Functional analysis of a chromosomal deletion associated with myelodysplastic syndromes using isogenic human induced pluripotent stem cells

Abstract

Chromosomal deletions associated with human diseases, such as cancer, are common, but synteny issues complicate modeling of these deletions in mice. We use cellular reprogramming and genome engineering to functionally dissect the loss of chromosome 7q (del(7q)), a somatic cytogenetic abnormality present in myelodysplastic syndromes (MDS). We derive del(7q)- and isogenic karyotypically normal induced pluripotent stem cells (iPSCs) from hematopoietic cells of MDS patients and show that the del(7q) iPSCs recapitulate disease-associated phenotypes, including impaired hematopoietic differentiation. These disease phenotypes are rescued by spontaneous dosage correction and can be reproduced in karyotypically normal cells by engineering hemizygosity of defined chr7q segments in a 20-Mb region. We use a phenotype-rescue screen to identify candidate haploinsufficient genes that might mediate the del(7q)- hematopoietic defect. Our approach highlights the utility of human iPSCs both for functional mapping of disease-associated large-scale chromosomal deletions and for discovery of haploinsufficient genes.

Figure 1. Generation of del(7q)- and isogenic karyotypically normal iPSCs from patients with MDS

(a) Scheme of strategy for the generation of del(7q)- and karyotypically normal iPSCs from patients with MDS. (b) Histology of representative teratomas derived from one normal (N-) and one del(7q)-MDS-iPSC line derived from each of the two patients (no. 2 and no. 3), showing trilineage differentiation (upper panels: endoderm, middle panels: mesoderm, lower panels: ectoderm). Scale bars, 100 μm. (c) Representative karyotypes of one normal (N-) and one del(7q)- iPSC line derived from each patient. (d) aCGH analysis of one representative iPSC line from each patient with a corresponding isogenic normal iPSC line as diploid control. The blue probe color indicates deletion (1 copy) and the white color normal diploid dosage. Both patients harbor large terminal chromosome 7q deletions starting at position 92,781,474 (patient no. 2) and 62,024,527 (patient no. 3). (e) Whole exome genetic characterization of del(7q)- and karyotypically normal iPSCs from MDS patient no. 2. Venn diagrams on the right: the black circle represents the somatic variants (n=34) identified in the patient BM; the blue and red circles represent the variants found in the MDS-2.13 del(7q)-iPSC line and the N-2.12 isogenic normal iPSC line, respectively. The former completely overlap with the variants of the MDS clone, indicating that this iPSC line captures the entire genetic repertoire of the MDS clone. There is no overlap between the MDS clone variants and the variants found in the normal isogenic line, demonstrating that the latter is derived from a normal residual hematopoietic cell that is unrelated to the cell that gave rise to the MDS clone.

Figure 2. MDS-iPSCs have diminished hematopoietic differentiation potential

(a) Left panels: CD34 and CD45 expression at days 10, 14 and 18 of hematopoietic differentiation in representative normal and del(7q)-MDS-iPSC lines. Right panels: CD34 and CD45 expression and co-expression at days 10, 14 and 18 of hematopoietic differentiation, as indicated, in all iPSC lines tested. Each graph shows the percentage of cells within the quadrants included in the corresponding red box. Mean and SEM are shown. Each line was tested in 1–4 independent differentiation experiments. For those lines that were differentiated more than once, the mean value is shown. ***P<0.001. (b) Hematopoietic colony assays in methylcellulose at day 14 of hematopoietic differentiation. The number of colonies from 5,000 seeded cells is shown. (CFU-GEMM: colony-forming unit-granulocyte, erythrocyte, monocyte, megakaryocyte; CFU-GM: colony-forming unit-granulocyte, monocyte; CFU-G: colony-forming unit-granulocyte; CFU-M: colony-forming unit-monocyte; BFU-E: burst-forming unit-erythrocyte) (c) Assessment of lineage markers CD33 (myeloid), GPA (Glycophorin A) or CD235 (erythroid) and CD41a (megakaryocytic) at day 10 of hematopoietic differentiation. (d) Cell viability measured by a luminescence assay based on ATP quantitation (upper panels) and by DAPI staining (lower panels) on days 10 and 14 of hematopoietic differentiation, as indicated. Viability in the upper panels is given relative to viability on day 1 of hematopoietic differentiation. Mean and SEM are shown. Each line was tested in 1–4 independent differentiation experiments. For those lines that were differentiated more than once, the mean value is shown. *P<0.05, **P<0.01. (e) May-Giemsa staining of cells cultured for an additional 12 days in erythroid differentiation media. In normal cells we can morphologically identify cells at several stages of maturation from proerythroblast (arrowhead) to basophilic, polychromatophilic (black arrows) and orthochromatic (white arrows) erythroblasts. No morphological changes of progression to maturation are seen in MDS cells. Scale bars, 10 μm.

Figure 3. Spontaneous compensation for chromosome 7q dosage imbalance rescues the hematopoietic defect of MDS-iPSCs

(a) Karyotype of line MDS-3.9, derived from patient no. 3, harboring a derivative chromosome from a 1;7 chromosomal translocation [der(1;7)(q10;p10), the entire long arm of one copy of chromosome 7q is missing and part of chromosome 1q is translocated in its place, identical to the translocation seen in all MDS-iPSC lines from patient no. 3, see also Fig. 1c, MDS-3.1], in addition to two normal chromosomes 7. (b) qPCR measurement of copy number of a region on 7q31.2 in the del(7q)-iPSC line MDS-2.13 at increasing passage numbers, as indicated. (c) Southern blot probing integration sites of the vector used for reprogramming of the MDS-2.13 line at passage number 12 (haploid for 7q) and 40 (diploid for 7q). (d) Karyotyping of MDS-2.13 (see Fig. 1c) at passage number 40 (MDS-2.13C) showing duplication of the normal chromosome 7 without additional karyotypic changes. (e) aCGH analysis confirming the karyotypic finding. The red color indicates amplification (3 copies) and the white color normal diploid dosage. (f) qPCR measurement of copy number with different probes along the length of chromosome 7, as indicated, in the del(7q)-iPSC line MDS-2.A3 at passage 10 and 24 (MDS-2.A3C). (g) Karyotype of line MDS-2.A3C. (h) aCGH analysis of the del(7q)-iPSC line MDS-2.A3 at passage 10 (MDS-2.A3) and passage 40 (MDS-2.A3C). The blue color indicates deletion (1 copy) and the white color normal diploid dosage. (i) CD34 and CD45 expression at days 10, 14 and 18 of hematopoietic differentiation. Representative panels of the normal isogenic line N-2.A2 and the dosage corrected MDS line MDS-2.A3C. (j) CD45 expression at day 14 of hematopoietic differentiation. Mean and SEM are shown. n.s.: not significant.

Figure 4. Engineering chr7q deletions in normal hPSCs

(a) Overview of strategy for engineering chromosome 7q deletions. An AAV carrying a positive (puro) and a negative (HSV-tk) selection gene is targeted into chr7q and correctly targeted clones are positively selected in a first step. In a second step, Cre recombinase is transiently expressed and clones that have lost the targeted copy of chr7q are selected with GCV. IDLV, integrase-deficient lentiviral vector. (b) aCGH specific for chromosome 7 in all engineered del(7q)-hPSC clones analyzed, as indicated. The blue color indicates deletion (1 copy), the red color amplification (3 copies) and the white color normal diploid dosage. Lower panel: chromosome 7 ideogram. The purple box indicates the ~40 Mb region (approximately 112,920,418 – 152,127,281) functionally mapped in the panel of clones with engineered chromosome 7q deletions. Its 5′ and 3′ borders are defined by the 3′ border of the deletion in clone N-2.12.D-6Cre6 and the 3′ end of the deletion in clone H1-D-2Cre6, respectively. (c, d) CD45 expression at day 14 of hematopoietic differentiation of del(7q)- engineered clones derived from the H1 hESC (c, upper panel and d, left panel) and the N-2.12 iPSC (c, middle and lower panels and d, right panel) line. Mean and SEM are shown. *P < 0.05, **P < 0.01, ***P < 0.001, n.s.: not significant. (e) Methylcellulose assays at day 14 of hematopoietic differentiation. The number of colonies from 5,000 seeded cells is shown. (CFU-GEMM: colony-forming unit-granulocyte, erythrocyte, monocyte, megakaryocyte; CFU-GM: colony-forming unit-granulocyte, monocyte; CFU-G: colony-forming unit-granulocyte; CFU-M: colony-forming unit-monocyte; BFU-E: burst-forming unit-erythrocyte) (f) Schematic summary of the approach to defining the chr7q critical region. The purple box indicates the ~40 Mb region functionally mapped in the panel of clones with engineered chromosome 7q deletions from b. The orange box denotes the ~30 Mb region duplicated in the rescued clone MDS-2.A3C, shown in Fig. 3h. Their overlap defines a critical region of ~20 Mb spanning cytobands q32.3-q36.1 (nucleotides approximately 131,706,336- 152,127,281).

Figure 5. A phenotype-rescue screen identifies candidate haploinsufficient genes in del(7q)-MDS

(a) Scheme of genetic screen. Expression microarray analyses of two isogenic pairs of iPSCs with one or two copies of chr7q were used to select candidate haploinsufficient genes, which were then each tagged to a unique barcode and cloned to construct a lentiviral ORF library. The library was transduced as a pool into del(7q)-MDS-iPSCs and genes that rescued hematopoiesis were identified by means of enrichment in sorted CD45⁺ progenitors differentiated from the cells. (b) Heat map of gene enrichment from 3–6 independent experiments. Fold enrichment was calculated from the sequencing reads as %representation in CD45⁺ cells over % representation in undifferentiated cells. Values between 0–1.0 are depicted as “not enriched”. Barcodes with read counts lower than 100 (marked as “low representation”) were not included in the analysis. Genes within the critical region (red box) that were recurrent (i.e. in at least 2 independent experiments) hits (i.e. had enrichment >1.5-fold) are shown in red. See also Supplementary Table 11. (c) Day 14 of hematopoietic differentiation of a del(7q)-iPSC line (MDS-2.12-D-8Cre23) transduced with lentiviral vectors expressing HIPK2, ATP6V0E2, LUC7L2, EZH2, ADCK2, AGK, GALNT11 or SSBP1, as indicated, compared to the untransduced (UT) line. One representative experiment is shown for each condition. (d) CD45 expression at day 14 of hematopoietic differentiation of the groups shown in (c). Mean and SEM are shown. *P<0.05.

Figure 6. Validation of chr7q haploinsufficient genes

(a) Experimental scheme. CB CD34+ cells were prestimulated for 48 h and transduced with a lentiviral vector encoding shRNA or scramble. 48 h later the cells were plated in colony-forming assays or in liquid erythroid induction culture. (b) Methylcellulose assays of CB CD34⁺ cells expressing shRNAs against HIPK2, ATP6V0E2, LUC7L2, EZH2, or scramble, as indicated. Number of colonies from 1000 seeded cells is shown. The mean of duplicate experiments (from two independent transductions) is shown. (CFU-GEMM: colony-forming unit-granulocyte, erythrocyte, monocyte, megakaryocyte; CFU-GM: colony-forming unit-granulocyte, monocyte; CFU-G: colony-forming unit-granulocyte; CFU-M: colony-forming unit-monocyte; BFU-E: burst-forming unit-erythrocyte) (c) Absolute number of GPA⁺ cells in vitro generated from CB CD34⁺ cells expressing shRNAs against HIPK2, ATP6V0E2, LUC7L2, EZH2 or scramble in erythroid media. Absolute numbers of GPA⁺ cells were calculated from the total cell counts and the percentage of GPA⁺ cells by flow cytometry. The mean values of duplicate experiments from two independent transductions are shown. (d) Schematic representation of the EZH2 locus with the position of the gRNA target sequence, the BstXI restriction sites and the primers used for screening of CRISPR/Cas9-targeted alleles indicated. The gRNA sequence is shown in green and the PAM motif in red. The sequence of the BstXI restriction site is underlined. The beginning of the gRNA sequence is 23 nt downstream of the ATG. (e) Representative image of RFLP analysis for screening of single cell clones. The 821 band (resistant to BstXI digest) indicates the presence of indels. Clones with indels in only one allele are shown in blue and clones with indels in both alleles are shown in green. Clones with both alleles intact are shown in black. (f) Sequences of the wild-type EZH2 locus and the two clones (clone 37 and clone 31) harboring monoallelic indels, as indicated. Sequences shown were obtained from the 821 bp band and therefore correspond to the disrupted allele. The other allele was confirmed to be intact by sequencing the 370 and 451 bp bands (not shown). Each clone harbors a different 14-nucleotide deletion introducing a frame-shift. (g) Western blot analysis (upper panels) and quantification (lower panels) of EZH2 expression in the two clones harboring monoallelic inactivation of EZH2, compared to that in the parental hESC line HUES8. (h) Day 14 of hematopoietic differentiation of the two clones with monoallelic inactivation of EZH2, compared to that of the parental hESC line HUES8 and of normal iPSCs. One of two replicate experiments is shown.