Elucidating the differences between individual human pluripotent stem cell lines: differentiation potential and malignancy
Human pluripotent stem cells represent a valuable in vitro research tool to study early human development and diseases, cell-based drug and toxicity screening and are also widely considered as a promising cell source for regenerative medicine. All of these applications require the availability of normal cells, which are able to reliably, efficiently and stably differentiate into the desired cell types.
Significant variation in differentiation potential and efficiency between individual hESC lines has been observed by several groups, often with the occurrence of a marked propensity to differentiate into a specific cell-lineage, e.g. pancreatic, hematopoietic or cardiac cells. This variation among hESC lines might be linked to different stages of embryonic development at derivation, (epi)genetic variation between individual human embryos, varying techniques for derivation, culturing and passaging, chromosomal or other genetic abnormalities and epigenetic changes acquired during prolonged in vitro culture.
Differentiation propensity can affect the intended use of a particular hESC line in biomedical research or regenerative medicine. As complete functional assays are time-consuming and costly, there is a need to develop easy-to-use assays for fast screening of differentiation propensity of hESC lines. Also, it is essential to gain deeper understanding in the phenomenon of early lineage specification bias in hESC for the further optimization and refinement of differentiation protocols.
Our aim is to chart the differentiation potential of individual hPSC lines and define markers, either genetic or epigenetic, that can reliably predict the differentiation propensity of a specific stem cell line. Similarly, we are interested in exploring whether and why certain stem cell lines display characteristics of a more malignant nature.
Recurrent chromosomal abnormalities have been detected in hPSC lines worldwide. Cells containing a specific aberration will only take over the culture because of a culture advantage that is conferred to them by this aberration. Culture advantages could for example be an increased proliferation rate or decreased apoptosis levels, but could also be linked to a change in differentiation potential of the subline.
We investigate whether there are specific chromosomal abnormalities that consistently change the differentiation capacity of hPSC or give them higher tumorigenic properties. The recurrent aberrations we are focusing on are the duplication of 20q11.21 and deletion of part of 18q.
Residual undifferentiated stem cells
One of the obstacles hampering the realization of the full potential of hPSC is that often during differentiation residual undifferentiated stem cells (rSC) are detected. These cells have lost the ability to differentiate, and remain mixed within a differentiated population. Upon transplantation into a patient receiving therapeutic cell treatment, they pose a significant tumorigenic risk. Our understanding of the characteristics and mechanisms of formation of these cells is still very poor, despite the danger they pose. A majority of the work involving rSC has focused on purification techniques, which do not yet show perfect efficiency. A sensitive a-priori screening for the presence of rSC in the cell populations prepared for transplantation could significantly increase the safety of hPSC-based regenerative medicine. Additionally, in-depth characterization of these cells could lead to better prevention methods in culturing techniques and differentiation protocols.
We aim at answering two main questions: how many rSC are present in differentiated populations, and what kind of phenotypic properties are inherent to this cell state? We are working on the development of ultra-sensitive detection methods to accurately quantify the number of rSC present in various differentiated populations. In parallel, we are characterizing isolated rSC both at the molecular as at the functional level in order to elucidate the ontology of these cells and asses the possible risk upon transplantation.
Collaborations: For this research topic, we collaborate with the research groups of
Prof. Peter Zandstra
(The Donnelly Centre for Cellular and Biomolecular Research, Toronto, Canada) and
Prof. Peter Andrews (Sheffield University, UK).
X chromosome inactivation in human pluripotent stem cells
While female cells have two X chromosomes, male cells have only one. To compensate for the difference in gene dosage between male and female cells one of the X chromosomes in female cells is transcriptionally inactivated by a process called X chromosome inactivation. X chromosome inactivation is an epigenetic event that happens very early during embryonic development. In the human, the X chromosome that will be inactivated is chosen randomly. Once X chromosome inactivation is set within a cell, all its daughter cells will display inactivation of the same X chromosome.
X chromosome inactivation starts with the transcription of the long non-coding RNA XIST. XIST transcripts will coat the X chromosome they were transcribed from and thereby form a first level of transcriptional silencing of this chromosome. The inactivated X chromosome further differs from the active X chromosome by its high DNA methylation content, the abundancy of repressive histone modifications (as for example H3K27Me3) and absence of activating histone marks (e.g. H3K9ac).
Variable appearances of the X chromosome inactivation status in female human pluripotent stem cells have been reported. Differences have been observed not only between different lines, but also within different sublines of the same stem cell line. Aberrant X chromosome inactivation patterns are known to occur in several cancers and have also been shown to influence the differentiation potential of human pluripotent stem cells. Therefore, we are interested in elucidating the stability of this epigenetic event during long-term culture and upon differentiation.
Collaborations: For the study of X chromosome inactivation in hPSC, we collaborate with the research groups of Prof. Josep Santalo
(Universitat Autonoma de Barcelona, Spain) and Prof. Anna Veiga
(Center of Regenerative Medicine, Barcelona, Spain).
Different cell sources for hPSC derivation: ethical and practical challenges
Exploring the potential of single blastomeres for hESC derivation
Embryonic stem cells are generally derived from the inner cell mass of blastocyst-stage human embryos, but also earlier stages of human preimplantation development show hESC derivation potential. In 2009, we reported the derivation of two hESC lines derived from single blastomeres of four-cell stage embryos. While this method would allow the derivation of hESC lines without the destruction of the embryo, it is not known whether the developmental stage of the embryo at derivation influences its pluripotent characteristics.
We want to further improve hESC derivation from single blastomeres of cleavage-stage human embryos, and investigate similarities and differences in the epigenetic landscape as well as in the differentiation potential of these cells.
Human iPSC reprogramming: influencing the epigenetic landscape
Even though hESC and hiPSC display a similar chromatin structure and gene expression pattern, observed differences at the epigenomic level and in the differentiation potential point towards fundamental differences and indicate variability in reestablishing the pluripotent epigenetic patterns throughout the genome. This reprogramming variability includes a somatic 'memory' (though this becomes less apparent after longer culture) as well as iPSC-specific aberrantly methylated loci, both at CG islands as in the non CG-context or regional enrichment of particular covalent histone modifications.
The conversion to pluripotency is an epigenomic event in which the ectopic expressed factors help generating a hyperdynamic chromatin state. The observed aberrant epigenetic marks may impart a regional chromatin conformation that is resistant to complete reprogramming. In this project, we aim at loosening the somatic cell chromatin structure in order to increase epigenetic reprogramming.
This research is part of a collaborative project with research groups from Universiteit Gent (Belgium) and Universiteit Maastricht (The Netherlands). The role of our group within this project is to obtain hiPSC in which the epigenetic landscape resembles as closely as possible that of standard inner cell mass-derived hESC, the golden standard in hPSC research. This research should lead to hiPSC that are better applicable for human germ cell derivation, which is the focus point of the collaborative project.
Collaborations: For this topic, we are collaborating with the Laboratory of Molecular and Cellular Therapy (Vrije Universiteit Brussel, Belgium), the Laboratory for Pharmaceutical Biotechnology (Universiteit Gent, Belgium).
Our research is supported by:
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Fonds voor Wetenschappelijk onderzoek Vlaanderen
Agentschap voor innovatie door wetenschap en techniek
Methusalem grant of the Research Council of the Vrije Universiteit Brussel
Scientific Research Fond Willy Gepts of Universitair Ziekenhuis Brussel, Vrije Universiteit Brussel