Chromosomal instability in human trophoblast stem cells and placentas

Danyang Wang, Andrew Cearlock, Katherine Lane, Ian Jan, Rajiv McCoy, Min Yang

Posted on: 31 December 2024

Preprint posted on 17 November 2024

The trophoblast: where instability is intrinsic

Selected by Carly Guiltinan

Categories: cell biology, developmental biology, molecular biology

Background

The trophoblast is the outer layer of cells of the blastocyst and precursor of the placenta. In humans, early placental development entails the emergence of specialized cell types from the cytotrophoblast (i.e., syncytiotrophoblast and extravillous trophoblast) that allow effective invasion and implantation into the uterine wall to support pregnancy establishment. Recently, the stabilization of trophoblast stem cells (TSCs) – the in vitro counterpart of cytotrophoblasts – from primary samples or pluripotent stem cells (PSCs) has allowed the study of trophoblast specialization and implantation. A limited number of reports have described aneuploidy of human trophoblasts/TSCs, however it remained unclear whether such chromosomal aberrations were associated with placental pathologies or a typical aspect of development.

Objectives

In the preprint highlighted here, the main goals were to:

Determine whether chromosomal instability is an inherent feature of human TSC and placental development,
Compare various methods of TSC establishment from an isogenic human PSC line.

Key findings

(1) Naïve PSCs are a superior starting point for the induction of bona fide TSCs. The authors used four approaches to derive TSCs from an isogenic primed human PSC line, including starting with cells that had been (1) epigenetically reprogrammed or (2) pre-differentiated into a trophectoderm-like intermediate, as well cells that were (3) reverted into a naïve state before TSC induction or (4) blastoid formation followed by TSC derivation. While initial analyses of the resulting TSC lines indicated similar morphology, proliferation rate, and trophoblast marker expression regardless of the induction method, naïve-derived TSCs (methods 3 and 4 above) had enhanced differentiation capacity into syncytio- and extravillous trophoblasts, as well as closer transcriptome and methylome profiles to cytotrophoblasts.

(2) TSCs have widespread genomic instability, but employ mechanisms to maintain stable growth and differentiation capacity. Karyotype analysis of TSC lines showed that around 25% of cells had aneuploidy by passage 12 for all derivation methods, unlike primed and naïve PSCs in which no aneuploidy was detected immediately before TSC induction. Notably, the heterogeneous nature of the karyotype aberrations and expression of DNA damage markers pointed to active chromosomal mis-segregation. However, despite ongoing – and in some cases, severe – chromosomal instability, TSCs demonstrated stable proliferation over 30 passages and differentiation capacity into advanced lineages of the trophoblast (with some differences based on induction method, described above). TSCs showed decreased marks associated with cell cycle safeguards and DNA damage repair accompanied by elevated autophagy, indicating that a metabolic shift may aid in TSC survival despite genomic abnormalities.

(3) Aneuploidy can be detected in the human placenta throughout healthy pregnancy. Using single-nuclei ATAC-sequencing data, the authors found an average of 8% aneuploid cells in first-trimester placentas and 4.4% in full-term placentas, compared to 0.7% in peripheral blood mononuclear cells. Furthermore, they speculated that this proportion is likely an underestimate due to limitations of dataset quality. Chromosomal changes could be detected across all 22 autosomes (but were most common for those with viable trisomies), indicating active DNA instability.

Importance

This preprint is the first demonstration that aneuploidy is a typical feature of TSCs and may even confer cells with a developmental advantage during placental development. The majority of pregnancy losses occur during the peri-implantation period, therefore further studies of these events may aid in the development of strategies to improve early pregnancy outcomes. Moreover, understanding how the placenta tolerates aneuploidy could be relevant to understanding cancer emergence, as placental development has many commonalities with and distinctions from tumor growth. Finally, this study highlights the importance of including a naïve-reversion step before the induction of TSCs from PSCs.

Questions for authors

Were you surprised by how considerable the differences among TSC lines were based on induction method?
Aside from G-banded karyotyping, did you perform any other analyses of chromosomal instability in naïve PSCs (similar to what is shown for primed PSCs in Figure 1 of the preprint)?
Hypothetically if chromosomal instability were inhibited in TSCs, do you anticipate that their developmental capacity would be hindered in any way? If so, what changes would you expect?

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Author's response

Min Yang shared

Questions for authors

1) Were you surprised by how considerable the differences among TSC lines were based on induction method?

The differences among TSC lines induced using various methods were not surprising. Our data, utilizing isogenic lines, reinforces the widely accepted view that naïve PSCs generate more bona fide trophoblasts. Among all the induction methods, the trophoblasts derived from blastoids appear to most closely resemble cytotrophoblasts in their characteristics.

2) Aside from G-banded karyotyping, did you perform any other analyses of chromosomal stability in naïve PSCs (similar to what is shown for primed PSCs in Figure 1 of the preprint)?

Yes, we performed FISH on naïve PSCs, including later passages (e.g., passage >15). Although these data are not included in the paper, they demonstrated the expected increase in chromosomal abnormalities in naïve PSCs at later passages. Naïve PSCs exhibit greater chromosomal stability compared to TSCs, which show significant abnormalities after only a few passages upon adopting the trophoblast fate. However, naïve PSCs are not entirely chromosomally stable, a feature that may reflect an intrinsic characteristic of preimplantation embryonic cells. This aspect is currently under investigation in our lab. We are also exploring cell fitness adaptations during the transition from the preimplantation (naïve) to the postimplantation (primed) epiblast stage. In this study, we focused on early-passage naïve ESCs, where detectable aneuploidy remains minimal at the resolution of karyotyping, to ensure that the abnormalities observed in TSCs were not inherited from the parental cells.

3) Hypothetically if chromosomal instability were inhibited in TSCs, do you anticipate that their developmental capacity would be hindered in any way? If so, what changes would you expect?

This is an excellent question, Carly. Our hypothesis is that forcing TSCs into a CIN-low (chromosomal instability low) state will compromise their proliferation. This is analogous to cancer cells, where reduced chromosomal instability is often associated with decreased metastatic potential (Bakhoum et al 2018 Nature). We are currently conducting experiments to test this hypothesis. We anticipate that stabilizing chromosomal integrity in TSCs may similarly constrain certain proliferative or even differentiation capacities.

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