Induced Pluripotent stem cells as a model system to study disease progression in myelodysplastic syndromes
Myelodysplastic syndromes (MDS) are a heterogeneous group of age-associated hematopoietic diseases characterised by abnormal blood cell maturation and a high propensity for leukemic transformation. It is a clonal disease thought to originate in the haematopoietic stem cell (HSC). Therapeutic strategies in high-risk patients include demethylating agents and cytotoxic drugs, however, 50-60% of these patients do not respond to the treatment and progress to the worst stage. Therefore, there is an unmet clinical need to better understand the mechanisms leading to these blood disorders with the ultimate aim to facilitate the development of improved diagnostic and therapeutic strategies. By making use of somatic reprogramming and CRISPR-Cas9 tools, our goal in this study was to generate an in vitro model of low-risk and high-risk MDS that could help to determine the molecular mechanisms leading to disease progression. Peripheral blood mononuclear cells were collected from patient MDS27 when he was diagnosed with low-risk MDS; with cells at this stage harbouring ASLX1, SRSF2 and RUNX1 mutations. These cells were used to generate hiPSC using the non-integrated methods; sendai virus and episomal. Several clones were confirmed to harbour the same somatic mutations as the cells of origin. The pluripotent characteristics of the iPSC clones generated from patient MDS27 as well as from a hiPSC control line were confirmed. Furthermore, the differentiation potential of iPSC into hematopoietic progenitor cells indicated the ability of iPSC to differentiate to hematopoietic stem/progenitor cells (HSPCs) (CD34+ CD43+, CD34+ CD45+). In addition, HSPCs derived from MDS27-iPSC were able to form the different colony-forming unit (CFU) in methylcellulose semi-solid medium with less potential when compared to the hiPSC control. Moreover, the study of the erythroid and myeloid lineage differentiation in liquid cultures indicated that HSPCs derived from MDS27-iPSC could differentiate to such lineages but with aberrant morphology, validating our in vitro system. To generate a model for high-risk MDS, a mutation in C/EBPα causing disruption of the DNA binding domain was generated by CRISPR in the MDS27-iPSC cells, mimicking the additional mutation observed in MDS27 cells when the patient progressed to high-risk MDS. The differentiation of the C/EBPα mutant line (high-risk MDS-iPSC) showed a significant reduction in the myeloid and erythroid colony forming units (CFUs) with a block in granulocytic CFU formation. Furthermore, study of the myeloid lineage indicated that the high-risk MDS-iPSC had an impaired myeloid differentiation due to altered expression of key genes required for myeloid differentiation such as PU.1, GATA2, LMO2 and RUNX1. The study of the erythroid lineage in high-risk MDS27 indicated that the four mutant genes induce erythroid differentiation, increasing the aberrant morphology. Our approach highlights the utility of human iPSCs to understand the molecular mechanisms leading to disease progression and their use as a platform for drug screening which will help to improve diagnostic and therapeutic strategies.