Our past research in a Nutshell
Our group was amongst the first to start working on mapping and understanding the extend of genome instability in hPSC. In 2008, we identified the gains of 20q11.21 as a mutational hotspot in hESC (Spits et al., 2008), a finding that was replicated in the large collaborative study published in 2011 by the International Stem Cell Initiative, of which we also are part (Amps et al., 2011). Later, we identified Bcl-xL as the driver gene behind the selective advantage of this mutation in culture (Nguyen et al., 2014) and studied its impact on differentiation capacity, publishing the first systematic study of this type (Markouli et al., 2019).
We have also worked on other aspects of genome integrity, amongst which de novo variation in the mitochondrial DNA of both hESC and hiPSC (Van Haute and Spits et al., 2013; Zambelli et al., 2018), identification of microsatellite instability in some hPSC lines (Nguyen et al., 2014), erosion and skewing of X-chromosome inactivation and extensive low-grade chromosomal mosaicism (Jacobs et al., 2014; Keller et al., 2019), which we found to be caused by the medium acidification associated to culturing the cells at high density (Jacobs et al., 2016).
Our most recent work is focused on understanding how these abnormalities modulate the ability of the cells to exit the pluripotent state and successfully commit to the cell type to which they are being directed. The work on gains of 20q11.21 revealed a TGF-beta mediated impairment of neuroectoderm commitment (Markouli et al., 2019), and research on lines that ‘misbehaved’ during differentiation showed an important role for WNT and BMP4 signaling in pushing the cells away from the definitive endoderm fate and into a mis-specified state (Markouli et al., 2021; Dziedzicka et al., 2021).
Ongoing research
The impact of chromosomal abnormalities in hPSC-derived retinal pigmented epithelial cells
Edouard Couvreu de Deckersberg, Yingan Lei
Age-related macular degeneration (AMD) is the leading cause of blindness and vision impairment worldwide and is a result of the degeneration of the retinal pigment epithelium (RPE). The most novel cell therapy to effectively cure AMD is based on the use of human pluripotent stem cells (hPSC) to make RPE for transplantation, and the first clinical trials yielded promising results. However, hPSC kept in culture acquire genetic abnormalities. We still have a poor understanding on the functional consequences of these abnormalities, which represents one of the major current bottlenecks for the safe transition of these therapies to the clinic. In our study, we will focus on understanding the impact of a chromosomal abnormalities on the differentiation of hPSC into RPE. We will research the functional effects of the chromosomal abnormalities most commonly found in hPSC lines worldwide, as well as of mosaic abnormalities that appear randomly in subpopulations of cells in culture. We will study the transcriptome of these cells to predict the impact of genetic abnormalities on the cells, and validate these findings with functional studies. This project will provide key information for a realistic risk-assessment of chromosomal abnormalities in the clinical translation of hPSC-derived RPE and novel insight to the role aneuploidy in modulating or altering the course of hPSC differentiation.
The impact of recurrent chromosomal abnormalities on growth advantage and tri-lineage differentiation capacity of human pluripotent stem cells
hPSCs can differentiate into any cell type of the adult human body and they hold a great promise in transplantation and regenerative medicine as well as in in vitro modeling of development and disease. hPSC cultures tend to acquire chromosomal abnormalities which provide them with a growth advantage. Currently, little is known on how genetic abnormalities affect the differentiation of the cells. In this project, we will elucidate whether the recurrent chromosomal changes identified in undifferentiated hPSC retain their selective advantage during differentiation and if they impact the quality of the differentiated progeny. We will mimic culture mosaicism by mixing fluorescently labelled pairs of hPSC lines with and without a chromosomal abnormality, and differentiate them to progenitor and mature cell types. With flow cytometry and single cell RNA sequencing we will study culture take-over and differentiation impairment to all 3 germ layers. Our results will represent the first information on how mosaic cultures can affect the end population of differentiated cells and will be the steppingstone to design assays for early detection of aneuploidies that are relevant for a specific cell type, which is essential to the transition of hPSC-derived cell types to a clinical setting. Future research will make use of the transcriptomic data of this project to investigate the mechanisms behind differentiation impairment in genetically unbalanced cells.
Establishing the effect of the loss of 18q on hPSC differentiation
Deletions of chromosome 18q are relatively rare structural chromosomal abnormalities in hPSCs. Our lab found 18q deletions in four different hESC lines at relatively early passages, in the form of derivative chromosomes, and routine G-banding of 7300 hPSC cultures at WiCell finds it in 4% of the lines (WiCell Cytogenetics Lab). The aim of this study is to examine the functional effects of the 18q deletions during the differentiation of hPSCs into the three embryonic germ layers and to determine the molecular mechanisms involved in impairment or bias, if any is detected.