Functional analysis of a chromosomal deletin 7q associated with MDS .

ARTICLE

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

Andriana G Kotini1–3, Chan-Jung Chang1–3,19, Ibrahim Boussaad4,5,19, Jeffrey J Delrow6, Emily K Dolezal7, Abhinav B Nagulapally5,8, Fabiana Perna9, Gregory A Fishbein5,10, Virginia M Klimek11, R David Hawkins5,8, Danwei Huangfu12, Charles E Murry5,13–16, Timothy Graubert17, Stephen D Nimer7 & Eirini P Papapetrou1–5,13,18©2015Nature America, Inc. All rights reserved.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 http://wendang.chazidian.comrge hemizygous deletions are found in most tumors and might be both hallmarks and drivers of cancer1. Hemizygous segmental 2chromosomal deletions are also frequent in normal genomes. Apart from rare prototypic deletion syndromes (e.g., Smith-Magenis, Williams-Beuren, 22q11 deletion syndromes), genome-wide asso-ciation studies (GWAS) have implicated genomic deletions in neuro-developmental diseases like schizophrenia and autism3, prompting the hypothesis that deletions might account for an important source of the ‘missing heritability’ of complex diseases3,4.Unlike translocations or point mutations, chromosomal deletions are difficult to study with existing tools because primary patient material is often scarce, and incomplete conservation of synteny (homologous genetic loci can be present on different chromosomes or in different physical locations relative to each other within a chromosome across species) complicate modeling in mice. Dissecting the role of specific chromosomal deletions in specific cancers entails, first, determin-ing if a deletion has phenotypic consequences; second, determining if the mechanism fits a “classic” recessive (satisfying Knudson’s “two-hit” hypothesis) or a haploinsufficiency model and, finally, identifying the specific genetic elements that are lost. Classic tumor suppressor genes were discovered through physical mapping of homozygous deletions5. More recent data suggest that sporadic tumor suppressor genes are more likely to be mono-allelically lost and to function through haploinsufficiency (wherein a single functional copy of a gene is insufficient to maintain normal function)6,7.MDS are clonal hematologic disorders characterized by ineffec-tive hematopoiesis and a propensity for progression to acute myeloid leukemia (AML)8. Somatic loss of one copy of the long arm of chro-mosome 7 (del(7q)) is a characteristic cytogenetic abnormality in MDS, well-recognized for decades as a marker of unfavorable progno-sis. However, the role of del(7q) in the pathogenesis of MDS remains elusive. The deletions are typically large and dispersed along the entire long arm of chr7 (ref. 9). Homology for human chr7q maps to four different mouse chromosomes.

1Department of Oncological Sciences, Icahn School of Medicine at Mount Sinai, New York, New York, USA. 2The Tisch Cancer Institute, Icahn School of Medicine at Mount Sinai, New York, New York, USA. 3The Black Family Stem Cell Institute, Icahn School of Medicine at Mount Sinai, New York, New York, USA. 4Division of Hematology, Department of Medicine, University of Washington, Seattle, Washington, USA. 5Institute for Stem Cell and Regenerative Medicine, University of Washington, Seattle, Washington, USA. 6Genomics Resource, Fred Hutchinson Cancer Research Center, Seattle, Washington, USA. 7Sylvester Comprehensive Cancer Center, Miller School of Medicine, University of Miami, Miami, Florida, USA. 8Division of Medical Genetics, Department of Medicine, University of Washington, Seattle, Washington, USA. 9Molecular Pharmacology and Chemistry Program, Memorial Sloan-Kettering Cancer Center, New York, New York, USA. 10Department of Pathology and Laboratory Medicine, David Geffen School of Medicine at UCLA, Los Angeles, California, USA. 11Department of Medicine, Memorial Sloan-Kettering Cancer Center, New York, New York, USA. 12Developmental Biology Program, Sloan-Kettering Institute, New York, New York, USA. 13Department of Pathology, University of Washington, Seattle, Washington, USA. 14Center for Cardiovascular Biology, University of Washington, Seattle, Washington, USA. 15Department of Bioengineering University of Washington, Seattle, Washington, USA. 16Division of Cardiology, Department of Medicine, University of Washington, Seattle, Washington, USA. 17MGH Cancer Center, Massachusetts General Hospital, Boston, Massachusetts, USA. 18Division of Hematology and Medical Oncology, Department of Medicine, Icahn School of Medicine at Mount Sinai, New York, New York, USA. 19These authors contributed equally to this work. Correspondence should be addressed to E.P.P. (eirini.papapetrou@mssm.edu).

Received 6 November 2014; accepted 13 February 2015; published online 23 March 2015; doi:10.1038/nbt.3178

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Genetic engineering of human pluripotent stem cells (hPSCs) has been used to model point mutations causing monogenic diseases in an isogenic setting10,11, but not disease-associated genomic deletions. We used reprogramming and chromosome engineering to model del(7q) in an isogenic setting in hPSCs. Using different isogenic pairs of hPSCs harboring one or two copies of chr7q, we characterized hematopoietic defects mediated by del(7q). We used spontaneous rescue and genome editing experiments to show that these pheno-types are mediated by a haploid dose of chr7q material, consistent with haploinsufficiency of one or more genes. We functionally map a 20-Mb fragment spanning cytobands 7q32.3–7q36.1 as the crucial region and identify candidate disease-specific haploinsufficient genes in BMMCs and fibroblasts, and variants with variant allele frequency >1% in fibroblasts or <10% in BMMCs (Fig. 1e and Supplementary Table 4). Similarly, 73 and 48 somatic SNVs were identified in the del(7q)- and isogenic normal iPSC line, respectively. The del(7q)- iPSC line contained all 34 variants of the MDS clone, whereas the iso-genic normal line harbored none. These variants included two genes found recurrently mutated in MDS, SRSF2 and PHF6. Resequencing of the rest of the iPSC lines derived from this patient (Supplementary Table 2) showed that all of the del(7q)- (6 out of 6) and none of the karyotypically normal iPSC lines (0 out of 15) harbored these mutations. These results unequivocally demonstrate that the isogenic iPSCs represent normal cells that contain no premalignant lesions and using a phenotype-rescue screen. Finally, we show that the hemato-poietic EZH2defect is mediated by the combined haploinsufficiency of date genes residing in this region. The strategy reported here could be , in cooperation with one or more out of three additional candi-applied to study the phenotypic consequences of segmental chromo-somal deletions and for haploinsufficient gene discovery in a variety of human cancers, and neurological and developmental diseases.

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devrRESULTS

esGeneration of del(7q)- and normal isogenic iPSCs

er sWe derived iPSC lines from hematopoietic cells of two patients (patients thno. 2 and no. 3) with del(7q)- MDS (girandFig. 1 and Supplementary Tables 1 llAlikely to co-exist with the MDS clone in the bone marrow of MDS 2). We reasoned that residual normal hematopoietic cells are .patients and that we could exploit this to reprogram both del(7q)- cnand isogenic karyotypically normal cells in parallel. We were able to I ,aderive del(7q)- and karyotypically normal (N-) iPSC lines at the same cirreprogramming round from both patients (emWe used an excisable lentiviral vector expressing Supplementary Table 3). APOU5F1 ergramming), KLF4OCT4 (also known as 12,13, c-MYC (also known as MYCBP) and SOX2 for repro-utaiPSC lines derived from the same starting cell from being considered and performed vector integration analysis to exclude Nindependent lines and thus obtain true biological replicate lines from 51each patient (02iPSC lines harbored identical deletions to those present in the starting Supplementary Fig. 1a,b). Karyotyping showed that the ©patient cells (tive genomic hybridization (aCGH) (Fig. 1c), which we mapped by array-based compara-all standard criteria of pluripotency, before and after excision of the Fig. 1d). These iPSC lines met reprogramming vector, including expression of pluripotency mark-ers, demethylation of the teratomas after injection into immunodeficient mice (OCT4 promoter and formation of trilineage Supplementary Fig. 1cFig. 1b and respectively, two and three del(7q)- iPSC lines (MDS-2.13, MDS-2.A3, –f). We selected from patients no. 2 and no. 3, MDS-3.1, MDS-3.4, MDS-3.5), as well as four and one karyotypically normal iPSC line (N-2.8, N-2.12, N-2.A2, N-2.A11, N-3.10) before or after vector excision, for further studies (Supplementary Table 1).goes genetic evolution through multiple cycles of mutation acquisi-Recent large-scale sequencing studies have shown that MDS under-tion14originate . To determine whether the karyotypically normal iPSC lines potential ‘founding’ clone harboring mutations that might have arisen from a completely normal hematopoietic cell or from a before the del(7q), we performed whole exome sequencing of bone marrow mononuclear cells (BMMCs) and fibroblasts (as paired nor-mal sample) from MDS patient no. 2, as well as of one del(7q)- and one normal iPSC line derived from BMMCs of this patient (MDS-2.13 and N-2.12, respectively) and identified the somatic variants. Thirty-four single-nucleotide variants (SNVs) were identified with confidence in the BMMCs by comparison to the fibroblast sample, after filtering out intronic variants, variants with less than 30 reads

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that the del(7q)- iPSCs accurately capture the genetic composition of the MDS clone.

Decreased hematopoietic differentiation of del(7q)- MDS iPSCsTo iPSCs we optimized an embryoid body–based differentiation pro-assess the hematopoietic differentiation potential of the MDS tocol hemato-endothelial marker, and CD45, the key marker of definitive and monitored the course of differentiation using CD34, a hematopoietic cells15embryonic stem cell (hESC) and iPSC lines typically express CD34 . In this differentiation protocol, normal human by day 14, and is followed by loss of CD34, as the cells become more day 6. Co-expression of CD45 appears by day 10, peaks at differentiated more assessed the intra- (than 90% along of cells hematopoietic are CD45+ lineages. By day 18, typically inter- (Supplementary Fig. 2a(Supplementary ,bFig. 2a). We differentiation process using normal iPSC lines derived from bone Supplementary Fig. 2a,b, panels III, IV) line variation in this , panels I, II and c) and marrow cells of patients no. 2 and no. 3, as well as one additional iPSC line derived previously from umbilical cord blood CD34+CB-3.1-Cre (ref. 13). On day 10 we observed some interexperimental cells, variation in expression of CD34 and CD45 markers, whereas little variation was present on day 14 and even less on day 18. Notably, increasing passage number did not alter the differentiation potential (from the same individual (in the same or different reprogramming Supplementary Fig. 2a,b, panel II) and distinct iPSC lines derived experiments) exhibited variation similar to the intra-line variation (tially lower than that seen in iPSC lines derived from different genetic Supplementary Fig. 2a,b, compare panels II and III) and substan-backgrounds on day 14 (and IV). These results show that different genetic background is the Supplementary Fig. 2a,b, compare panels III main source of variation and that variation among different lines from the same individual is no greater than the intra-line variation.

ation in the hematopoietic differentiation of normal iPSCs, we then Having established the reproducibility and range of normal vari-tested lines exhibited greatly reduced hematopoietic differentiation poten-the hematopoietic potential of MDS iPSCs. All MDS-iPSC tial (Fig. 3aFig. 2a) and clonogenic capacity (Fig. 2b and Supplementary MDS-iPSC lines also showed increased cell death during differentia-) affecting all myeloid hematopoietic lineages (Fig. 2c). The tion compared to their isogenic and nonisogenic normal iPSC lines (decrease in the efficiency of differentiation, MDS-iPSC lines differ-Fig. 2d and Supplementary Fig. 3b). In addition to an absolute entiated with slower kinetics (vation was corroborated by assessing the kinetics of the emergence Supplementary Fig. 3c,f). This obser-and disappearance of a transient wave or primitive-like hematopoi-esis, marked by co-expression of CD41a and CD235 (glycophorin A; GPA)16tion along the erythroid lineage, MDS-iPSC lines, unlike their normal (Supplementary Fig. 3d,e). Furthermore, upon differentia-isogenic lines, did not acquire cell-surface markers or morphological

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d

Cartilage

Cartilage

Cartilage

Cartilage

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q11.21q11.23q21.11

q21.3q22.1

q31.1

q33q34q35q36.1

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Present in MDS-2.13 iPSCs?

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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 (nos. 2 and 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 (one copy) and white, 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 bone marrow; 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.

features of erythroid cells (Fig. 2e and Supplementary Fig. 3g,h). These phenotypes of ineffective hematopoiesis, reduced or absent clonogenicity, and increased cell death are consistent with the pheno-type of ex vivo cultured primary MDS cells from bone marrow or peripheral blood17,18.

MDS-iPSC hematopoiesis rescue by chr7q dosage correctionApart from the profound hematopoietic defects, at least some of the MDS-iPSC lines exhibited slower growth in culture at the undifferenti-ated state compared to their isogenic normal iPSCs, as observed by smaller colony size (Supplementary Fig. 4a) and confirmed by growth competition assays against a GFP-marked normal iPSC line derived from N-2.12 (Supplementary Fig. 4b,c). Because hPSCs can spontaneously

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acquire chromosomal abnormalities and those that provide a growth

advantage can be selected over time in culture (as the clone that har-bors them outgrows the rest of the population), we reasoned that we might be able to isolate clones that spontaneously acquired a second copy of chr7q material. Indeed, by screening additional iPSC lines derived from patient no. 3 for chr7q dosage using a qPCR assay, we identified one line, MDS-3.9, that had acquired an additional chro-mosome 7, an event not detectable in the starting bone marrow cells (Fig. 3a). Additionally, by monitoring chr7q dosage in the del(7q)- MDS iPSC lines over time, we were able to detect spontaneous dosage cor-rection in one del(7q)- MDS iPSC line derived from patient no. 2, MDS-2.13, at passage 40 (Fig. 3b). We excluded the possibility of con-tamination of the culture with an unrelated line by reprogramming

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ARTICLESa

CD33

©2015Nature America, Inc. All rights reserved.

GPCD41Figure 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 s.e.m. are shown. Each line was tested in one to four 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: CFU-granulocyte, erythrocyte, monocyte, megakaryocyte; CFU-GM: CFU-granulocyte, monocyte; CFU-G: CFU-granulocyte; CFU-M: CFU-monocyte; BFU-E: burst-forming unit–erythrocyte). (c) Assessment of lineage markers CD33 (myeloid), GPA 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 s.e.m. are shown. Each line was tested in one to four 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.

vector integration analysis (Fig. 3c). Karyotyping and aCGH showed that dosage correction had again occurred through duplication of the normal intact chromosome 7 (Fig. 3d,e). As expected, the deriva-tive line MDS-2.13C grew faster than its parental line in subsequent culture, albeit not as fast as its isogenic karyotypically normal iPSCs (Supplementary Fig. 4d). The same phenomenon of spontaneous acquisition of an extra chromosome 7 occurred in two additional independent clones of the same iPSC line, obtained after single-cell subcloning of early-passage cells (before or after reprogramming vector excision), selection of a chr7q haploid clone and subsequent expansion for more than ten passages. Notably, a large chromosome 3q deletion also initially present in this iPSC line (and in the patient bone marrow cells it was derived from) remained unchanged (Figs. 1c and 3d). Moreover, we never observed acquisition of extra chr7 copies in normal iPSC lines by close qPCR monitoring over more than 50 passages, in line with reports from systematic studies

19,20of chromosomal aberrations commonly observed in hPSCs.

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These events of spontaneous compensation of chr7q dosage imbal-ance provided evidence for strong in vitro selection for these events.

We therefore screened additional del(7q)- MDS iPSC lines using more probes along the length of chr7 to potentially find clones with dupli-cations of only part of chr7q. We were able to identify an informa-tive clone derived from a second del(7q)- iPSC line from patient 2, MDS-2.A3, which over time acquired a second copy of a telomeric part of chr7q without duplication of the entire chr7 (Fig. 3f,g). aCGH analysis showed that the duplicated region was approximately 30 Mb and spanned bands 7q32.3–7qter (Fig. 3h and Supplementary Table 5). The hematopoietic differentiation potential of this ‘corrected’ line, MDS-2.A3C, was fully restored to levels comparable to those of normal iPSCs (Fig. 3i,j). The corrected line maintained the SRSF2 and PHF6 mutations. These data strongly suggest that reduced chr7q dosage is responsible for the hematopoietic defect of MDS iPSCs, as

the latter is fully rescued by acquisition of a second copy of chr7q material derived from duplication of the existing copy, and pinpoint

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Figure 3 Spontaneous compensation for

MDS-3.9

chromosome 7q dosage imbalance rescues MDS-2.13C p40the hematopoietic defect of MDS iPSCs.

(a) Karyotype of line MDS-3.9, derived 12345from patient no. 3, harboring a derivative 12345

chromosome from a 1;7 chromosomal 6789101112

translocation (der(1;7)(q10;p10), the entire 6789101112

131415161718

long arm of one copy of chromosome 7q

131415161718is missing and part of chromosome 1q is 19202122XY

translocated in its place, identical to the

19202122XY

translocation seen in all MDS-iPSC lines from

N-2.A2MDS-2.A3Cpatient no. 3, see also Fig. 1c, MDS-3.1),

06.10.21.1in addition to two normal chromosomes 7.

MDS-2.13C(b) qPCR measurement of copy number of a Day 10

region on 7q31.2 in the del(7q)- iPSC line

–1.0001.0041.052.939.459.3

MDS-2.13 at increasing passage numbers, as

22.251.015.648.6indicated. (c) Southern blot probing integration 7q31.27p14.3

sites of the vector used for reprogramming 7q357q11.22Day 14MDS-2.A3C p27

7q21.12of the MDS-2.13 line at passage number 12

16.610.110.725.0(haploid for 7q) and 40 (diploid for 7q).

16.093.95.284.0

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(d) Karyotyping of MDS-2.13 (see Fig. 1c) at

passage number 40 (MDS-2.13C) showing Day 18

6789101112duplication of the normal chromosome 7 0.70.200

without additional karyotypic changes. (e) aCGH 131415161718

analysis confirming the karyotypic finding. The 19202122XYp10p24red color indicates amplification (three copies) n.s.

100

and the white color, normal diploid dosage.

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(f) qPCR measurement of copy number with

60chr7

different probes along the length of q11.21q11.23q21.11q21.3q22.1q31.1q33q34q35q36.140chromosome 7, as indicated, in the 20MDS-2.A3del(7q)- iPSC line MDS-2.A3 at passage 010 and 24 (MDS-2.A3C). (g) Karyotype of

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line MDS-2.A3C. (h) aCGH analysis of the del(7q)- iPSC line MDS-2.A3 at passage 10

–1.00

(MDS-2.A3) and passage 40 (MDS-2.A3C).

The blue color indicates deletion (one 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 s.e.m. are shown. n.s.: not significant.

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©2015Nature America, Inc. All rights reserved.

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an ~30 Mb 7q32.3–7qter fragment (nucleotides approximately 131,706,336–159,128,530) as the critical region.

Engineering heterozygous chr7q loss in normal hPSCs

To further determine the impact of chr7q hemizygosity on the cellular phenotype, we generated perfectly isogenic pairs of hPSCs harboring one or two copies of chr7q by engineering chr7q deletions in normal hPSCs. To this end, we combined a recently reported strategy for deleting supernumerary chromosomes using adeno-associated vec-tor (AAV)-mediated gene targeting of an HSV-tk transgene21 with a previously described strategy using Cre-mediated recombination of inverted loxP sites22,23 (Fig. 4a). We constructed an AAV vector containing two loxP sites in inverted orientation and a positive (puro) and negative (HSV-tk) selection marker. The vector was targeted to a near-telomeric region of chromosome 7 (7q36.3) by ATG-trap of the DNAJB6 gene, highly expressed in hPSCs (Supplementary Fig. 5a). The hESC line H1, as well as the normal iPSC line N-2.12, were transduced with the vector. Fourteen and five puromycin-resistant clones from line N-2.12 and H1, respectively, were picked, initially screened by PCR specific for the targeted allele and subsequently by Southern blot analysis (Supplementary Fig. 5b). Thirteen of the 14 N-2.12 clones were found to be correctly targeted and one to have a random vector integration. Five out of the five H1 clones were correctly targeted, one of which harbored an additional random integration. One targeted clone from each line (H1-D-1-1 and N-2.12-D-1-1, respectively, hereafter referred to as H1-D and N-2.12-D)

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was selected on the basis of lowest background resistance to ganciclovir

and transduced with an integrase-deficient lentiviral vector tran-siently expressing Cre recombinase12,13. After single-cell subcloning and selection with ganciclovir, clones were first screened by qPCR probing different regions along the length of chromosome 7. Three H1-derived and seven N-2.12-derived clones (H1-D-Cre1, H1-D-2Cre6, H1-D-Cre7, N-2.12-D-Cre10, N-2.12-D-Cre32, N-2.12-D-2Cre4, N-2.12-D-8Cre21, N-2.12-D-8Cre23, N-2.12-D-Cre44 and N-2.12-D-6Cre6) were selected after screening 23 and 46 clones, respectively, and after excluding clones with additional chromosomal abnormali-ties by karyotyping (Supplementary Fig. 5c). aCGH showed that these clones harbored different deletions spanning variable lengths along the entire chromosome 7q (Fig. 4b and Supplementary Table 6). Clones N-2.12-D-Cre-44 and N-2.12-D-6Cre6 were among the selected ganciclovir-resistant clones, although the chr7q deletion in these clones does not include the tk gene. Ganciclovir resistance may have been conferred by a mutation or other mechanisms. Clones N-2.12-D-Cre10 and N-2.12-D-Cre32 shared the exact same deletion and hence were presumably derived from the same clone, but were treated as separate clones.

Based on our previous phenotypic characterization data (Fig. 2 and Supplementary Fig. 2), we selected the percentage of CD45+ cells at day 14 of hematopoietic differentiation as the most appropriate readout. Eight of the ten clones showed markedly diminished hemato-poietic differentiation potential and clonogenic capacity (Fig. 4c–e), similarly to our del(7q)-MDS iPSCs. Notably, two of the seven clones

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