Chromosome-specific aberrations, rather than general aneuploidy, may drive mouse embryonic stem cell-derived teratoma metastasis

Abstract

Introduction

Pluripotent stem cells (PSC) are known to recurrently acquire genetic aberrations including chromosomal aneuploidy during long-term culture of which the consequences remain largely unknown. A recent study reported that xenografting of aneuploid mouse embryonic stem cells (mESC) gives rise to teratomas containing undifferentiated elements with metastatic capacity. This contrasts with benign teratomas composed of fully differentiated tissues from the three embryonic germ layers obtained by xenografting diploid mESCs. The highlighted study thus proposes that general aneuploidy may drive metastasis of PSC-derived teratomas, thus directly correlating it with malignant potential of PSCs.

Main

The aneuploid mESC lines used in the aforementioned study showed trisomies of chromosomes 6, 8, 11, 15 or combinations thereof. Interestingly, several of these trisomies, specifically in chromosomes 6, 8, and 11, constitute well-known chromosomal aberrations recurrently acquired in mESCs upon (long-term) in vitro culture. This is suggested to be driven by numerous (proto)oncogenes in these regions. Notably, recurrent chromosomal aberrations are also observed in human PSCs (hPSCs), mostly in chromosome 1, 12, 17, 20 and X, and have similarly been associated with the acquisition of a malignant phenotype in vitro, posing a risk to their potential clinical application. There is synteny between the chromosomes recurrently affected by such aberrations in mouse and human PSCs, namely mouse chromosome 6 and human chromosome 12 as well as mouse and human chromosome 1, suggesting a chromosome-specific phenomenon that may transcend the species barrier. Additionally, in the field of cancer, malignant (but not benign) human germ cell tumors (hGCTs), pathologically similar to the tumors derived from the aforementioned aneuploid mouse PSCs, are characterized by supernumerical copies of chromosome 12, highlighting the additional clinical relevance of these findings and the aneuploid mESC models for GCT research and treatment.

Conclusions

We suggest that chromosome-specific trisomies, rather than general aneuploidy, might drive teratoma metastasis upon mESC xenografting in vivo. We also observed indications of synteny between the recurrent chromosomal aberrations among human and mouse PSCs, suggesting potentially common intraspecies selection mechanisms. We finally reiterate the similarities observed between the PSC and GCT field related to chromosomal aberrations and malignancy, highlighting the relevance of these experimental models in both research fields.

Main text

Safe clinical application of pluripotent stem cells (PSCs) has been hampered by their propensity to acquire recurrent genetic aberrations upon long-term culture which are reminiscent of those observed in cancer [1, 2]. Intense research efforts are thus ongoing aiming to unravel the underlying mechanisms and consequences of these aberrations. In recent years, notable studies have been published on this topic, including a recent study conducted by Xiao et al. [3] (hereinafter referred to as Xiao) who showed that aneuploid (trisomic for specific chromosomes) mouse embryonic stem cells (mESCs) give rise to malignant metastatic teratomas when xenografted into mice. This contrasts the benign teratomas with fully differentiated tissues obtained by xenografting diploid mESCs. Based on their extensive data, the authors concluded that general aneuploidy may be a universal driver of teratoma metastasis.

The aneuploid mESC lines studied by Xiao originate from their previous work where they generated lines with trisomies of chromosomes 6, 8, 11 or 15, as well as double trisomies in chromosomes 6/8 and 8/15 [3, 4]. It is crucial to highlight that mouse chromosomes 1, 6, 8 and 11 are the most recurrent chromosomal aneuploidies observed in mESCs upon (long-term) in vitro culture [5,6,7] (Fig. 1A). This parallelism may be underappreciated by the authors, as it suggests that the observed teratoma metastases may be consequent to the aneuploid status of these specific chromosomes rather than general aneuploidy. This is supported by studies which show that aberrations affecting these specific chromosomes confer a rapid selective growth advantage (i.e. culture adaptation) in PSC culture due to increased proliferation, decreased differentiation potential in vitro and in vivo, as well as decreased dependency on growth factors for self-renewal [1, 2, 4, 5, 7], recapitulating characteristics of malignant transformation.

Human PSCs (hPSCs) are similarly known to recurrently acquire chromosomal gains, most commonly affecting chromosome 1, 12, 17, 20 and X [1, 2]. In hPSCs, these aberrations are suggested to be driven by specific (proto)oncogenes located in those regions, whose resulting overexpression have been shown to lead to culture advantages and malignant behavior [2, 8]. For instance, the recurrently gained chromosome 20q11.21 contains several oncogenes, namely BCL2L1 and TPX2, which are considered to be the drivers of the aberration [9]. Similarly, MDM4 is considered the driver gene for the 1q gain frequently observed in hPSCs [8]. Indications of this in mESCs were also shown in the figures shared by Xiao in the Peer review file, where an upregulated transcription of the genes located within the trisomic mESC chromosomes was shown [3]. On the protein level, the authors also show an upregulation of many known and putative oncogenes within the gained chromosomal regions when compared to the isogenic diploid cells [3]. These include Kras (1.27-fold), Nanog (1.41-fold) and Lrp6 (1.29-fold) on chromosome 6; Idhd (1.15-fold) on chromosome 8; and C-myc (1.58-fold) on chromosome 15. However, several tumor suppressor genes located in the same aneuploid chromosomal regions were also upregulated, namely Tp53 (1.77-fold), C53 (1.30-fold) and Rgs22 (2.40-fold). Thus, as overexpression of both (proto)oncogenes and tumor suppressor genes was observed, Xiao conclude that it is not possible to accurately calculate their functional effects related to the acquired metastatic capacity of mESCs.

Despite this conclusion from the authors, we evaluated whether the most recurrently aberrant chromosomes in hPSCs (1, 12, 17, 20, and X) are syntenic to those in mESCs and observed in Xiao’s aneuploid mESC lines (1, 6, 8, 11), by using The Jackson Laboratory Synteny Browser (syntenybrowser.jax.org) and the NIH Comparative Genome Viewer (ncbi.nlm.nih.gov/cgv). Interestingly, synteny was observed between mouse chromosome 6 and the long arm of human chromosome 12, where certain potential driver genes are located such as Nanog, Kras and Gdf3, although their specific involvement as driver genes requires further investigation (Fig. 1B). Moreover, synteny was observed between mouse and human chromosome 1 (specifically the short arm), which contains the potential driver gene MDM4 [8]. This therefore indicates a potentially conserved mechanism underlying the recurrent acquisition of aberrations in specific chromosomes in PSCs of both species which may be driven by specific proto-oncogenes. We did not observe similarly significant synteny between mouse chromosomes 8 or 11 with any region of the human genome previously reported as recurrently aberrant in hPSCs. Of note, a previous report observed synteny between mouse chromosome 11qE2 and human chromosome 17q25, containing the putative driver gene Birc5 [6]. Additionally, for mouse chromosome 8, several putative driver genes have been suggested such as Jun-b and Lyl-1 [10]. Together, this may indicate that additional species-specific mechanisms may underlie the observed recurrent aberrations as well and as previously suggested [6].

Furthermore, the functional relevance of these recurrent chromosomal aberrations is also highlighted by their similarities to the aberrations observed in many human cancers, including human germ cell tumors (hGCTs). These neoplasms originate from the deregulation of ESCs and the stem cells of the germ cell lineage [1, 11]. In the context of the Xiao study, the type I and II hGCTs are most relevant [11]. The mature teratomas obtained by Xiao by xenografting diploid mESCs reflect type I mature benign teratomas as both contain fully differentiated tissues derived from the three embryonic germ layers and lack chromosomal aberrations. In contrast, the aneuploid mESC-derived tumors obtained by Xiao reflect type II hGCTs as both have metastatic potential and contain multiple malignant histological elements, including embryonal carcinoma which is considered the malignant counterpart of ESCs. Moreover, type II hGCTs exclusively harbor gains of the short arm of chromosome 12 (mostly in the form of isochromosome 12p which is acquired during progression to invasive growth) which parallels the tumors derived from the triploid chromosome 6 mESCs by Xiao based on the conserved synteny (Fig. 1B) [11].

In conclusion, the models presented by Xiao are highly relevant to both the PSC and hGCT fields as they aid in further understanding the mechanisms underlying their recurrent chromosomal aberrations and the malignant transformation. Further light needs to be shed on whether these effects on PSC behavior are caused by chromosome-specific events or through general aneuploidy. This may, in part, be achieved by inducing aneuploidy in chromosomes that are not recurrently aberrant in mESCs and hPSCs and investigating the effects in vivo (related to e.g. teratoma metastasis). This was in part achieved by Xiao related to mouse chromosome 15 which is not recurrently aberrant in mESCs yet also resulted in teratoma metastasis. However, aneuploidy in other chromosomes will need to be investigated. More investigations are also warranted to confirm the potential synteny between the chromosomal aberrations in both mouse and human PSCs to determine whether conserved mechanisms are at play. Ultimately, this will guide future developments in: (1) unravelling the mechanisms underlying chromosomal aberrations in PSCs and consequently refining safety guidelines for their clinical application; (2) the surveillance of hGCT patients, stratification of those at risk for metastasis, and the development of new treatment options for hGCT patients.

Fig. 1

The synteny between recurrently gained chromosomes in mESCs, hPSCs and hGCTs. A. Amplification plot indicating the recurrent chromosomal aberrations observed in mESCs (mus musculus), pertaining specifically to gains in chromosome 1 (5.88%), 6 (4.71%), 8 (20.59%), and 11 (14.12%). Data was obtained from⁹. The X-axis indicates mouse chromosomes 1–19. The Y-axis indicates the frequency (in percentage) of the chromosomal aberrations. The blue shaded chromosomes indicate the aneuploid mouse chromosomes investigated by Xiao et al., 2024 (chromosome 6, 8, 11, 15). B. Highlight of mouse chromosome 6 (trisomic in Xiao mESC lines which gave rise to metastatic tumors) followed by the syntenic regions within human chromosomes and the frequencies of aberrations within those chromosomes in hPSCs and hGCTs (red indicates chromosomal gains and blue indicates chromosomal loss). Potential driver (onco)genes located in this syntenic region (in both mouse and human cells) are highlighted at the bottom right. PSC (human and mouse) data was obtained from [8, 9] using KaryoBrowser (https://karyo.group.shef.ac.uk/) and hGCT data was obtained from [12] using cBioPortal (https://www.cbioportal.org/study/summary?id=gct_msk_2016). The X-axis indicates the frequency (in percentage) of the observed chromosomal aberrations. The Y-axis indicates chromosome number. All plots were generated using RStudio (version 4.4.0). Abbreviations used: ESC, embryonic stem cell; PSC, pluripotent stem cell; GCT, germ cell tumor