation factor of human ESC to trophoblast is BMP4, a member of the TGFβ family (9).

In culture, murine trophoblast cells were studied to see what factors are essential in differentiating into trophoblast subtypes.  FGF4, heparin, and embryonic fibroblasts (EMFIs) are required to maintain “stemness” and the removal of any one of these factors will induce differentiation into trophoblast subtypes (4).  Factors such as FGF4, Nodal, Activin, and TGFβ suppress TGC formation and withdrawal of these factors results in differentiation into TGCs (5).

Although there are 23 FGF family members, FGF4 provides essential signaling that is necessary for both TS cells and preimplantation embryos (10).  There are some differences between species.  For example, FGF4 is expressed in the ICM and ESC in murine ESC, but not in human ESC (10).  There is evidence that FGF4 expression is induced by the TGFβ-related protein, Nodal (11).  Nodal acts with FGF4 on extraembryonic ectoderm (ExE) to sustain a microenvironment that inhibits differentiation of TS cells (11).

Differentiation of TS cell derivatives are dependent on many factors, such as Mash2 for spongiotrophoblast (SpT) and trophoblast giant cells, Hand1 for trophoblast giant cells (TG) cells, and Gcm1 for syncytiotrophoblast (SynT) (12).  Mash2 promotes transient FGF4-independent amplification to TS cells that are progressing towards the SpT and TG fate (12).  Hand1 and Stra13 promote the cell cycle exit and restrict cells towards the TG fate (12).  Gcm1 also promotes the cell cycle exit and restriction towards SynT fate and can suppress the ability of TS cells to form TG cells (12).

Suppressor of cytokine signaling-3 (SOCS3) is an essential negative regulator of the leukemia-inhibitory factor receptor (LIFR) signaling in trophoblast differentiation.  Disruption of SOCS3 leads to detrimental embryonic consequences with placental defect (3).  In some studies, TS cell supplementation has been shown to rescue placental defects in SOCS3-deficient embryos and therefore, may lead to an avenue of treatment for placental defects (3).

Foxd3, a transcriptional repressor, is required for maintenance of the epiblast and in vitro establishment of embryonic stem cell lines and is also required for the trophoblast lineage (13).  Studies show that in vivo, Foxd3 negative ExE differentiates almost exclusively to trophoblast giant cells (13). 
             

Implication for disease and medicine
Trophoblast stem cells are important in establishing implantation and placental development.  It has been shown that defects in placental development lead to pregnancies that are at risk for miscarriage and intrauterine growth retardation, and are associated with preeclampsia, a leading cause of maternal death and premature birth (14).  Gultice, et al. showed that impaired placental formation may be associated with alterations in a specific trophoblast lineage, such as the invasive trophoblast giant cells (14). 

In this experiment, Rcho-1 cells represent an isolated trophoblast population committed to the giant cell lineage (14).  Undifferentiated Rcho-1 cells express the trophoblast stem cell marker, Id2, and other trophoblast giant cell markers (14).  Upon differentiation, Id2 is downregulated and Csh1, a TG cell marker, is up regulated (14).  Also in this experiment, researchers showed how the role of oxygen deficiency in tissue, hypoxia, is relevant to the trophoblast development.  Differentiating Rcho-1 cells in hypoxia did not alter the expression of lineage-specific markers, however, hypoxia did inhibit the downregulation of Id2 and blocked induction of CSH1 (14).  This shows that hypoxic culturing conditions are similar to the environment of early pregnancy when the placenta is still forming, but prevents the formation of fully functional TG cells (14).  Trophoblast cells remain proliferative and fulfill their first roles in implantation under low oxygen conditions (14).  As pregnancy progresses, trophoblast giant cells differentiate, invade the maternal deciduas and migrate toward the uterine spiral arteries (14).  Although this study provides a picture of the environment of early pregnancy, the molecular mechanisms by which oxygen mediates trophoblast differentiation have not been fully understood (14).  Seemingly, hypoxia, along with other environmental factors, induces proliferation of trophoblast stem cells and stimulates the rapid growth needed to accumulate the large quantity of cells required for formation of the placenta during the early phase of pregnancy (14).  It is also thought that hypoxia may prevent DNA damage from oxidative metabolites (14).  Increasing oxygen availability could act as a signal in the differentiation process, while extended hypoxia would not be the preferred physiological condition and could lead to placental defects (14).

Trophoblast stem cells could be used to rescue mutations associated with placental defect (3).  Studying trophoblast stem cells will help not only our understanding of placental and birth defects, but also in understanding what factors contribute to implantation.  This could provide us with useful tools that can be applied to solving infertility problems and birth defects.  Another area where TS cells could provide some understanding is in the recruitment of natural killer cells (NK) in the first trimester of pregnancy (15).  One study showed that first trimester trophoblast cells express CXCL12, which is a chemo attractant for NK cells (15).  CXCL12 allows trophoblast cells to direct migration of CD56brightCD16- NK cells (15).  “This could contribute to the recruitment of the decidual leukocytes or may be used to modulate the immune milieu at the maternal-fetal interface so as to keep the pregnancy going smoothly” (15).

References

1. Cross JC. How to Make a Placenta: Mechanisms of Trophoblast Cell Differentiation in Mice. Placenta 2005,A:S3-9.
2. Rossant, Janet. Stem Cells from the Mammalian Blastocyst. Stem Cells 2001; 19;447-82.
3. Takahashi Y, Carpino N, Cross JC et al. SOCS3: an essential regulator of LIF receptor signaling in trophoblast giant cell differentiation. EMBO J 2003;22:372-84.
4. Kunath T, Strumpf D, Rossant J. Early trophoblast determination and stem cell maintenance in the mouse--a review. Placenta 2004;25:S32-8.
5. Simmons DG, Cross JC. Determinants of trophoblast lineage and cell subtype specification in the mouse placenta. Dev Biol 2005;284:12-24.
6. Hay DC, Sutherland L, Clark J, Burdon T. Oct-4 knockdown induces similar patterns of endoderm and trophoblast differentiation markers in human and mouse embryonic stem cells. Stem Cells 2004;22:225-35.
7. Niwa H, Toyooka Y, Daisuke S et al. Interaction between Oct3/4 and Cdx2 determines trophoectoderm differentiation. Cell 2005;123:917-29.
8. Tolkunova E, Cavaleri F, Sigrid E et al. The Caudal-related protein Cdx2 promotes trophoblast differentiation and mouse embryonic stem cells. Stem Cells 2006;24:139-44.
9. Xu RH, Chen X, Li DS et al. BMP4 initiates human embryonic stem cell differentiation to trophoblast. Nat Biotech 2002;20:1261-4.
10. Zhong W, Wang QT, Sun T et al. FGF ligand family mRNA expression profile for mouse preimplantation embryos, early gestation human placenta, and mouse trophoblast stem cells. Mol Reprod. Dev 2006;73:540-50.
11. Guzman-Ayala M, Ben-Haim N, Severine B et al. Nodal protein processing and fibroblast growth factor 4 synergize to maintain a trophoblast stem cell microenvironment. Proc Natl Acad Sci USA 2004;101:.
12. Hughes M, Dobric N, Ian SC et al. The Hand1, Stra13 and Gcm1 transcription factors override FGF signaling to promote terminal differentiation of trophoblast stem cells. Dev Biol 2004;271:26-37.
13. Tompers DM, Foreman R K,  Qiaohong W et al. Foxd3 is required in the trophoblast progenitor cell lineage of the mouse embryo. Dev Biol 2005;285:126-37.
14. Gultice AD, Selesniemi K L, and Thomas L. Brown. Hypoxia Inhibits differentiation of lineage-specific Rcho-1 trophoblast giant cells. Biol of Reprod 2006;74:1041-50.
15. Wu X, Jin LP, Yuan MM et al. Human first-trimester trophoblast cells recruit CD56brightCD16- NK cells into decidua by way of expressing and secreting of CXCL12/stromal cell-derived factor 1. J Immunol 2005;175:61-8.

Fig. 1. Overview of the trophoblast lineage and the expression patterns of trophoblast cell subtype specific genes