Methylation status reveals the purity of derived cells that may one day be used for hemophilia A therapy
Epigenetic regulation across the genome, and methylation patterns in particular, are important modulators of basic biological processes, as well as the switch between health and disease. In this blog post, we’ll explain how understanding the methylation status of specialized endothelial cells in the liver may one day lead to a long-term treatment for hemophilia – read on to learn how.
Hemophilia is a debilitating inherited blood disorder recently estimated to affect over 1 million people globally . Individuals with hemophilia A, caused by a mutation in the F8 gene, lack sufficient blood levels of the essential clotting protein factor VIII (FVIII) . As a result, these individuals experience prolonged bleeding from wounds and, in severe cases, spontaneous internal bleeding . Currently, the standard treatment for hemophilia A is clotting factor replacement therapy, which requires life-long, regularly-scheduled injections of recombinant FVIII protein [1,2].
While this expensive treatment is available in higher-income countries, it is often entirely inaccessible in countries with more limited medical resources . Alternative therapeutic approaches that provide a long-term and more cost-effective solution are therefore sorely needed. One promising option is a cell-based therapy using iPSC-derived cells expressing FVIII protein. In humans, liver sinusoidal endothelial cells (LSECs) naturally produce the vast majority of FVIII protein, making this a good target for iPSC differentiation. While this approach has already proven successful in resolving a mouse model of hemophilia A , many hurdles remain before this technique can be used therapeutically in humans.
A major open question is how best to effectively assess the quality and characteristics of a differentiated therapeutic cell population at a high-throughput scale. One solution is to identify a universal set of biomarkers that are indicative of the correct differentiation stage – basically, a fingerprint for the correct cell type. In a recent study published in the International Journal of Molecular Sciences, researchers from the Institute of Experimental Hematology and Transfusion Medicine, University of Bonn, including team leader Osman El-Maarri and co-first authors Muhammad Ahmer Jamil and Heike Singer, describe the use of methylation status as such a biomarker .
DNA methylation, which is predominantly found at CpG sites in the mammalian genome, is essential for establishing cell identity during events like differentiation and development . Indeed, distinct methylation patterns are associated with specific tissues, developmental stages and times during differentiation. According to El-Maarri and Jamil, “Every cell has its own epigenome that is its specific fingerprint.”
With this in mind, El-Maarri and Jamil analyzed the methylation status in LSECs at different stages in their maturation, along with more traditional biomarkers like gene expression patterns. They first examined the methylation pattern in fetal LSECs, which have been shown to exhibit better engraftment in mice than adult cells following transplantation , and compared this to the pattern in adult LSECs. To do this, they used a variety of complementary approaches for methylation detection.
“It was a long study that started with an Illumina 450k methylation array, and then for further understanding, we used EPIC arrays. After that, as the genomic coverage of arrays is less than sequencing, we performed WGBS [using the QIAseq Methyl Library Kit],” said El-Maarri and Jamil.
Once they had identified differentially methylated regions between the adult and fetal LSECs, they moved on to confirming the methylation differences between the cell types using a more targeted approach. “After identifying targets from every technique, it was essential to see the single-read methylation status for specific regions. Therefore, we used the QIAseq Targeted Methyl Panels for further validation of our differentially methylated regions,” said the group. “The idea behind the use of the targeted methyl panel was to validate the targets from different techniques like WGBS and arrays, and also to provide a panel of regions to identify the differentiation stage.”
Using these techniques, the group was able to define a methylation fingerprint that was unique to fetal LSECs. They found that methylation status acted as a stable, accurate marker of the developmental stage: “There are expression markers, physiological markers and of course methylation markers. For example, in terms of LSECs, they have expression markers like FCN3, CD36, LYVE1 or others, and are also known to have high endocytic activity and fenestrae, whereas methylation markers were not studied earlier. We observed in our analysis that methylation markers are much more stable for analysis, whereas expression markers are sensitive for handling conditions.”
As one of the most effective routes for therapeutic cell production would involve using patient-derived iPSCs differentiated into FVIII-expressing cells, the group then analyzed their genomic regions of interest in different endothelial cell types and their precursors. “We identified that progenitor endothelial cells are closer to fetal LSECs and need more maturation and changes in methylation to possess specific properties of matured LSECs. A methylation profile provides a quantitative measurement for differentiation.”
According to El-Maarri and Jamil, “We now have in our hands methylation markers that can be used as stable markers to analyze the progression of differentiated cells into functional LSECs, which otherwise is difficult to follow precisely on the mRNA level. In addition, methylation bisulfite sequencing can reveal purity/heterogeneity of the derived/obtained cells that otherwise cannot be revealed by RNA-seq.”
El-Maarri and Jamil’s work in developing a protocol for characterizing the molecular profile of isolated and in vitro-differentiated LSECs represents an important step towards developing a cell-based therapeutic for hemophilia A. “LSECs are a very special type of cell and molecular profiling of LSECs could help researchers and doctors understand these cells better, which can further be used for developing new diagnostics or therapies.”
By characterizing these cells and identifying their methylation fingerprint, the group has laid the groundwork for future studies of LSECs. And because methylation status can be used as a universal biomarker, a similar approach can be applied to studying other inherited diseases in the future.