Modeling Myelodysplastic Syndromes with human iPSCs

Figure 1.  Generation of MDS- and isogenic normal iPSCs. a, By reprogramming hematopoietic cells from the bone marrow of patients with MDS we can derive both iPSCs from the MDS clone and normal iPSCs from residual normal hematopoietic cells. b, A teratoma comprising tissues of all three germ layers generated from a patient-derived iPSC line.

Myelodysplastic syndromes (MDS) are clonal hematologic disorders characterized by ineffective hematopoiesis and a propensity for progression to acute myeloid leukemia (AML). Once thought to be rare disorders, MDS are now recognized to be among the most common blood cancers, but their pathogenesis remains poorly understood. Current tools to study MDS are limited: animal models do not faithfully recapitulate human MDS, no cell lines exist and primary cells are heterogeneous and hard to grow ex vivo.

Figure 2. MDS-iPSCs recapitulate disease phenotypes. Left panels: MDS-iPSCs generate fewer hematopoietic progenitors than normal isogenic iPSCs. Middle: MDS-iPSCs do not mature along the erythroid lineage. Right: Undifferentiated MDS-iPSCs grow slower and form smaller colonies than normal iPSCs.

We have recently established the first iPSC models of MDS that offer exciting new possibilities for the study of the cellular and molecular pathogenesis of MDS and its genetics and clonal evolution. They also provide a powerful platform for phenotype-based genetic and chemical screens to identify new therapeutic targets.

Modeling disease-associated chromosomal deletions with CRISPR

Chromosomal deletions associated with human disease are common in normal and cancer genomes and may constitute an important component of the “missing heritability” of complex diseases and the “dark matter” of cancer genetics. Unlike translocations or point mutations, chromosomal deletions are difficult to study because physical mapping in primary patient material is limited by the rarity of informative cases and incomplete conservation of synteny complicates their modeling in mice.

Figure 3. Engineering chromosomal deletions in human iPSCs. a, A strategy combining AAV-mediated gene targeting of an HSV-tk transgene for negative selection with a modified Cre-loxP strategy. Upon transient expression of Cre-recombinase, loss of the targeted chromosome is selected with ganciclovir. b, c, Selection of iPSC clones with deletions of chromosome 7 by karyotyping and aCGH. 

We developed an approach to model disease-related chromosomal deletions in human iPSCs.  By using modified Cre-loxP and CRISPR/Cas9 technologies we can engineer hemizygous deletions of specific chromosomal fragments. These allow us to functionally map disease phenotypes and identify candidate disease genes through phenotype-rescue screens.

Figure 4. Chromosome engineering using CRISPR. Using CRISPR/Cas9 with one or more gRNAs we can engineer terminal and interstitial chromosomal deletions.