Abstract
Myelodysplastic syndromes (MDS) are a heterogeneous group of clonal stem cell diseases characterized by inefficient hematopoiesis and risk of progression to acute myeloid leukemia with poor prognosis. Although massive parallel sequencing studies have revealed a number of genomic alterations associated with MDS, functional consequences of these alterations remain poorly understood, mainly due to a difficulty in the ex vivo culture of primary MDS cells and a lack of good animal models. Induced pluripotent stem cells (iPSCs) from MDS patients are expected to provide a new platform for elucidation of the pathogenesis of MDS.
We attempted to generate iPSCs from peripheral blood mononuclear cells of a MDS patient (RAEB-1 by WHO classification) with chromosome 20q deletion, using episomal methods. We successfully established more than 30 iPSC lines derived from Non-T cells as well as 6 iPSC lines derived from T cells at the same time. Karyotyping and SNP-CGH analysis revealed that most of the Non-T-cell-derived iPSC lines (Del20q-iPSC lines) have the isolated 20q deletion at q11.2-13.1 identical to those of the primary MDS cells, whereas all T-cell-derived iPSC lines (NK-T-iPSC lines) have normal karyotype. In order to evaluate chromosome stability, we validated karyotype of 3 randomly selected Del20q-iPSC lines after 30 passages and found no additional chromosomal aberrations other than deletion 20q. Del20q-iPSC lines displayed characteristic morphology and expressed pluripotent stem cell markers at the levels comparable to those in isogenic NK-T-iPSC lines and ES cell lines. Nine randomly selected Del20q-iPSC lines and all 6 NK-T-iPSC lines formed teratomas.
Next, we performed microarray analysis in CD34+38-CD43+lineage- hematopoietic progenitor cells (HPCs) re-induced from 6 Del20q-iPSC lines and 3 NK-T-iPSC lines. 315 genes were up-regulated (fold change >2) and 437 genes were down-regulated (fold change <0.5) in Del20q-iPSC-derived HPCs compared to isogenic NK-T-iPSC-derived HPCs. In particular, expression levels of 48 genes located on 20q11.2-13.1 had reduced expression by at least 2 fold (76 genes by 1.5 fold).
Finally, we investigated the potential of hematopoietic differentiation in 9 Del20q-iPSC lines and 6 isogenic NK-T-iPSC lines. The efficiency of HPC production assessed by the OP9 co-culture system and the embryoid body differentiation culture system was comparable between Del20q-iPSC lines and NK-T-iPSC lines. However, colony forming capacity of iPSC-derived HPCs in methylcellulose culture and granulocyte and erythroid differentiation of iPSCs were severely impaired in all tested Del20q-iPSC lines (CFU-C numbers: 23±4 vs 114±16 per 2,500 HPCs, p< .001; CD66b+/CD11b+ cells: 3.9±1.2% vs 49.0±6.7%, p< .001; CD235a+ cells: 3.8±2.9% vs 32.2±5.2%, p< .001, in Del20q-iPSC lines vs NK-T-iPSC lines respectively). These results indicate that Del20q-iPSC lines are capable of exhibiting the identical feature of the MDS patient.
This iPSC-based system could be useful for studying the precise molecular mechanisms of MDS and may also allow testing new therapeutic compounds under genetically defined conditions.
Disclosures
Yamanaka: iPS Academia Japan: Consultancy.
Generation of SIV resistant T cells and Macrophages from Nonhuman Primate Induced Pluripotent Stem Cells with Edited CCR5 locus
