Differences in Signaling Requirements for Trophoblast Stem Cells between Humans and Marmosets

Background and Research Motivation

Embryo implantation and placenta formation are critical features of eutherian mammalian development. The trophoblast is a group of outer cells of the embryo that mediates the connection between the embryo and maternal tissue. Trophoblast cells originate from the polar trophectoderm at the pre-implantation stage and undergo primitive cell fusion during embryo implantation to form invasive cells, which then penetrate the uterine epithelium to form three major cell lineages: cytotrophoblast, syncytiotrophoblast, and extravillous trophoblast. In early human development, the syncytiotrophoblast secretes human chorionic gonadotropin to maintain pregnancy, while the extravillous trophoblast deeply invades the uterus to promote vascular remodeling and immune regulation.

However, due to ethical and technical constraints, obtaining samples of early human embryos is challenging, and the molecular mechanisms of trophoblast cell development remain unclear. Non-human primate models, such as the common marmoset (Callithrix jacchus), exhibit developmental characteristics similar to humans but have different trophoblast implantation methods. Marmoset embryos implant on the surface of the central cavity of the uterus, which is comparatively shallow compared to humans. Studies on the trophoblast development of these New World monkeys may provide insights into human trophoblast development and its abnormalities. This paper, completed by scientists including Dylan Siriwardena, is published in the journal Cell Stem Cell.

Research Process

This study focused on tracing the development of trophoblast cells from the early implantation to the post-implantation stages in marmoset embryos, exploring the signaling requirements under different conditions, and culturing marmoset trophoblast stem cells (TSCs) in vitro to reveal differences in invasion patterns between human and marmoset trophoblast cells.

Experimental Methods

1. In Vivo Study of Marmoset Trophoblast Development:

   • The research team used spatiotemporal embryo analysis technology to further analyze trophoblast attachment, invasion, and placenta formation in marmoset embryo samples from Carnegie stages (CS) 5 to 7.
   • Through immunofluorescence labeling, they discovered that in CS5, the polar trophectoderm near the inner cell mass attaches to the uterine endometrium and begins dismantling the uterine epithelium. By CS6, the trophoblast cells establish a secondary implantation site at the opposite uterine wall, forming a cell layer primarily consisting of cytotrophoblast and syncytiotrophoblast.

2. In Vitro Culture of Marmoset Trophoblast Stem Cells:

   • The research team differentiated marmoset pluripotent stem cells (PSCs) into trophoblast stem cells and explored different signaling requirements.
   • They found that human TSC culture conditions could not support the formation of marmoset TSCs but rather led these cells to differentiate towards the extraembryonic mesoderm.
   • By co-inhibiting signals such as MEK, TGF-β/Nodal, and HDAC (histone deacetylase), they stabilized the “post-implantation” characteristics of marmoset trophoblasts, while under these conditions, human TSCs tended to differentiate into extravillous trophoblasts.

3. Comparison of Signaling Pathway Requirements:

   • The study found that Wnt signaling plays a crucial role in inhibiting human TSCs’ differentiation into invasive extravillous trophoblasts, whereas marmoset trophoblast cells do not rely on Wnt signaling.
   • Transcriptome analysis showed unique Wnt signaling characteristics in marmoset trophoblasts post-implantation, indicating evolutionary adaptations different from humans.

Data Analysis and Technical Highlights

The research team used high-throughput techniques such as single-cell RNA sequencing and methylomics for spatial identity mapping of differentiated samples. Particularly, through dimensionality reduction analysis like Uniform Manifold Approximation and Projection (UMAP), they revealed the uniqueness in the differentiation path of marmoset trophoblasts post-early implantation. Marmoset TSCs can form multinucleated structures in vitro, showcasing the potential to differentiate into syncytiotrophoblasts and are more likely to form this structure under low-density conditions. Additionally, marmoset trophoblast stem cells have the potential to implant in “human-marmoset chimeric embryo” models, further confirming the cross-species applicability of this model.

Major Findings and Conclusions

1. Species Differences in Trophoblast Development:

   • Marmoset trophoblasts exhibit shallow invasiveness post-early implantation, differing from the deep invasion patterns in humans. The study suggests that this is due to evolutionary differences between species.
   • Marmoset trophoblast cells in vitro show strong differentiation potential, forming major trophoblast cell lineages, but exhibit distinct signaling requirements reflecting evolutionary adaptations.

2. Role of Wnt Signaling in Trophoblast Development in Different Species:

   • Wnt signaling is not seen to play a significant role in marmoset TSCs but inhibits spontaneous differentiation of extravillous trophoblasts in human TSCs. This difference in requirement may be a result of regulation for uniquely human invasion patterns.

3. Establishment of TSC Model and Cross-Species Application:

   • The study established an in vitro culture model of marmoset TSCs, revealing significant potential in maintaining trophoblast stem cell characteristics and differentiating into multinucleated syncytiotrophoblasts. This model holds broad application potential in exploring early embryo-uterine interactions and deeper understanding of placenta-related diseases, such as placental abnormal implantation and hypertensive disorders of pregnancy.

Scientific Value and Application Significance

By systematically comparing signaling requirements and invasion characteristics of human and marmoset TSCs, this study provides a novel perspective for understanding evolutionary differences in primate trophoblast development. This contributes not only to exploring the molecular mechanisms during early embryo implantation but may also provide a theoretical basis for treating placenta-related diseases. Through the marmoset model, researchers can better study the dynamic behavior of trophoblasts in the uterine environment, offering new research ideas and experimental platforms for human placental development and related pathologies.