Trophoblast specification and differentiation potential in early human and non-human primate embryogenesis

Change log
Siriwardena, Dylan 

Implantation is mediated by trophoblast, the outer layer of the early embryo. Trophoblast is an extraembryonic tissue that gives rise to the placenta, which sustains fetal growth and development. Human embryo implantation and early trophoblast development have remained elusive due to the inaccessibility of early implantation stages. The conservation of trophoblast development between humans and non-human primates provides an exciting opportunity to understanding of the pathophysiology of implantation and related pregnancy disorders. This thesis examines trophoblast development in the common marmoset (Callithrix jacchus) to investigate the molecular regulation of (1) trophoblast specification and (2) trophoblast differentiation, and ultimately the conservation of these processes in human. To delineate the molecular framework of marmoset trophoblast development, a single-cell RNA sequencing dataset was generated from zygote to postimplantation embryos at Carnegie stage (CS) 7. Postimplantation samples were isolated from in utero embryos using laser capture dissection, enabling spatial reconstruction of trophoblast expression patterns and virtual reconstruction of implanted marmoset embryos. I identify and molecularly characterize three distinct trophoblast populations: preimplantation trophectoderm, postimplantation luminal cytotrophoblast, and postimplantation syncytiotrophoblast. Marmoset trophectoderm differentiated into syncytiotrophoblast upon implantation but did not form invasive extravillous trophoblast. Staining of in utero embryos demonstrated extensive cytotrophoblast proliferation but superficial invasion of syncytiotrophoblast by CS7. To determine if these molecular regulators were conserved in human trophoblast development, I generated a continuous pseudotime trajectory of human trophoblast development from zygote to mid-gestation using publicly available single cell RNA sequencing datasets. The results from this analysis showed that core transcription factors controlling trophectoderm, cytotrophoblast and early syncytiotrophoblast identity are conserved between marmoset and human. To interrogate regulators of trophoblast differentiation, I developed a protocol to derive marmoset trophoblast stem cells (TSCs) from pluripotent stem cells (PSCs) in a naïve (preimplantation) like state. Screening of culture conditions for trophoblast derivation revealed that human TSC medium (OKAE) did not promote trophoblast from embryos and naïve PSCs, in contrast to human. However, OKAE conditions with MEK inhibition generated marmoset TSCs. Single-cell RNA sequencing of marmoset TSCs suggested a postimplantation trophoblast identity. In contrast, OKAE conditions promoted an extraembryonic mesenchyme (ExMes) phenotype. WNT signalling induced ExMes fates in the absence of MEK and TGFβ inhibition. Marmoset TSCs form syncytiotrophoblast by endoreduplication as determined by live imaging. Removal of EGF promotes a peri-implantation like phenotype that can form correctly polarized trophoblast spheroids. Moreover, implantation of marmoset TSC spheroids into extracellular matrix marmoset form syncytiotrophoblast. Together, this study established a non-human model system to investigate non-human primate trophoblast development. The study of conserved and divergent regulators of early trophoblast differentiation represents an exciting opportunity to delineate the molecular mechanisms underlying pathophysiological changes in early human implantation and placental development.

Boroviak, Thorsten
Trophoblast, Implantation, Marmoset, Preimplantation, Primate
Doctor of Philosophy (PhD)
Awarding Institution
University of Cambridge