Trophoblast Stem Cell - The Current State in Research

A Scientific Review

Introduction:

The first differentiation event that occurs in the blastocyst after fertilization is between the outer layer of the blastocyst, called trophoectoderm (TE), and the inner cell mass (ICM) (1). Embryonic stem cells (ESCs) are located in the inner cell mass where embryo begins to develop (1). However, they can only do so if the trophoectoderm, which derives from the Trophoblast Stem cells (TSC), is first able to implant the developing blastocyst into the uterine wall of the mother and stimulate placental formation (1) (Figure 1). This "connection" between the mother and the early embryo is essential in providing the embryo with nutrients and oxygen (1).

Figure 1

The trophoblast stem cells are found in the early embryo and play a vital role in supporting the implantation and nourishment of the developing conceptus (1). TSCs are actively proliferating, diploid cells that have the potential to develop into any of the cells that form the placenta (1). Although TSCs have been isolated from mice, there has been no conclusive identification of homologous cells in primates (5). This dichotomy may be due in part to differences in the developmental processes of early human and early mouse embryos, a subject that warrants a brief discussion.

Murine vs. Human:

While both human and mouse embryos have distinct cell populations that comprise the inner cell mass (ICM) and trophoectoderm (TE), the relationship between these populations differs in the two species (5). In a human embryo, ESC from the inner cell mass has been induced to differentiate into mature trophoblast tissue following incubation with bone morphogenic protein 4 (BMP4) (9). Even though this result has only been obtained in vitro, it fuels the speculation that an in vivo pathway for this differentiation exists (9). In a mouse embryo no such physiologically relevant differentiation has been observed. In chimeric models, murine ESCs formed embryonic tissues but did not form extra-embryonic (ExE) tissues (3). Similarly, murine TE cells only contributed tissue to the trophoectoderm and not to the embryo proper (3).

The formation of murine TE cells from ESCs was only possible through genetic manipulation (5; 3). The transcription factor Oct4 plays a major role in maintaining the ICM/ESCs by repressing several genes that would normally cause trophoblast differentiation (3). When Oct4 expression was repressed in the ESCs, the cells differentiated into trophoblast giant cells (3). Similarly, mutant mouse embryos lacking Oct4 expression there was upregulated expressions of the TE markers, H19 and Mash2 (3). Thus, downregulation of Oct4 is necessary for trophoblast development in vitro and perhaps, in vivo.

Although researchers have established the presence of trophoblast lineage cells within human blastocyst, the location or even the existence of human TSCs has yet to be established. In contrast, within the murine model, TSCs can be isolated from the mouse embryo at the early streak stage and reach their maximum numbers at the four-somite pair stage (8). At 3.5 days postcoitum (dpc), pre-implantation of TSCs can be derived from the blastocyst, and at 6.5 dpc post-implantation, TSCs can be derived from the chorion, which is the fetal placental organ responsible for nourishing the developing embryo (7). With the above in mind, the remaining information focuses on research conducted using murine TSCs.

Maintenance of TS cells:

Undifferentiated TSCs can be maintained in vitro up to 50 passages when cultured on a feeder layer of primary mouse embryonic fibroblasts in media supplemented with Fibroblast growth factor 4 (FGF4)) and heparin (7) (Figure 2A). Removing FGF4 from the media promotes TSC terminal differentiation to trophoblast giant cells, which are ultimately responsible for invading and attaching to the uterine wall (6). In vivo, FGF4 is produced by the inner cell mass and diffuses locally to bind its receptor FGFR2, which is present on the trophoectoderm cells (7) (Figure 2B).

Figure 2A

Figure 2B

Receptor binding leads to activation of the MAP kinase pathway and eventually to the activation of the transcription factors Cdx2 and Eomes (6). Both of these transcription factors are required for early trophoblast development and a null mutation of either transcription factor blocks development at the blastocyst/peri-implantation stage (6). Cdx2 and Eomes also act through an unknown mechanism to help maintain the TSCs in a proliferating, undifferentiated state (2).

The cells of the ExE, which are proximal to the inner cell mass, receive high concentrations of FGF4 and remain undifferentiated TSCs, while the more distal cells of the extraplacental cone (EPC), do not receive FGF4 and differentiate into trophoblast giant cells (7). Transplantation of proximal ExE tissue to the EPC similarly induces giant cell differentiation (6) To further highlight the importance of FGF4 signaling in trophoblast proliferation, FGF4(-/-) or FGFR2 (-/-) mutations are lethal due to failure of implantation (6).

Trophoblast Determination

While trophoblast giant cell differentiation appears to be the end result following the removal of FGF4, TSCs can also differentiate into the other cell types comprising the placenta. In mice, these cells include spongiotrophoblasts (SpT), syncytiotrophoblasts (SynT) or glycogen trophoblasts (GlyT) (2), and the choice depends on which transcription factors are present. Specifically, Hand1 and Stra13 promote cell cycle exit and restrict cells towards the TG fate (2) (Figure 3). Gcm1 also promotes cell cycle exit and restriction towards the SynT fate, cells that form the middle part of the placenta (2). When expressed ectopically in TS cells, these transcription factors may 'override' FGF4 signaling and cause the TSCs to differentiate (2). While this mechanism is unclear, it is thought that their expression directly suppresses one of the phosphorylation steps involved in the FGF4/MAP kinase pathway (2).

Current Research:

Stem cells can be potentially used as therapeutic agents for degenerative diseases and tissue damage. The study of stem cells, specifically TSCs and the mature cells that they generated are also relevant to general developmental biology. For example, these stem cells have been used in new technique, altered nuclear transfer to enhance the field of stem cell research (4). A current mainstay of embryonic stem cell research is nuclear transfer, a process in which donor cells are fused with an enucleated egg to form a new blastocyst. Nuclear transfer is used to derive embryonic stem cells; however, this procedure requires the destruction of the cloned blastocyst, which theoretically has the potential to develop into a mature embryo and fetus. To circumvent this dilemma, Meissner and Jaenisch have developed a procedure called altered nuclear transfer in which the derived blastocyst lacks the ability to implant into the uterus and form the placenta but retains the ability to produce fully functional embryonic stem cells (4). By eliminating the fetal-maternal interface, the blastocyst has no potential to develop into a fetus, and the embryonic stem cells can be harvested with mimimal ethical concerns and or regulations.

In order to block implantation and placental formation, Meissner and Jaenisch used an RNAi technique to knockout the expression of Cdx2 in the murine fibroblast donor cells used for nuclear transfer (4). Cdx2 is a transcription factor that is expressed in the 8-cell embryo and is vital for trophoectoderm lineage differentiation and function. The Cdx2-deficient blastocysts did not develop trophoblast cells, and were not able to successfully implant when transferred into pseudo-pregnant mice. The blastocytes were, however, able to produce pluripotent ESCs when explanted into culture (4).

Conclusion:

It is clear that the process by which the trophoblast invades and connects with the maternal blood circulation is sensitive to mutations and errors, which can greatly affect embryonic development. In fact, the major cause of infertility is due to the failure of the early embryo to develop and implant into the uterus (1). Studies with murine TSCs allows developmental biologists to further research and elucidate the mechanisms by which the process of implantation and placental formation occurs. However, there still exist fundamental morphological and developmental differences in the early human and mouse embryos, and applying what was discovered from working with murine TSCs to human TSCs has proven to be a challenge. For instance, with the altered nuclear transfer method proposed by Meissner and Jaenisch, there are concerns that Cdx2's expression and function in a human embryo is not conclusively known and may be different from the mouse system (4). Another minor concern is that the retroviral vector used to introduce the RNAi might cause insertional mutagenesis and possible leukemia (4). Clearly, scientists need to conduct more research to understand placental development and implantation, and murine TSCs provide researchers with the tools to do so.

Acknowledgements:

This review was prepared by the following graduate students in the Stem Cell Biology Class, Graduate School of Biomedical Sciences, University of Medicine and Dentistry of New Jersey: Roger Diaz, Thomas Finocchio, Shannon Henning, Gaurav Gandhi, Nicole Pannucci (in alphabetical order).

Teaching Assistant: Elaine Wong

The review was edited by two stem cell biologists.